key: cord- -sc ersky authors: zhang, wei-ying; xu, fu-qing; shan, chang-liang; xiang, rong; ye, li-hong; zhang, xiao-dong title: gene expression profiles of human liver cells mediated by hepatitis b virus x protein date: - - journal: acta pharmacol sin doi: . /aps. . sha: doc_id: cord_uid: sc ersky aim: to demonstrate the gene expression profiles mediated by hepatitis b virus x protein (hbx), we characterized the molecular features of pathogenesis associated with hbx in a human liver cell model. methods: we examined gene expression profiles in l-o -x cells, an engineered l-o cell line that constitutively expresses hbx, relative to l-o cells using an agilent k human -mer oligonucleotide microarray representing more than , unique, well-characterized homo sapiens genes. western blot analysis and rna interference (rnai) targeting hbx mrna validated the overexpression of proliferating cell nuclear antigen (pcna) and bcl- in l-o -x cells. meanwhile, the brdu incorporation assay was used to test cell proliferation mediated by upregulated cyclooxygenase- (cox- ). results: the microarray showed that the expression levels of genes were remarkably altered; of the genes were upregulated and genes were downregulated in l-o -x cells. the altered genes were associated with signal transduction pathways, cell cycle, metastasis, transcriptional regulation, immune response, metabolism, and other processes. pcna and bcl- were upregulated in l-o -x cells. furthermore, we found that cox- upregulation in l-o -x cells enhanced proliferation using the brdu incorporation assay, whereas indomethacin (an inhibitor of cox- ) abolished the promotion. conclusion: our findings provide new evidence that hbx is able to regulate many genes that may be involved in the carcinogenesis. these regulated genes mediated by hbx may serve as molecular targets for the prevention and treatment of hepatocellular carcinoma. hepatitis b virus (hbv) infection is one of the major risk factors for the development of hepatocellular carcinoma (hcc) [ ] . however, the mechanism of hbv-induced hcc remains unclear [ ] . the hbv genome is a partially doublestranded dna molecule with four open reading frames (orfs) termed s, c, p, and x [ ] . the x gene of hbv (hbx) encodes a amino acid polypeptide that is essential for viral infection and replication and plays a crucial role in the development of hcc [ ] . although hbx does not bind to dna, it exerts a pleiotropic effect on diverse cellular functions as a transcriptional co-activator. hbx exhibits many activities in vitro. in a cell culture system, hbx is able to activate the transcription of host genes, such as c-myc, as well as viral genes [ , ] . hbx is also involved in different cytoplasmic signaling pathways. the majority of hbx is localized in the cytoplasm, where it interacts with and stimulates protein kinases such as protein kinase c, janus kinase/stat, ikk, pi- -k, stress-activated protein kinase/jun n-terminal kinase, and protein kinase b/akt. hbx induces centrosome hyperamplification and mitotic aberration by activation of the ras-mek-mapk pathway, which may contribute to genomic instability during hepatocarcinogenesis [ ] . hbx can transactivate various cellular transcriptional elements, such as ap- , ap- , nf-κb, and camp response element (cre) sites. consequently, hbx can affect the activities of various transcriptional elements in the host cell and elicit different cellular responses. previously, we demonstrated that hbx upregulated both the expression and the activity of htert in hepatoma [ ] . we also found that hbx was able to upregulate the expression of survivin, which suggests that survivin may be involved in the carcinogenesis of hcc that is mediated by hbx [ ] . cdna microarray is a powerful tool for identifying disease-related gene expression profiles in biological samples. using cdna microarray, we examined gene expression profiles in a model of hepatoma cells stably transfected with hbx [ ] . these data from the hepatoma cell model showed progressive changes during tumor development. however, the mechanism of hepatoma mediated by hbx remains unclear. in the present study, we examined the gene expression profiles of l-o -x cells by cdna microarray analysis. when compared with the gene expression profiles of h -x cells [ ] , our findings provide new insight into the molecular mechanism of carcinogenesis mediated by hbx in human liver cells. cell culture the human liver l-o cell line (purchased from nanjing keygen biotech co ltd, nanjing, china), which originated histologically from normal human liver tissue that had been immortalized by stable transfection of the htert gene, was used previously [ , [ ] [ ] [ ] . l-o -p (cell line from l-o cells stably transfected with empty pcdna plasmid) and l-o -x (cell line from l-o cells stably transfected with the hbx gene) were established as described previously [ ] . l-o , l-o -p, and l-o -x cells were cultured in rpmi- medium (gibco, usa) containing u/ ml penicillin, μg/ml streptomycin and % fetal calf serum. cultures were incubated in a humidified atmosphere with % co at °c. gene expression profiling analysis an agilent k human -mer oligonucleotide microarray (capitalbio corp, china) containing more than unique, well-characterized homo sapiens genes was used. double-stranded cdnas (containing the t rna polymerase promoter sequence) were synthesized from µg of total rna using the cbcscript reverse transcriptase with cdna synthesis system according to the manufacturer's protocol (capitalbio corp, china) with the t oligo (dt). cdna labeled with a fluorescent dye (cy or cy -dctp) was produced by eberwine's linear rna amplification method and the subsequent enzymatic reaction and was improved by capitalbio. the klenow enzyme labeling strategy was adopted after reverse transcription using cbcscript ii reverse transcriptase. all procedures for hybridization and slide and image processing were carried out according to the manufacturer's instructions. the slides were washed, dried, and scanned using a confocal luxscan tm scanner and the obtained images were then analyzed using luxscan tm . software (both from capitalbio corp, china). the procedures were repeated for a replicate experiment with independent hybridization and processing. for individual channel data extraction, faint spots for which the intensities were below units after background subtraction in both channels (cy and cy ) were removed. a space-and intensity-dependent normalization based on a lowess program was employed. to avoid false positive results, multiple testing corrections were considered. in each experiment, three types of positive controls (hex, four housekeeping genes, and eight yeast genes) and two types of negative controls ( % dmso and twelve negative control sequences from the operon oligo database) were used. each probe was printed in triplicate. furthermore, we performed the fluorescence-exchange microarray analysis. we performed three independent cdna microarray experiments to obtain more precise data. for two-color designs, intensity reproducibility was calculated both within and across the two different dyes to assess the impact of the dye on the resulting measurement. for within-dye calculations, the technical replicates of samples labeled with the same dye across the microarrays were considered, and for across-dyes calculations, all replicates for a given sample labeled with either dye were evaluated. comparisons between the combinations of p values and fold-change thresholds were given, with the differentially expressed genes identified using a one-sample t-test of the sample b to sample a (b/a) ratio data, including five replicates for each site. two p values (p< . and p< . ) and three fold-change (fc) thresholds (fc> . , fc> . and fc> . ) were acceptable in the microarray analysis. rna interference the hbv x gene was cloned into psilencer . to create psilencer . -x. the following primers were used: sense, ′-gatcccggtcttaca-taagaggactttcaagagaagtcctcttatgt-aagaccttttttggaaa- ′; antisense, ′-agc tttt-ccaaaaaaggtcttacataa gaggacttctctt-gaaagtcctccttatgtaagaccggg- ′ [ ] . the purified vector was transfected using lipofectamine into l-o -x cells for h, as described previously [ ] . the expression level of the hbx protein in l-o -x cells was examined by western blot analysis. treatment with an inhibitor of cox- according to the data, we found that the ptgs gene was upregulated in l-o -x cells ( table ). the ptgs gene encodes cox- [ ] . therefore, we investigated the effect of hbx on cox- using an inhibitor of cox- . the l-o , l-o -p, and l-o -x cells were cultured as described above in a -well plate for h, and then the cells were re-cultured in serum free medium for h. briefly, l-o -x cells were treated with μmol/l indomethacin (indo, sigma-aldrich, usa, inhibitor of cox- ) for h. the level of cox- in the treated l-o cells was examined by western blot analysis. meanwhile, the proliferation of l-o -x cells treated with indo was examined by the brdu incorporation assay. western blot analysis l-o -x, l-o -p, and l-o cells were lysed in protein lysis buffer ( . mmol/l tris-hcl, ph . , % sds, % -mercaptoethanol, % glycerol) and the protein concentration was determined using the -d quant kit (amersham biosciences, buckinghamshire, uk). following electro-transfer onto polyvinylidene difluoride membranes, the membranes were blocked with % non-fat dry milk in . % triton x -tbs (ttbs) and incubated overnight at °c with specific primary antibodies. the following primary antibodies were used as described previously [ , ] : pcna (neomarkers, fremont, ca, usa, : dilution); bcl- (neomarkers, fremont, ca, usa, : dilution); hbx (obtained from the fox chase institute for cancer research, philadelphia, pa, usa, : dilution); cox- (neomarkers, fremont, ca, usa, : dilution); and β-actin (neomarkers, fremont, ca, usa, : dilution). staining was performed with an hrp-linked secondary antibody (ge healthcare bio-science, usa). the protein bands were visualized by an enhanced chemiluminescence (ecl) kit according to the manufacturer's specifications (ge healthcare bio-sciences, usa). brdu incorporation assay dna synthesis was measured using a ′-bromodeoxyuridine (brdu, sigma, usa) incorporation assay. briefly, l-o , l-o -p, and l-o -x cells were seeded in -well plates for h, and then the cells were serum starved in defined medium overnight. additionally, l-o , l-o -p, and l-o -x cells were treated with μmol/l indo for h. the brdu incorporation assay was performed according to a previously published protocol [ ] . the brdu labeling index was assessed by point counting through a nikon te inverted microscope (nikon, tokyo, japan) using a ×objective lens. a total of - nuclei were counted in - representative fields. the labeling index was expressed as the number of positively-labeled nuclei/total number of nuclei. all groups (n= in each group) were performed. statistical analysis all experiments were repeated independently, at least three times. values are given as means±sd. the analyzed data from two groups were compared using student's t test. a p< . was considered statistically significant. differential expression profiles in l-o -x cells previously, we investigated progressive changes in hepatoma cells stably transfected with hbx and found some differentially expressed genes [ ] . however, those data only showed the distinctive gene pattern in the context of heptoma. to distinguish the differential expression of genes in normal human liver l-o cells and hepatoma cells mediated by hbx, we examined the differential expression profiles in l-o -x cells by cdna microarray analysis ( figure ). the cdna microarray showed that the expression levels of genes were remarkably altered, of those genes were upregulated and genes were downregulated in the l-o -x cells. the altered genes were associated with signal transduction pathways, cell cycle, metastasis, transcriptional regulation, immune response, metabolism, and other processes (table ) . to avoid false positive results, each probe was printed in triplicate using a smartarray tm microarrayer (capitalbio corp beijing, china). the cdna microarray the black points represented ratios that ranged from . to . and belonged to the no difference group. the red points represented the ratios that were > . and the green points represented the ratios that were < . , both of which indicate that gene expression was most probably altered. used was the fluorescence-exchange microarray. additionally, we performed three independent cdna microarray analyses to get more precise data. in table , the expression of the selected candidates was either up-or down-regulated by at least two folds. when compared with the differential expression profiles in h -x cells [ ] , we found that the data from the differential expression profiles in l-o -x cells differed substantially. only three genes, pcna, bmp , and il- , were altered in both h -x cells and l-o -x cells. hbx was responsible for the upregulation of pcna and bcl- to further validate the candidate genes in the cdna microarray and to preliminarily investigate the molecular alterations of proliferating cell nuclear antigen (pcna) and bcl- in the l-o -x cell line, we examined the regulation of pcna and bcl- at the protein level by western blot analysis. the data showed that the expression levels of pcna and bcl- were increased in l-o -x cells (figure a) . we further confirmed the findings by applying glyco band-scan software (prozyme, san leandro, ca, usa; figure c ). moreover, we investigated the effect of hbx on the regulation of pcna and bcl- by rna interference (rnai) targeting hbx mrna. after transfection, the rnai resulted in the decrease of pcna and bcl- within h ( figure b ), suggesting that hbx was responsible for the upregulation of pcna and bcl- . these data were further confirmed by glyco bandscan software (prozyme, san leandro, ca, usa; figure d ). the upregulation of cox- , mediated by hbx, contributed to the proliferation as shown in table , the cox- gene was upregulated in l-o -x cells. we provided evidence by western blot analysis that the expression of cox- at the protein level was higher in l-o -x cells than in l-o cells ( figure a ). the downregulation of hbx mediated by rnai in l-o -x cells abolished the upregulation of cox- ( figure a) . we further confirmed the findings by applying glyco bandscan software (prozyme, san leandro, ca, usa; figure b ). next, we demonstrated the effect of cox- on proliferation using the brdu incorporation assay. the data showed that the percentage of cells in the s phase significantly increased in l-o -x cells (p< . , vs l-o cells, student's t test). however, enhanced proliferation in l-o -x cells was abolished by treatment with indomethacin (indo, an inhibitor of cox- , sigma-aldrich, usa) (figure ), suggesting that hbx was able to upregulate cox- , which contributed to the proliferation. no statistically significant difference was observed between l-o cells and cells transfected with empty pcdna vector (termed l-o -p). hbx plays a crucial role in hbv-related pathogenesis. some research groups have investigated the gene expression profiles associated with hbx. however, our data differ markedly from that data in these reports [ , ] . hbx can lead to contradictory findings, largely because of the use of different cell types and transformed cells. in the present study, we chose an immortalized human liver cell line as a model to show the basic response of host cells to the hbx gene. using a cdna microarray technique, we identified and classified the genes that were altered as a result of their involvement in an hbx-mediated process (figure ). our findings showed that genes were upregulated and genes were downregulated in l-o -x cells (table ) . most were involved in the cell cycle, signal pathways, metastasis, immune response, metabolism, and other processes. table shows that many genes that have not been reported to associate with hbx in the literature were found by microarray assay, which provides us with valuable clues for the further investigation of hbx. our data showed that cyclin a , pcna and bcl- were upregulated, whereas p was simultaneously downregulated. furthermore, the altered expression of pcna and bcl- was verified by western blot analysis (figure ). hbx upregulates pcna by increasing the recruitment of cbp/p to endogenous pcna promoters [ ] . our microarray data were consistent with this report. cyclin a , a member of the cyclin a family, is related to some types of carcinogenesis [ ] . p is a negative regulator of the cell cycle. bcl- family members form hetero-or homodimers and act as anti-or pro-apoptotic regulators that are involved in a wide variety of cellular activities. cyclindependent kinases (cdks) and cyclin-dependent kinase inhibitors (ckis) play important roles in controlling cell proliferation, differentiation, and apoptosis. defects in cell cycle regulation are common causes of the abnormal proliferation of cancer cells. those proteins, which are mentioned above and are involved in the cell cycle and apoptosis, may greatly influence tumorigenesis. another study indicates that many mapk family members are upregulated in hbvrelated hcc [ , ] . our present data showed that map k , map k and max were upregulated, whereas gadd a was downregulated in l-o -x cells; these changes may be related to rapid proliferation. all biological activities of c-myc require its binding partner, max [ ] . c-myc has been assigned roles in hepatocyte proliferation during liver development and regeneration, control of hepatic metabolism, and the dysregulated growth that occurs during heptocarcinogenesis [ ] [ ] [ ] [ ] . therefore, the enhancement of max may be consistent with the induction of c-myc in l-o -x cells to promote hepatocyte growth and proliferation. in our experiments, gadd a, a p -regulated and dna damage inducible protein, was downregulated. gadd plays a role in g -m arrest in response to dna damage. gadd can bind to multiple important cellular proteins such as pcna, p protein, mtk/mekk -an upstream activator of the jnk pathway -and cdc protein kinase. furthermore, some reports show that gadd can inhibit cell transformation and tumor progression [ , ] . signaling by the wnt family of secreted glycoproteins is essential both in normal embryonic cell-to-cell interactions and in the pathogenesis of a variety of diseases, including cancer [ ] . first, wnt proteins bind to receptors of the frizzled and lrp families on the cell surface. then, through several cytoplasmic relay components, the signal is transduced to activate transcription of wnt target genes. here, we found that both fzd (one of the frizzled and lrp families) and ctbp , which are involved in wnt signaling, were increased. additionally, btrc, which may be a candidate for tumor-suppressive activities [ ] , was decreased in l-o -x cells. indeed, rna interference-mediated knockdown of ctbp (c-terminal binding protein family proteins) decreases cell invasion, and ectopic expression of ctbp enhances tumor cell migration and invasion. importantly, the overexpression of fzd (a cell-surface receptor for molecules in the wnt pathway) acts as a potential contributor to synovial sarcomas. moreover, a humanized antibody against fzd might be a promising treatment for patients with tumors that overexpress fzd [ ] . secreted-type glycoprotein wnts can bind to fzd to transduce signals to the β-catenin-tcf pathway, the jnk pathway, or the calcium signaling pathway. accordingly, variation in the wnt pathway and other signals affected by the wnt pathway that were seen in our study may contribute to the early carcinogenesis mediated by hbx. interactions between cells and their surrounding extracellular matrix are essential for the proper execution and regulation of survival, proliferation, cytoskeletal organization, and migration. furthermore, cell-extracellular matrix contact regulates physiological and pathological processes, such as development, differentiation, and metastasis. laminins, a family of extracellular matrix glycoproteins, are the major noncollagenous constituents of the basement membrane. some reports suggest that a strong correlation between the high expression of lama (one of laminin family) and tumor invasion and metastasis indicates that it is a novel marker for the processes of tumor invasion and metastasis [ ] . our microarray data showed that lama was upregulated in l-o -x cells. the chemokine rantes (regulated on activation normal t-cell expressed and secreted; ccl ) is a member of the cc-chemokine family, a group of small proteins with a highly conserved structure [ ] . using the cdna microarray technique, we found that ccl was highly expressed in l-o -x cells. other studies indicate that ccl s are inflammatory mediators with pro-malignancy activities in breast cancer and that they are shown to mediate many types of tumor-promoting cross-talk between the tumor cells and cells of the tumor microenvironment. tumor-derived ccl can inhibit the t cell response and enhance the growth of murine mammary carcinoma in vivo [ ] . interleukin- (il- ) induces either differentiation or growth of a variety of cells and also plays a role in cell interaction. in our study, il- was upregulated in l-o -x cells relative to l-o cells. binding of il- to its receptor initiates cellular events including activation of jak kinases and activation of ras-mediated signaling. in addition, it has been suggested that il- participates in the malignant progression of prostate cancer [ ] . adhesion of tumor cells to host cell layers and subsequent migration are pivotal steps in cancer invasion and metastasis. organization of the cytoskeleton and cell adhesion molecules plays an important role in this event. some candidates in our study were related to these cell processes, including cldn , cldn , cav , and cav . the family of more than claudin (cldn) proteins comprises one of the major structural elements within the apical tight junction apparatus, a dynamic cellular nexus for maintenance of a luminal barrier, paracellular transport, and signal transduction. loss of normal tight junction functions constitutes a hallmark of human carcinomas. cldn proteins are maintained or elevated in breast, ovarian, pancreatic, and prostate cancers, whereas cldn proteins are diminished in head, neck, and breast tumors [ ] . the down-regulation of caveolin- (encoded by cav ) and caveolin- (encoded by cav ) is found in various types of primary tumors and cancer cell lines, indicating that these are candidate tumor suppressor genes [ , ] . in our data, we also found that hbx greatly affected cellular metabolism, in which hbx upregulated the ptgs gene that encodes cyclooxygenase- (cox- ) (figure ). cox- is overexpressed in various cancers, including esophageal, gastric, colon, and pancreatic cancers [ ] . it is possible that both tumor-and stroma-derived cox- could influence tumor proliferation, angiogenesis and/or immune function. using the brdu incorporation assay, we observed that hbx enhanced the proliferation of l-o cells by upregulating cox- ( figure ). our findings are concurrent with a report that hbx induces cox- through the activation of nf-at to promote tumor cell invasion and metastasis [ ] . loxl catalyzes the crosslinking of collagen and elastin in the extracellular matrix and is assumed to be involved in tumor progress and cell adhesion. some findings support the presumption that loxl plays a role in malignant transformation [ , ] . in this study, we found that only three genes, pcna, bmp and il- , were altered in both h -x cells and l-o -x cells. we considered the possibility that hbx may affect gene expression in different ways in hepatoma h cells and l-o liver cells because the genetic background is different. the gene expression profiles mediated by hbx in l-o cells may reflect alterations in the early stage of carcinogenesis. our study has revealed some novel candidate genes by cdna microarray analysis ( table ) that provide new insight into the pathogenesis mediated by hbx. consequently, an understanding of the molecular mechanisms involved in this dynamic process will aid in identifying novel potential targets for earlier diagnosis and more specific therapies. prof xiao-dong zhang and li-hong ye designed the research; wei-ying zhang performed the research; prof rong xiang discussed the research; fu-qing xu and chang-liang shan help to perform part of the research. wei-ying zhang analyzed the data and wrote the paper. prof xiao-dong zhang revised the paper. pathogenesis of hepatitis b virus infection genetic mechanisms of hepatocarcinogenesis effects of hepatitis b virus x protein on the development of liver cancer hepatitis b virus transcriptional activators: mechanisms and possible role in oncogenesis x protein of hepatitis b virus functions as a transcriptional corepressor on the human telomerase promoter mitotic aberration coupled with centrosome amplification is induced by hepatitis b virus x oncoprotein via the ras-mitogen-activated protein/extracellular signal-regulated kinase-mitogen-activated protein pathway effects of hepatitis b virus x protein on human telomerase reverse transcriptase expression and activity in hepatoma cells hepatitis b virus x protein upregulates survivin expression in hepatoma tissues progressive changes in hepatoma cells stably transfected with hepatitis b virus x gene a specific splicing variant of svh, a novel human armadillo repeat protein, is upregulated in hepatocellular carcinomas upstream binding factor up-regulated in hepatocellular carcinoma is related to the survival and cisplatin-sensitivity of cancer cells identification of a natural mutant of hbv x protein truncated amino acids at the cooh terminal and its effect on liver cell proliferation farnesoid x receptor antagonizes nuclear factor kappab in hepatic inflammatory response promotion of cell proliferation by hbxip via upregulation of human telomerase reverse transcriptase in human mesenchymal stem cells monitoring s phase progression globally and locally using brdu incorporation in tk (+) yeast strains cdna microarray analysis of early gene expression profiles associated with hepatitis b virus x protein-mediated hepatocarcinogenesis altered proteolysis and global gene expression in hepatitis b virus x transgenic mouse liver the hepatitis b virus x protein promotes tumor cell invasion by inducing membrane-type matrix metalloproteinase- and cyclooxygenase- expression the role of the phospho-cdk /cyclina recruitment site in substrate recognition insight into hepatocellular carcinogenesis at transcriptome level by comparing gene expression profiles of hepatocellular carcinoma with those of corresponding noncancerous liver myc and max associate in vivo c-myc is required for the glucose-mediated induction of metabolic enzyme genes induction of ribosomal genes and hepatocyte hypertrophy by adenovirus-mediated expression of c-myc in vivo overexpression of c-myc in diabetic mice restores altered expression of the transcription factor genes that regulate liver metabolism sequential protooncogene expression during rat liver regeneration gadd a function in suppressing cell transformation and tumor malignancy the gadd and myd genes define a novel set of mammalian genes encoding acidic proteins that synergistically suppress cell growth wnt signaling in disease and in development the btrc gene, encoding a human f-box/wd -repeat protein, maps to chromosome q -q therapeutic potential of antibodies against fzd , a cell-surface protein, for synovial sarcomas lama , highly expressed in human hepatocellular carcinoma from chinese patients, is a novel marker of tumor invasion and metastasis chemokines: a new classification system and their role in immunity ccl -ccr interaction provides antiapoptotic signals for macrophage survival during viral infection interleukin- and prostate cancer progression loss of the tight junction protein claudin- correlates with histological grade in both ductal carcinoma in situ and invasive ductal carcinoma of the breast caveolin- is down-regulated in human ovarian carcinoma and acts as a candidate tumor suppressor gene caveolin- inhibits anchorage independent growth, anoikis and invasiveness in mcf- human breast cancer cells altered proteolysis and global gene expression in hepatitis b virus x transgenic mouse liver the hepatitis b virus x protein functionally interacts with crebbinding protein/p in the regulation of creb-mediated transcription reduction of lox-and loxl -mrna expression in head and neck squamous cell carcinomas mapping of a deletion interval on p - in prostate cancer by gene dosage pcr table . genes significantly altered in l- -x cells and their classifications in signal pathway. abbreviation up-or down-name regulation key: cord- - i mj jw authors: chen, xue-han; xu, yu-jia; wang, xiao-ge; lin, peng; cao, bi-yin; zeng, yuan-ying; wang, qi; zhang, zu-bin; mao, xin-liang; zhang, tie title: mebendazole elicits potent antimyeloma activity by inhibiting the usp /c-maf axis date: - - journal: acta pharmacol sin doi: . /s - - - sha: doc_id: cord_uid: i mj jw c-maf is a critical oncogenic transcription factor that contributes to myelomagenesis. our previous studies demonstrated that the deubiquitinase usp stabilizes c-maf and promotes myeloma cell proliferation and survival; therefore, the usp /c-maf axis could be a potential target for myeloma therapy. as a concept of principle, the present study established a usp /c-maf-based luciferase system that was used to screen an fda-approved drug library. it was found that mebendazole, a typical anthelmintic drug, preferentially induced apoptosis in c-maf-expressing myeloma cells. moreover, oral administration of mebendazole delayed the growth of human myeloma xenografts in nude mice but did not show overt toxicity. further studies showed that the selective antimyeloma activity of mebendazole was associated with the inhibition of the usp /c-maf axis. mebendazole downregulated usp expression and disrupted the interaction between usp and c-maf, thus leading to increased levels of c-maf ubiquitination and subsequent c-maf degradation. mebendazole inhibited c-maf transcriptional activity, as confirmed by both luciferase assays and expression measurements of c-maf downstream genes. in summary, this study identified mebendazole as a usp /c-maf inhibitor that could be developed as a novel antimyeloma agent. multiple myeloma (mm) is a hematological malignancy derived from plasma cells that produce antibodies [ ] . mm features gene mutations and chromosomal aberrations, including deletions, inversions, interchanges, and translocations. these genetic abnormalities lead to discrepant responses of mm patients to individual therapeutic agents [ ] . there are at least six types of chromosomal translocations, among which t ( : ) results in the overexpression of the transcription factor gene c-maf [ , ] . c-maf modulates the transcription of several key genes, including cyclin d (ccnd ), integrin b (itgb ), ampk-related protein kinase (ark ), and c-c chemokine receptor (ccr ), by binding to its specific recognition element on the upstream regulatory region of these genes [ ] . among these c-maf-modulated genes, ccnd is a key factor in cell cycle progression and thus promotes mm cell proliferation, while itgb mediates mm cell adhesion to bone marrow stromal cells and facilitates mm cell migration and invasion, thereby conferring chemoresistance [ ] . therefore, c-maf promotes mm cell proliferation and survival. the expression level of the c-maf protein is negatively associated with the overall survival of mm patients [ , ] . therefore, targeting c-maf degradation is proposed as a promising therapeutic approach for mm [ , ] . our previous studies show that c-maf can be degraded through the ubiquitin-proteasome pathway [ ] , while the deubiquitinase usp abolishes k -linked polyubiquitination, thus stabilizing the c-maf protein and promoting mm cell survival [ ] . in contrast, knockdown of usp leads to c-maf degradation and mm cell apoptosis [ ] . therefore, targeting the usp /c-maf axis may be a potential therapy for mm. in the present study, we established a usp /c-maf luciferase system and applied it to screen a library composed of small molecule chemical drugs approved by the us food and drug administration. from this screen, we identified mebendazole, an agent widely used for the treatment of human gastrointestinal parasitic infections [ ] , selectively induces mm cell apoptosis by targeting usp /c-maf signaling. cell culture human embryonic kidney cells (hek t) were maintained in dulbecco's modified eagle's medium. mm cell lines, including rpmi- , lp , and u , were cultured in iscove's modified dulbecco's medium. all media were supplemented with % fetal bovine serum, glutamine and antibiotics, as described previously [ ] . the c-maf and usp plasmids were constructed as described previously [ , ] . a nucleotide sequence containing six tandem maf recognition elements (mares) was chemically synthesized by genewiz, inc. (suzhou, jiangsu, china) and inserted into a pgl vector to generate the mare.luci reporter plasmid (pmare.luci) [ , ] . gene transfection one day before transfection, hek t cells were seeded in six-well plates. when grown to % confluence, cells were subjected to gene delivery using polyethyleneimine (pei) as described previously [ ] . screening of the fda-approved drug library hek t cells were cotransfected with the c-maf, usp , and pmare.luci plasmids. forty-eight hours later, cells were isolated and reseeded into -well plates at a density of × cells/well for h before each drug (final concentration: μm) was added and were then incubated for another h. cells were then collected for the luciferase activity assay, which was performed according to the manufacturer's instructions, as described previously [ ] . the drugs that decreased the luciferase activity by more than % were selected for further evaluation as candidate usp /c-maf axis inhibitors [ ] . immunoblot (ib) assays after cell lysates were prepared, protein concentrations were determined by a bca assay (beyotime). equal amounts of protein ( µg) were separated by sds-page and transferred to polyvinylidene difluoride membranes. after normalization, the blots were probed with appropriate antibodies, as described previously [ ] . immunoprecipitation (ip) after being treated with mebendazole for h, lp and rpmi- cells were harvested to prepare whole cell lysates, and the ip assay was conducted as described previously [ ] . after ip, proteins were resuspended in ripa buffer and used for an ib assay [ ] . apoptosis detection via flow cytometric analysis cells were treated with mebendazole for h followed by staining in dark for min with annexin v-fitc and propidium iodide (pi, multi sciences biotech co., ltd., hangzhou, china). cells were then subjected to analysis on a bd ® flow cytometer as described previously [ ] . reverse transcription polymerase chain reaction (rt-pcr) total rna was extracted using trizol (sangon biotech, shanghai, china). total rna ( µg) was reverse transcribed using an easyscipt first-strand cdna synthesis kit (vazyme biotech co., ltd., nanjing, china) according to the manufacturer's instructions. pcr amplification was carried out using the following primers: usp , '-cgg att tga cct tag cg- ' (forward) and '-ctg cca tcg aag tag cg- ' (reverse); c-maf, '-aaa aag gaa ccg gtg gag ac- ' (forward) and '-ggt agc cgg tca tcc agt ag- ' (reverse); and gapdh, '-aat ccc atc acc atc ttc c- ' (forward) and '-cat cac gcc aca gtt tcc- ' (reverse). the primers used for ccnd , itgb , and ark were described previously [ ] . pcr products were visualized by gold-view ® staining (transgen biotech co., ltd., beijing, china) following electrophoresis on % agarose gels. myeloma xenografts in nude mice female balb/c nude mice ( - weeks old) purchased from shanghai slac laboratory animal co. ltd. were used to establish xenograft models with the human mm cell lines lp and rpmi- . when tumors were palpable, mice were randomly divided into three groups. one group received vehicle as the control, and the other two groups were orally administered mebendazole at a dosage of or mg/kg body weight once daily for days. body weight and tumor volumes were measured every other day (tumor volume = tumor length × width / ). at the end of this experiment, tumors and blood samples were collected for further studies. all experiments involving mice were reviewed and approved by the review board of animal care and use of soochow university. statistical analyses statistical differences between the control and the experimental groups were analyzed by student's t-test. identification of c-maf/usp inhibitors by a mid-throughput screen c-maf is sufficient to induce myelomagenesis [ ] and confers chemoresistance to proteasomal inhibitors such as bortezomib [ ] , the mainstay anti-mm agent. our previous studies showed that targeting usp leads to c-maf degradation and mm cell death, suggesting that the usp /c-maf axis is a potential therapeutic target in mm [ ] . to identify anti-mm inhibitors of the usp /c-maf axis, we established a screening system in hek t cells based on mare-driven luciferase activity in the presence of c-maf and usp (fig. a) . these cells were treated for h with each drug from an fda-approved drug library before luciferase activity was measured in cell lysates. the results showed that out of the drugs decreased luciferase activity by > % in the presence of usp and the transcription factor c-maf (fig. b) . these drugs included the anti-mm agent daunorubicin (dnr), the antileukemia agent vincristine (vcr), and other cytotoxic fda-approved anticancer drugs. several drugs that have not yet been approved for cancer treatment were also identified. these drugs included artesunate (art), an antimalarial agent; mebendazole (mbz), an antiparasitic agent; nicergoline (ncg), for the treatment of cognitive, affective, and behavioral disorders of older people; and tolazamide (tlz), for the treatment of type diabetes. in the second screen, the mm cell line lp , which expresses a high level of c-maf [ , ] , was treated with all of the above drugs for h, followed by ib assays to evaluate parp cleavage, a hallmark of apoptosis, and c-maf degradation. the results showed that the classic cytotoxic anticancer drugs daunorubicin and vincristine induced mm cell apoptosis at nanomolar concentrations, in association with c-maf degradation (fig. c) . however, further studies showed that these two drugs downregulated the transcription of c-maf at the mrna level (fig. d) . nicergoline and tolazamide did not show marked parp cleavage activity. artesunate and mebendazole induced mm cell apoptosis as evidenced by parp cleavage and c-maf degradation. because artesunate has been well studied in mm cell apoptosis [ , ] and it downregulated c-maf mrna expression (fig. d) , mebendazole was selected for further studies. mebendazole decreased c-maf protein expression but had no effects on its mrna level (fig. d) , and its potential application in myeloma treatment has not yet been reported. mebendazole inhibits the usp /c-maf axis and induces c-maf degradation via the ubiquitin-proteasome pathway previous studies have demonstrated that the deubiquitinase usp stabilizes c-maf by binding to c-maf and suppressing its polyubiquitination [ ] . the above results showed that mebendazole downregulated c-maf protein expression but had no effect on its mrna expression (fig. ) ; thus, we sought to determine whether mebendazole could suppress usp activity via c-maf ubiquitination. to this end, we first measured the c-maf and usp levels in mebendazole-treated mm cells. the results showed that mebendazole downregulated the protein expression of both usp fig. identification of mebendazole as a promoter of c-maf protein degradation. a the system for screening c-maf/usp inhibitors. b hek t cells were plated in -well plates ( cells per well) h before being cotransfected with plasmids, including the c-maf, usp and pmare.luci plasmids. on the third day, cells were treated with each compound from the drug library for h before being lysed for luciferase assays. the activities of the compounds were expressed as log (sample luciferase rlu/control luciferase rlu). drugs associated with a value of log < - were considered potential inhibitors. c, d lp cells were treated with candidate drugs at the indicated concentrations for h, followed by ib assays and rt-pcr for the indicated proteins/genes and c-maf in both lp and rpmi- cells (fig. a, b) . as expected, mebendazole failed to downregulate c-maf mrna expression, as shown by the rt-pcr assay results (fig. a, b) . notably, we found that usp mrna expression was also decreased by mebendazole, suggesting that mebendazole might suppress usp transcription. because c-maf turnover is processed via the proteasome, we sought to determine whether mebendazole-induced c-maf fig. mebendazole inhibits usp expression and induces c-maf degradation via the ubiquitin-proteasome pathway. a lp and rpmi- cells were treated with mbz at the indicated concentrations for h, followed by ib assays for c-maf and usp . total rna was extracted for rt-pcr to assess c-maf and usp expression. gapdh was used as the internal control. b the c-maf and usp expression levels from a were analyzed by densitometry. c lp and rpmi- cells were treated with mbz or mg at the indicated concentrations for h, followed by immunoblotting for c-maf and gapdh. d hek t cells were cotransfected with the ha-c-maf and myc-usp plasmids for h, followed by mbz treatment for h and subsequent ip/ib assays. e lp and rpmi- cells were treated with mbz at the indicated concentrations for h, followed by cell lysate preparation and ip/ib assays. f hek t cells were cotransfected with the ha-c-maf, myc-usp and flag-ub plasmids for h. cells were then treated with mbz for h before being lysed and subjected to ip/ib assays as indicated. g lp and rpmi- cells were treated with mbz at the indicated concentrations for h, followed by ip/ib assays degradation could be reversed by proteasomal inhibitors. to this end, lp and rpmi- cells were treated with mebendazole followed by treatment with mg . the ib assay results showed that mebendazole downregulated c-maf but that this downregulation was abolished by mg , a typical proteasomal inhibitor (fig. c) , suggesting that mebendazole induces c-maf degradation via the ubiquitin-proteasome pathway. because usp , as a deubiquitinase of c-maf, binds to and suppresses c-maf polyubiquitination, we sought to determine whether mebendazole could interfere with the association of c-maf and usp . therefore, we first evaluated the interaction between usp and c-maf in the presence of mebendazole. the results showed that mebendazole decreased the usp content in the c-maf immunoprecipitates in both exogenous and endogenous contexts, as shown in hek t and mm cells, respectively (fig. d, e) , suggesting that the interaction between usp and c-maf was disrupted by mebendazole. next, we evaluated the polyubiquitination levels of c-maf in hek t, lp and rpmi- cells in the presence of mebendazole. the ip/ib assay results showed that mebendazole increased the polyubiquitination levels of c-maf in all three cell lines (fig. f, g) . these findings thus confirmed the hypothesis that mebendazole prevented usp -mediated c-maf deubiquitination and led to c-maf degradation. mebendazole suppresses the transcriptional activity of c-maf c-maf is involved in myelomagenesis as a transcription factor by promoting the transcription of its target genes, including ccnd , itgb , and ark [ ] . the above results demonstrated that mebendazole suppressed the usp /c-maf axis and promoted c-maf degradation; therefore, mebendazole might downregulate the transcriptional activity of c-maf. to confirm this hypothesis, we used firefly luciferase as the reporter under the regulation of a c-maf recognition element (mare). hek t cells cotransfected with mare.luci, c-maf, and usp were treated with mebendazole for h, followed by a luciferase assay. ectopic expression of usp significantly increased the transcriptional activity of c-maf fig. mebendazole inhibits the transcriptional activity of c-maf. a hek t cells were cotransfected with the ha-c-maf, pmare.luci and myc-usp or flag-usp plasmids. after h, cells were treated with mbz in a dose-dependent manner, as indicated, for another h. cells were harvested for luciferase activity measurements and ib assays. b rpmi- , lp , and u cells were treated with mbz in a dose-dependent manner for h, followed by total rna extraction and rt-pcr analysis. c-g lp and rpmi- cells were treated with mbz at the indicated concentrations for h, followed by quantitative real-time pcr to measure the expression of genes, including usp (c), c-maf (d), ccnd (e), itgb (f), and ark (g). *p < . , **p < . , ***p < . in terms of the measured luciferase activity (fig. a) . notably, this activity was markedly inhibited by mebendazole in a concentration-dependent manner. in contrast, another deubiquitinase, usp , also increased the transcriptional activity of c-maf, but luciferase activity driven by usp was much less affected by mebendazole than luciferase activity driven by usp (fig. a) . to confirm this finding, c-maf-expressing lp and rpmi- and c-maf-deficient u cell lines were treated with mebendazole for h at increasing concentrations, followed by rt-pcr to measure the gene transcription levels. the results showed that mebendazole decreased the mrna levels of all three genes-ccnd , ark , and itgb -that were under the control of c-maf in lp and rpmi- cells that express c-maf but not in u cells that lack c-maf (fig. b) . to confirm these findings, we next performed quantitative real-time pcr to measure the expression of all genes. consistent with the results of the above assays, mebendazole fig. mebendazole preferentially induces the apoptosis of c-maf-expressing mm cells. a lp , rpmi- , and u cells were treated with mbz at the indicated concentrations for h, followed by ib assays to evaluate the expression of apoptotic proteins, as indicated. b the ratio of cleaved parp to pro-parp and c the ratio of cleaved caspase- to pro-caspase- . d mm cells were treated with mbz ( . µm) for h, followed by annexin v-fitc and pi staining and flow cytometric analyses. ***p < . downregulated the transcription of usp (fig. c), ccnd (fig. e) , itgb (fig. f) , and ark ( fig. g) but had no effects on c-maf transcription (fig. d) . therefore, mebendazole probably affects c-maf transcriptional activity by inducing c-maf degradation via the inhibition of usp . because the usp /c-maf axis plays a critical role in promoting mm cell proliferation and survival, and interference with usp and c-maf leads to mm cell apoptosis [ ] , we next evaluated the effects of mebendazole on mm cell survival and apoptosis. to this end, the mm cell lines lp , rpmi- , and u were exposed to mebendazole for h at increasing concentrations, followed by ib assays. as shown in fig. a , the cleavage and activation of the apoptotic markers parp and caspase- were significantly increased in both lp and rpmi- cells that express c-maf. in contrast, a lower level of parp cleavage fragments was detected in u cells that lack c-maf (fig. a, b) . caspase- , one of the major apoptotic executor enzymes, was not activated in u cells (fig. a-c) . these findings suggest that mebendazole probably preferentially induces the apoptosis of c-maf-expressing mm cells. to further confirm this hypothesis, all three mm cell lines were exposed to mebendazole at a very low concentration ( . μm) followed by annexin v/pi staining and flow cytometric analyses. as shown in fig. d , . μm mebendazole induced marked apoptosis in lp and rpmi- cells but not in u cells, suggesting that u cells are less sensitive to mebendazole due to their deficiency in c-maf expression. therefore, mebendazole, especially at low concentrations, preferentially induces mm cell apoptosis in a manner dependent on the expression of c-maf. mebendazole exerts synergistic antimyeloma effects with a usp inhibitor previous studies identified wp as a small molecule compound that inhibits the deubiquitinase activity of usp [ ] . in the above studies, mebendazole was demonstrated to downregulate usp expression and disrupt the interaction between usp and c-maf. we sought to determine whether these two agents could synergize to induce mm cell apoptosis. to this end, lp and rpmi- cells were treated with wp and mebendazole alone or in combination (at a very low concentration; μm for wp and . μm for mebendazole), followed by ib assays to investigate parp cleavage, a hallmark of cell apoptosis. the results showed that parp was not markedly cleaved by wp or mebendazole due to the very low concentrations used; however, it was significantly cleaved by cotreatment with these two agents. notably, c-maf expression was also decreased by cotreatment (fig. a, b) . therefore, combined treatment with mebendazole and wp demonstrated synergy against mm. we next examined the effects of mebendazole in combination with daunorubicin, a typical anti-mm drug. these two drugs alone induced limited parp cleavage, but combined treatment significantly increased parp cleavage (fig. c, d) . collectively, these results thus showed marked synergy between mebendazole and usp inhibitors or anti-mm drugs such as daunorubicin. mebendazole delays the growth of human multiple myeloma xenografts in a nude mouse model the above studies provided strong evidence that mebendazole inhibits the usp /c-maf axis and induces mm cell apoptosis. to further investigate the effects of mebendazole against human mm in vivo, two myeloma xenograft models were established by subcutaneous injection of lp or rpmi- cells in the right flanks of nude mice. when the tumors were~ mm in diameter, mice in each model were randomly divided into three groups. each group was orally administered vehicle or mebendazole at a dose of or mg/kg for a continuous days. the tumor sizes and mouse body weights were monitored every other day. tumor growth was significantly decreased by mebendazole, and this effect was dose-dependent (fig. a, b) . at the dose of mg/kg, mebendazole almost completely suppressed the growth of myeloma xenografts in both models (fig. a, b) . however, mebendazole did not show toxicity to mice, as shown via body weight measurements (fig. c, d) and biochemical analyses of blood. there were no marked alterations in the levels of alanine aminotransferase (alt), aspartate aminotransferase (ast), alkaline the ratio of cleaved parp to pro-parp from a. c lp and rpmi- cells were treated with mbz ( . µm) and dnr ( nm) alone or in combination for h, followed by ib assays for c-maf, parp, and gapdh. d the ratio of cleaved parp to pro-parp from c. **p < . , ***p < . fig. mebendazole delays myeloma tumor growth in nude mice without toxicity. human multiple myeloma cells (lp and rpmi- ) were injected subcutaneously into nude mice at a density of million cells/site. when tumors were palpable, mice were orally administered vehicle or mbz ( mg/kg or mg/kg body weight) in pbs containing % sesame oil daily for a continuous days. tumor volumes (a, b) and body weights (c, d) were monitored every other day. **p < . ; ***p < . . e, f tumor samples from each model were subjected to ib analyses for usp , c-maf, caspase- , and parp with specific antibodies phosphatase (alp), urea, creatinine (crea), or albumin in mice treated with mebendazole compared with those levels in vehicletreated mice (tables and ). collectively, these results suggested that mebendazole was well-tolerated. subsequently, we analyzed the effects of mebendazole on mm xenografts. in the ib assays on tumor tissues from treated mice, usp and c-maf were decreased in tumor tissues from mebendazole-treated mice but not in tumor tissues from untreated mice (fig. e, f) . moreover, caspase- and parp were also cleaved in tissues from mebendazole-treated mice (fig. e, f) , suggesting that mebendazole also induces mm cell apoptosis in vivo. taken together, these results indicated that mebendazole displays significant anti-mm activity in vivo by inhibiting the usp /c-maf axis. the above studies identified mebendazole as an inhibitor of the usp /c-maf axis and indicated that it displays potent antimyeloma activity in vitro and in vivo. mebendazole, a benzimidazole, is a well-tolerated, highly effective broad-spectrum anthelmintic used for the treatment of various intestinal infections with pinworms, hookworms, or roundworms. recent studies found that mebendazole also displays anticancer activity against several types of cancer, including colon cancer [ , ] , brain tumor [ ] , glioblastoma multiforme [ ] , non-small cell lung cancer [ ] , and melanoma [ ] . the initial anticancer mechanistic studies focused on its antimicrotubule activity [ ] , but mebendazole probably operates through diverse mechanisms dependent on the cancer type. it was found that mebendazole can stabilize p , p , and mdm in lung cancers [ , ] but downregulates the prosurvival protein bcl- through phosphorylation in chemoresistant melanoma cells [ ] . mebendazole blocks the hedgehog/smo pathway in basal cell carcinoma [ ] but inhibits the traf -and nck-interacting kinase in colorectal cancer [ ] . all of the above studies suggest that mebendazole elicits diverse anticancer effects. in the present study, we found that mebendazole displays potent antimyeloma activity at low concentrations by inhibiting the usp /c-maf axis. c-maf is an oncogenic transcription factor that is frequently dysregulated in mm and contributes to myelomagenesis by promoting the expression of several important genes involved in cancer cell proliferation, survival and metastasis, including ccnd , itgb , ccr , and ark [ ] . inhibiting c-maf thus leads to regression of mm tumors and inhibition of mm cell proliferation [ , ] . our recent studies demonstrated that the c-maf protein is processed via the ubiquitin-proteasome pathway under the control of the ubiquitin-conjugating enzyme ube o [ ] , the ubiquitin ligase herc [ ] or tmepai/nedd [ ] . c-maf ubiquitination can be reversed by the deubiquitinase usp because usp prevents c-maf from polyubiquitination and degradation [ ] . moreover, inhibiting usp by genetic shrna or small molecule inhibitors such as wp leads to mm cell apoptosis [ ] . collectively, these findings thus establish a rationale for treating mm by targeting the usp /c-maf axis. as a principle of concept, the present study identified mebendazole as an agent with potent antimyeloma activity from the fda-approved drug library. the mechanistic investigation showed that mebendazole not only prevents the interaction between usp and c-maf but also suppresses usp transcription, thus inducing c-maf proteasomal degradation. notably, mebendazole is cytotoxic to various cancer cells, including mm and leukemia cells, at higher concentrations (data not shown); however, it elicits selective anti-mm activity at low concentrations. as shown in the present study, at low molar concentrations, such as . µm, mebendazole induced the apoptosis of lp and rpmi- cells that express c-maf (fig. ) but was less effective in u cells that lack c-maf, suggesting that c-maf is a key factor in mm cell death induced by low concentrations of mebendazole. we also found that . µm mebendazole can markedly enhance the anti-mm activity of other agents. mebendazole synergizes with wp , an inhibitor of usp , because mebendazole downregulates usp expression and wp inhibits usp deubiquitinase activity. mebendazole also enhances antimyeloma activity in combination with daunorubicin, a major antimyeloma drug. these results indicate that mebendazole exerts potent antimyeloma activity alone or in combination with other anticancer agents. however, mebendazole does not show high toxicity, as seen in the tumor-bearing nude mice. actually, as an oral antiparasitic agent, mebendazole has historically been well demonstrated to be safe in humans. in summary, the present study identified mebendazole as a potent and promising antimyeloma agent via its inhibition of the usp /c-maf axis. this report is the first on treating myeloma by targeting c-maf ubiquitination and proteasomal degradation. given its high safety and potent anti-mm activity alone or in combination with other agents, mebendazole is guaranteed to be repurposed for personalized mm therapy. the study was also partly supported by the suzhou key laboratory for pediatric leukemia (szs to xm) and the suzhou key medical center multiple myeloma genomic complexity of multiple myeloma and its clinical implications risk stratification and targets in multiple myeloma: from genomics to the bedside overexpression of c-maf is a frequent oncogenic event in multiple myeloma that promotes proliferation and pathological interactions with bone marrow stroma integrin beta -mediated regulation of multiple myeloma cell adhesion, migration, and invasion maf protein mediates innate resistance to proteasome inhibition therapy in multiple myeloma ubiquitination of the transcription factor c-maf is mediated by multiple lysine residues inhibition of the deubiquitinase usp leads to c-maf protein degradation and myeloma cell apoptosis the role of the cytoskeletal protein, tubulin, in the mode of action and mechanism of drug resistance to benzimidazoles the ubiquitin ligase herc mediates c-maf ubiquitination and delays the growth of multiple myeloma xenografts in nude mice the ubiquitin-conjugating enzyme ube o modulates c-maf stability and induces myeloma cell apoptosis a novel transgenic mouse model of the human multiple myeloma chromosomal translocation t( ; )(q ; q ) lymphoma and myeloma cells are highly sensitive to growth arrest and apoptosis induced by artesunate artesunate overcomes drug resistance in multiple myeloma by inducing mitochondrial stress and non-caspase apoptosis deubiquitinase inhibition by small-molecule wp triggers aggresome formation and tumor cell apoptosis repositioning of the anthelmintic drug mebendazole for the treatment for colon cancer mebendazole and a non-steroidal anti-inflammatory combine to reduce tumor initiation in a colon cancer preclinical model brain penetration and efficacy of different mebendazole polymorphs in a mouse brain tumor model antiparasitic mebendazole shows survival benefit in preclinical models of glioblastoma multiforme the anthelmintic drug mebendazole induces mitotic arrest and apoptosis by depolymerizing tubulin in non-small cell lung cancer cells mebendazole induces apoptosis via bcl- inactivation in chemoresistant melanoma cells the antihelmintic flubendazole inhibits microtubule function through a mechanism distinct from vinca alkaloids and displays preclinical activity in leukemia and myeloma mebendazole elicits a potent antitumor effect on human cancer cell lines both in vitro and in vivo predicting new indications for approved drugs using a proteochemometric method repurposing the antihelmintic mebendazole as a hedgehog inhibitor comprehensive modeling and discovery of mebendazole as a novel traf -and nck-interacting kinase inhibitor c-maf oncogene dysregulation in multiple myeloma: frequency and biological relevance the transmembrane protein tmepai induces myeloma cell apoptosis by promoting degradation of the c-maf transcription factor this work was partly supported by the national natural science foundation of china ( to xm, to zz, and to bc), the natural science foundation of jiangsu higher education institutes of china ( kja to xm), the key: cord- - c rxg authors: wu, xiao-li; lu, shou-sheng; liu, meng-ru; tang, wei-dong; chen, jun-zi; zheng, yan-rong; ahsan, anil; cao, ming; jiang, lei; hu, wei-wei; wu, jia-ying; chen, zhong; zhang, xiang-nan title: melatonin receptor agonist ramelteon attenuates mouse acute and chronic ischemic brain injury date: - - journal: acta pharmacol sin doi: . /s - - - sha: doc_id: cord_uid: c rxg melatonin receptors (mts) are potential drug targets for stroke therapy. ramelteon is a selective melatonin receptor agonist used to treat insomnia. in this study we investigated whether ramelteon could attenuate cerebral ischemia in mice. acute focal cerebral ischemia was induced in mice via middle cerebral artery occlusion (mcao). we found oral administration of ramelteon ( . mg/kg) significantly attenuated ischemic injury even when it was given h after the onset of ischemia. we showed that administration of ramelteon ( . mg/kg) displayed comparable protective efficacy and length of effective time window as administration of edaravone ( mg/kg, i.p.), which was used in clinic to treat ischemic stroke. chronic ischemic brain injury was induced in mice using photothrombosis. oral administration of ramelteon ( . mg · kg(− ) · d(− )) for days after ischemia significantly attenuated functional deficits for at least days. the neuroprotection of ramelteon was blocked by -p-pdot, a specific mt antagonist. we further revealed that ramelteon significantly inhibited autophagy in the peri-infarct cortex in both the mouse ischemia models via regulating ampk/mtor signaling pathway. intracerebroventricular injection of rapamycin, an autophagy activator, compromised the neuroprotection of ramelteon, suggesting ramelteon might attenuate ischemic injury by counteracting autophagic cell death. these data demonstrate for the first time the potential benefits of ramelteon in the treatment of both acute and chronic ischemic brain injury and provide the rationale for the application of ramelteon in stroke therapy. cerebral ischemia is one of the leading causes of mortality and disability worldwide [ , ] . the administration of tissue plasminogen activator (tpa) within the therapeutic time window is one of the few available therapies for stroke. however, only a small portion of patients benefit from this thrombolytic treatment [ ] [ ] [ ] . after the occlusion of arteries, approaches to increase collateral flow may attenuate the ischemic injury [ ] . therefore, intensive efforts have been made to find therapeutic targets and related drugs for stroke treatment. melatonin protects against ischemic brain injury by pleiotropic mechanisms, including anti-oxidation, anti-inflammation, anti-apoptosis and the reduction of autophagic cell death [ ] [ ] [ ] [ ] [ ] . these findings suggest that melatonin can be a novel therapy for ischemic stroke treatment [ ] . it has become clear in recent years that melatonin confers its neuroprotection, at least partly, by activating its receptors, namely, mt and mt , in ischemic brains [ , ] . mts activate a variety of signaling cascades rapidly and subsequently trigger the protective effect, i.e., the reduction of ischemia-induced inflammation, oxidative stress and mitochondrial dysfunction [ , ] . in addition, the recent discoveries that mt agonists attenuate ischemic brain injury further shed light on mts as promising drug targets for stroke therapy [ , ] . ramelteon is an fda-approved mt /mt agonist used for insomnia. ramelteon acts selectively on mts and shows limited affinity to the receptors of other neurotransmitters in the central nervous system, including gaba, serotonin, dopamine, noradrenaline and acetylcholine [ , ] . ramelteon has been proven effective in preventing delirium and is safe for long-term use. emerging studies have shown the cardioprotective effect of ramelteon in reducing ischemic injury, suggesting additional pharmacological activities of ramelteon other than being a sleep agent [ ] . however, whether ramelteon can serve as a potential drug to ameliorate ischemic brain injury has never been evaluated. male c bl/ mice weighing - g were used. all experiments were approved by and conducted in accordance with the ethical guidelines of the zhejiang university animal experimentation committee and were in complete compliance with the national institutes of health guide for the care and use of laboratory animals. efforts were made to minimize any pain or discomfort, and the minimum number of animals was used. mcao mouse model and the determination of ischemic injury focal cerebral ischemia was induced by middle cerebral artery occlusion (mcao) [ ] . in brief, mice were anesthetized by the inhalation of isoflurane for surgery. cerebral blood flow (cbf) was determined in the area of the middle cerebral artery (mca) by laser doppler flowmetry (model moor vms-ldf , uk). a probe was attached to the skull over the cortex supplied by the right mca ( mm caudal to bregma and -mm lateral to midline). a - nylon monofilament suture with a rounded tip was inserted~ mm into the internal carotid to occlude the origin of the mca. animals with less than an % reduction in cbf were excluded from the study. mice underwent mcao for h or h. body temperature was maintained at °c until the animals recovered from the anesthesia. to analyze protein expression, the penumbral tissue of ischemic brains was rapidly collected after h or h of mcao, as previously described [ ] . infarct volumes were measured at h or h after surgery, and coronal mouse brain slices at -mm intervals were stained with . % ttc (sigma, t ). infarcted areas were analyzed using image pro plus . and measured by the indirect method, which corrects for edema [ ] . neurological deficit scores were evaluated at h or h after surgery as follows: , no deficit; , flexion of the contralateral forelimb upon lifting the whole animal by the tail; , circling to the contralateral side; , falling to the contralateral side; and , no spontaneous motor activity [ ] . photothrombotic model of stroke in mice to induce chronic focal cerebral ischemia, photothrombosis was performed in mice as described previously [ ] . mice were anesthetized with isoflurane and mounted in a stereotaxic apparatus (stoelting, ). a cold light source (diameter . mm; , lux) was placed in close contact with the skull surface at . mm lateral from bregma. rose bengal solution (sigma; ) was administered intraperitoneally at a dose of mg/kg. five minutes after injection, the brain was illuminated for min through the intact skull to initiate photothrombosis. sham mice were treated with the same procedure without light illumination. for protein analysis, days after photothrombosis, the brain cortex around the infarct core was rapidly dissected as previously described [ ] . drug administration ramelteon (mce, a ) was dissolved in saline containing . % carboxymethylcellulose (cmc). animals were given ramelteon ( . , . or . mg/kg, i.g.) at the onset of mcao. for the treatment after ischemia, ramelteon ( . mg/kg, i.g.) was administered at the onset of or at and h after mcao. edaravone (sigma, m ) was intraperitoneally injected at doses of , , mg/kg at the indicated time after mcao. for the treatment of chronic cerebral ischemia, ramelteon ( . mg/kg, i.g.) was administered daily after photothrombosis for days. the mts antagonist -p-pdot (tocris, ) was administered ( . mg/kg, i.p.) . h before ramelteon administration [ ] . rapamycin (sigma, r ) was dissolved in saline and intracerebroventricularly ( μl, μmol/l) injected at the onset of ischemia. grid-walking task the grid-walking task apparatus was manufactured, as previously described [ ] . the grid area was cm × cm × cm (length × width × height) with a mm square wire mesh. mice walked on a wire grid for min while being video-recorded. foot faults for the forelimb and the total normal steps were counted. the foot fault index was calculated as the number of foot faults/(foot faults + the number of non-foot-fault steps) × %. foot faults were considered to occur when the forelimbs passed through the grid hole. the video analysis was performed by individuals who were blinded to these experiments. cylinder task mice were placed in a plexiglas cylinder ( cm in height with a diameter of cm) and video-recorded for min to determine forelimb symmetry in exploratory rearing [ ] . the video footage was analyzed by calculating the time (in seconds) that each forelimb or both forelimbs were placed on the cylinder wall. the asymmetry index is as follows: (% ipsilateral use)−(% contralateral use). the video analysis was performed by individuals who were blinded to these experiments. toluidine blue staining toluidine blue staining was performed on day after photothrombosis [ ] . brain sections were cut into μm sections on a cryostat (leica, germany) and were collected every μm. the slices were stained with . % toluidine blue solution (sigma, ) for - min. then, sections were washed in distilled water, decolorized in % ethyl alcohol and dehydrated in % and % ethyl alcohol for to min in each step. the sections were cleared in xylene for min. finally, the sections were mounted and observed under a light microscope (olympus, vs ). toluidine blue staining was traced and quantified by imagej. western blot analysis western blot analysis was performed as described previously [ ] . briefly, peri-infarct cortex tissues were homogenized in ripa buffer ( mmol/l tris-hcl, ph . , mmol/l nacl, mmol/l edta, % triton x- , . % sodium deoxycholate (sigma, ), and % protease inhibitor cocktail tablets (roche, )). the primary antibodies were as follows: sqstm ( : , ; cell signaling technology, ), lc ( : , ; sigma, l ), ampk ( : , ; cell signaling technology, ), p-ampk (t ) ( : , ; cell signaling technology, ), p-mtor (s ) ( : ; cell signaling technology, ), p-p s ( : ; cell signaling technology, ), il- β ( : ; cell signaling technology, ), tnf-α ( : ; cell signaling technology, ) and gapdh ( : , ; kang chen, kc- g ). secondary antibodies were as follows: hrp against either rabbit or mouse igg ( : , ; cell signaling technology, and ). statistical analysis all data were collected and analyzed in a blinded manner. data are presented as the mean ± sem. the data were evaluated by a one-way anova (analysis of variance) with dunnett t post hoc tests. for behavioral testing, differences between treatment groups were analyzed using a two-way anova with tukey's multiple comparisons test for post hoc tests. p < . was considered statistically significant. to explore the neuroprotective effect of ramelteon against acute ischemic brain injury, mice were subjected to middle cerebral artery occlusion (mcao) (fig. a) . ramelteon was administered ( . , . and . mg/kg, i.g.) at the onset of mcao. brain infarct volumes and neurological deficits were determined h after ischemia. the results showed that ramelteon reduced brain infarct volumes in a dose-dependent manner. ramelteon at . mg/kg significantly reduced the brain infarct volumes from . % ± . % to . % ± . % (fig. b, c) . in line with this finding, . mg/kg ramelteon ameliorated mcao-induced neurological deficit (ram . . ± . vs vehicle . ± . , p < . , fig. d ). these data revealed the neuroprotective role of ramelteon against mcao-induced acute brain injury. ramelteon attenuates ischemic brain injury xl wu et al. to further clarify the involvement of mts in neuroprotection, -p-pdot was employed as a specific mt antagonist. the results showed that . mg/kg -p-pdot (i.p.) significantly increased the infarct volumes ( -p-pdot + ram . % ± . % vs ram . % ± . %, fig. e , f) and neurological deficit score ( -p-pdot + ram . ± . vs ram . ± . , p < . , fig. g ) in the ramelteon group, while -p-pdot alone showed no impact on these determinations. these data indicated that -p-pdot abolished ramelteon-conferred neuroprotection. we next determined the fig. ramelteon protected against middle cerebral artery occlusion (mcao)-induced acute ischemic brain injury. a experimental design of acute ischemia is shown. b ramelteon at . , . or . mg/kg was administered orally at the onset of mcao. representative brain slices are shown after ttc staining. c the infarct volumes and d neurological deficit scores were accessed at h after mcao. e mice were treated with ramelteon at . mg/kg, -p-pdot at . mg/kg or cotreatment after surgery. -p-pdot was injected . h before ramelteon administration. n = to for each group. f the brain infarct volumes and g neurological deficit scores were measured as mentioned previously. h, i the brain infarct volumes and j neurological deficit scores were measured after h. n = to for each group. the data are shown as the mean ± sem. statistical comparisons were performed with a one-way anova followed by dunnett's multiple comparisons test. *p < . , **p < . ; n.s. (not significant) vs the indicated group neuroprotective effect of ramelteon after days of mcao. the results showed that ramelteon significantly reduced the infarct volumes (vehicle . % ± . % vs ram . % ± . %, p < . , fig. h , i) and neurological deficit scores (vehicle . ± . vs ram . ± . , p < . , fig. j ). we found that ramelteon still had a neuroprotective role in prolonged ischemic brain injury. effective time window of ramelteon in reducing mcao-induced brain injury to explore the time window during which ramelteon can reduce acute ischemic brain injury. ramelteon at . mg/kg was administered at the onset ( h) or at h and h after ischemia onset. ramelteon significantly reduced brain infarct volumes as late as h after ischemia (ram h . % ± . % vs vehicle . % ± . %, p < . , fig. d , e). consistent with this observation, ramelteon significantly reversed neurological deficit when administered as late as h after mcao (ram h . ± . vs vehicle . ± . , p < . , fig. f) . we further prolonged the administration time to h after mcao. the results showed that . mg/kg ramelteon failed to rescue infarct volumes (ram h . % ± . % vs vehicle . % ± . %, p = . , fig. e ) and neurological deficit (ram h . ± . vs vehicle . ± . , p = . , fig. f ). these data indicated that the effective time window of ramelteon can be as long as h in mcao mice. representative ttc-stained slices are shown. e brain infarct volumes and f neurological deficit scores were determined h after ischemia. data are expressed as the mean ± sem, n = to for each group. statistical comparisons were performed with a one-way anova followed by dunnett's multiple comparisons test. *p < . , **p < . , ***p < . ; n.s. (not significant) vs the indicated group comparison of the neuroprotective effect of ramelteon and edaravone in mcao-induced acute brain injury we next compared the neuroprotection of ramelteon with edaravone, a well-accepted therapy for stroke patients, in the mcao model. the neuroprotective effect of edaravone against ischemia was reported as previously described [ , ] . to optimize the dose of edaravone in our model, mice were intraperitoneally administered edaravone at , , and mg/kg at the onset of mcao. edaravone at . mg/kg showed the maximal efficacy in reducing infarct volumes (eda . % ± . % vs vehicle . % ± . %, p < . , fig. a, b) and nds (eda . ± . vs vehicle . ± . , p < . , fig. c) . the results showed that mg/kg edaravone significantly reduced brain infarct volumes and neurological deficits; nevertheless, edaravone showed no significant advantages in ameliorating mcao-induced brain injury compared with ramelteon. we further determined the neuroprotective effect of edaravone and ramelteon by prolonging the administration time to and h. edaravone showed no advantage over ramelteon in reducing mcao-induced injury by delayed administration, as reflected by brain infarct volumes and nds (fig. e, f) . these data suggested that ramelteon provided a similar effective time window as edaravone for stroke therapy. to determine whether ramelteon may also attenuate chronic ischemic brain injury, mice were subjected to photothrombosis. ramelteon ( . mg/kg) was administered daily after photothrombosis for days, and the volumes of lesioned brains were evaluated days after ischemia (fig. a) . the results showed that ramelteon significantly reduced the brain lesion from . ± . mm to . ± . mm (p < . ). additionally, the protective effect of ramelteon was counteracted by -p-pdot, suggesting the involvement of mts in the neuroprotection of ramelteon (fig. b, c) . to determine the impairment of motor function, we measured the number of foot faults in the grid-walking task and forelimb symmetry in the cylinder task every other day. we found that ramelteon significantly reduced the number of foot faults and forelimb symmetry during the days after pt onset. likewise, the functional recovery with ramelteon treatment was abolished by -p-pdot treatment (fig. d, e) . taken together, these data clearly indicated that ramelteon protected against chronic cerebral ischemia and promoted functional recovery in an mt-related manner. the ampk/mtor signaling pathway and autophagy may be involved in ramelteon-conferred neuroprotection ischemic brain injury is intimately related to autophagy cell death. it has been shown that mt activation regulates the ampk/mtor signaling pathway [ ] . as expected, ramelteon significantly decreased the cerebral-ischemia-induced phosphorylation of ampk (t ) and increased the phosphorylation of mtor (s ) and p s kinase, which were abolished by -p-pdot (fig. a, b) . ampk/mtor signaling has been accepted to activate autophagy in the context of cerebral ischemia [ ] , and there are studies indicating that mt agonists regulate autophagy in ischemic brains [ , ] . we thus hypothesized that ramelteon conferred its neuroprotection by modulating autophagy. to this end, autophagy-related proteins sqstm and lc were determined. autophagy activation was detected in the brain tissue subjected to mcao, as reflected by reduced sqstm and accumulated lc -ii. ramelteon treatment significantly prevented these alterations, indicating that ramelteon inhibited autophagy activation in acute ischemic brain tissue. these effects were reversed by -p-pdot, further suggesting the involvement of mts in autophagy regulation (fig. c, d) . these data support the notion that ramelteon may regulate autophagy activation by modulating the ampk/mtor signaling pathway. to further determine the involvement of autophagy inhibition in the neuroprotection of ramelteon, rapamycin was administered as an autophagy activator. the neuroprotection of ramelteon was abolished by rapamycin, as demonstrated by increased infarct volumes (ram . % ± . % vs rapa . % ± . %, p < . ) and nds (ram . ± . rapa vs . ± . , p < . , fig. e, f) . we next asked whether the ampk/mtor signaling pathway and autophagy activation can also be regulated by ramelteon in chronic ischemia. we found that p-ampk (t ) was upregulated and p-mtor (s ) and p-p s were downregulated at days after ischemia. ramelteon prevented the phosphorylation of ampk (t ) and the decrease in p-mtor (s ) and p-p s , while these effects were abrogated by -p-pdot (fig. a, b) . autophagy was still activated, as reflected by increased lc -ii and decreased sqstm . ramelteon counteracted autophagy activation, which was further abolished by -p-pdot (fig. c, d) . overall, these data supported the involvement of the ampk/mtor signaling pathway and autophagy in ramelteon-conferred neuroprotection in either acute or chronic ischemic brain injury. mtor signaling was reported to regulate neuroinflammation [ , ] , which is involved in the pathological process of cerebral ischemia [ ] . melatonin attenuated ischemic brain injury through its antiinflammatory properties [ ] . we also examined whether ramelteon protected cerebral ischemia through anti-inflammation. after ischemia, the increased levels of il- β and tnf-α were suppressed by ramelteon treatment (fig. s ) . the results indicated that ramelteon might attenuate inflammation in cerebral ischemia. b mice were treated with ramelteon at . mg/kg daily for days after photothrombosis. -p-pdot was injected . h before ramelteon treatment. fifteen days after pt onset, brain sections were stained with toluidine blue. representative images are shown, and the dashed lines outline the brain lesion in the cortex. data are expressed as the mean ± sem, n = for each group. scale bar: mm. c the infarct volumes were quantified and expressed as mm . statistical comparisons were performed with a one-way anova followed by dunnett's multiple comparisons test. d motor functions were accessed by foot fault test and e cylinder task. behavioral tests were performed every two days after stroke. n = - for each group. statistical comparisons were performed with a two-way anova followed by tukey's multiple comparisons test. *p < . , ***p < . vs the indicated group the molecular mechanism of ramelteon in anti-inflammation needs to be investigated in future studies. our results showed that ramelteon protected against both acute and chronic ischemic injury. ramelteon may activate the ampk/mtor signaling pathway and inhibit ischemia-induced autophagy in a melatonin receptor-related manner (fig. ) . ramelteon is a hypnotic agent that activates melatonin receptors. some emerging studies have implied other clinical applications beside sedation [ ] ; however, the neuroprotective effect of ramelteon against ischemic injury has never been tested. in the present study, we reported for the first time that ramelteon attenuated brain injury caused by either acute or chronic brain ischemia. mcao was the model used to mimic acute brain ischemia. ramelteon showed significant neuroprotection in reducing mcao-induced infarct volume and nds in a dosedependent manner. we also found that ramelteon still protected against ischemia in the brain days after mcao onset (fig. ) . likewise, ramelteon attenuated brain lesions induced by chronic ischemia, and neuroprotection was observed at least days after ischemia onset (fig. ) . we cannot exclude the ineffectiveness of ramelteon in the time range longer than days in our study. to evaluate the neuroprotection of ramelteon more comprehensively, we compared its efficacy with that of edaravone (fig. ) , which is an approved drug for stroke therapy [ , ] . we found that ramelteon showed neuroprotective effects that were comparable with those of edaravone against acute ischemic injury. this observation suggested that ramelteon might have equivalent efficacy with edaravone for stroke therapy. considering the safety of ramelteon in clinical application, our data indicated fig. ramelteon activated the ampk/mtor signaling pathway and inhibited autophagy in acute ischemic brain injury. a mice were administered ramelteon at . mg/kg or were cotreated with -p-pdot at . mg/kg after mcao. the expression of ampk, p-ampk (t ), p-mtor (s ), p-p s and gapdh in the ischemic penumbra was measured by western blot analysis h after mcao. n = for each group. b semiquantitative analyses of ampk, p-ampk (t ), p-mtor (s ), and p-p s are shown. c the expression of sqstm and lc b was determined by western blot analysis. d semiquantitative analyses of sqstm and lc -ii are shown. e mice were treated with ramelteon at . mg/kg or were cotreated with rapamycin at . μmol at the onset of ischemia. rapamycin was dissolved in saline and intracerebroventricularly (i.c.v.) injected. f infarct volumes and nds were accessed as previously described. n = - . the data are expressed as the mean ± sem. statistical comparisons were performed with a one-way anova followed by dunnett's multiple comparisons test. *p < . , **p < . , ***p < . ; n.s. (not significant) vs the indicated group that ramelteon can be a novel potential therapeutic drug for cerebral ischemia. we optimized the dose of ramelteon and found that . mg/kg showed the maximal efficacy (data not shown). we found that . mg/kg of ramelteon was able to attenuate both acute and chronic brain ischemia. by converting body surface area, this dose is~ mg in humans ( kg body weight). this dose is equivalent to that for insomnia therapy, which ranges from to mg/day [ , ] . approximately % of stroke patients suffered from sleep problems, including delirium and insomnia [ , ] . given the hypnotic purpose of ramelteon at this dose, ramelteon may have advantages in treating patients with destabilized circadian rhythms after stroke [ , ] . in addition, a narrow therapeutic window is one of the most challenging issues in stroke therapy [ , ] . ramelteon showed an effective therapeutic time window as long as h in this mouse mcao model. this effective therapeutic window was equal to that of edaravone (fig. ) [ ] , which further supported the potential clinical application of ramelteon in stroke therapy. lines of evidence documented the neuroprotective effect of melatonin against cerebral ischemia [ ] . mts are the primary targets of melatonin; however, it is controversial whether mts are required for melatonin to rescue ischemic brains [ , ] . here, we used -p-pdot to block mts [ , ] . although -p-pdot has been frequently used as a selective mt antagonist, its selectivity between mt and mt was poor at the current dose [ ] . we found that the neuroprotection of ramelteon was almost totally abolished by -p-pdot both in acute and chronic ischemia (figs. , ) , which supported the requirement of mts for the neuroprotection of ramelteon. some recent studies indicated that mt and mt may conduct diverse signaling in ischemic brains [ , ] . due to the lack of selective mt antagonists, it remains unclear how mts may contribute to the neuroprotection of ramelteon. this issue could be addressed by employing mt and mt gene knockout mice in future studies. additionally, previous studies indicated the requirement of melatonin pretreatment before an ischemic episode [ ] , and a delay of administration abolished the neuroprotection of melatonin against acute ischemia [ ] . it took fig. ramelteon activated the ampk/mtor signaling pathway and suppressed autophagy in chronic ischemic brain injury. a mice were subjected to photothrombosis, and days later, the expression of ampk, p-ampk (t ), p-mtor (s ), p-p s and gapdh in the periinfarct cortex was examined by western blot analysis. b semiquantitative analyses are shown. n = for each group. c the expression of sqstm and lc b was determined by western blot analysis. d semiquantitative analyses of sqstm and lc -ii are shown. the data are expressed as the mean ± sem. statistical comparisons were performed with a one-way anova followed by dunnett's multiple comparisons test. *p < . , **p < . , ***p < . vs the indicated group fig. diagram of the neuroprotective effects of ramelteon on cerebral ischemic injury. ramelteon inhibited the cerebral-ischemiainduced activation of autophagy through the ampk/mtor signaling pathway. these effects were blocked by the melatonin receptor inhibitor -p-pdot hours for melatonin to reach its therapeutic level in a nonrodent stroke animal model [ ] . given the sudden occurrence of ischemic stroke, melatonin may still have disadvantages as a rescuing agent for ischemic brain injury. as a comparison, we found that ramelteon was effective even h after mcao onset. this result suggested that ramelteon could be a therapeutic drug, while the prophylactic effect of melatonin should be emphasized for acute ischemia intervention. the discrepancy may be attributed to the advantages in pharmacokinetics and to the fact that ramelteon has a higher affinity than melatonin to mts [ , , ] . autophagy activation has been accepted as a critical mechanism underlying ischemic neuronal death, particularly in ischemic brains without reperfusion [ , ] . additionally, ampk/mtor signaling was accepted to activate autophagy during cerebral ischemia [ ] . our data confirmed autophagy activation in ischemic brains by showing upregulated lc -ii and decreased sqstm and ampk/mtor inhibition (fig. a, c) . ramelteon suppressed the autophagy activation induced by ischemia. in addition, rapamycin, an autophagy activator, abolished the ramelteon-conferred neuroprotection (fig. e, f) . it remains a controversial issue whether rapamycin is neuroprotective in cerebral ischemia, yet a variety of studies have confirmed its role as an autophagy activator [ ] [ ] [ ] . nevertheless, due to the lack of specific approaches to activating autophagy, we cannot exclude other mechanisms underlying the neuroprotection of ramelteon in addition to autophagy inhibition. nevertheless, the present study indicated that autophagy may be involved in ramelteon-conferred protection. likewise, melatonin has been demonstrated to regulate autophagy activation in ischemic rats [ ] . however, it was unclear whether mts are required for autophagy regulation by melatonin. it seems that mts regulate neuronal autophagy via ampk/mtor signaling, which is activated in ischemic conditions [ ] . here, we found that mt blockage by -p-pdot largely prevented ampk/mtor activation and autophagy induction (figs. and ) . therefore, the present study supported the involvement of mts in autophagy regulation. in addition, melatonin was reported to counteract ischemia-induced autophagy [ , ] . in these studies, pretreatment with melatonin was required for its neuroprotection, and the benefits of melatonin against chronic stroke have not been determined. we found that ramelteon could be neuroprotective as late as h after acute ischemia and be beneficial in chronic cerebral ischemia. these data implied the advantages of ramelteon over melatonin as a therapeutic drug for stroke. taken together, we provide the first evidence indicating that ramelteon can be a promising therapeutic drug for stroke therapy. the neuroprotective effect of ramelteon may be attributed to its agonism on mts and its inhibition of autophagy in ischemic brains. neuroprotective mechanisms of hypothermia in brain ischaemia current practice and future directions in the diagnosis and acute treatment of ischaemic stroke normobaric hyperoxia slows bloodbrain barrier damage and expands the therapeutic time window for tissue-type plasminogen activator treatment in cerebral ischemia impact of the extended thrombolysis time window on the proportion of recombinant tissue-type plasminogen activator-treated stroke patients and on door-to-needle time acute ischemic stroke therapy overview neurovascular regulation in the ischemic brain pre-ischemia melatonin treatment alleviated acute neuronal injury after ischemic stroke by inhibiting endoplasmic reticulum stress-dependent autophagy via perk and ire signalings melatonin prevents increases in neural nitric oxide and cyclic gmp production after transient brain ischemia and reperfusion in the mongolian gerbil (meriones unguiculatus) effect of melatonin on ischemia reperfusion injury induced by middle cerebral artery occlusion in rats visualizing peroxynitrite fluxes in endothelial cells reveals the dynamic progression of brain vascular injury ischemic injury promotes keap nitration and disturbance of antioxidative responses in endothelial cells: a potential vasoprotective effect of melatonin the protective effect of melatonin on brain ischemia and reperfusion in rats and humans: in vivo assessment and a randomized controlled trial melatonin's protective action against ischemic neuronal damage is associated with up-regulation of the mt melatonin receptor melatonin pretreatment improves the survival and function of transplanted mesenchymal stem cells after focal cerebral ischemia neuroprotective mechanism of the novel melatonin derivative neu-p in brain ischemia related models challenging behaviour and sleep cycle disorder following brain injury: a preliminary response to agomelatine treatment ramelteon (rozerem) a novel approach for insomnia treatment a review of suvorexant, doxepin, ramelteon, and tasimelteon for the treatment of insomnia in geriatric patients melatonin receptor agonist ramelteon reduces ischemia-reperfusion injury through activation of mitochondrial potassium channels cerebral ischemiareperfusion-induced autophagy protects against neuronal injury by mitochondrial clearance park -dependent mitophagy induced by acidic postconditioning protects against focal cerebral ischemia and extends the reperfusion window bnip l/nix-mediated mitophagy protects against ischemic brain injury independent of park opening a new time window for treatment of stroke by targeting hdac ampa receptor-induced local brain-derived neurotrophic factor signaling mediates motor recovery after stroke melatonin attenuates memory impairment induced by klotho gene deficiency via interactive signaling between mt receptor, erk, and nrf -related antioxidant potential comparison of unbiased estimation of neuronal number in the rat hippocampus with different staining methods edaravone attenuates oxidative stress induced by chronic cerebral hypoperfusion injury: role of erk/nrf /ho- signaling pathway edaravone with and without . mg/kg alteplase within . h after ischemic stroke: a prospective cohort study (protect . ) melatonin suppresses milk fat synthesis by inhibiting the mtor signaling pathway via the mt receptor in bovine mammary epithelial cells a combination of four active compounds alleviates cerebral ischemia-reperfusion injury in correlation with inhibition of autophagy and modulation of ampk/mtor and jnk pathways n-acetyl-serotonin offers neuroprotection through inhibiting mitochondrial death pathways and autophagic activation in experimental models of ischemic injury the multiple protective roles and molecular mechanisms of melatonin and its precursor n-acetylserotonin in targeting brain injury and liver damage and in maintaining bone health early molecular and behavioral response to lipopolysaccharide in the wag/rij rat model of absence epilepsy and depressive-like behavior, involves interplay between ampk, akt/mtor pathways and neuroinflammatory cytokine release mtor signaling inhibition modulates macrophage/microglia-mediated neuroinflammation and secondary injury via regulatory t cells after focal ischemia inflammatory responses in brain ischemia methazolamide and melatonin inhibit mitochondrial cytochrome c release and are neuroprotective in experimental models of ischemic injury clinical effects of early edaravone use in acute ischemic stroke patients treated by endovascular reperfusion therapy edaravone improves functional and structural outcomes in animal models of focal cerebral ischemia: a systematic review an efficacy, safety, and doseresponse study of ramelteon in patients with chronic primary insomnia delirium in acute stroke: a systematic review and meta-analysis disorders of sleep and wake in patients after subarachnoid hemorrhage ischemic stroke destabilizes circadian rhythms addition of suvorexant to ramelteon therapy for improved sleep quality with reduced delirium risk in acute stroke patients evidence that membrane-bound g protein-coupled melatonin receptors mt and mt are not involved in the neuroprotective effects of melatonin in focal cerebral ischemia melatonin ameliorates neural function by promoting endogenous neurogenesis through the mt melatonin receptor in ischemic-stroke mice selective mt melatonin receptor antagonists block melatonin-mediated phase advances of circadian rhythms cardiac assistance and substitutes for cardiocirculatory function melatonin regulates parp to control the senescence-associated secretory phenotype (sasp) in human fetal lung fibroblast cells pretreatment with melatonin reduces volume of cerebral infarction in a rat middle cerebral artery occlusion stroke model administration of melatonin after onset of ischemia reduces the volume of cerebral infarction in a rat middle cerebral artery occlusion stroke model melatonin as an adjunct to therapeutic hypothermia in a piglet model of neonatal encephalopathy: a translational study age and gender effects on the pharmacokinetics and pharmacodynamics of ramelteon, a hypnotic agent acting via melatonin receptors mt and mt clinical pharmacokinetics of melatonin: a systematic review inhibition of autophagy prevents hippocampal pyramidal neuron death after hypoxicischemic injury neuronal injury in rat model of permanent focal cerebral ischemia is associated with activation of autophagic and lysosomal pathways inhibition of amp-activated protein kinase alleviates focal cerebral ischemia injury in mice: interference with mtor and autophagy rapamycin in ischemic stroke: old drug, new tricks? sevoflurane postconditioning inhibits autophagy through activation of the extracellular signal-regulated kinase cascade, alleviating hypoxic-ischemic brain injury in neonatal rats neuroprotective effects of pinocembrin on ischemia/reperfusion-induced brain injury by inhibiting autophagy inhibition of autophagy contributes to melatonin-mediated neuroprotection against transient focal cerebral ischemia in rats melatonin attenuates myocardial ischemia/reperfusion injury by inhibiting autophagy via an ampk/ mtor signaling pathway regulation of the ischemia-induced autophagy-lysosome processes by nitrosative stress in endothelial cells zc and xnz designed the project; xlw and ssl performed most of the surgical experiments, imaging and immunoblotting; mrl, jzc, yrz and wdt contributed to analyzing the data in a blinded manner; aa, mc and lj helped to perform the mcao model and to raise mice; wwh and jyw contributed to organizing the results and discussion; and zc and xnz wrote the manuscript with input from all other authors. the online version of this article (https://doi.org/ . /s - - - ) contains supplementary material, which is available to authorized users.competing interests: the authors declare no competing interests. key: cord- -pwlybr h authors: liu, yuan-yuan; chen, liang-jun; zhong, yan; shen, meng-xin; ma, nian; liu, bing-yu; luo, fan; hou, wei; yang, zhan-qiu; xiong, hai-rong title: specific interference shrna-expressing plasmids inhibit hantaan virus infection in vitro and in vivo date: - - journal: acta pharmacol sin doi: . /aps. . sha: doc_id: cord_uid: pwlybr h aim: to investigate the antiviral effects of vectors expressing specific short hairpin rnas (shrnas) against hantaan virus (htnv) infection in vitro and in vivo. methods: based on the effects of shrnas targeting different regions of htnv genomic rna on viral replication, the most effective rna interference fragments of the s and m genes were constructed in psilencer- . -h vectors, and designated psilencer-s and psilencer-m, respectively. the antiviral effect of psilencer-s/m against htnv was evaluated in both htnv-infected vero-e cells and mice. results: in htnv-infected vero-e cells, psilencer-s and psilencer-m targeted the viral nucleocapsid proteins and envelope glycoproteins, respectively, as revealed in the immunofluorescence assay. transfection with psilencer-s or psilencer-m ( , , μg) markedly inhibited the viral antigen expression in dose- and time-dependent manners. transfection with either plasmid ( μg) significantly decreased htnv-rna level at day postinfectin (dpi) and the progeny virus titer at dpi. in mice infected with lethal doses of htnv, intraperitoneal injection of psilencer-s or psilencer-m ( μg) considerably increased the survival rates and mean time to death, and significantly reduced the mean virus yields and viral rna level, and alleviated virus-induced pathological lesions in lungs, brains and kidneys. conclusion: plasmid-based shrnas potently inhibit htnv replication in vitro and in vivo. our results provide a basis for development of shrna as therapeutics for htnv infections in humans. hantaviruses represent one of the important emerging and re-emerging viral groups and have become a global threat to public health in different areas of the world [ , ] . hantaviruses lead to two severe febrile diseases worldwide, ie, hemorrhagic fever with renal syndrome (hfrs) in eurasia due to old world hantavirus and hantavirus pulmonary syndrome (hps) in the americas due to new world hantavirus [ ] . approximately - hfrs cases are reported each year worldwide, and the mortality rate can reach % [ , ] . hantaviruses belong to the genus hantavirus of the bunyaviridae family, and they are enveloped, single-strand, negative-sense, tri-segmented rna viruses. the viral genomes consist of large (l), medium (m) and small (s) segments that encode an rna-dependent rna polymerase and two envelope glycoproteins (gn and gc) and a nucleocapsid (n) protein. variations in the m and s segments may influence the antigenicity and virulence of hantaviruses [ , ] . extensive efforts have been made in the search for effective preventative and treatment measures for hantavirus infections [ , ] . ribavirin is a broad-spectrum synthetic nucleoside analog that has been reported to effectively inhibit the replication of the hantaan virus (htnv, an old world hantavirus) and andes virus (andv, a new world hantavirus) in vitro and in vivo [ ] . however, there are currently no prophylactic vaccines or therapeutic antivirals for hantavirus infection that are approved by the fda. therefore, the exploration of novel treatment strategies for hantavirus infection is urgent and necessary. rna interference (rnai) is a short, double-strand rna induced-process that can target the mrna of a specific sequence for degradation [ , ] . post-transcriptional gene silencing can be mediated by endogenous micrornas (mirnas), exogenous small interfering rnas (sirnas), and short hairpin rnas (shrnas). rnai technology is recognized as a promising, novel nucleic acid-based tool for use against various diseases, including cancer, infectious diseases and genetic disorders. rnai reagents have also been reported to potently and specifically inhibit the replication of different viruses, including hiv, hbv [ ] , hsv [ ] , sar-cov [ ] , influenza virus [ , ] , coxsackievirus [ ] and others. thus, the development of rnais as possible antiviral agents for hantavirus infection is of considerable interest [ ] . in the present study, we examined the effects of shrnas on htnv replication using shrnas that targeted different regions of the htnv genomic rna. we further transfected the two most effective shrnas into the shrna expression vector, psilencer- . -h and named the resultant vectors psilencer-s and psilencer-m. the antiviral effect of psilencer-s/m against htnv was ultimately evaluated in tissue culture and a mouse model. vero-e cells were obtained from the china center for type culture collection (cctcc) and maintained in dmem supplemented with % fetal bovine serum (fbs, gibco, carlsbad, ca, usa), . % l-glutamine, u/ml penicillin and streptomycin. dmem containing % fbs was used to maintain the medium after viral infection. the stocks of hantaan virus strain - were obtained from the institute of virology of the chinese academy of preventive medicine (beijing, china) and were propagated in vero-e cells. htnv titration was performed regularly based on indirect immunofluorescence assay (ifa) as described previously [ ] . the tcid was determined to be / . ml. shrna design and selection two individual targeting sites were selected for the s (genbank accession number af ) and m genes (genbank accession number af ) of htnv. the design of the shrna sequences was performed according to methods referred to in the literature [ ] and involved the use of the webbased block-it rnai designer program (http://rnaidesigner. thermofisher.com/rnaiexpress/). a blast search (http:// www.ncbi.nlm.nih.gov/blast) was performed to exclude the possible homologous sequences. the sequences and positions of the shrnas are illustrated in table . the designed structure contained the following sequences: '-aa-sense strand sirna, cttgcttc (loop region); and antisense strand sirna, tatagtga (t -oligo promoter)- '. the target gene is presented in table . a negative control was chosen to target positions to of leishmania crk , the sequence of which was '-aatcgggcagttgtttgagat- '. the dna template was synthesized by sangon biotech (shanghai) co, ltd, and the shrna was generated using the messagemuter™ shrna production kit (qiagen) as described previously [ ] . the vero-e cells were grown in -well plates to %- % confluence and transfected with specific or control shrna ( nmol/l) with rnaifect transfection reagent (qiagen, germantown, md, usa). at h after transfection, the cells were infected with tcid htnv - ( μl per well), and viral rna was detected by qrt-pcr at or hour post-infection (hpi) to determine the interference efficiency. the viral titers were determined at hpi. the shrna s and shrna m were found to be the most effective shrnas in terms of the inhibition of htnv and were selected for use in the subsequent experiments. construction of shrna vectors targeting the s and m genes of htnv synthesized oligonucleotides containing the s and m shrna sequences with bamh i or hind iii sequences at the ' or ' termini were designed as illustrated in table according to the instructions of the manufacturer of rnai designer (invitrogen, carlsbad, ca, usa). the two oligos sequences npg complementary oligonucleotides were annealed and inserted into the bamh i/hind iii site of the psilencer- . -h vector. after transformation, clone pcr and enzyme digestion, the identified shrna expression plasmids were sent for sequence analysis for further confirmation and were subsequently designated psilencer-s and psilencer-m. one day before transfection, the vero-e cells were seeded into -well plates at × cells per well. the %- % confluent cells were then transfected with . , . , , , or μg of the psilencer-s, psilencer-m or psilencer . h (as psilencerblank) vectors with lipofectamine (invitrogen) according to the manufacturer's instruction. the transfection efficiencies were according to gfp expression of the co-transfected plasmid pegfp-c . after hpi, the cells were infected with tcid / . ml of htnv - . at , , , , , and day post-infection (dpi), antigen slides were prepared to detect the htnv protein expressions by ifa. in other parallel experiments, the cells from individual wells ( -μg plasmid transfection groups) were collected at dpi for viral rna detection. at dpi, the cultures were subjected to two cycles of freezing and thawing followed by centrifugation at low speed ( ×g), and the supernatants were then serially diluted, inoculated on the cells, and finally titrated by ifa. this study was approved by the ethics committee of wuhan university school of medicine. all work with infected mice was performed in the animal biosafety level (absl- ) laboratory of the animal research center at wuhan university and was humanely conducted in compliance with the chinese animal protection act and the national research council criteria. specific pathogen-free pregnant balb/c mice were obtained from the animal research center of wuhan university. suckling mice ( d old) were intracranially inoculated with μl htnv - viral stock at ld . the mice were randomly divided into groups ( mice per group) and intraperitoneally injected with μl of the plasmids (psilencer-s, psilencer-m or psilencer . h vectors at concentrations of μg/μl) or μl pbs for the normal controls and viral controls at and dpi, respectively. the treatments were performed for the survival rate study (n= ); these mice were observed daily for d following infection, and the mortality rate and mean time to death (mtd) were estimated. four mice from each group were sacrificed at , , , and dpi, and brain tissues were collected for viral rna detection by qrt-pcr. at dpi, the brains, lungs and kidneys were dissected from the sacrificed animals (n= ) and used for pathological examinations with h&e staining. at the experimental point of dpi, the brains, lungs and kidneys were harvested, weighed and homogenized into a ~ % (weight/volume) suspension in test medium. the homogenates were frozen and thawed twice and centrifuged at rounds per minute for min. the virus titers were determined as described above. at the experimental point of dpi, blood samples were collected and pooled for each group. tnf-α was detected in the sera with quantikine quantitative sandwich enzyme immunoassays according to the manufacturer's instructions. qrt-pcr analysis total rnas were extracted from the cell or tissues using trizol reagents (invitrogen, usa) following the manufacturer's instructions. first-strand cdna was synthesized by reverse transcription of ng of total rna using random primers (promega) and m-mlv reverse transcriptase (promega) at ºc for min, ºc for min, ºc for min and ºc for min. the amplification was performed using the following primer sets: htnv forward: '-tctagtt-gtatccccatcgactg- ', htnv reverse: '-acatgc-ggaatacaattatggc- '; human gapdh forward: '-ggtggtcctctgacttcaaca- ', human gapdh reverse: '-gttgctgtagccaaattcgttgt- '; and mouse gapdh forward: '-acccagaagactgtgga tgg- ', mouse gapdh reverse: '-acacattgggggtagga-a ca- '. the plasmid pgem-t/htnv, pgem-t/human gapdh, and pgem-t/mouse gapdh were stored in our laboratory and used to generate the standard curves. the absolute quantification of the htnv viral gene was performed as previously described [ ] . the data are presented as vrna copies/ng of gapdh mrna. immunofluorescence assay (ifa) ifa was performed to detect the hantavirus antigens in the infected vero-e cells as previously reported [ ] with monoclonal antibody a , which targets the nucleocapsid protein ( : dilution, provided by the institute of virology, chinese academy of preventive medicine, beijing, china), or rabbit polyclonal antibody targeting glycoprotein g ( : dilution, provided by prof john w huggins, virology division, united states army medical research institute of infectious disease). after incubation, fitc-conjugated goat anti-mouse or anti-rabbit igg (sigma aldrich, : dilution) secondary antibodies were used. the cell cytoplasm was stained red with evans blue. the images were collected using a fluorescence microscope (nikon te ). all of the data were analyzed with spss . software. the data are expressed as the mean±sd for all experiments. oneway anovas were used to assess the significance of the differences between the means. p values below . were considered statistically significant. inhibition of htnv production in vero-e cells by shrnas shrnas were designed to specifically target the s and m genes of htnv. the efficacies of the shrnas in the inhibition of htnv replication were evaluated by transfecting the vero-e cells with nmol/l of s , s , m and m shrnas fol-liu yy et al acta pharmacologica sinica npg lowed by the infection of the cells with tcid / . ml of htnv - . as illustrated in figure a and b, the transfections of the shrnas resulted in the inhibitions of viral rna transcription at hpi of . %± . % (s ), . %± . % (s ), . %± . % (m ) and . %± . % (m ) (p< . vs the virus group). when the cells were treated with the shrnas at hpi, the efficiencies of viral gene inhibition were . %± . % (s ), . %± . % (s ), . %± . % (m ) and . %± . % (m ) (p< . vs virus group). no protective effect was observed in the vero-e cells treated with shcrk. for the yield-reduction assay, culture samples were collected at hpi, and the titers were determined. the inhibitory effects of the four shrnas against htnv replication were evaluated. the four shrnas reduced the htnv yields by approximately log (s) and log (m) compared with the control ( figure c ). based on these results and the viral antigen expression results detected by ifa (data not shown), we concluded that the shrnas that targeted the s and m segments of the htnv gene were able to inhibit rna transcript and virus production in the htnv-infected cells and that shrna-s and shrna-m exhibited a stronger inhibitory effect against htnv. thus, shrna-s and shrna-m were further selected for insertion into the psilencer- . -h of the rnai vector, and the resultant vectors were designated psilencer-s and psilencer-m. the rnai psilencer-s and psilencer-m plasmids were constructed, and their antiviral effects were further evaluated by detecting the viral protein synthesis and rna transcript and progeny virus titers in the htnv-infected cells. the transfection efficiencies were evaluated according to the gfp expression of the co-transfected plasmid pegfp-c and reached approximately %- %. treatment with psilencer-s or psilencer-m at concentrations of , , and μg significantly decreased viral the antigen expression in the htnv-infected vero-e cells at dpi ( figure ). the percentages of positive cells declined with increases in the dose of transfected rnai plasmids ( figure h and i) . only . %± . % of the cells were positive for nucleocapsid protein staining in the -μg psilencer-s treatment group compared to . %± . % in the psilencer-blank treatment group ( figure h) . similarly, the corresponding glycoprotein g positive staining rates were . %± . % and . %± . % in the -μg psilencer-m treatment group ( figure i ). these results indicate that the -μg plasmid transfection achieved the most effective inhibition of viral protein synthesis. we further detected reductions of viral antigen expression in the htnv-infected vero-e cells treated with μg plasmid throughout the course of a -d infection. the greatest inhibitory rates were noted at dpi ( . %± . % for psilencer-s and . %± . % for psilencer-m, figure j) . moreover, the antiviral effect lasted at least d ( . %± . % for psilencer-s and . %± . % for psilencer-m at dpi). the efficacies of the inhibitions of htnv replication by psilencer-s and psilencer-m were also evaluated by detecting viral rna transcription at hpi following -μg plasmid treatment. as illustrated in figure a , when the cells were treated with μg of the rnai plasmid, the efficiencies of s and m gene inhibition reached . %± . % and . %± . % at hpi, respectively. at hpi, the culture samples were harvested, serially diluted and assayed to determine the viral titers. as illustrated in figure b , the two shrna expression plasmids reduced the htnv yields by approximately log compared with the control ( figure b ). taken together, these results indicate that psilencer-s and psilencer-m significantly reduced viral rna transcripts, viral antigen synthesis and progeny virus production in the htnv- infected vero-e cells. suckling mice were infected with htnv as described above and treated with psilencer-s or psilencer-m at and dpi, respectively. compared with the psilencer-blank group, the psilencer-s-and psilencer-m-treated animals died later and exhibited reduced clinical signs including weight loss, ruffled fur, huddling tendencies, paralysis of the hind legs and spasms. all placebo-treated and psilencer-blank-treated mice died with an mtd of d, whereas the treatments with the rnai plasmids increased the survival rates and mtds ( figure a ). the rnai plasmid treatment increased the survival rate to . % and the mtd to - d (p< . ). the rnai plasmid therapy resulted in significant reductions in the viral titers from the brain, lung, and kidney samples collected on dpi ( figure b ), which were homogenized, serially diluted and assayed to determine the viral titers by ifa. no infectious viral particles were detected in the lungs, kidneys, or brains of the survivors at dpi. the dynamic viral loads in the brains were analyzed by qrt-pcr, which indicated that psilencer-s and psilencer-m significantly decreased htnv gene expression on , and dpi ( figure c and d) . four animals from each group were sacrificed at dpi, and the brains, lungs and kidneys were collected for pathological examination. as illustrated in figure , in the viral control group, the lung sections displayed thickened alveolar walls, interstitial lymphocyte and macrophage infiltrations, and hemorrhages. the brain tissues exhibited scattered hemorrhages, congestion, edema, and focal necrosis. the kidney exhibited focal renal interstitial hemorrhages and congestion. both the psilencer-s and psilencer-m treatments decreased the severities of the viral lesions in the brain, lung and kidney tissues. htnv infection has emerged as one of the severest infectious diseases threatening global health and has a significant worldwide economic influence [ ] . in the present study, we demonstrated that vector-based shrnas targeting specific sequences in the s and m segments of htnv inhibited the expression and replication of htnv in vitro and in vivo. our results provide a new therapeutic candidate for the treatment of htnv infections. since the discovery of rnai, synthetic sirna duplexes that generally consist of - nucleotides with -nt ' overhangs have been used to target viral rnas in a sequence-specific manner following transfection [ , ] . however, the application of sirnas has been limited by their short half-lives, the high costs of synthetic sirnas and instability due to degradation by nucleases. another method for inducing rnai is via the transfection of plasmids that express antiviral short-hairpin rnas (shrnas), and this method is also an efficient means of eliciting rnai in vivo [ , ] . our results indicated that the with the exception of ribavirin, no antiviral drugs for the treatment of hantavirus infection have been identified. several members of the bunyaviridae family, particularly htnv, are sensitive to ribavirin [ ] . we have reported that ribavirin can induce an up to . -fold decrease in the vrna level in htnv infection at dpi [ ] , which is equivalent to the in vitro effects of psilencer-s and -m observed in our experiments. with regard to the in vivo administration, the rnai plasmid treatments increased the survival rate to . % in a lethal htnvinfected suckling mouse model. zhou et al reported that the np-specific sirna expression plasmid pbabe-np protected two of the eight mice ( / ) challenged with the lethal dose of avian influenza virus (h n ) that killed all of the control mice [ ] ; this result is similar to the antiviral effects of the other sirna expression plasmids against htnv observed in our in vivo experiments. however, we noticed that ribavirin has been reported to be capably of affording % protection against lethal andes virus infections in hamsters [ ] and also increases the survival rate to . % in seov-infected suckling icr mice [ ] . the explanation of these phenomena may be related to the delivery of sirna. the shrna expression plasmid was distributed in the brain because the blood-brain barrier (bbb) of newborn mice is immature [ ] . however, as a nonviral vector, the psilencer shrna expression vector does not readily cross the cellular membrane and is not stably introduced into the cells. further studies are required to solve this problem, which is frequently considered a hurdle for the development of sirna-based therapeutics [ ] . to our knowledge, this is the first report of the inhibition of hantavirus infection with an shrna; thus, this report enriches the antiviral spectrum of rnai therapy. in recent years, known and emerging viruses have posed increasingly serious threats to public health. effective vaccines and antiviral drugs are not available for the majority of these viruses. the transfection of shrna-encoding plasmids is probably best-suited for the treatment of acute viral infections, particularly among people infected with virus strains that are resistant to conventional antivirals and in cases of severe or re-emergent disease. however, a number of barriers to medical application remain to be solved, eg, improvements in delivery strategies and the safety and stability of sirna and other issues. a global perspective on hantavirus ecology, epidemiology, and disease hantaviruses--globally emerging pathogens hantaviruses: immunology, treatment, and prevention hantavirus infection in east asia hantavirus-induced immunity in rodent reservoirs and humans new ecological aspects of hantavirus infection: a change of a paradigm and a challenge of prevention-a review drug targets in infections with other emerging viruses: influenza viruses, metapneumovirus and hantaviruses in vitro and in vivo activity of ribavirin against andes virus infection rnai therapeutics: principles, prospects and challenges advances, nuances, and potential pitfalls when exploiting the therapeutic potential of rna interference stable inhibition of hepatitis b virus expression and replication by expressed sirna short hairpin rna-mediated inhibition of hsv- gene expression and function during hsv- infection in vero cells sirna targeting the leader sequence of sars-cov inhibits virus replication rna interference of influenza virus production by directly targeting mrna for degradation and indirectly inhibiting all viral rna transcription rna interference of avian influenza virus h n by inhibiting viral mrna with sirna expression plasmids inhibition of coxsackievirus b replication by small interfering rnas requires perfect sequence match in the central region of the viral positive strand progress in rnai-based antiviral therapeutics efficacy of arbidol on lethal hantaan virus infections in suckling mice and in vitro rational sirna design for rna interference establishment of sybr green-based qpcr assay for rapid evaluation and quantification for anti-hantaan virus compounds in vitro and in suckling mice therapeutic potentials of gene silencing by rna interference: principles, challenges, and new strategies rna interference against viruses: strike and counterstrike characterization of in vitro and in vivo antiviral activity of lactoferrin and ribavirin upon hantavirus the rights and wrongs of blood-brain barrier permeability studies: a walk through years of history sirna as a tool to improve the treatment of brain diseases: mechanism, targets and delivery this work was supported by the national natural science yuan-yuan liu, zhan-qiu yang and hai-rong xiong designed the research plan; yuan-yuan liu, liang-jun chen, yan zhong, meng-xin shen, nian ma, bing-yu liu and fan luo performed the research; yuan-yuan liu and wei hou analyzed the data; and yuan-yuan liu, zhan-qiu yang and hai-rong xiong wrote the paper. key: cord- -rhpfpku authors: zhong, hui-hai; wang, hui-yuan; li, jian; huang, yong-zhuo title: trail-based gene delivery and therapeutic strategies date: - - journal: acta pharmacol sin doi: . /s - - - sha: doc_id: cord_uid: rhpfpku trail (tumor necrosis factor-related apoptosis-inducing ligand), also known as apo l, belongs to the tumor necrosis factor family. by binding to the death receptor (dr ) or dr , trail induces apoptosis of tumor cells without causing side toxicity in normal tissues. in recent years trail-based therapy has attracted great attention for its promise of serving as a cancer drug candidate. however, the treatment efficacy of trail protein was under expectation in the clinical trials because of the short half-life and the resistance of cancer cells. trail gene transfection can produce a “bystander effect” of tumor cell killing and provide a potential solution to trail-based cancer therapy. in this review we focus on trail gene therapy and various design strategies of trail dna delivery including non-viral vectors and cell-based trail therapy. in order to sensitize the tumor cells to trail-induced apoptosis, combination therapy of trail dna with other drugs by the codelivery methods for yielding a synergistic antitumor efficacy is summarized. the opportunities and challenges of trail-based gene delivery and therapy are discussed. nucleic acid-based therapy has been considered one of the most promising strategies for the treatment of various diseases [ ] . tumor necrosis factor (tnf) plays an important role in the homeostatic regulation of the immune system [ ] . although tnf is potent in causing tumor necrosis, the first two clinical trials of tnflike molecules for cancer therapy failed because of lethal inflammatory shock syndrome and fulminant liver toxicity [ , ] . subsequentlyx, a novel tnf family member, tnf-related apoptosis-inducing ligand (trail), was found [ , ] ; this protein is a type ii transmembrane protein and can be released from the cell surface in soluble form via proteolysis [ ] . soluble trail is nontoxic to normal cells, and in fact, there is a trace amount of endogenous trail (~ pg/ml) in healthy adult plasma [ , ] . the trail protein is expressed in various tissues-predominantly in the spleen, lung, and prostate-and on the surface of cytotoxic t cells and natural killer (nk) cells [ ] . its death receptors (drs), dr and dr , are overexpressed in many types of cancer cells. importantly, trail is capable of killing tumor cells without causing lethal adverse effects [ , ] . apoptosis is an essential function of the maintenance of cellular homeostasis and prevents a number of diseases, including cancer [ ] . tumorigenesis is associated with defects in apoptosis regulation [ ] . there are two major apoptotic pathways: the intrinsic, or mitochondrial, pathway usually induced by chemotherapy [ ] , and the extrinsic, or dr, pathway that mediates extrinsic programs of cell death, such as trail-induced apoptosis. however, these two pathways usually associate with each other downstream via "crosstalk" [ ] . the extrinsic pathway is activated by extracellular proapoptotic stimulators that bind to cell surface receptors [ ] . there are five homologous human receptors for trail: the full-length intracellular death domain (dd)-containing receptors dr [ ] and dr (trail-r ) [ ] [ ] [ ] ; the decoy receptor (dcr or trail-r ), which lacks an intracellular domain; [ , , ] dcr (trail-r ), which contains a truncated dd; [ , ] and the soluble receptor osteoprotegerin (opg). among these receptors, only the binding of trail to dr or dr -because of their integrated intracellular structure-can induce an apoptotic effect; dr has the highest affinity for trail [ ] . however, trail/dcr binding cannot induce downstream signaling, because dcr lacks an intracellular domain, while dcr and opg act as nf-κb ligand receptors, which induce nf-κb activation but not apoptosis. after dr or dr binding to trimeric trail, the intracellular dd structure of the drs is altered, and binding to fas-associated death domain-containing protein (fadd) then occurs. then, fadd binds to procaspase- /- via the death effector domain (ded) on the n-terminus, thereby forming dr /dr -fadd-procaspase- /- , which is called the death-inducing signaling complex (disc). oligomerization and autocatalysis of procaspase- leads to the activation of caspase- , which consequently triggers cleavage of the effector caspases- /- /- to induce apoptosis. furthermore, caspase- promotes the release of cytochrome c, inducing intrinsic apoptosis via the mitochondrial pathway in type ii cells [ ] . the trail-induced apoptosis process is summarized in fig . the ability of the tumor-specific action of trail to induce the apoptosis of cancer cells while sparing normal cells is attractive and renders trail signaling a potential therapeutic target. to date, clinical trials of trail have focused on recombinant trail protein and antibodies against trail-r (table ) . however, clinical trials have shown inadequate treatment outcomes [ ] . the recombinant form of trail and anti-trail-r antibodies, as well as their combination with other components, have not achieved the expected efficacy [ ] . for example, the recombinant form of trail did not exhibit significant antitumor effects, partially due to its short half-life [ , ] , while tas , an antibody targeting dr , showed acute toxicity in a phase i clinical study [ ] . there are three major limitations of trail-based therapy: its short in vivo half-life [ , ] , its poor tumor-targeting efficacy, and resistance to trail monotherapy [ , ] . the emergence of nanotechnology has provided a useful tool to address these problems. trail-based nanotherapies offer the potential to improve the stability of trail and prolong its half-life in the bloodstream, to specifically deliver trail to target sites and to overcome resistance to trail [ ] . compared with direct administration of trail proteins, trail gene therapy also has the unique advantages of delivering trail-encoding dna into tumor cells to locally secrete the trail protein on the membrane or into the tumor microenvironment, thereby overcoming the limitations of recombinant trail protein. notably, combination therapies of trail with other anticancer agents via a codelivery system may solve the problem of trail resistance. trail gene therapy also benefits from the "bystander" effect, by which not only the host cancer cells but also the neighboring cancer cells can be killed by both secreted and membrane-bound trail [ ] . trail shows a unique advantage over other cell deathinducing ligands (e.g., fas ligand, fasl), of which only the membrane-bound form can induce apoptosis, while the intrinsic soluble form cannot [ ] . liposome-bound trail induces even more efficient apoptosis than the soluble form [ ] . trail-based gene therapy has been investigated in various types of tumors, such as hepatocellular carcinoma and cervical cancer. since the completion of the human genome project, the development of gene therapy has accelerated. gene therapy depends on the success of delivering specific nucleic acids to target sites by overcoming a series of biobarriers; in other words, it relies on the efficient delivery of vectors. viral vectors are well known for their high transfection efficiency, which mainly relies on their ability to integrate genes into the genome of host cells. this approach increases the risk of insertion mutations at the integration site, especially if there are hotspots near prooncogenes. thus, there is an urgent need to find a highly efficient vector with low genotoxicity and immunogenicity. nonviral vectors delivering genes typically via a nanosized platform are another option, with obvious advantages of safety and the high packing capacity of nucleic acids. in addition, advances in nanotechnology have provided good insight into rational designs for targeted delivery. although numerous nonviral vectors have been developed, the amount of data from clinical trials has been very limited due to their low transfection efficiency [ ] . thus, there is a pressing need to develop nonviral vectors with increased efficiency. nonviral vectors include cationic lipids, cationic polymers, and inorganic nanoparticles. polyethyleneimine (pei), for example, has high transfection efficiency in various cell lines. for the systemic delivery of gene therapeutics, delivery systems must cross a series of barriers, which is an important consideration in the design of delivery systems. regarding trail-based therapy, viral vectors are prominently used for cell therapy, while nonviral vectors deliver plasmidencoded trail (ptrail) to targets. in this review, we summarize trail-based gene therapeutic strategies and discuss the challenges facing clinical trials of trail. most gene therapy clinical trials involved viral vectors [ ] . modified viruses such as retroviruses, lentiviruses, adenoviruses, and adeno-associated viruses (aavs) are commonly used to deliver genes. viruses are able to transfer genes into many cell types, with highly efficient transfection. griffith et al. constructed a trail-encoded adenovirus and found that rapid expression of the trail protein and apoptosis of tumor cells were triggered by the activation of caspase- [ ] . oncolytic adenoviruses (oads) can selectively replicate in cancer cells while sparing normal cells; thus, these viruses have been used in clinical trials for anticancer therapy. el-shemi et al. applied systemic therapy with ad-Δb/ing (inhibitor of growth ) plus ad-Δb/trail in an orthotopic human hepatocellular carcinoma (hcc) -bearing mouse model. this study found that the combination of these agents elicited potent eradicative effects by inducing apoptosis and immune responses, and suppressing tumor angiogenesis without causing obvious overlapping toxicity [ ] . in addition, the use of viral vectors has been explored for trailbased cell therapy, which will be introduced in section . because of the inherent shortcomings of viral vectors, including their limited dna packaging capacity, complicated production processes, broad tropism, cytotoxicity, immunogenicity, and tumorigenicity, nonviral vectors have also been widely investigated as an option for gene therapy [ ] . in contrast to viral vectors, nonviral vectors have the advantages of low immunogenicity, high delivery capacity, and easy preparation [ , ] . before they reach the target cells, delivery systems need to cross a series of biobarriers. in the physiological environment, positively charged complexes are more prone to bind serum proteins and aggregate and thus be cleared rapidly [ , ] . shielding the positive charge using polyethylene glycol (peg) or anionic materials such as γ-pga can be helpful. another challenge is selective accumulation at the target tissue, which requires specific designs, such as arming targeted ligands. subsequently, fig. pathway of trail-induced apoptosis. trail binds to five receptors, including four membrane-bound receptors (i.e., dr , dr , trail-r , and trail-r ) and one soluble receptor (opg). only binding to dr or dr results in receptor trimerization and recruitment of fadd via the dds of dr or dr . after further recruitment of caspase- , these proteins form a complex named disc, which can activate caspase- . in type i cells, caspase- activates caspase- and triggers apoptosis via the extrinsic pathway. however, in type ii cells, the intrinsic pathway is triggered via caspase- /bid/tbid, and consequently, bax/bak on the mitochondrial membrane is activated to induce the release of cytochrome c, which promotes the formation of the apoptosome with apaf and procaspase- . subsequently, activation of caspase- and caspase- is induced. figure adapted from fig. in ref. [ ] the dna of interest needs to be delivered to the nucleus for transcription [ ] . thus, the design of gene delivery systems should account for these considerations. polymers. cationic polymers are an important class of nonviral vectors. poly(l-lysine) (pll) and polyethyleneimine (pei) were developed for gene delivery in the s. subsequently, numerous cationic polymers have been developed and used, including poly [( -dimethylamino) ethyl methacrylate] (pdmaema), poly(β-amino ester)s, and various carbohydrate-based polymers and dendrimers. pei and its derivatives are the most commonly used polymeric vectors for gene delivery, with the advantage of the "proton sponge" effect, which facilitates the endosomal escape of gene drugs [ ] . however, the major hurdles to overcome for the in vivo use of pei are the substantial cytotoxicity related to its strong positive charge and the issue of its stability in the bloodstream [ ] . to address these issues, modification of pei and the surface coating have been investigated. for example, a γ-pga corona can shield the positive charge of the pei/dna complex, thereby decreasing its toxicity and increasing its stability. it has been reported that the γ-pga-coated branched pei/ptrail complex can efficiently transfect pancreatic stellate cells expressing fibroblast growth factor receptors [ ] . although pei k is the gold standard for polymer transfection, its high molecular weight generally causes high toxicity. therefore, many studies focusing on low-molecular-weight pei have been reported. for instance, a gene delivery system for brain targeting was established by using pei k modified with myristic acid (mc); mc-pei k /dna nanoparticles could interact with the cell membrane via the hydrophobic segment on mc that can be incorporated into the phospholipid bilayer. mc-pei k /porf-htrail nanoparticles were effective against the growth of intracranial tumors [ ] . furthermore, cell-penetrating peptide (cpp)-modified and mannosylated low-molecular-weight pei (termed man-pei k -cpp) was constructed as a vector to deliver ptrail for colorectal cancer treatment. man-pei k -cpp increased the cellular uptake efficiency and improved the efficiency of transfection [ ] . then, a ternary complex system was developed by a γ-pga-based γ-glutamyl transpeptidase (ggt)-targeting and surface camouflage strategy via a layer-bylayer self-assembly method. biodegradable polyanionic γ-pga protected the pei/pdna complexes from interaction with body fluid components and interacted with tumor-overexpressed ggt, which can mediate the endocytosis of nanoparticles for cervical cancer gene therapy (fig. ) [ ] . hyaluronic acid (ha) also acts as a biocompatible polyanionic biomaterial for shielding positive charges. an ha-decorated polyethyleneimine-poly(d, l-lactide-co-glycolide) nanoparticle (pei-plga np) system was established for targeted codelivery of ptrail and gambogic acid (ga) for triple-negative breast cancer (tnbc) therapy [ ] . ga was encapsulated into the hydrophobic core of pei-plga nps, while ptrail was adsorbed onto the positively charged np surface. the ha coating on the pei-plga nps not only functioned as a shell to neutralize the excess positive charge on the nps but also served as a targeting ligand by binding to the cd receptor on tnbc cells. a series of terpolymers were synthesized via the enzyme-catalyzed copolymerization of lactone with dialkyl diester and aminodiol. targeted delivery of ptrail to the tumor by the terpolymers resulted in significant inhibition of tumor growth with minimal toxicity in vivo, and the transfection efficiency was associated with the high molecular weight and increased hydrophobicity [ ] . intriguingly, it was found that preparation via a high concentration the clinical trials can be found at https://www.clinicaltrials.gov trail-based gene delivery and therapeutic strategies hh zhong process (i.e., a small reaction volume) resulted in large pei/dna complexes that had a higher gene transfection efficiency than their small counterparts prepared at a low concentration (fig. ) [ ] . the mechanisms were associated with macropinocytosis and fast dissociation. accordingly, large-sized pei/ptrail complexes exhibited increased anticancer efficacy via local or regional administration in subcutaneous xenograft and peritoneal xenograft mouse models. poly(beta-amino ester)s (pbaes) are a class of cationic synthetic polymers that can be used as nonviral gene carriers. these compounds are easy to synthesize, bind effectively to dna, and are hydrolytically degradable under physiological conditions. tzeng et al. reported the use of pbae nanoparticles containing ptrail for the selective transfection of cancer cells and the induction of apoptosis in several human cancer cells [ ] . additionally, shen et al. developed an ingenious vector comprising quaternary amines carrying n-propionic -acetoxybenzyl ester substituents; the -acetoxybenzyl ester group can undergo rapid intracellular esterase-catalyzed hydrolysis, which subsequently triggers a reversal of the polymer's charge from cationic to zwitterionic [ ] . due to the high cytosolic esterase activity in cancer cells but low activity in fibroblasts, the loaded ptrail can be delivered specifically into cancer cells without damaging fibroblasts, preventing the expression of wnt b (fig. ) [ ] . the same group also synthesized another enzyme-responsive cationic polymer, poly (pqdea), which is rapidly hydrolyzed by intracellular esterases to form anionic poly(acrylic acid) to confer low cytotoxicity and fast release of dna [ ] . pqdea/dna polyplexes were further coated with a lipid layer to generate serum-stable lipidic polyplexes (lpqdea/dnas) for in vivo use. lpqdea/ptrail strongly inhibited tumor formation as effectively as paclitaxel but gave less tumor relapse and longer survival. in addition, some inorganic materials have been modified with polymers for ptrail delivery. for example, a sandwich-type peicoated gold nanocomposite coloaded with ptrail and nucleartargeted dexamethasone (dexa) (termed au-pei/ptrail/pei-dexa) exhibited efficient transfection and significantly inhibited the growth of hep b tumors [ ] . a targeted iron oxide np coated with a chitosan-peg-pei copolymer and chlorotoxin was developed; this np efficiently delivered ptrail into human t g gbm cells and induced the secretion of trail [ ] . further, systemic administration to mice bearing t g-derived flank xenografts resulted in almost imperceptible tumor growth and induced apoptosis in tumor tissue. to overcome treatment limitations of adenoid cystic carcinoma, the fe o -pei-plasmid complex (fpp) was generated, in which iron oxide nps were modified by positively charged pei to enable them to carry pactert-trail [ ] . the efficiency of fpp-mediated transfection was sixfold higher than that of pei alone or of lipo , and fpp-mediated trail gene transfer efficiently inhibited sacc- tumor growth. dendrimers. dendrimers, which are characterized by their welldefined size and low polydispersity index, have been widely used for drug and gene delivery [ ] . jiang et al. reported a gene vector generated by polyamidoamine (pamam) and angiopep- another strategy has been reported in which transferrin (tf) was conjugated to a generation diaminobutyric polypropylenimine (dab) dendrimer; this delivery system harbors plasmids encoding for tnf-α, trail, or il- and leads to therapeutic effects on prostate tumors following intravenous administration [ ] . in addition, lactoferrin (lf)-bearing dab dendriplexes showed similar efficacy [ ] . later, coumarin-anchored low-generation dendrimers (g pamam dendrimers) were established to improve dna binding and gene delivery [ ] . the coumarin moieties endowed these materials with light-responsive drug delivery behaviors, and the drug-loaded nanoparticles exhibited complementary anticancer activity through the codelivery of -fluorouracil and ptrail. a triazine-modified dendrimer, g -dat , was synthesized and used as a vector for ptrail therapy, showing higher transfection efficacy than commercial transfection reagents such as lipo and superfect [ ] . furthermore, in vivo studies demonstrated that g -dat /ptrail efficiently inhibited tumor growth in osteosarcoma-bearing mice. pishavar et al. synthesized a vector composed of pamam dendrimers modified with alkyl-carboxylate chains, peg, and cholesteryl chloroformate for delivering ptrail, and these pamam g -alkyl-peg ( %)-chol ( %)-trail complexes inhibited ct tumor growth in mice [ ] . peptides and proteins. cationic peptides rich in residues such as lysines or arginines are able to condense dna. furthermore, conjugation of peptide ligands to delivery systems allows targeting to specific types of cancer cells [ ] . a biomimetic vector was developed for delivering ptrail to tumor; it contained an adenovirus μ peptide for pdna condensation, a synthetic cyclic peptide for tumor-targeting and intracellular delivery, a phresponsive synthetic fusogenic peptide for endosome escape, and a nuclear localization signal from human immuno-deficiency virus for intranuclear delivery [ ] . up to % of zr- - breast cancer cells were killed after exposure to ptrail in complexation with this vector. a dimerized hiv- tat peptide was used to formulate fig. illustration of the esterase-responsive charge-reversal polymer (erp) and its lipid-coated esterase-responsive polyplexes with trail plasmid for cancer gene therapy. a the erp is a pei whose amines are quaternized with propionic -acetoxybenzyl ester. hydrolysis of the phenolic acetate triggers the elimination of p-hydroxymethylphenol and consequent conversion of the cationic polymer into a zwitterionic form. b erp condenses plasmid dna into the polyplexes, which are easily coated with dc-chol/dope lipids to form lipidic esterase-responsive polyplexes (lerps). after i.p. injection into nude mice bearing hela cell-derived tumors, tumor cells internalize lerps into the cytosol, which is rich in esterases. the lerps disassemble and release the polyplexes to allow the esterases to trigger the charge reversal of the erp and thus plasmid release. these free plasmids enter the nucleus for effective gene expression, inducing apoptosis when delivering the trail gene. in tumor fibroblasts, the low esterase level cannot efficiently induce the charge reversal process, and the trail plasmids will not be expressed, preventing wnt b production. reprinted with permission from [ ] a nanoparticle vector (dtat np) to leverage the efficiency of this cell penetration strategy for tumor-targeted gene delivery [ ] . in cell culture, dtat np was an effective pdna transfection vector with negligible cytotoxicity. gene expression in tumor tissues lasted for > days after intratracheal administration. bolus administration of dtat np-encapsulated ptrail markedly attenuated the growth of tumors derived from lewis lung carcinoma cells. phosphatase and tensin homolog (pten) and trail genes loaded into zein nanoparticles showed antiproliferative activity against hepg cell lines, indicating their potential for gene therapy for the treatment of hcc [ ] . liposomes. cationic lipids have been widely used for nucleic acid delivery and for advances in gene delivery through their molecular design. cationic lipids can spontaneously form specific types of complexes for condensing and encapsulating dna into particles [ ] . huang et al. reported a novel lipid ( , -di-( z-octadecenoyl)- biguanide-propane (dobp)) that was elaborately designed by utilizing biguanide as the cationic head group [ ] . this novel cationic lipid acted as a gene carrier and had a metformin-like antitumor activity via activation of the ampk and inhibiting the mtor pathways. dobp-lpdtrail nps showed potent efficacy against tumor progression. then, these nps were used to transfer a secreted form of trail (strail) to tumor-associated fibroblasts in order to secrete cytotoxic proteins to tumor cells via lipidcoated protamine [ ] . strail triggered apoptosis in tumor cell nests adjacent to tafs. furthermore, strail converted the residual fibroblasts to a quiescent state, thus arresting the tumor growth and remodeling the microenvironment to facilitate the secondwave nanotherapy. the system showed good efficacy in an orthotopic xenograft model of human pancreatic cancer, where the desmoplastic stroma is a major barrier to the delivery of therapeutic nanoparticles. chen et al. developed a tumor-targeted lcpp (lipid/calcium/ phosphate/protamine) np to deliver ptrail into hcc cells in a mouse model of hcc [ ] . ptrail was entrapped in a phresponsive calcium phosphate (cap) core, and protamine was included to direct intranuclear delivery. trail resistance could be reversed by intracellular release of ca + from the cap core that induced dr up-regulation through camkii activation (fig. ) . other vectors. gong et al. developed a well-tailored and versatile "core-shell" ternary system (rrphc) for systemic gene delivery to treat aggressive melanoma [ ] . this system consisted of a core of fluorinated polymers (pfs) that bound to a plasmid (pdna) and a negatively charged multifunctional rrph (rgd-r -peg-ha) shell constructed by grafting a hyaluronan polymer with peg side chains, which were further conjugated with the r -rgd tandem peptide on the distal side, simultaneously targeting the cd receptors and integrin α v β receptors overexpressed on the neovasculature and most malignant tumor cells. systemic injection of the proapoptotic mtrail plasmid by rrphc ternary complexes inhibited the melanoma growth, without noticeable side toxicity. next, this group reported a similar system of an artificial virus core-shell to target cancer stem cell-like cells [ ] . the intravenously injected nanoparticles accumulated at the tumor sites while reducing the exposure to the normal tissues, and efficiently arrest the tumor growth, without obvious systemic toxicity. mesenchymal stem cells (mscs) are a population of fibroblast-like cells originally isolated from bone marrow and other tissues, including adipose tissue, peripheral blood, umbilical cord blood, and wharton's jelly, among others [ , ] . mscs have therapeutic potential in several pathological conditions and unique immunological features [ ] [ ] [ ] . in recent years, the cellular vehicle function of mscs has been used to transfer trail to the tumor parenchyma [ ] [ ] [ ] . lee et al. reported a novel application of magnetic core-shell nanoparticles for the dual purpose of delivering and activating a heat-inducible gene vector that encodes trail in adipose-derived mesenchymal stem cells (ad-mscs) [ ] . this group developed a plasmid harboring the heat shock protein b' (hsp b ) promoter. the magnetic core-shell nanoparticles (mc nps) was composed of znfe o magnetic nanoparticle core and mesoporous silica shell, as well as a surface coating of pei for dna binding. the mc nps facilitated the intracellular delivery of the heat-inducible plasmid into ad-mscs via magnetic guidance, and after systemic injection, the engineered ad-mscs could home to tumors/metastases. trail expression could be specifically activated via the induction of mild magnetic hyperthermia (~ °c). this system enhanced control over the activation of stem cell-based gene therapies. it was reported that human mscs were transduced with trail and the ires-egfp reporter gene under the control of the tetracycline promoter using a lentiviral vector [ ] . the transduced and activated mscs led to the apoptosis and death in various cancer cells in coculture experiments. the in vivo studies demonstrated that the i.v. injected trail-expressing mscs significantly arrested the tumor growth. gao et al. transfected ptrail into mscs with a nonviral vector, pei -cyd, prepared by linking low-molecular-weight polyethyleneimine (pei) and β-cyclodextrin (β-cd) [ ] . the lung tumor homing ability of mscs expressing trail in vivo proved to be efficient for lung metastasis therapy. fig. shows a schematic of the specific process of this cell-based trail therapy. human umbilical cord-derived mesenchymal stem cells (humscs) were transfected by lentiviral vectors coding the strail with the alpha-fetoprotein (afp) promoter, and the treatment efficacy of these engineered humscs on orthotopically implanted hepatocarcinomas in mice was examined [ ] . humscs could migrate to the hepatocarcinoma, where the afp promoter was triggered by the early hepatic differentiation of humscs, and expressed strail at the cancer cells and yielded significant antitumor activity. dominici et al. transduced human ad-mscs with a retroviral vector encoding full-length human trail [ ] . ad-mscs could target various cancer cell lines in vitro, and reverse the trail resistance by coadministration of bortezomib. these ad-mscs targeted to tumors and induced apoptosis, without apparent side toxicity. the engineered mscs with trail expression also induced apoptosis by cell-to-cell contact in the trailresistant ewing sarcoma (ews) that is insensitive to amg , an antibody against the death receptor dr , too, and the treatment effect was confirmed in two orthotopic models of ews [ ] . despite these encouraging results, there is some concern regarding the safety of inoculating both wild-type and genetically modified mscs, especially regarding their possible damaging effects on normal organs, malignant transformation, and promotion of cancer growth [ ] . then, researchers incorporated suicide genes-the herpes simplex virus thymidine kinase (hsv-tk) and cytosine deaminase genes-into mscs to control their fate once infused [ ] . this approach is based on a variant of human caspase- that binds with high affinity to a synthetic, bioinert small molecule (ap ), leading to cell death [ ] . conventionally, mscs have been genetically modified for cancer therapy by using viral vectors that can elicit oncogenicity, thus limiting their use in clinical trials. chen et al. used nonviral agents such as a polylysine-modified polyethyleneimine (pei-pll) copolymer to generate genetically engineered mscs with suicide genes, namely, hsv-tk and trail [ ] . the mscs armed with suicide genes along with prodrug ganciclovir can induce significant antitumor effect to glioblastoma by intratumoral injection both in vitro and in vivo. another study on delivering the trail gene for stem cell-mediated gene therapy was conducted by using nonviral vectors (a less efficient but safer method). na et al. prepared the polyplexes of ptrail and bpei, and photochemical internalization (pci) was applied to improve the polyplex entrapping in hmscs and enhance the transfection efficiency of ptrail; the tumor-homing hmscs could also increase the trail secretion in the tumors [ ] . pci-mediated polyplex loading significantly enhanced trail expression in stem cells, and that homing ability enhanced cancer targeting. exposure of polyplex-loaded hmscs (ptrail/bpei@hmscs) to laser irradiation resulted in a beneficial therapeutic antitumor effect in a xenograft mouse model. in addition to mscs, hu et al. reported a dc cell-based therapy for colon cancer cells [ ] . tyrosine kinase receptor ligand (fl) and trail plasmids were constructed for combination therapy. fl, a hemopoietic growth factor, is important in progenitor cell proliferation and differentiation, which can enhance the proliferative and antitumor effect of dcs. these two plasmids were transfected into dcs by lipo . the combination of fl-carrying b trail expression and secretion were quantified by confocal microscopy and elisa, respectively. c sp -lcpp nps without pdna significantly increased dr expression in a dose-dependent manner in hcc cells. d the camkii inhibitor k a prevented the effects of lcpp nps on dr upregulation. e, f treatment with trail pdna loaded into sp -lcpp nps showed higher cytotoxicity in hcc cells than to control cells. however, these nps exhibited slight cytotoxicity in murine hepatocyte fl b cells. reprinted with permission from [ ] dcs and trail-carrying dcs showed a good level of apoptosis in colon cancer [ ] . although trail-induced apoptosis is an attractive therapeutic target, resistance to trail readily develops during treatment. this therapeutic resistance derives from two sources: intrinsic resistance in some highly malignant tumors [ ] [ ] [ ] and acquired resistance after repeated exposure to trail [ ] . resistance to trail is conferred by multiple receptors and involves a series of signaling pathways and activation of inhibitory molecules [ ] . trail signaling begins with the binding of trail to the death receptors. first, binding to decoy receptors (e.g., trail-r and r ) will not lead to the activation of downstream trail-signaling. second, mutation or downregulation of the functional drs (e.g., dr or dr ) can also result in resistance. for instance, transfection of mutated dr into sw colon cancer cells caused a lower efficacy of cell killing than transfection of the wild-type counterpart [ ] . low expression of dr contributed to trail resistance in anti-dr antibody therapy [ ] . in this case, upregulation of dr is a therapeutic strategy for sensitization to trail treatment [ ] . third, the multifunctionality of downstream trail signaling also induces resistance. the assembly of the disc can be inhibited by apoptosis inhibitors. for example, cflip, with a similar structure to caspase- , can inhibit caspase- activation after binding to fadd [ ] . the ratio of cflip/caspase- is correlated with trail resistance in various tumors, such as hepatocellular carcinoma and burkitt lymphoma [ , ] . in type ii cells, trail-initiating apoptosis is mainly mediated by the mitochondrial pathway (fig. ) . upregulation of the proapoptotic proteins bax and bak promotes cell death, while the antiapoptotic proteins bcl- and bcl-x l determine cell survival [ , ] . furthermore, inhibitors of apoptosis proteins (iaps) can block apoptosis by inhibiting the activity of the effector caspases (e.g., caspase- and caspase- ), and overexpression of iaps can confer trail resistance [ , ] . however, this effect can be reversed by iap antagonists such as smac/diablo, which promotes apoptosis by interacting with iaps [ , ] . the mechanisms are summarized in fig. . in addition, epigenetic changes play a role in trail resistance via the regulation of caspase- gene expression, and dna methylation can restore apoptosis in trail-resistant tumor cells. trail resistance is also related to nf-κb and mitogen-activated protein (map) kinases. however, these signaling pathways showed both proapoptotic and antiapoptotic effects [ ] . in conclusion, dr dysfunction and overexpression of cflip, bcl- , bcl-x l , and iaps contribute to trail resistance, and therefore, regulation of drs and inhibition of antiapoptotic proteins resensitizes cells to trail treatment [ ] . the resistance of cancer cells to trail has encouraged the investigation of combination therapy. in recent years, many multifunctional drug delivery systems, including liposomes, micelles, and polymeric nanoparticles, have been developed for the codelivery of genes and drugs. here, we summarize the delivery and therapeutic strategies of combinations of ptrail and different types of drugs. combination with trail sensitizers the combination of trail with its sensitizers is a promising strategy to overcome trail resistance. it was found that polyether ionophore antibiotics (e.g., monensin) can overcome trail resistance via endoplasmic reticulum stress induction, dr fig. the nonviral vector pei -cyd was used to transduce trail plasmid into mscs. trail-armed mscs showed a lung tumor-homing ability and an antitumor effect on lung metastasis-bearing c bl/ mice after i.v. injection. reprinted with permission from [ ] fig. inhibition of trail signaling results in trail resistance. when the disc assembles, flip (flice-like inhibitory protein) can competitively bind to fadd and limit the recruitment of caspase- . while bcl- and bcl-x l can inhibit bax and bak, tbid plays roles in inhibiting bcl- and bcl-x l and activating bax and bak in type ii cells. iaps can strongly inhibit the activation of effector caspases, as smac/ diablo is needed to interact with iaps to release effector caspases. figure adapted from fig. in ref. [ ] upregulation and cflip downregulation [ ] . huang et al. constructed a biocompatible nanosystem for the codelivery of ptrail and monensin, in which low-molecular-weight pei . k (lmw-pei) was intermolecularly crosslinked via disulfide bonds using sulfhydryl β-cyclodextrin as a linker (fig. ) [ ] . the resulting β-cd-sspei carrier, which can bind efficiently with ptrail, can serve as a carrier for codelivery, while monensin is encapsulated inside the cavities of the β-cyclodextrin molecules. the mo β-cd-sspei ptrail nanocomplex can further be modified by a polyanionic polymer γ-pga, thus protecting the nanocomplex from interaction with serum proteins and consequently extending its half-life in the bloodstream. furthermore, γ-pga/ mo β-cd-sspei ptrail can achieve tumor targeting via specific binding between γ-pga and tumor-overexpressed ggt, which can mediate the endocytosis of the nanocomplex. inside cancer cells, the acidic endosomal environment facilitates the detachment of γ-pga, whose conformation is ph-dependent, and the exposed pei facilitates endosomal escape. importantly, the intermolecular crosslinks of pei can be degraded, and ptrail and monensin can be released. monensin can increase intracellular ros levels and induce apoptosis. moreover, dr expression is upregulated, thus synergizing with trail-based treatment for colon cancer gene therapy. combination with chemotherapeutic drugs trail exhibits improved efficacy in combination with chemotherapy because chemotherapeutic agents can sensitize tumors to trail-induced apoptosis via crosstalk between the intrinsic and extrinsic pathways of cell death [ ] . the chemotherapeutic drugs doxorubicin (dox) and paclitaxel (ptx) were explored to determine their synergistic antitumor effect with ptrail. ebrahimian et al. reported a vector composed of polypropylenimine (ppi) modified with -bromodecanoic acid for the codelivery of ptrail and dox for tumor therapy [ ] . in addition, a host-guest conjugated nanoparticle for the codelivery of dox and ptrail was designed [ ] . the adamantane-conjugated dox (ad-dox, guest component) and the pei-cyclodextrin conjugates (pei-cd, host) were selfassembled into the supramolecular pei-cd/ad-dox, which further bound with trail dna to form the pei-cd/ad-dox/pdna snps. the snps exhibited the enhanced therapeutic efficacy with the significantly increased survival rate of the tumor-bearing mice. jiang et al. investigated the codelivery of the htrail-encoding plasmid open reading frame (porf-htrail) and dox using a tumor-targeting carrier, a peptide haiyprh (t )-conjugated polyethylene glycol-modified polyamidoamine dendrimer (pamam-peg-t ) [ ] . in this system, approximately dox molecules were bound to one porf-htrail molecule, and t served as a ligand targeting tumor cell-overexpressed transferrin receptors. this codelivery system induced apoptosis of tumor cells and efficiently inhibited bel- tumor growth in vivo. furthermore, the combination therapy strategy was further applied to glioma, in which dendrigraft poly-l-lysine (dgl), modified by the t peptide and conjugated with dox via a ph-sensitive hydrazone bond, was used to deliver porf-htrail to glioma tissue [ ] . in addition, other dual targeting systems have also been developed for the codelivery of dox and porf-htrail for glioma treatment [ , ] . regarding the combination of ptx and trail, an angiopep- peptide-modified cationic liposome (ang-clp) for the efficient codelivery of pegfp-htrail and ptx to glioma was reported. angiopep- can target the low-density lipoprotein receptorrelated protein (lrp) overexpressed on the bbb and on glioma cells [ ] . lu et al. developed two kinds of nanoparticles for the delivery of porf-htrail and ptx to glioma tissues [ , ] . porf-htrail was delivered by c(rgdyk)-poly(ethylene glycol)-polyethyleneimine (rgd-peg-pei). rgd can bind to integrin α v β , which is overexpressed in the neovasculature and on u glioblastoma cells. ptx was loaded in a cdx-poly(ethylene glycol)-block-poly(lactic acid) micelle. cdx is a peptide derived from the loop ii region of the snake neurotoxin candoxin, with a high-binding affinity to nicotinic acetylcholine receptors (nachrs). dominici et al. investigated the combination of ptrail and ptx for msc therapy [ ] . ptx restored the sensitivity of pancreatic cancer to msc-delivered trail by reverting its prosurvival gene expression profile. additionally, a combination of cisplatin and trail with high anticancer activity was found [ ] . fig. the process of codelivery of ptrail and monensin nanocomplexes. as a trail sensitizer, monensin upregulates dr and sensitizes tumor cells to trail. reprinted with permission from [ ] trail-based gene delivery and therapeutic strategies hh zhong these results indicate that trail gene therapy in combination with chemotherapy can be promising for ovarian cancer therapy. although it is not fully known how chemotherapy sensitizes cells to trail-induced apoptosis, the effect may be related to a p independent pathway, in addition to changes in the expression of proteins involved in trail signaling, which also play an important role [ ] . combination with intracellular apoptosis-induced agents as trail induces extracellular apoptosis, its combination with agents mediating the intracellular apoptosis pathway can yield a synergistic effect. histone deacetylase inhibitors (hadci) induce cell cycle arrest and apoptosis in tumor cells, and hadci can resensitize trail-resistant cancer cells. codelivery of ptrail and vorinostat (saha) with a reactive oxygen species (ros)-triggered charge reversal polymer (b-pdeaea) produced a good antitumor effect [ ] . the increased transfection efficiency of ptrail and saha-induced ros accumulation caused significant apoptosis of the cancer cells. the mscs-based gene therapy for antiglioma was developed, in which hat-mscs was transfected by pires -egfp-strail [ ] . the engineered mscs effectively inhibited the proliferation of malignant glioma cells and upregulated drs, yielding a potent antiglioma effect in combination with panobinostat. iaps play an important role in cancer cell resistance to trail, and the combination of trail with iap antagonists can help sensitize cells to trail-induced apoptosis. ge et al. demonstrated that an oncolytic adenovirus coexpressing trail and smac, in combination with the cyclin-dependent kinase (cdk) inhibitor sns- , synergistically reinforced their individual anti-pancreatic cancer activities, and sns- enhanced zd -trail-ietd-smac-induced apoptosis [ ] . shi et al. developed an aav-mediated gene therapy that was characterized by coexpression of trail with mir- -zip, which produced a synergistic effect on the enhanced apoptosis induction via the sensitizing effect of mir- -zip by upregulation of pten and downregulation of survivin [ ] . compared with trail monotherapy, combination therapy provides enhanced treatment outcomes. therefore, combinations including trail-mediated therapy are a promising approach for cancer therapy. trail is a promising drug candidate for the treatment of many cancers. the investigation of trail-based therapy in clinical trials focuses on recombinant trail proteins and anti-trail antibodies, but delivery and drug resistance issues are the main hurdles in the successful translation of these results. trail-based gene therapy is another potential treatment approach. importantly, the codelivery systems loaded with ptrail and drugs combined with trail have been actively explored. the development of safe and efficient gene vectors is the central issue for gene therapy. the elegant design of advanced systems provides the tumor-targeted codelivery of both agents, thus achieving synergistic treatment effects. in addition, it is necessary to identify proper biomarkers for maximizing trail-based therapy, which can also provide helpful information for the appropriate selection of drug combinations. however, clinical trials have made little progress. efforts should be directed toward the rational design of appropriate formulations of trail to improve its in vivo pharmacokinetic profile and the exploration of biomarkers specific to trail-based therapy. the future application of trail may benefit from a better understanding of the anticancer mechanisms of trail as well as its combination with trail sensitizers. it is expected that as precision medicine progresses, trail-mediated antitumor functions will be better understood to allow the development of precise and effective trail-based treatments for patients. overcoming cellular barriers for rna therapeutics the cd (apo- /fas) and the trail (apo- l) apoptosis systems cachetin/tnf-alpha in septic shock and septic adult respiratory distress syndrome lethal effect of the anti-fas antibody in mice identification and characterization of a new member of the tnf family that induces apoptosis induction of apoptosis by apo- ligand, a new member of the tumor necrosis factor cytokine family exploring the trails less travelled: trail in cancer biology and therapy rankl/ opg/trail plasma levels and bone mass loss evaluation in antiretroviral naive hiv- -positive men apo l/ trail and the death receptor agonist antibody amg cooperate to promote receptor clustering and antitumor activity targeting trail in the treatment of cancer: new developments tumoricidal activity of tumor necrosis factor-related apoptosis-inducing ligand in vivo safety and antitumor activity of recombinant soluble apo ligand a super way to kill cancer cells apoptosis-its significance in cancer and cancer-therapy cross-talk in cell death signaling proapoptotic activation of death receptor on tumor endothelial cells disrupts the vasculature and reduces tumor growth an antagonist decoy receptor and a death domain-containing receptor for trail the receptor for the cytotoxic ligand trail control of trail-induced apoptosis by a family of signaling and decoy receptors trail-r : a novel apoptosis-mediating receptor for trail cloning and characterization of trail-r , a novel member of the emerging trail receptor family the novel receptor trail-r induces nf-kappab and protects against trailmediated apoptosis, yet retains an incomplete death domain a novel receptor for apo l/trail contains a truncated death domain temperature-sensitive differential affinity of trail for its receptors. dr is the highest affinity receptor structural determinants of disc function: new insights into death receptor-mediated apoptosis signalling getting trail back on track for cancer therapy preclinical studies to predict the disposition of apo l/tumor necrosis factor-related apoptosisinducing ligand in humans: characterization of in vivo efficacy, pharmacokinetics, and safety unexpected hepatotoxicity in a phase i study of tas , a novel tetravalent agonistic nanobody(r) targeting the dr receptor phase i dose-escalation study of recombinant human apo l/trail, a dual proapoptotic receptor agonist, in patients with advanced cancer phase b study of dulanermin (recombinant human apo l/trail) in combination with paclitaxel, carboplatin, and bevacizumab in patients with advanced non-squamous nonsmall-cell lung cancer trailing trail resistance: novel targets for trail sensitization in cancer cells trail and apoptosis induction by tnf-family death receptors nanocarriers for trail delivery: driving trail back on track for cancer therapy antitumor activity and bystander effects of the tumor necrosis factor-related apoptosis-inducing ligand (trail) gene membrane-bound fas ligand only is essential for fas-induced apoptosis liposomes decorated with apo l/trail overcome chemoresistance of human hematologic tumor cells polymers for gene delivery across length scales overcoming gene-delivery hurdles: physiological considerations for nonviral vectors adenoviralmediated transfer of the tnf-related apoptosis-inducing ligand/apo- ligand gene induces tumor cell apoptosis efficacy of combining ing and trail genes in cancer-targeting gene virotherapy strategy: first evidence in preclinical hepatocellular carcinoma non-viral vectors for gene-based therapy design and development of polymers for gene delivery nonviral vectors for gene delivery barriers to nonviral gene delivery progress in developing cationic vectors for non-viral systemic gene therapy against cancer a versatile vector for gene and oligonucleotide transfer into cells in culture and in vivo: polyethylenimine toxicity of cationic lipids and cationic polymers in gene delivery new path to treating pancreatic cancer: trail gene delivery targeting the fibroblast-enriched tumor microenvironment the use of myristic acid as a ligand of polyethylenimine/dna nanoparticles for targeted gene therapy of glioblastoma a mannosylated pei-cpp hybrid for trail gene targeting delivery for colorectal cancer therapy poly-gamma-glutamic acid-based ggt-targeting and surface camouflage strategy for improving cervical cancer gene therapy co-delivery of gambogic acid and trail plasmid by hyaluronic acid grafted pei-plga nanoparticles for the treatment of triple negative breast cancer biodegradable poly(amine-co-ester) terpolymers for targeted gene delivery nano-structural effects on gene transfection: large, botryoid-shaped nanoparticles enhance dna delivery via macropinocytosis and effective dissociation polymeric nanoparticle-based delivery of trail dna for cancer-specific killing esterase-activated chargereversal polymer for fibroblast-exempt cancer gene therapy enzyme-responsive charge-reversal polymer-mediated effective gene therapy for intraperitoneal tumors sandwich-type au-pei/dna/pei-dexa nanocomplex for nucleus-targeted gene delivery in vitro and in vivo nanoparticlemediated target delivery of trail as gene therapy for glioblastoma antitumor effect of human trail on adenoid cystic carcinoma using magnetic nanoparticle-mediated gene expression discovery of dendrimers and dendritic polymers: a brief historical perspective dual targeting effect of angiopep- -modified, dna-loaded nanoparticles for glioma therapeutic efficacy of intravenously administered transferrin-conjugated dendriplexes on prostate carcinomas regression of prostate tumors after intravenous administration of lactoferrinbearing polypropylenimine dendriplexes encoding tnf-alpha, trail, and interleukin- a self-assembled coumarin-anchored dendrimer for efficient gene delivery and light-responsive drug delivery triazine-modified dendrimer for efficient trail gene therapy in osteosarcoma modified pamam vehicles for effective trail gene delivery to colon adenocarcinoma: in vitro and in vivo evaluation peptide-guided gene delivery development of a genetically engineered biomimetic vector for targeted gene transfer to breast cancer cells intratracheal administration of a nanoparticle-based therapy with the angiotensin ii type receptor gene attenuates lung cancer growth pten and trail genes loaded zein nanoparticles as potential therapy for hepatocellular carcinoma advancing nonviral gene delivery: lipid-and surfactant-based nanoparticle design strategies a novel cationic lipid with intrinsic antitumor activity to facilitate gene therapy of trail dna targeting tumor-associated fibroblasts for therapeutic delivery in desmoplastic tumors a multifunctional nanocarrier for efficient trail-based gene therapy against hepatocellular carcinoma with desmoplasia in mice core-shell" nanoparticles-based gene delivery for treatment of aggressive melanoma multifunctional nucleus-targeting nanoparticles with ultra-high gene transfection efficiency for in vivo gene therapy minimal criteria for defining multipotent mesenchymal stromal cells human bone marrow and adipose tissue mesenchymal stem cells: a user's guide mesenchymal stem/stromal cells as a delivery platform in cell and gene therapies how do mesenchymal stromal cells exert their therapeutic benefit? mesenchymal stem cell therapy for autoimmune disease: risks and rewards understanding tumor-stroma interplays for targeted therapies by armed mesenchymal stromal progenitors: the mesenkillers adiposederived mesenchymal stem cells as stable source of tumor necrosis factorrelated apoptosis-inducing ligand delivery for cancer therapy trail delivered by mesenchymal stromal/stem cells counteracts tumor development in orthotopic ewing sarcoma models stem cell-based gene therapy activated using magnetic hyperthermia to enhance the treatment of cancer mesenchymal stem cell delivery of trail can eliminate metastatic cancer mesenchymal stem cells as a novel carrier for targeted delivery of gene in cancer therapy based on nonviral transfection suppression of orthotopically implanted hepatocarcinoma in mice by umbilical cord-derived mesenchymal stem cells with strail gene expression driven by afp promoter an inducible caspase suicide gene to improve the safety of mesenchymal stromal cell therapies inducible caspase -mediated suicide gene for msc-based cancer gene therapy an inducible caspase safety switch for t-cell therapy polylysine-modified polyethylenimine polymer can generate genetically engineered mesenchymal stem cells for combinational suicidal gene therapy in glioblastoma trail-secreting human mesenchymal stem cells engineered by a non-viral vector and photochemical internalization for pancreatic cancer gene therapy the roles of flt in hematopoiesis and leukemia nanoliposomemediated fl/trail double-gene therapy for colon cancer: in vitro and in vivo evaluation bcl-xl protects pancreatic adenocarcinoma cells against cd -and trail-receptormediated apoptosis sensitization for death receptor-or drug-induced apoptosis by re-expression of caspase- through demethylation or gene transfer resistance to tumor necrosis factor-related apoptosis-inducing ligand (trail)-induced apoptosis in neuroblastoma cells correlates with a loss of caspase- expression mechanisms involved in development of resistance to adenovirus-mediated proapoptotic gene therapy in dld human colon cancer cell line molecular determinants of response to trail in killing of normal and cancer cells tumoricidal activity of a novel anti-human dr monoclonal antibody without hepatocyte cytotoxicity flice-inhibitory proteins: regulators of death receptor-mediated apoptosis cellular flice/caspase- -inhibitory protein as a principal regulator of cell death and survival in human hepatocellular carcinoma modulation of caspase- and flice-inhibitory protein expression as a potential mechanism of epstein-barr virus tumorigenesis in burkitt's lymphoma induction of apoptotic program in cell-free extracts: requirement for datp and cytochrome c the release of cytochrome c from mitochondria: a primary site for bcl- regulation of apoptosis synergy is achieved by complementation with apo l/trail and actinomycin d in apo l/trail-mediated apoptosis of prostate cancer cells: role of xiap in resistance x-linked inhibitor of apoptosis (xiap) blocks apo ligand/ tumor necrosis factor-related apoptosis-inducing ligand-mediated apoptosis of prostate cancer cells in the presence of mitochondrial activation: sensitization by overexpression of second mitochondria-derived activator of caspase/direct iap-binding protein with low pl (smac/diablo) smac, a mitochondrial protein that promotes cytochrome c-dependent caspase activation by eliminating iap inhibition identification of diablo, a mammalian protein that promotes apoptosis by binding to and antagonizing iap proteins mechanisms of resistance to trail-induced apoptosis in cancer loss of caspase- protein expression correlates with unfavorable survival outcome in childhood medulloblastoma monensin, a polyether ionophore antibiotic, overcomes trail resistance in glioma cells via endoplasmic reticulum stress, dr upregulation and c-flip downregulation. carcinogenesis targeting death receptors for drug-resistant cancer therapy: codelivery of ptrail and monensin using dualtargeting and stimuli-responsive self-assembling nanocomposites trail and chemotherapeutic drugs in cancer therapy evaluation of efficiency of modified polypropylenimine (ppi) with alkyl chains as non-viral vectors used in co-delivery of doxorubicin and trail plasmid in vivo treatment of tumors using host-guest conjugated nanoparticles functionalized with doxorubicin and therapeutic gene ptrail plasmid porf-htrail and doxorubicin co-delivery targeting to tumor using peptide-conjugated polyamidoamine dendrimer gene and doxorubicin codelivery system for targeting therapy of glioma choline-derivate-modified nanoparticles for brain-targeting gene delivery choline transporter-targeting and codelivery system for glioma therapy co-delivery of pegfp-htrail and paclitaxel to brain glioma mediated by an angiopep-conjugated liposome candoxin, a novel toxin from bungarus candidus, is a reversible antagonist of muscle (alphabetagammadelta) but a poorly reversible antagonist of neuronal alpha nicotinic acetylcholine receptors co-delivery of trail gene enhances the anti-glioblastoma effect of paclitaxel in vitro and in vivo msc-delivered soluble trail and paclitaxel as novel combinatory treatment for pancreatic adenocarcinoma in vitro and in vivo growth inhibition of drug-resistant ovarian carcinoma cells using a combination of cisplatin and a trail-encoding retrovirus is trail the holy grail of cancer therapy? saha (vorinostat) facilitates functional polymer-based gene transfection via upregulation of ros and synergizes with trail gene delivery for cancer therapy histone deacetylase inhibitor panobinostat potentiates the anti-cancer effects of mesenchymal stem cell-based strail gene therapy against malignant glioma synergistic antitumor effects of cdk inhibitor sns and an oncolytic adenovirus coexpressing trail and smac in pancreatic cancer combination of aav-trail with mir- -zip therapeutic strategy overcomes the resistance to trail induced apoptosis in liver cancer competing interests: the authors declare no competing interests.open access this article is licensed under a creative commons attribution . international license, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the creative commons license, and indicate if changes were made. the images or other third party material in this article are included in the article's creative commons license, unless indicated otherwise in a credit line to the material. if material is not included in the article's creative commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. to view a copy of this license, visit http://creativecommons. org/licenses/by/ . /. key: cord- - bbd ud authors: wu, lei; liu, ting-ting; jin, ying; wei, shuang; qiu, chun-yu; hu, wang-ping title: endothelin- enhances acid-sensing ion channel currents in rat primary sensory neurons date: - - journal: acta pharmacol sin doi: . /s - - -z sha: doc_id: cord_uid: bbd ud endothelin- (et- ), an endogenous vasoactive peptide, has been found to play an important role in peripheral pain signaling. acid-sensing ion channels (asics) are key sensors for extracellular protons and contribute to pain caused by tissue acidosis. it remains unclear whether an interaction exists between et- and asics in primary sensory neurons. in this study, we reported that et- enhanced the activity of asics in rat dorsal root ganglia (drg) neurons. in whole-cell voltage-clamp recording, asic currents were evoked by brief local application of ph . external solution in the presence of trpv channel blocker amg . pre-application with et- ( − nm) dose-dependently increased the proton-evoked asic currents with an ec( ) value of . ± . nm. pre-application with et- ( nm) shifted the concentration–response curve of proton upwards with a maximal current response increase of . % ± . %. we showed that et- enhanced asic currents through endothelin-a receptor (et(a)r), but not endothelin-b receptor (et(b)r) in both drg neurons and cho cells co-expressing asic and et(a)r. et- enhancement was inhibited by blockade of g-protein or protein kinase c signaling. in current-clamp recording, pre-application with et- ( nm) significantly increased acid-evoked firing in rat drg neurons. finally, we showed that pharmacological blockade of asics by amiloride or apetx significantly alleviated et- -induced flinching and mechanical hyperalgesia in rats. these results suggest that et- sensitizes asics in primary sensory neurons via et(a)r and pkc signaling pathway, which may contribute to peripheral et- -induced nociceptive behavior in rats. endothelin- (et- ) is an endogenous vasoactive peptide. it is derived from endothelial cells and is also produced by other cell types, including inflammatory cells, tumor cells, and neurons [ ] [ ] [ ] [ ] . et- exerts its biological effects by binding to two specific g-protein-coupled receptors, endothelin-a receptor (et a r) and endothelin-b receptor (et b r) [ , ] . in the peripheral nervous system, et a r is expressed in nociceptive primary sensory neurons, including the dorsal root ganglion (drg) neurons, whereas et b r is mainly found in satellite glial cells [ ] . numerous studies have demonstrated that et- plays an important role in peripheral pain signaling. in humans, exogenous et- causes tactile allodynia and severe pain [ ] . in rodents, an intraplantar injection of et- produces mechanical and thermal hyperalgesia and spontaneous pain-like behaviors [ ] [ ] [ ] [ ] . paw withdrawal thresholds to mechanical stimuli and heat are significantly lower in conditional et- knockout mice [ ] . peripheral et- acts on nociceptors through its cognate receptors, which subsequently modify pain-related ion channels to amplify signal generation [ ] . in drg neurons, et- increases neuronal excitation by hyperpolarizing tetrodotoxinresistant (ttx-r) na + channels and suppressing the delayed outward rectifier current (i k ) via et a r [ , ] . in contrast, et- decreases the excitability of drg neurons through the activation of et b r [ ] . et- potentiates trpv -mediated currents by an et a r-mediated and protein kinase c (pkc)-dependent mechanism in both the expression system and sensory neurons [ , ] . et- induced pain-like behavior is attenuated in trpv -deficient mice [ ] . trpv also contributes to et- -induced thermal and mechanical hyperalgesia [ , ] . et- sensitizes trpa in primary sensory neurons via an et a r and protein kinase a (pka) pathway [ ] . trpa is involved in et- -induced mechanical hyperalgesia and spontaneous pain-like behavior [ , ] . however, et- induced nociceptive responses are not completely inhibited in trpv -deficient mice or by trpv and trpa antagonists, suggesting that other mechanisms are also involved. acid-sensing ion channels (asics), such as ph sensors, are also pain-related ion channels. asics are expressed in both drg cell bodies and sensory terminals, where they contribute to protonevoked pain signaling [ , ] . asics, rather than trpv , are shown to mainly mediate pain sensation induced by a moderate (up to ph . ) peripheral ph [ ] [ ] [ ] [ ] . asics were found to be the major player in pain associated with tissue acidosis, such as inflammation, tissue damage, ischemia, and tumors [ ] [ ] [ ] . thus, asics represent a novel therapeutic target for pain [ ] [ ] [ ] . et- and protons activate their cognate receptors expressed in nociceptors and separately contribute to the nociceptive process. since both endothelin receptors and asics are distributed in drg neurons, we hypothesized that there may be an interaction between endothelin receptors and asics in the same drg neuron. herein, we showed that et- enhanced the electrophysiological activity of asics in drg neurons through et a r, which may contribute to et- -induced spontaneous flinching and mechanical hyperalgesia in rats. all experimental protocols were approved by the animal research ethics committee of hubei university of science and technology. all procedures were performed to minimize the suffering of animals. male sprague-dawley rats ( -to -week-old) were sacrificed. the drgs were removed and minced with fine spring scissors. the ganglion fragments were placed in a flask containing ml of dulbecco's modified eagle's medium (dmem, sigma). dmem contained trypsin (type ii-s, sigma) . mg/ml, collagenase (type i-a, sigma) . mg/ml and dnase (type iv, sigma) . mg/ml, and were incubated at °c in a shaking water bath for - min. soybean trypsin inhibitor (type ii-s, sigma) . mg/ml was then added to stop trypsin digestion. electrophysiological experiments were carried out as described previously [ ] . whole-cell patch clamp and voltage-clamp recordings were carried out at room temperature ( - °c) using a multiclamp- b amplifier and digidata- a a/d converter (axon instruments, ca, usa). dissociated neurons were placed into a -mm petri dish and were bathed in an external solution containing (mm): nacl , kcl , cacl . , mgcl , hepes , and d-glucose , and the ph and osmolarity were adjusted to . with naoh and to mosm/l with sucrose, respectively. cells were kept for at least min in normal external solution before the start of electrophysiological experiments. the neurons selected for electrophysiological experiments were - μm in diameter, which are thought to be nociceptive neurons. recording pipettes were pulled using a sutter p- puller (sutter instruments, ca, usa). the micropipettes were filled with internal solution containing (mm): kcl , mgcl . , hepes , egta and atp ; the ph of the solution was adjusted to . with koh and its osmolarity was adjusted to mosm/l with sucrose. the resistance of the recording pipette was in the range of - mΩ. to establish a whole-cell configuration, a small patch of membrane underneath the tip of the pipette was aspirated to form a giga seal, and then, negative pressure was applied to rupture it. the series resistance was compensated for by %- %. the capacitance compensation was also adjusted before recording the membrane currents. the membrane voltage was maintained at − mv in all voltage-clamp experiments. current-clamp recordings were obtained by switching to currentclamp mode after a stable whole-cell configuration was formed in voltage-clamp mode. only cells with a stable resting membrane potential (more negative than − mv) were used in the study. signals were sampled at to khz and filtered at to khz, and the data were stored in a compatible pc computer for offline analysis using pclamp acquisition software (axon instruments, ca, usa). drug application drugs were obtained from sigma chemical co. (st. louis, mo, usa), including hydrochloric acid, et- , bq- , bq- , amiloride, apetx , capsaicin, and amg . different ph values were configured with hydrochloric acid and external solution. working et- and other drugs were freshly prepared in normal external solution and held in a series of independent reservoirs. the pipette tips connecting the reservoirs were positioned ∼ μm away from the recorded neurons. the application of each drug was driven by gravity and controlled by the corresponding valve. in some experiments where gdp-β-s (sigma) or gf x (rbi) was applied for intracellular dialysis through recording patch pipettes, they were dissolved in the internal solution before use. to ensure that the cell interior was perfused with the dialysis drug, there was at least a min interval between the establishment of whole-cell access and current measurement. to functionally characterize asic activity, we used amg ( μm) to block trpv in the extracellular solution [ ] . cell culture and transfection asic and et a r complementary dna (cdnas) were used for heterologous expression in cho cells as described previously [ ] . in brief, cho cells were cultured at °c in a humidified atmosphere of % co and % o and passaged twice a week. transient transfection of cho cells was performed using a hilymax liposome transfection reagent (dojindo laboratories). cho cells were maintained in f- nutrient mixture (added . g of nahco /l medium) supplemented with % fetal bovine serum and % gluta-maxtm- ( ×; invitrogen). when asic and et a r cdna was cotransfected, the ratio was maintained at : . all plasmids contained, in addition to the desired asic cdna, the coding sequence for enhanced green fluorescent protein to aid in the identification of transfected cells. electrophysiological measurements were performed - h after transfection. animal behavioral assay rats were placed in a × × cm plexiglas chamber and allowed to habituate for at least min before nociceptive behavioral experiments. a double-blind experiment was carried out. to assess et- -induced flinching, rats were divided randomly into seven groups, with seven rats in each group. separate groups of rats were coded and administered drugs. et- ( ng/paw) or vehicle (phosphate-buffered saline) was injected into the hind paw of rats using μl syringe and -gauge needle in a volume of μl, with bq- ( µg/paw), bq- ( ng/paw), amiloride ( µm, µl/paw) or apetx ( μm, µl/ paw) co-injected with et- . the other experimenters measured nociceptive behavior. flinching (time spent licking, biting, and lifting the injected paw) was counted for min starting immediately after the injection. to assess et- -induced mechanical hyperalgesia, rats were divided randomly into groups, with seven rats in each group. separate groups of rats were coded and administered drugs as above. paw withdrawal thresholds (pwts) were determined thereafter, at , . , and . h after the injection. paw withdrawal thresholds (pwts) were tested using the up and down method [ ] . a series of von frey filaments (stoelting, wood dale, il) were used to vertically press against the injected hind paw until the filaments were bent for s. quick withdrawal, licking or paw flinching were counted as positive reactions. the test was carried out many times in each rat, and the interval between tests was at least min. all rats were tested only in one behavioral experiment, either for flinching or mechanical hyperalgesia. the data were statistically compared using student's t-test or analysis of variance (anova), followed by bonferroni's post hoc test. statistical analysis of concentration-response data was performed using the nonlinear curve-fitting program allfit. the data are expressed as the mean ± s.e.m. in the present study, amg ( μm) was added to the external solution to block proton-induced trpv activation [ ] . as shown in fig. a , a perfusion of ph . external solution to drg neurons for s caused a rapid inward current (i ph . ). this i ph . could be completely blocked by both μm amiloride, a broad-spectrum asic channel blocker, and μm apetx , an asic blocker. however, capsaicin ( nm) failed to evoke any membrane currents in the presence of amg . thus, these protoninduced currents were considered to be pure asic currents after amg blocked proton-induced trpv activation. in some drg neurons sensitive to acid stimuli ( . %, / ), we observed that the pre-application of et- for min increased the peak amplitude of the asic currents. as shown in fig. b , c, the i ph . was enhanced by et- pretreatment, and the potentiation of the i ph . was dependent upon the concentration of et- . fig. b shows that the peak amplitude of the i ph . increased as the concentration of pretreated et- increased from to nm in a representative drg neuron. the potentiation of the i ph . was reversible after the washout of et- . fig. c shows the concentration-response curve for et- with an ec (half-maximal effective concentration) value of . ± . nm. the results indicated that et- increased asic currents in rat drg neurons in a concentration-dependent manner. we then investigated whether the et- potentiation of asic currents was dependent upon ph values. asic currents were measured by applying a range of different low ph values in the absence and presence of et- ( nm). fig. a shows that the peak amplitudes of i ph . , i ph . , and i ph . increased after the pre- fig. potentiation of proton-gated currents by et- in rat drg neurons. a representative current traces were produced by the application of a ph . acidic solution for s on a drg neuron. the proton-gated current (i ph . ) could be blocked by μm amiloride, a broad-spectrum asic channel blocker. this current was also blocked by μm apetx , an asic blocker. the proton-gated currents were recorded in the presence of amg ( μm) to block proton-induced trpv activation. under such conditions, capsaicin ( nm) failed to evoke any membrane currents. the membrane potential was clamped at - mv. b the sequential current traces illustrate that the i ph . amplitude was enhanced by different concentrations of et- in a representative drg neuron. et- was preapplied to external solution for min. c the graph shows et- concentration-dependently increased the i ph . with an ec of . ± . nm. each point represents the mean ± s.e.m of - cells. fig. b shows the effect of et- ( nm) on the concentration-response curve to protons. first, the pre-application of et- shifted the concentration-response curve to protons upwards, as indicated by an increase of . % ± . % in the maximal current response to protons in the presence of et- . second, neither the hill coefficient nor slope of the curve was significantly changed by et- (ph: n = . ± . ; et- + ph: n = . ± . ; p > . , bonferroni's post hoc test). third, the ph . (ph for half-maximal activation) values of both curves also showed no significant difference (ph: ph . = . ± . ; et- + ph: ph . = . ± . ; p > . , bonferroni's post hoc test). we therefore concluded that the potentiation of the asic current by et- was not due to a change in the apparent affinity of protons for asics. to verify whether the et- potentiation of asic currents was mediated by et receptors, we observed the effects of the selective et a r antagonist bq- and the selective et b r antagonist bq- on the et- enhancement of asic currents. as shown in fig. a , b, the amplitude of the i ph . increased . % ± . % after et- ( nm) pretreatment alone. however, the amplitude of the i ph . increased only . % ± . % when both bq- ( nm) and et- ( nm) were coapplied to drg neurons (p < . , compared with et- alone, bonferroni's post hoc test, n = ; fig. a, b) . in contrast, et- ( nm) caused an increase of . % ± . % on the i ph . after bq- ( nm) was coapplied with et- (p > . , compared with et- alone, bonferroni's post hoc test, n = ; fig. a, b) . these results indicated that et a r, but not et b r, is involved in the et- potentiation of asic currents in rat drg neurons. we further explored the signaling pathway downstream of et a r in the et- potentiation of asic currents using an intracellular dialysis technique. et a r is most commonly associated with the g q/ g-protein subtype, which activates plc and leads to a cascade of events, including the activation of pkc [ , ] . thus, we internally applied gdp-β-s (a nonhydrolyzable gdp analog) or gf x (a selective pkc inhibitor) to drg cells through recording patch pipettes. unlike the increase of . % ± . % on the i ph . produced under the normal internal solution condition, the preapplication of et- ( nm) for min produced only increases of . % ± . % and . % ± . % on the i ph . when gdp-β-s ( μm) or gf x ( μm), respectively, was included in the pipette solution (p < . , compared with normal internal solution, post hoc bonferroni's test, n = ; fig. c, d) . these results indicated that the potentiation of asic currents by et- was dependent upon g-protein and pkc signaling. to further verify whether et- could enhance acid-evoked currents mediated by asic , asic was coexpressed with et a r in cho cells. we observed that a rapid reduction in the extracellular ph from . to . for s also caused a rapid inward current. the acid-evoked currents were asic currents, since they were blocked by μm apetx , an asic blocker. similar to that observed in drg neurons, the asic currents were enhanced by the pre-application of et- ( nm) in cho cells co-expressing asic and et a r (fig. a, b) . the et- potentiation of asic currents was also inhibited after pharmacological blockade of et a r by bq- (fig. a, b) . in contrast, et- had no effect on asic currents at a concentration of nm in cho cells expressing asic alone, but not expressing et a r (fig. c, d) . under current-clamp conditions, a stimulus of ph . could trigger bursts of action potentials (aps) in drg neurons in the presence of μm amg to block proton-induced trpv activation (fig. a) . similar to that observed under voltage-clamp conditions, et- also increased the number of aps evoked by acidic stimuli in drg neurons (fig. a, b) . as shown in fig. b , the number of aps evoked by a stimuli of ph . increased in seven drg neurons with et- ( nm) pretreatment for min (p < . , paired t-test, n = , fig. b) . however, pretreatment with vehicle had no effect on the number of aps evoked by stimuli of ph . (p > . , paired t-test; n = , fig. c ). these results indicated that et- exerted an enhancing effect on acid-evoked action potentials in rat drg neurons. attenuation of et- -induced flinching and mechanical hyperalgesia by the pharmacological blockade of et a r, et b r, or asics in rats we finally examined the contribution of asics to et- -induced nocifensive behavior in vivo. rats displayed an obvious flinching response after et- ( ng/paw) was injected subcutaneously into the ipsilateral hind paw compared with rats receiving vehicle injection only (fig. a) . the et- -induced flinching was almost completely blocked by subcutaneous cotreatment with bq- ( ng/paw), but not with bq- ( ng/paw). as shown in fig. a , when asics blocker amiloride ( µm, µl/paw) or asic blocker rapetx ( μm, µl/paw) was coadministered subcutaneously with et- ( ng/paw), it significantly attenuated hind paw flinching (p < . , bonferroni's post hoc test, compared with et- alone group, n = rats). rats injected with et- into the hind paw ( ng/paw) also displayed obvious mechanical hyperalgesia, measured . and . h after injection (fig. b, c) . et- -induced mechanical hyperalgesia disappeared h after injection. fig. b shows that et- -induced mechanical hyperalgesia was partially attenuated by subcutaneous cotreatment with bq- ( ng/paw) or bq- ( ng/paw). furthermore, the intraplantar application of both bq- ( ng/paw) and bq- ( ng/paw) together almost completely blocked the mechanical hyperalgesia induced by et- (fig. b) . as shown in fig. c , amiloride ( µm, µl/paw) or apetx ( μm, µl/paw) also significantly attenuated the mechanical hyperalgesia induced by et- (p < . and . , bonferroni's post hoc test, compared with et- alone group, n = rats). the injection of these antagonists alone at the same dosage used above did not produce any effects on the number of flinches or the pwt (data not shown). together, these results demonstrated that asics mediated, at least partially, et- -induced flinching and mechanical hyperalgesia. b et- -induced mechanical hyperalgesia was attenuated by bq- or bq- alone and was blocked by the combined application of bq- and bq- . c et- -induced mechanical hyperalgesia was also attenuated after the pharmacological blockade of asics by amiloride or apetx . flinching and mechanical hyperalgesia were induced by the injection of et- subcutaneously into the rat plantar hind paw. spontaneous flinching was evaluated by the time spent licking/lifting the injected paw (in seconds). the paw withdrawal threshold (pwt, in g) of rats was measured by von frey hair test before injection ( h) and . , . , h after injection. bq- ( ng/paw) and/or bq- ( ng/ paw), amiloride ( µm, µl/paw), or apetx ( μm, µl/paw) were co-injected with et- ( ng/paw) into the hind paw of rats. n = rats in each group. *p < . , **p < . , ***p < . , bonferroni's post hoc test, compared with the et- alone group. et- enhances asic currents l wu et al. we demonstrated here that et- sensitized asics through et a r. et- increased asic-medicated currents and action potentials in rat primary sensory neurons, which may contribute to et- induced spontaneous flinching and mechanical hyperalgesia in rats. low ph-evoked currents are mediated by proton-gated ion channels, including asics and trpv [ ] . in the present study, the activation of trpv channels was completely blocked by amg , so capsaicin failed to evoke any membrane currents. we believe that asics mediated these low ph-evoked currents, which could be completely blocked by asic channel blockers; these evoked currents were, therefore, considered to be asic currents, although precise asic subunits need to be identified. to date, at least seven asic subunits have been identified in mammals. there are six asic subunits (asic a and b, asic a and b, asic , and asic ) expressed in both drg cell bodies and sensory terminals [ , ] . these asic subunits exist as homomeric or heteromeric channels in drg neurons. the asic subunit is the most abundant in drg and has emerged as a critical ph sensor [ , ] . the current data demonstrated that et- can enhance the electrophysiological activity of asics in rat drg neurons. et- increased not only asic-mediated currents in voltage-clamp experiments but also the number of action potentials evoked by extracellular acid stimuli in current-clamp experiments. obviously, the two results corroborated each other. et- shifted the proton concentration-response curve upward without changing the ph . . thus, the sensitization of asics by et- did not change the apparent affinity of protons for asics. we found that et- sensitized asics in rat drg neurons through et a r, but not through et b r, because the et- potentiation of asic currents was completely blocked by the selective et a r antagonist bq- and was not affected by the selective et b r antagonist bq- . in cho cells co-expressing asic and et a r, et- also sensitized asic through et a r. together, et a r, but not et b r, appeared to contribute to the et- potentiation of asic currents. the results were consistent with the expression characteristics of et a r and et b r in primary sensory neurons. et a r is present in a subset of small-and medium-sized drg neurons and their axons, whereas et b r is mainly expressed by satellite glial cells and nonmyelinating schwann cells, but not neurons [ ] . it has been found that et a r is mainly expressed in isolectin b -negative (ib − ) drg neurons [ ] . asics are also more prevalent in ib − drg neurons than in ib + neurons [ ] . thus, the et- potentiation of asics may mainly occur in peptidergic and ib -drg neurons. et a r belongs to a subfamily of gpcrs and is coupled to multiple second messenger systems. although et a r couples to g s in other tissues, et- does not increase intracellular camp levels in isolated drg neurons [ ] . et a r also couples to g q/ to activate plcβ, which, in turn, produces ip and dag, followed by intracellular ca + release and the activation of pkc [ , , ] . et- can trigger intracellular calcium mobilization through et a r in drg neurons [ ] . et a r, but not et b r, activates pkc in peptidergic, ib − sensory neurons [ ] . in the present study, the lack of et- potentiation in drg neurons treated with gdp-β-s or the pkc inhibitor gf x indicated that a g-protein-and pkc-dependent pathway was involved in the sensitization of asics by et- . it has been shown that pkc is involved in the et- -mediated enhancement of capsaicin-induced ca + increases in sensory neurons. et- potentiates trpv currents via the et a r-mediated activation of pkc in both drg neurons and the trigeminal ganglion neurons [ , ] . the stimulation of et a r by et- inhibits inward rectifier k + channels via the pkc pathway [ ] . however, et- sensitizes trpa via the et a r and pka pathways in primary sensory neurons [ ] . our previous studies show that asics are modulated by intracellular pkc signaling [ , ] . initial studies show that exogenous et- elicits spontaneous pain and hyperalgesia. in the present study, rats displayed flinching responses and mechanical hyperalgesia after et- was injected subcutaneously into the ipsilateral hind paw. et- induced flinching was almost completely blocked by subcutaneous cotreatment with bq- , but not bq- , suggesting this behavior is mediated by et a r, but not et b r. a combined intraplantar injection of bq- and bq- completely blocked et- -induced mechanical hyperalgesia, suggesting it is mediated by both et a r and et b r. previous studies have shown that a subcutaneous injection of et- into the rat plantar hind paw induces flinching behavior and spike responses in nociceptors through et a r [ , ] . it has been shown that et a r activation contributes to et- -induced pain-like behavior and thermal hyperalgesia [ , , ] . in contrast, et- -induced mechanical hyperalgesia requires both et a r and et b r activation [ , , ] . et- has been implicated in inflammation, so we cannot rule out et- -elicited nociceptive behavior that may be due to the induction of inflammatory responses after hind paw injection. however, some studies also show that et- elicits nociceptive behavior due to et- 's direct action on primary afferents, since this nociceptive behavior is mediated by et a r located immunocytochemically on primary afferents [ , ] . in this work, we used cell bodies of drg neurons as a simple and accessible model to examine the characteristics of the membrane of peripheral terminals. the sensitization of asics by et- may also occur in peripheral terminals. the pharmacological blockade of asics significantly alleviated et- -induced flinching and mechanical hyperalgesia in rats, suggesting that asics mediated, at least partially, et- -induced nociceptive behavior. it is possible that et- sensitizes asics on primary afferents, which underlies et- -induced nociceptive behavior. during tissue damage and inflammation, et- is generated and released in a variety of cells, such as endothelial cells, monocytes and macrophages, which results in an increase of local et- levels [ , ] . drg neurons are also an important source of et- [ ] . et- can excite these sensory neurons in a paracrine or autocrine manner. et- levels are higher within a cancerous microenvironment, and the administration of an et a r antagonist alleviates cancer pain [ , , , ] . thus, an endogenous concentration of et- in diseased tissue is required to cause pain. the present tested concentration of et- ( nm) falls within the endogenous concentration of et- ranges reported [ ] . et- is implicated in the pathogenesis of a variety of types of pain, such as inflammatory, neuropathic, and tumoral pain [ , , ] . during tissue damage and inflammation, protons are released from damaged cells and the degranulation of mast cells. these released protons result in a drop in the local extracellular ph. for instance, the local extracellular ph drops to approximately . during acute inflammation, which is low enough to activate asics and cause pain [ , ] . an acidic ph is commonly associated with cancerous microenvironments, contributing to cancer-related pain via the activation of asics and trpv [ , ] . under inflammatory and cancer states, once both et- and protons are locally released together in diseased tissue, and they activate their cognate receptors expressed in nociceptors to initiate and/or sensitize nociceptive processes. furthermore, the present study showed that released et- could sensitize asics through et a r. et- and protons may potentially work synergistically to produce more intense pain and mechanical hyperalgesia. in summary, the major finding of this study was that et- enhanced the electrophysiological activity of asics in drg neurons via the intracellular pkc signaling pathway, which may contribute to et- -induced nociceptive behavior in rats. our results demonstrated that asics act as novel targets of et- , which may provide a strategy for the treatment of pain associated with et- , such as inflammatory pain and cancer-related pain. et- enhances asic currents l wu et al. endothelin , an endothelium-derived peptide, is expressed in neurons of the human spinal cord and dorsal root ganglia a novel potent vasoconstrictor peptide produced by vascular endothelial cells identification of endothelin- in the pathophysiology of metastatic adenocarcinoma of the prostate endothelins, peptides with potent vasoactive properties, are produced by human macrophages evidence for the endothelin system as an emerging therapeutic target for the treatment of chronic pain expression and localization of endothelin receptors: implications for the involvement of peripheral glia in nociception regional haemodynamic effects of endothelin- in rat and man: unexpected adverse reaction local injection of endothelin- produces pain-like behavior and excitation of nociceptors in rats contribution of transient receptor potential vanilloid subfamily to endothelin- -induced thermal hyperalgesia roles of endothelin eta and etb receptors in nociception and chemical, thermal and mechanical hyperalgesia induced by endothelin- in the rat hindpaw mechanical hyperalgesia induced by endothelin- in rats is mediated via phospholipase c, protein kinase c, and map kinases increased sensitivity to acute and persistent pain in neuron-specific endothelin- knockout mice new perspectives on the endothelin axis in pain endothelin- raises excitability and reduces potassium currents in sensory neurons endothelin- (et- ) selectively enhances the activation gating of slowly inactivating tetrodotoxin-resistant sodium currents in rat sensory neurons: a mechanism for the pain-inducing actions of et- endothelin- decreases excitability of the dorsal root ganglion neurons via etb receptor endothelin potentiates trpv via eta receptor-mediated activation of protein kinase c endothelin receptor-mediated responses in trigeminal ganglion neurons involvement of transient receptor potential vanilloid subfamily in endothelin- -induced pain-like behavior tactile allodynia initiated by local subcutaneous endothelin- is prolonged by activation of trpv- receptors etar and protein kinase a pathway mediate et- sensitization of trpa channel: a molecular mechanism of et- -induced mechanical hyperalgesia involvement of trpa in et- -induced pain-like behavior in mice functional implications of the localization and activity of acid-sensitive channels in rat peripheral nervous system heteromultimers of deg/enac subunits form h + -gated channels in mouse sensory neurons asic , a sensor of acidic and primary inflammatory pain acid-sensing ion channels in sensory perception amiloride-blockable acid-sensing ion channels are leading acid sensors expressed in human nociceptors acid-sensing ion channels in pain and disease tissue acidosis in nociception and pain acid-sensing ion channels and nociception in the peripheral and central nervous systems roles of asics in nociception and proprioception acid-sensing ion channels: a novel therapeutic target for pain and anxiety asics as therapeutic targets for migraine potentiation of acid-sensing ion channel activity by the activation of -ht( ) receptors in rat dorsal root ganglion neurons ], a novel vanilloid receptor (trpv ) antagonist with antihyperalgesic properties sensitization of asic by proteinase-activated receptor signaling contributes to acidosis-induced nociception quantitative assessment of tactile allodynia in the rat paw subcellular mechanisms of endothelin action in vascular system differential regulation of proton-sensitive ion channels by phospholipids: a comparative study between asics and trpv functional endothelin receptors are selectively expressed in isolectin b -negative sensory neurons and are upregulated in isolectin b -positive neurons by neurturin and glia-derived neurotropic factor differential ph and capsaicin responses of griffonia simplicifolia ib (ib )-positive and ib -negative small sensory neurons endothelin- enhances capsaicin-induced peptide release and cgmp accumulation in cultures of rat sensory neurons et- activates ca + sparks in pasmc: local ca + signaling between inositol trisphosphate and ryanodine receptors endothelin- enhances capsaicin-evoked intracellular ca + response via activation of endothelin a receptor in a protein kinase cepsilon-dependent manner in dorsal root ganglion neurons endothelin- inhibits inward rectifier k + channels in rabbit coronary arterial smooth muscle cells through protein kinase c age-and sex-specific nociceptive response to endothelin- endothelin- -induced et(a) receptor-mediated nociception, hyperalgesia and oedema in the mouse hindpaw: modulation by simultaneous et(b) receptor activation endothelins induce etb receptor-mediated mechanical hypernociception in rat hindpaw: roles of camp and protein kinase c effect of peripheral endothelin- concentration on carcinoma-induced pain in mice effects of selective endothelin et(a) receptor antagonists on endothelin- -induced potentiation of cancer pain localization and endogenous concentration of endothelin-like immunoreactivity in human placenta endothelin- -induced pain and hyperalgesia: a review of pathophysiology, clinical manifestations and future therapeutic options acid-sensing ion channels (asics): therapeutic targets for neurological diseases and their regulation emerging factors in the progression of cancer-related pain acidic microenvironment created by osteoclasts causes bone pain associated with tumor colonization this work was supported by the national natural science foundation of china (no. and no. ). wph designed this research. lw, ttl, yj, sw, and cyq performed the experiments. lw and ttl participated in data analysis. wph, lw, and ttl wrote the paper. all authors contributed substantially to this research and reviewed this manuscript. competing interests: the authors declare no competing interests. key: cord- -eoovm gh authors: liu, qi; zuo, rui; wang, kai; nong, fei-fei; fu, ya-jun; huang, shao-wei; pan, zeng-feng; zhang, yi; luo, xia; deng, xiang-liang; zhang, xiao-xue; zhou, lian; chen, yang title: oroxindin inhibits macrophage nlrp inflammasome activation in dss-induced ulcerative colitis in mice via suppressing txnip-dependent nf-κb pathway date: - - journal: acta pharmacol sin doi: . /s - - - sha: doc_id: cord_uid: eoovm gh oroxindin is a flavonoid isolated from the traditional chinese medicine huang-qin, which has shown various pharmacological activities including anti-inflammatory, antitumor, antioxidant, etc. thus far, the effect of oroxindin on colonic inflammation and the underlying mechanism remain unknown. in this study, we investigated the tissue distribution of oroxindin and its therapeutic effects on ulcerative colitis (uc) as well as the underlying mechanisms. uc model was established in mice by administrating dextran sulfate sodium (dss) in drinking water for d. we first showed that oroxindin was largely absorbed by the colon as an active ingredient after normal mice received huang-qin-tang, a traditional chinese medicine decoction. uc mice were then treated with oroxindin ( . , , mg ·kg(− ) ·d(− ), i.g.) for d. we found that oroxindin treatment greatly suppressed massive macrophages infiltration and attenuated pathological changes in colonic tissue. furthermore, oroxindin treatment significantly inhibited the generation of il- β and il- in the colon via inhibiting the nucleotide-binding oligomerization domain-like receptor (nlrp ) inflammasome formation and activation. in cultured macrophages, lps induced nlrp inflammasome formation and caspase- activation, which were suppressed by oroxindin ( . – μm). in lps-treated macrophages, oroxindin dose-dependently restored the expression of txnip protein, leading to suppressing txnip-dependent nf-κb activation. in conclusion, these results demonstrate that oroxindin could be absorbed by the colon and attenuate inflammatory responses via inhibiting nlrp inflammasome formation and activation, which is related to the inhibitory effect on txnip-dependent nf-κb-signaling pathway. hence, oroxindin has the potential of becoming an effective drug for treating uc. ulcerative colitis (uc) is a chronic and relapsing inflammatory disease that occurs in the colon with abdominal pain, rectal bleeding, and diarrhea [ ] . moreover, patients with uc have an increased risk of developing colorectal carcinoma [ , ] . the precise mechanisms involved in the initiation and development of uc have not yet been completely clarified. numerous studies have indicated that genetic alterations and the impaired balance in the intestinal immune system may contribute to the development of uc [ , ] . in particular, imbalance in the mucosal immune system causes the generation of pro-inflammatory cytokines, which in turn leads to chronic inflammation, ulceration, and lesions in the colonic mucosa [ ] [ ] [ ] [ ] . the proinflammatory cytokines il- β and il- are considered important pathogenic factors during the development of uc [ ] [ ] [ ] . meanwhile, the secretion of il- β and il- is regulated by activated caspase- , which is controlled by the nucleotide-binding oligomerization domain-like receptor (nlr) inflammasome. intestinal inflammation has been studied in nlr-deficient mice [ ] , and evidence has confirmed the pathogenic role of the nlrp inflammasome in uc [ , ] , which makes the nlrp inflammasome a potential target for uc treatment. thioredoxin-interacting protein (txnip) possesses various functions including regulating cell metabolism, cell death, tumor formation, and inflammation [ , ] . txnip was shown to compete with migration inhibitory factor (mif) for nf-κb activation [ ] . lps is known to inhibit the expression of txnip mrna and protein levels and activate the nf-κb-signaling pathway [ ] . a previous study demonstrated that when treated with lps or tnf-α, the expression of txnip was significantly decreased and the inhibition of tnf-α-induced nf-κb activation was mostly dependent on txnip in hepatocarcinogenesis mice [ ] , which suggested a protective role of txnip. recently, some studies showed that txnip was down-regulated in patients with inflamed colonic mucosa and suggested that thioredoxin (trx) might be a potential therapeutic agent for the treatment of inflammatory bowel disease (ibd) [ , ] . in summary, these studies indicated that monomers that regulate txnipdependent nf-κb activation and inhibit nlrp inflammasome activation have the potential to become new anti-inflammatory drugs for treating uc. at present, uc has evolved into a global disease and is severely threatening people's health in many asian countries [ ] , while existing drugs for uc are usually associated with serious side effects [ ] [ ] [ ] . as a traditional chinese medicine decoction, huang-qin-tang (hqt) is used for treating uc in china with a more than -year history [ ] [ ] [ ] . to determine the bioactive ingredients in hqt, in this study, we measured the accumulated constituents of hqt in colon tissue. consequently, we chose oroxindin and explored its protective mechanism against uc. oroxindin is a natural bioflavonoid isolated from huang-qin (one of the major ingredients in hqt). some studies have demonstrated that oroxindin exhibits various bioactivities including antiinflammatory, antitumor, antioxidant, etc. [ ] [ ] [ ] . however, the protective effects of oroxindin on regulating the function of colonic macrophages during uc and the inhibitory mechanism of oroxindin on the nlrp inflammasome remain unclear. in the present study, we investigated the protective effects of oroxindin and the underlying mechanisms, and proved that oroxindin attenuates uc by inhibiting nlrp inflammasome activation via suppressing the txnip-dependent nf-κb-signaling pathway in colonic macrophages. reagents and antibodies oroxindin (> % purity; chengdu pufei de biotech co., ltd., china) was dissolved in dimethyl sulfoxide (dmso) at a concentration of . m and stored at − °c. the stock was then diluted with dulbecco's modified eagle's medium (dmem; gibco, suzhou, china) to the final concentration used in each experiment. lipopolysaccharide (lps) from escherichia coli serotype o :b , -( , -dimethylthiazol- -yl)− , -di-phenyltetrazolium bromide (mtt), -aminosalicylic acid, and triton x- were purchased from sigma-aldrich (st. louis, mo, usa). ′, -diamidino- -phenylindole was purchased from invitrogen (carlsbad, ca, usa). dextran sulfate sodium (dss) and dmso were purchased from mp biomedicals (solon, oh, usa). β-actin and hematoxylin were obtained from boster biological technology co., ltd. (wuhan, china). antibodies against caspase- , mouse epidermal growth factor-like module-containing mucinlike hormone receptor-like (also known as f / ), and apoptosis-associated speck-like protein containing a caspaseassociated recruitment domain (asc) were purchased from santa cruz biotechnology (santa cruz, ca, usa). nlrp , txnip, and p-p primary antibodies were purchased from cell signaling technology (danvers, ma, usa). an immunofluorescent nlrp antibody was purchased from abcam (cambridge, ma, usa). an alexa fluor (abcam) secondary antibody kit for immunohistochemistry was obtained from cell signaling technology. protein a/g plus-agarose beads were from santa cruz biotechnology (sc- ). an immunohistochemistry application solution kit was obtained from cell signaling technology. liquid chromatography-mass spectrometry (lc-ms) analysis the lc-ms system consisted of a triple tof™ + mass spectrometer (ab sciex, foster city, ca, usa) and a prominence ultra-high-performance lc system (nexera lc- a; shimadzu, kyoto, japan). data were analyzed using multiquant . software. after overnight fasting, mice were administered hqt decoctions at a dose of g/kg, and tissue samples were collected at . , , , and h. colon tissue samples were washed in normal saline and homogenized with methanol to a concentration of mg/ml. the supernatant of the homogenate ( μl) was deproteinized using a previously described method. then, μl of an internal standard solution (final concentration: ng/ml) and μl of methanol were added. finally, μl samples were injected into an agilent sb-c column ( . mm × mm, . μm) for analysis, using a mobile phase of (a) acetonitrile and (b) . % formic acid in water at a flow rate of . ml/min. paeoniflorin was detected by an electrospray ionization (esi) anion, and the remaining constituents were measured by esi-positive-ion detection and multiple reaction monitoring. to prepare standard samples, stock solutions of baicalin, oroxindin, glycyrrhizin, glycyrrhizinate, and paeoniflorin were diluted with methanol to nominal concentrations of . - , , . - , . - , . - , and . - ng/ml, respectively. adult male c bl/ mice ( - weeks old, - g) were supplied by and maintained at the laboratory animal services center of guangzhou university of chinese medicine (guangzhou, china). the animal study followed the guide for the care and use of laboratory animals of the national institutes of health and the animal protocol was approved by the animal ethics committee of guangzhou university of chinese medicine. the animals were housed in cages under standard environmental conditions of a temperature of - °c under a -h light/dark cycle and were provided free access to standard food and water. they were randomly divided into six groups: control, dss, oroxindin ( . , , or mg/kg) +dss group, and sulfasalazine ( mg/kg) +dsstreatment groups. to establish the uc model, the model group received % (w/v) dss in drinking water for d, followed by regular water provided for d, while the control group received regular drinking water. oroxindin was dissolved in water to prepare an oral suspension, and oroxindin and sulfasalazine were administered via gastric intubation from d to d after dss treatment until the termination of the experiment. the mice were sacrificed on d . the pieces of colonic tissues were fixed with % paraformaldehyde, embedded in paraffin and cut into -μm-thick sections. hematoxylin-eosin (h&e) staining was performed. colon histopathological changes were observed by a light microscope (olympus, tokyo, japan). the extent of histopathological changes was scored according to previous studies [ ] (table ) . immunohistochemical analysis of colon tissues five-micrometer-thick paraffin sections were used for immunohistochemical experiments. for antigen retrieval, the samples were placed in citric acid solution (ph . ) and heated in a microwave oven for min. subsequently, the sections were incubated with an animal-free blocking solution for h, prior to incubation with the f / monoclonal antibody overnight at °c. after that, the sections were labeled with secondary antibodies for h. ′-diaminobenzidine was used to produce a brown signal, and hematoxylin was used as a counterstain. more detailed steps were performed according to the immunohistochemistry application solutions kit. the immunohistochemical images were visualized by a microscope (bx ; olympus corp., tokyo, japan) and analyzed by image-pro plus software. cell culture and drug treatment thp- cells were maintained in a humidified atmosphere with % co at °c in dmem supplemented with % fetal bovine serum (gibco, carlsbad, ca, usa), u/ml benzylpenicillin, and mg/ml streptomycin. when cells reached~ % confluence, they were plated in six-well plates at a density of~ × cells/well and treated with various concentrations of oroxindin ( . , , and μm) for h. then, μg/ml lps was added, and the cells were incubated for an additional h. nitric oxide (no) was measured according to the manufacturer's protocols. co-immunoprecipitation experiment co-immunoprecipitation experiments were performed according to the user's protocol (santa cruz biotechnology, inc. sc- ). ice-cold immunoprecipitation lysis buffer was added to thp- cells before shaking for min. after incubation on ice for min, the cell lysate was centrifuged ( , ×g, min, °c) and the supernatant was incubated with μl of pbssuspended protein a/g plus-agarose beads and incubated for min at °c. subsequently, the supernatant was incubated with μg of asc antibody for h at °c. immunoprecipitates were then collected by centrifugation at ×g for min, and the pellet was resuspended in × sample buffer and boiled. the bound proteins of nlrp and pro-casp were detected by western blot. western blot analysis when cells reached~ % confluence, they were cultured in sixwell dishes and treated with various concentrations of oroxindin ( . , , and μm) for h. then, μg/ml lps was added, and the cells were incubated for additional h to detect nlrp , asc, txnip, and caspase- protein. to detect phospho-nf-κb p (p-p ) protein, lps was incubated for an additional h. cellular proteins were extracted using a cytoplasmic and nuclear extraction kit (beyotime institute of biotechnology, shanghai, china). the bradford method was used to determine the protein concentration. subsequently, the proteins were separated in sodium dodecyl sulfate-polyacrylamide gels and transferred to a polyvinylidene difluoride membrane (millipore, bedford, ma, usa). the membranes were incubated at room temperature with % non-fat milk or % bovine serum albumin (bsa) in tris-buffered saline with tween (tbst) to block non-specific binding for h, followed by incubation with the specific antibodies targeting nlrp ( : ), asc ( : ), txnip ( : ), p-p ( : ), caspase- ( : ), and inos ( : ) at °c overnight. on the second day, the membranes were washed with tbst three times, followed by incubation with an appropriate secondary antibody for h at room temperature. protein bands were visualized using enhanced chemiluminescence. measurement of il- β in cells cells were cultured at × cells/well in -well plates. experiment groups and drug treatment have been stated above in the "cell culture and drug treatment" section. the culture supernatant was collected to determine the concentration of il- β by an elisa kits following the manufacture's instructions. immunofluorescence microscopic analysis of colon tissues eight-micrometer-thick frozen colon tissue sections were used for immunofluorescence experiments. briefly, the sections were placed in acetone for min, permeabilized with . % triton x- , and blocked with % bsa for h at room temperature. for immunofluorescence, the sections were incubated with anti-f / ( : ) plus anti-nlrp ( : ) or anti-caspase- ( : ) antibodies overnight. on the next day, the slides were washed and labeled with corresponding conjugated secondary antibodies (alexa fluor and dylight ) for h. then, slides were visualized sequentially using a laser scanning confocal microscope (zen . , zeiss) [ , ] . settings for image acquisition were identical for both control and experimental tissues. the colon tissues were homogenized in cold normal saline buffer and centrifuged at ×g for min at °c. the culture supernatant was collected to determine the concentration of il- β and il- by elisa kits following the manufacturer's instructions. subsequently, the protein concentration of the supernatant was detected using a bca kit. the results are shown as the ratio of the amount of cytokines to the protein concentration. statistical analysis statistical analysis was performed using graphpad prism and spss . software. data are expressed as the mean ± standard deviation (sd) or mean ± standard error of the mean (sem) and are representative of at least three experiments. p < . was considered to indicate a statistically significant difference. concentration of baicalin, oroxindin, glycyrrhizin, and paeoniflorin in the colon we analyzed the main ingredients of hqt and measured the concentration of four principal constituents (baicalin, oroxindin, glycyrrhizin, and paeoniflorin) in the colon tissue. after being fed with hqt ( g/kg), normal mice were sacrificed at predetermined time points ( . , , , and h), and the results were shown in fig. a . fig. a shows that large amounts of baicalin and oroxindin accumulated in colon tissue. subsequently, we calculated the ratio of monomer concentration in the colon tissue to the monomer concentration in hqt, and the ratio of oroxindin was higher than that of other compounds (fig. b) . oroxindin ameliorated dss-induced uc to determine the protective effect of oroxindin against uc, we established the acute colitis model by administering dss [ ] . specifically, c bl/ mice were treated with % dss in drinking water for d and were then provided regular water for d. oroxindin ( . , , and mg/kg) and sulfasalazine ( mg/kg) were orally administered from d to d . based on the data obtained, we found that oroxindin treatment attenuated body weight loss (percentage of that on d ) compared with that of the model group (fig. a) . dss caused colonic shortening, while the symptom was markedly improved by oroxindin treatment during colitis progression (fig. b, c) . as shown in fig. d , we also measured the spleen index in each group, which was raised in the model group and reduced by sulfasalazine or oroxindin administration. oroxindin also significantly decreased the white blood cell (wbc) count, an inflammation-related marker in peripheral blood (fig. e ). in addition, the ratios of monocytes in lymphocytes were significantly decreased in the oroxindintreated groups (fig. f) . as shown in fig. a , c, colonic inflammation and ulceration were evaluated by histopathological analysis using hematoxylin and eosin staining, and histological scores were determined by a standard method. mucosal oroxindin protects against ulcerative colitis q liu et al. inflammation, missing epithelium, and submucosal infiltration were observed in colon tissues in the model group. oroxindin remarkedly relieved these severe symptoms in the colon. in addition, oroxindin diminished dss-induced massive colonic macrophage infiltration. to assess the extent of intestinal inflammation in the colons of the dss-treated mice, colon tissue sections were stained with an anti-f / antibody to detect macrophage infiltration (fig. b, d) . these data indicated that oroxindin is able to suppress pathological changes and massive macrophage infiltration in colonic tissues, which proves the protective effects of oroxindin against uc. oroxindin inhibited nlrp inflammasome formation in macrophages macrophages are known to play critical roles in the pathogenesis of uc. in order to clarify the specific mechanism, we subsequently performed immunoprecipitation in cultured macrophages to determine whether oroxindin inhibits the formation of the nlrp inflammasome, one of the core protein complexes in inflammatory responses (the nlrp -asc-pro-casp assembly). as shown in fig. a -c, after incubation with asc primary antibody, the colocalization coefficient was increased in the model group based on the ratio of nlrp and pro-casp to asc, and this tendency could be significantly reversed by oroxindin, which indicated that inflammasome formation was suppressed by oroxindin. oroxindin inhibited lps-induced nlrp expression and activation in macrophages we further explored whether the protective effect of oroxindin was related to the inhibitory effect on nlrp inflammasome activation in vitro. as illustrated in fig. a , b, we measured the protein level of nlrp in the cytoplasm of macrophages. interestingly, the nlrp protein increased in the model group and was significantly suppressed by oroxindin treatment as expected. in addition, the expression level of asc was not changed in any of the groups (fig. c, d) . previous studies have indicated that the nlrp inflammasome activation could induce robust caspase- activation, as based on the equal amount of the cleaved and pro-enzyme form [ ] . importantly, the activation of caspase- was significantly inhibited by oroxindin (fig. e, f) . the concentration of il- β indicates the activation of the nlrp inflammasome. the results showed that the concentration of active il- β in the supernatant was increased in the model group and could be effectively reduced by oroxindin (fig. g) . oroxindin suppressed txnip-dependent nf-κb activities in macrophages to further elucidate the molecular mechanisms that regulate nlrp inflammasome activation, we evaluated the levels of the txnip protein (fig. a, b) , which is involved in nf-κb activation. txnip was markedly reduced in macrophages cytoplasm in the model group and could be restored by oroxindin in a concentration-dependent manner. this result was especially confusing until we found the protective role of txnip in cells, because decreased txnip leads to txnip-dependent nf-κb activation. therefore, we analyzed the levels of p-p in the cytoplasm. as shown in fig. c , d, p-p expression was markedly up-regulated in the model group and was inhibited by oroxindin, suggesting that the protective effect of oroxindin is possibly related to the txnip-dependent nf-κb-signaling pathway. oroxindin lessened uc by inhibiting the nlrp inflammasome nlrp is initially activated during inflammatory injury, and its activation has been confirmed to play a crucial role in dssinduced colitis [ ] . to further confirm our hypothesis in vitro, we examined whether oroxindin could suppress nlrp inflammasome activation in uc mice. as shown in fig. a, b, we examined the co-localization of f / (red) and nlrp (green) or caspase- (green) in colon tissues. according to the results, the expression of nlrp and caspase- was increased in macrophages in the dss-treated mice, while oroxindin exerted specific protective effects in the inflamed tissue. these findings indicated that oroxindin suppressed nlrp inflammasome formation in macrophages in colonic tissue. to further determine whether nlrp inflammasome activation was repressed in vivo, the concentrations of il- β and il- (caspase- downstream effectors) were measured in colon tissue supernatants. the results showed that oroxindin could reduce the generation of il- β and il- , suggesting that oroxindin could quell the pathologic inflammation and ameliorate inflammatory symptoms in uc mice (fig. c, d) . previous studies have identified nlrp inflammasome-mediated inflammatory responses that underlie the critical mechanism of intestinal inflammation in a dss colitis model [ ] . thus, inhibition of nlrp inflammasome formation and activation may be an effective approach for uc treatment. in the present study, we confirmed that oroxindin, one of the important pharmacological substances in hqt, exerted protective effects against uc, and the b macroscopic appearances of the colon. c colon length changes in each group. d the spleen index was measured in each group. e wbc counts in peripheral blood. f mononuclear cells were detected in peripheral blood using an automatic blood analyzer. values are the mean ± sd (n = ). ## p < . , ### p < . vs. the control group; *p < . , **p < . , ***p < . vs. the dss-treated group. oroxindin protects against ulcerative colitis q liu et al. the results are representative of three independent experiments and expressed as the mean ± sd. # p < . vs. the control group; *p < . vs. the lps-treated group. oroxindin protects against ulcerative colitis q liu et al. underlying mechanism was associated with inhibition of the nlrp inflammasome. according to the introduction in traditional medicine, hqt could be used to prevent and treat uc. our results showed that dss administration to mice led to the loss of body weight (fig. s a) and induced mucosal inflammation, missing epithelium, and macrophage infiltration in colon tissues (fig. s b, c) . oral administration of hqt ( , , g/kg) relieved mice weight lose and ameliorated the pathological changes and macrophage infiltration in colon tissues, which confirmed the protective effect of hqt against uc (fig. s a-c) . however, the specific bioactive monomer in hqt that exerts protective effect on uc has not yet been reported. the tissue distribution of drugs measured by lc-ms is an important index in pharmacological effectiveness studies. our data showed that the concentrations of baicalin and oroxindin were the highest monomers absorbed by the colon tissue (fig. a) . on the other hand, it is important to take the proportion of baicalin and oroxindin in hqt into consideration, because the concentration of baicalin is four times higher than that of oroxindin (data not shown). therefore, we analyzed the relative content of oroxindin (the concentration in the colon relative to that in hqt), and the results suggested that the colon's absorption of oroxindin was better than that of other constituents (fig. b) . this indicated that oroxindin could be one of the bioactive compounds in hqt with high colon absorption. therefore, we were interested in whether oroxindin could prevent the development of uc. as one of the innate immune cells, macrophages are vital in protecting the human body from luminal bacteria during uc [ ] . in this study, we found that oroxindin could ameliorate colonic inflammatory symptoms caused by dss and we focused on the numbers of macrophages in the blood (fig. f) and colon. intriguingly, oroxindin decreased the number of macrophages in the blood and reversed the infiltration of macrophages (f / + cells) in colon tissues. sun et al. have also demonstrated that oroxindin could ameliorate the infiltration of inflammatory cells in colon tissues, as well as the histopathological features of dssinduced colitis [ , ] . thus, we aimed to determine the underlying mechanism of the regulating effect of oroxindin on macrophage function. when exposed to significant interference, the antigenic receptors on the surface of macrophages interact with the antigenic receptors in the cytoplasm. in particular, a previous study has suggested the pivotal role of the nlrp inflammasome in the pathogenesis of uc [ ] , and it may be a potential target for developing novel therapeutics for uc [ ] . the gastrointestinal microbiota is known as one of the pathogenic factors of uc. in particular, gram-negative bacteria, with an lps-containing outer membrane, account for a large part of the gut microbiota. to further clarify the results of our in vivo study, we evaluated the anti-inflammatory effects of oroxindin in lps-stimulated thp- cells in vitro. first, we performed a cell proliferation assay to assess the effects of oroxindin in macrophages. compared to the control group, the proliferation of lps-stimulated cells was significantly decreased, whereas oroxindin ( . , , μm) could alter this effect (fig. s ) . therefore, we chose these three doses in our in vitro experiments. it was shown that oroxindin reduced nlrp inflammasome protein expression in lps-stimulated thp- cells (fig. a) . nlrp is a critical component of the nlrp inflammasome. it can form a single oligomeric structure, recognizing pathogen-associated or damage-associated molecular patterns. once the inflammasome is formed, asc, a significant adaptor protein for pyrin domain (pyd)-pyd interaction, is recruited, and intracellular redistribution of asc is essential for nlrp inflammasome activation [ ] . in our present study, the asc proteins were found in the cytoplasm at a consistent level. asc subsequently bridges to pro-caspase- through caspase-associated recruitment domain (card)-card interaction and, thereby, to the assembly of a cytosolic protein complex called the "inflammasome" [ , ] , which induces autocleavage of caspase- into a cleaved form [ ] . additionally, our results showed that the amount of the multiprotein complex fig. oroxindin inhibited nlrp inflammasome activation in a txnip-dependent manner. a protein expression of txnip was analyzed by western blotting and shown as cumulative data from multiple experiments. b densitometric analysis was used to determine the relative protein expression, normalized to that of β-actin. c protein expression of p-p was analyzed by western blotting. d densitometric analysis was used to determine the relative protein expression, normalized to that of β-actin. the results are representative of three independent experiments. values are the mean ± sd (n = ). # p < . vs. the control group; *p < . , **p < . vs. the lps-treated group. oroxindin protects against ulcerative colitis q liu et al. (nlrp -asc-caspase- assembly), as well as the activation ratio of caspase- (cleaved form to pro-enzyme form), was significantly diminished by oroxindin (figs. a and e ). nlrp inflammasome activation leads to the generation of the proinflammatory cytokine il- β. elisa analysis revealed that oroxindin suppresses the concentration of il- β in macrophages (fig. g) . thus, the in vitro study indicated that oroxindin could inhibit nlrp inflammasome formation and activation. in this study, the expression of the nlrp inflammasome proteins was repressed by oroxindin in vitro, and we attempted to clarify the upstream mechanism. previous studies have demonstrated that txnip expression is related to nf-κb activity. lps stimulation decreased the protein level of txnip, which could regulate txnip-dependent nf-κb activation and prime nlrp , pro-il- β, and pro-il- expression. therefore, we detected the expression of txnip and nf-κb in macrophages [ ] [ ] [ ] . the results showed that the txnip protein level was significantly reduced in the lps-stimulated group and that the level was restored by oroxindin (fig. a, b) . correspondingly, the activation of nf-κb was repressed by oroxindin, suggesting the involvement of the txnip-dependent nf-κb-signaling pathway. as shown in fig. , all results indicated that oroxindin suppressed the nlrp inflammasome in a txnip-dependent manner, suggesting that oroxindin could be a powerful tool for inhibiting txnip-dependent nf-κb activation and repressing nlrp inflammasome activation. in addition, it has been reported that the expression of txnip is closely related to the inducible nitric oxide synthase (inos)-nosignaling pathway [ , ] . forrester et al. [ ] proved that txnip expression in macrophages is suppressed by endogenously synthesized no, which is produced by inos. therefore, to further fig. oroxindin ameliorated dss-induced acute colitis via inhibition of the nlrp inflammasome. a sections of colonic tissue were immunostained with f / (red) and nlrp (green) antibodies and observed under a confocal laser scanning microscope and shown as representative data. b sections of colonic tissue were immunostained with f / (red) and caspase- (green) antibodies and observed under a confocal laser scanning microscope and are shown as representative data. enlarged areas of interest are shown in merged images. c the concentration of il- β in colonic homogenates was determined by an enzyme-linked immunosorbent assay (elisa) in triplicate. d the concentration of il- in colonic homogenates was determined by elisa in triplicate. data are presented as the mean ± sd. ## p < . compared with the normal mice; *p < . , **p < . compared with the dss-induced colitis mice. confirm the role of txnip, we investigated the effect of oroxindin on the inos-no signaling pathway. it has been proven that txnip expression in macrophages is suppressed by inos-induced no production [ ] . therefore, we measured the concentration of no in the cell supernatant, and the results showed that lps induced robust no production, while this effect could be suppressed by oroxindin (fig. s a) . subsequently, we detected the expression of inos, and the results showed that the increase in inos induced by lps could be down-regulated by oroxindin (fig. s b, c) . to further identify the participance of the inos-no signaling pathway, nicaraven, a well-known no scavenger, was used to abate no production. the results showed that nica could obviously decrease the expression of inos (fig. s d, e) , while the expression of txnip was correspondingly up-regulated (fig. s f, g) . oroxindin has a similar effect as nica (fig. s d-g) , suggesting that oroxindin has an inhibitory effect on the inos-no signaling pathway, followed by the restoration of txnip expression and suppression of nf-κb activities. to further confirm our hypothesis in vitro, we examined whether oroxindin could suppress nlrp inflammasome activation in uc mice by immunofluorescence co-localization. we found that the nlrp inflammasome components, nlrp and the downstream protein, caspase- , were upregulated in macrophages in colonic tissue in the uc model and were dramatically suppressed by oroxindin (fig. a, b) . the levels of il- β and il- , central mediators of the inflammatory responses, were also decreased, which confirmed the anti-inflammatory effects of oroxindin (fig. c, d) . taken together, these data supported our in vivo study and confirmed our hypothesis that oroxindin inhibits colonic inflammation by inhibiting nlrp inflammasome formation and activation. in conclusion, this study demonstrated that oroxindin was absorbed into the colon at a high level and ameliorated acute intestinal inflammation in a mouse uc model by suppressing nlrp inflammasome activation. oroxindin could also suppress nlrp inflammasome activation by restoring txnip expression and inhibiting the txnip-dependent nf-κb-signaling pathway induced by lps in macrophages. considering the effective protective effect of oroxindin on inflammatory responses, this compound could be regarded as a potential therapeutic drug for uc. however, the relationship between txnip and the uc model remains to be further investigated. fig. schematic representation of the protective mechanism of oroxindin against uc. dss-induced intestinal epithelium bacterial translocation results in large amounts of lps entering the cytoplasm, leading to a dramatic decrease in txnip, followed by txnip-dependent nf-κb activation. nf-κb activation, which is also regarded as the first signal that activates the nlrp inflammasome, is responsible for the increased transcription levels of pro-nlrp , pro-il- β, and pro-il- . the activation of the nlrp inflammasome complex induces auto-cleavage of caspase- from pro-caspase- into cleaved caspase- and clips cleavage of pro-inflammatory cytokines pro-il- β and pro-il- into their active forms, which results in intestinal inflammation and tissue injury. therefore, the protective effect of oroxindin on restoring txnip expression and abating the downstream nf-κb-signaling pathway helps to reduce inflammatory cytokine release and ameliorate pathological changes during colitis. oroxindin protects against ulcerative colitis q liu et al. inflammatory bowel disease: etiology and pathogenesis clonal evolution of colorectal cancer in ibd inflammatory networks underlying colorectal cancer the genetics and immunopathogenesis of inflammatory bowel disease inflammatory bowel disease review article: the role of oxidative stress in pathogenesis and treatment of inflammatory bowel diseases. naunyn-schmiedeberg's arch pharmacol proteus mirabilis: the enemy within interleukin β mediates intestinal inflammation in mice and patients with interleukin receptor deficiency expression levels of proinflammatory cytokines and nlrp inflammasome in an experimental model ofoxazolone-induced colitis the role of pattern recognition receptors in intestinal inflammation inflammasome complexes: emerging mechanisms and effector functions nlrp inflammasome plays a key role in the regulation of intestinal homeostasis the nlrp inflammasome protects against loss of epithelial integrity and mortality during experimental colitis diverse functions of vdup in cell proliferation, differentiation, and diseases thioredoxin/txnip: redoxisome, as a redox switch for the pathogenesis of diseases macrophage migration inhibitory factor interacts with thioredoxin-interacting protein and induces nf-κb activity thioredoxin-interacting protein gene expression via mondoa is rapidly and transiently suppressed during inflammatory responses vitamin d upregulated protein suppresses tnf-α-induced nf-κb activation in hepatocarcinogenesis human thioredoxin- ameliorates experimental murine colitis in association with suppressed macrophage inhibitory factor production decreased expression of thioredoxin interacting protein mrna in inflamed colonic mucosa in patients with ulcerative colitis epidemiology of inflammatory bowel diseases from west to east inflammatory bowel disease: clinical aspects and established and evolving therapies herbal medicine in the treatment of ulcerative colitis cerebral sinus thrombosis occurring in a patient with ulcerative colitis treated with the chinese herbal medicine yannan baiyao huangqin-tang and ingredients in modulating the pathogenesis of ulcerative colitis wogonoside induces growth inhibition and cell cycle arrest via promoting the expression and binding activity of gata- in chronic myelogenous leukemia cells new therapeutic aspects of flavones: the anticancer properties of scutellaria and its main active constituents wogonin, baicalein and baicalin wogonoside displays anti-inflammatory effects through modulating inflammatory mediator expression using raw . cells dimethyl fumarate ameliorates dextran sulfate sodium-induced murine experimental colitis by activating nrf and suppressing nlrp inflammasome activation aspirin alleviates endothelial gap junction dysfunction through inhibition of nlrp inflammasome activation in lps-induced vascular injury puerarin inhibits hyperglycemiainduced inter-endothelial junction through suppressing endothelial nlrp inflammasome activation via ros-dependent oxidative pathway animal models of intestinal inflammation: ineffective communication between coalition members potentiation of caspase- activation by the p x receptor is dependent on tlr signals and requires nf-kappab-driven protein synthesis colitis induced in mice with dextran sulfate sodium (dss) is mediated by the nlrp inflammasome diversity of intestinal macrophages in inflammatory bowel diseases wogonoside protects against dextran sulfate sodium-induced experimental colitis in mice by inhibiting nf-κb and nlrp inflammasome activation apoptosisassociated speck-like protein containing a caspase recruitment domain is a regulator of procaspase- activation nlrp gene is associated with ulcerative colitis (uc), but not crohn's disease (cd), in chinese han population inflammasomes and intestinal inflammation structure and interdomain dynamics of apoptosis-associated specklike protein containing a card (asc) regulatory molecules involved in inflammasome formation with special reference to a key mediator protein activation of the native -kda precursor form of interleukin- -converting enzyme canonical and non-canonical activation of nlrp inflammasome at the crossroad between immune tolerance and intestinal inflammation inflammasomes: mechanism of action, role in disease, and therapeutics cutting edge: nf-κb activating pattern recognition and cytokine receptors license nlrp inflammasome activation by regulating nlrp expression txnip deficiency exacerbates endotoxic shock via the induction of excessive nitric oxide synthesis thioredoxin-interacting protein (txnip) is a feedback regulator of s-nitrosylation ql and rz contributed to the animal experiments, cell experiments, and data analysis. kw, ql and ffn contributed to the lc-ms experiments. yjf and xl contributed to the study analysis and data analysis. ql, zfp, and xld, swh, yz, and xxz contributed to the revision of the manuscript. yc and lz contributed to the conception and design of the project. the online version of this article (https://doi.org/ . /s - - - ) contains supplementary material, which is available to authorized users.competing interests: the authors declare no competing interests. key: cord- - fvumvdc authors: chen, bao-yi; huang, cheng-cui; lv, xiao-fei; zheng, hua-qing; zhang, ya-juan; sun, lu; wang, guan-lei; ma, ming-ming; guan, yong-yuan title: sgk mediates the hypotonic protective effect against h( )o( )-induced apoptosis of rat basilar artery smooth muscle cells by inhibiting the foxo a/bim signaling pathway date: - - journal: acta pharmacol sin doi: . /s - - -y sha: doc_id: cord_uid: fvumvdc serum- and glucocorticoid-inducible kinease- (sgk ) is a serine/threonine kinase regulated by hypotonic stimuli, which is involved in regulation of cell cycle and apoptosis. our previous study shows that activation of volume-regulated cl(−) channels (vrccs) protects rat basilar artery smooth muscle cells (basmcs) against hydrogen peroxide (h( )o( ))-induced apoptosis. in the present study, we investigated whether sgk was involved in the protective effect of vrccs in basmcs. we showed that hypotonic challenge significantly reduced h( )o( )-induced apoptosis, and increased sgk phosphorylation, but did not affect sgk protein expression. the protective effect of hypotonic challenge against h( )o( )-induced apoptosis was mediated through inhibiting mitochondria-dependent apoptotic pathway, evidenced by increased bcl- /bax ratio, stabilizing mitochondrial membrane potential (mmp), decreased cytochrome c release from the mitochondria to the cytoplasm, and inhibition of the activation of caspase- and caspase- . these protective effects of hypotonic challenge against h( )o( )-induced apoptosis was diminished and enhanced, respectively, by sgk knockdown and overexpression. we further revealed that sgk activation significantly increased forkhead box o a (foxo a) phosphorylation, and then inhibited the translocation of foxo a into nucleus and the subsequent expression of bcl- interacting mediator of cell death (bim). in conclusion, sgk mediates the protective effect of vrccs against h( )o( )-induced apoptosis in basmcs via inhibiting foxo a/bim signaling pathway. our results provide compelling evidences that sgk is a critical link between vrccs and apoptosis, and shed a new light on the treatment of vascular apoptosis-associated diseases, such as vascular remodeling, angiogenesis, and atherosclerosis. ion channel activation is essential for regulating apoptosis through the process of cell shrinkage, which is also called apoptotic volume decrease (avd). in addition to ca + -activated k + channels, the most important cation channels involved in avd, anion channels are also required for the process of avd [ ] . among these anion channels, volume-regulated cl − channels (vrccs) are crucial for cell volume regulation in almost all types of cell types [ ] ; however, their basic components, including lrrc a [ , ] , bestrophin- [ ] , and clc- [ , ] , are still under debate. in addition to their roles in cell volume homeostasis, vrccs have been implicated in various physiological and pathophysiological processes, such as proliferation, apoptosis, migration, angiogenesis, inflammation, neuronal excitation, and cancer [ ] . our previous work showed that the activation of vrccs induced by hypotonic challenge protected against hydrogen peroxide (h o )-induced apoptosis in rat basilar artery smooth muscle cells (basmcs) [ ] . however, how the cl − fluxion induced by the opening of vrccs, as cl − channels, affects the process of apoptosis remains largely unclear. because vrccs have various functions, we propose that alterations in intracellular cl − concentration might activate one or more cl − -sensitive kinases, subsequently resulting in a signaling cascade. the ubiquitously expressed serum-and glucocorticoid-inducible kinase- (sgk ) is a serine/threonine kinase that has been shown to be regulated by osmotic pressure [ ] . sgk was first cloned as a gene activated by serum and glucocorticoids [ ] . sgk has since been implicated in the regulation of cell growth, proliferation, survival, apoptosis, and migration, is regulated by numerous cytokines, growth factors, and cell stresses, and was identified as a cell volume-regulated gene [ ] . it was reported that hypotonic challenge increased sgk levels in distal nephritic cells of xenopus leavis [ , ] and canine pulmonary artery smooth muscle cells [ ] . in addition to being involved in the regulation of cell cycle arrest and apoptosis [ , ] , it is possible that sgk might serve as a mediator between vrccs and downstream signaling and play a role in the protective effect of vrccs against h o -induced apoptosis. in this study, we were interested in studying the relationship between vrccs and sgk in apoptosis of basmcs and examined whether hypotonic challenge plays a protective role in h o induced apoptosis via sgk in basmcs. moreover, as a downstream target of sgk , forkhead box o a (foxo a) is phosphorylated by sgk and subsequently inactivated and translocates from the nucleus to cytoplasm, making it unable to stimulate its proapoptotic gene targets, such as bcl- interacting mediator of cell death (bim) [ , ] . thus, we further explored whether the underlying mechanism of the protective effect of sgk on h o induced apoptosis is related to the inhibition of the foxo a/bim signaling pathway in basmcs. reagents and antibodies cell culture medium (dulbecco's modified eagle's medium: nutrient mixture f- (dmem/f- )), fetal calf serum, bovine serum albumin (bsa), and protease inhibitor cocktail were obtained from gibco/invitrogen (carlsbad, ca, usa). anti-sgk and anti-phospho-sgk (ser ) were purchased from merck (darmstadt, germany). an anti-clc- antibody was obtained from alomone labs (jerusalem, israel). anti-bcl , anti-bax, anticaspase- , anti-cleaved caspase- , anti-caspase- , anti-cleaved caspase- , anti-cytochrome c (cyt-c), anti-bim, anti-foxo a, antiphospho-foxo a (ser ), anti-poly adp-ribose polymerase (parp), anti-cleaved parp, anti-histone h and anti-cyt-c oxidase subunit iv (cox iv) antibodies were purchased from cell signaling technology (boston, ma, usa). hyperfect transfection reagent was purchased from qiagen (valencia, ca, usa cell culture male sprague-dawley rats were supplied by the experimental animal center of sun yat-sen university in guangzhou, china. all procedures complied with the standards for the care and use of animal subjects as stated in the guide for the care and use of laboratory animals (issued by the ministry of science and technology of china, beijing) and were approved by the ethics committee of zhongshan school of medicine on laboratory animal care (no. - ). rat basmcs were cultured as described previously [ ] . briefly, rats were anesthetized with intraperitoneal phenobarbital (merck, darmstadt, germany; mg· kg − ) and decapitated. the basilar arteries were isolated aseptically and placed in krebs buffer. after the fat and connective tissues were removed, the artery strips were cut into - mm pieces and then plated in dmem/f- containing % fetal calf serum (gibco brl, grand island, ny, usa) with μg· ml − streptomycin and u· ml − penicillin. basmcs were maintained at °c in a humidified incubator with a % o plus % co atmosphere. the cells were identified as smooth muscle cells based on spindle morphology under a light microscope, and immunostaining was performed with a monoclonal antibody specific for smooth muscle α-actin. basmcs from passages to were used when they reached %- % confluence. solution preparation a hypotonic solution was prepared with ml h o and ml dmem/f- medium as previously described [ ] . the osmolality of the solution was mosm· kg − h o (measured by a freezing point depression osmometer, osmomat , gonotec, gmbh, berlin, germany). the isotonic solution contained ml dmem/ f- medium with ml mannitol solution ( mmol· l − ), and the final osmotic pressure was mosm· kg − h o. basmcs were treated with hypotonic solution for different durations, and the reactions were stopped using liquid nitrogen. western blot analysis western blot analysis was performed as described previously [ ] . basmcs were lysed for min in lysis buffer with % protease inhibitor cocktail. after centrifugation at , r· min − for min at °c, the supernatant was obtained, and the protein concentration was measured with a bca kit (beyotime, nanjing, china). samples containing μg of protein were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes. the membranes were blocked at room temperature for h in tris-buffered saline tween- with % bovine serum albumin. then, the cells were incubated with primary antibodies overnight at °c and with appropriate secondary peroxidase-conjugated antibodies for h at room temperature. the blots were detected by a chemiluminescence system and qualified using a computer aided -d gel analysis system (bio-red, hercules, ca). the intracellular cl − concentration ([cl − ] i ) was measured using methoxy-n-ethylquinolinium iodide (meq) as described previously [ ] . briefly, meq was first reduced to its cell-permeable derivative -methoxy-n-ethyl- , -dihydroquinoline (dihydro-meq). meq reduction was carried out by the addition of mol· l − sodium borohydride to meq solution at room temperature under flowing nitrogen in the dark for min. dihydro-meq was freshly prepared, and cells were incubated with dihydro-meq for min before the experiment. in the cytoplasm, dihydro-meq is quickly oxidized to meq, which is sensitive to the [cl − ] i . fluorescence quenching induced by cl − was monitored by metafluor imaging software (universal imaging systems, chester, pa) at a -nm excitation wavelength and a -nm emission wavelength. transfection of adenovirus/sirna into basmcs an sgk overexpression adenovirus was purchased from vigene biosciences (rockville, usa), and the adenovirus expressed human sgk (nm_ . ) with an rfp (red fluorescent protein) tag. basmcs were transfected with the adenovirus according to a previously described method [ ] . sgk small interfering rna (sirna) ( ′-gguuaucugcacuccc uaa- ′) was designed and synthesized by qiagen. scrambled rna ( ′-uucuccgaacgugucacgu- ′) was used as a negative control. sgk sirna and negative control sirna were transfected into basmcs by using hiperfect transfection reagent according to a previously described protocol [ ] . cck- assay for cell viability cell viability was measured by the cck- according to a previously described protocol [ ] . at % confluence, basmcs from passages to were growth arrested by incubation in dmem/ f- with . % fetal calf serum for h before the experiments. then, μl of cck- solution was added to each well, and the cells were incubated for another h. the absorbance at nm was measured using a microplate reader (bio-tek, winooski, vt). briefly, basmcs were washed with phosphate-buffered saline (pbs) and stained with annexin v-apc and -aad. after incubation for min in the dark at room temperature, the stained cells were counted by flow cytometry (coulter, hialeah, fl) as described previously [ ] . the percentages of cells in the lower and upper right corners represented the early and late apoptosis rates, respectively, and the percentages of cells in the lower and upper left corners represented the survival and necrosis rates, respectively. mmp measurement jc- exposed to basmcs can be detected by the presence of both cytoplasmic jc- monomers and mitochondrial jc- aggregates. jc- forms monomers and emits green fluorescence at a low mitochondrial membrane potential (mmp), while it forms aggregates and emits red fluorescence at a high mmp. jc- fluorescence is usually excited by a wavelength of nm by flow cytometry, and the approximate emission peaks of monomeric and aggregated jc- are nm (green) and nm (red), respectively. thus, the fluorescence intensity ratio of monomeric jc- to aggregated jc- (green/red) represents a loss of mmp. in the experiments, cells were seeded into -well plates and exposed to μmol· l − h o for h with or without hypotonic medium. the cells were incubated with jc- dye for min at °c in the dark and then analyzed by flow cytometry as previously described [ ] . isolation of mitochondria intact mitochondria were isolated using a mitochondria isolation kit for cultured cells (thermo fisher scientific inc.) according to the manufacturer's protocol. a pellet containing × cells was obtained by centrifuging the cell suspension for min, and the suspension was discarded. mitochondria isolation reagent a ( μl) was added to the pellet, and the pellet was centrifuged at a medium speed for s and incubated for min on ice. next, μl mitochondria isolation reagent b was added to the tube, and the tube was centrifuged at maximum speed for s and incubated on ice for min. then, μl mitochondria isolation reagent c was added to the tube and centrifuged at × g for min at °c. the supernatant, which was the cytosolic fraction, was transferred to a new tube and centrifuged at , × g for min at °c. the pellet, which contained isolated intact mitochondria, was washed with μl reagent c and centrifuged at , × g for min at °c. the cytosolic and mitochondrial fractions were used for western blotting. cox iv was used as a loading control for the mitochondrial fraction. basmcs were fixed with % paraformaldehyde for min. the cells were washed twice in pbs for min at room temperature and permeabilized in pbs containing . % triton x- for min. nonspecific binding sites were blocked by % bsa in pbs for h. a primary monoclonal antibody was added and incubated overnight at °c. then, the cells were washed twice with pbs and incubated with a fluorescein isothiocyanate (fitc)-conjugated secondary antibody for h. finally, the cells were counterstained with hoechst to simultaneously evaluate nuclear condensation. cell micrographs were obtained with a ccd camera on a zeiss lsm imaging microscope (zeiss, oberkochen, germany). statistical analysis data analysis was performed using graphpad prism . (graphpad software inc., san diego, ca). all data are expressed as the means ± sem, and the n value represents the number of independent experiments. statistical significance was tested by one-or two-way anova followed by bonferroni multiple comparisons test. p < . was considered statistically significant. effects of hypotonic solution and h o on sgk activation sgk expression and phosphorylation were detected by immunoblotting after basmcs were incubated with isotonic or hypotonic solution for different durations. as shown in fig. a , the sgk phosphorylation level was increased by treatment with hypotonic solution for min and then returned to the normal level after min. the sgk protein level was not obviously altered within min under hypotonic stimulation. h o is one of the major reactive oxygen species (ros) and is widely used to establish a rapid and sensitive oxidative stressinduced apoptosis model [ , ] . therefore, sgk activity was also measured after basmcs were treated with μmol· l − h o . as shown in fig. b (fig. d) . after switching to the isotonic solution, the [cl − ] i rapidly returned to the normal level. to further verify the relationship between the [cl − ] i and sgk activation, sgk expression and phosphorylation were measured in basmc lysates in a low-cl − environment. as shown in fig. s a , the phosphorylation of sgk was upregulated in a solution containing or mmol· l − nacl, which represented a low-cl − environment, compared with that in a solution containing mmol· l − nacl, which represented a normal cl − environment. when cl − was substituted with gluconate, the phosphorylation level of sgk was not remarkably changed. moreover, when na + was substituted with k + , as shown in fig. s b , a low-cl − environment, but not a lowgluconate environment, remarkably increased the phosphorylation of sgk . taken together, these results suggest that sgk is activated by treatment with hypotonic solution and inhibited by with treatment of h o , which is related to the [cl − ] i . next, we performed sgk sirna or adenovirus transfection to increase or decrease the corresponding protein expression level, respectively. endogenous sgk protein expression was remarkably reduced to . % ± . % by transfection with nmol· l − sgk sirna for h (fig. s a ) and increased . % ± . -fold by transfection with multiplicity of infection (moi) sgk adenovirus for h (fig. s b) . to analyze whether sgk affects h o -induced cell injury, the viability rate of basmcs was evaluated by the cck- assay. as shown in fig. a next, the cell apoptosis rate was analyzed by flow cytometry. cells were double-stained with annexin v-fitc and propidium iodide, and the percentage in the lower right corner represented the early apoptosis rate. as shown in fig. c , d, h o remarkably increased the early apoptosis rate from . % ± . % to . % ± . % under isotonic conditions, and the apoptosis rate was sgk mediates the hypotonic protective effect on apoptosis by chen et al. alleviated to . % ± . % by hypotonic challenge. the protective effect of hypotonic stimulation was ameliorated by sgk silencing and potentiated by sgk overexpression. these data suggest that sgk is a crucial signaling molecule that mediates the protective effect of vrccs against h o -induced apoptosis in basmcs. hypotonic challenge increases the bcl- /bax ratio via sgk the anti-apoptotic protein bcl- and the pro-apoptotic protein bax regulate the mitochondrial pathway of apoptosis. an increased or decreased bcl- /bax ratio appears to determine the survival or death of cells under apoptotic stimulation. as shown in fig. , h o decreased bcl- protein expression and increased bax protein expression, resulting in a decline in the bcl- /bax ratio. hypotonic solution reversed the effects of h o and induced an upregulation in the bcl- /bax ratio, which was alleviated and strengthened by silencing and overexpressing sgk , respectively. hypotonic challenge prevents the loss of mmp and the release of cyt-c via sgk h o -induced apoptosis is regulated by the loss of mmp. to test whether hypotonic challenge protected h o -induced apoptosis through the sgk -mediated mitochondrial pathway, the mmp level was measured using jc- fluorescent dye. this dye usually accumulates in the mitochondria as aggregates and emits red fluorescence in healthy cells. however, it flows out into the cytoplasm as monomers and emits green fluorescence in apoptotic cells when mmp decreases. thus, a decrease in the red/green fluorescence ratio represents a loss of mmp. as shown in fig. a, b , the xand y-axis in the flow cytometry plot represent the green and red fluorescence intensity, respectively. h o decreased the red/green fluorescence ratio, indicating a promoting effect of mmp loss, which was protected in cells treated with hypotonic solution. the protective effect of hypotonic solution was reversed and enhanced by transfection with sgk sirna and sgk adenovirus, respectively. mmp loss induces cyt-c release from the mitochondria into the cytosol. therefore, cyt-c expression in both the cytosol and mitochondria was determined by western blotting. as shown in fig. c , d, treatment with h o enhanced the release of cyt-c from the mitochondria into the cytosol, resulting in an increased cyt-c level in the cytosol and a reduced cyt-c level in the mitochondria. these effects were prevented by hypotonic challenge. silencing sgk attenuated the effect of hypotonic solution on cyt-c release, whereas overexpressing sgk further potentiated this effect. hypotonic challenge reduces caspase activation via sgk an increase in the cytosolic cyt-c level leads to the activation of caspases, such as caspase- and its downstream caspase- , which are crucial proteases that cleave cellular proteins and, as a consequence, induce mitochondria-dependent apoptosis. as shown in fig. , the cleavage of caspase- and caspase- was promoted by treatment with h o and was restored to a normal level by hypotonic challenge. the effects of hypotonic stimulation were remarkably attenuated and strengthened by silencing and overexpressing sgk , respectively. accordingly, the cleavage of parp, one of the downstream targets of caspase- , induced the same changes as those in cleaved caspase- . taken together, these results demonstrate that sgk mediates the protective effect of hypotonic challenge on h o -induced mitochondrial pathway-dependent apoptosis. hypotonic challenge inhibits foxo a activation via sgk foxo a, a downstream target of sgk , is phosphorylated by sgk . this process inhibits the translocation of foxo a from the cytosol to the nucleus and results in the disruption of foxo adependent transcription, cell cycle arrest, and apoptosis. in this study, hypotonic challenge induced an increase in foxo a phosphorylation within min, and the protein expression of foxo a was not affected (fig. s ) . the phosphorylation of foxo a was upregulated by hypotonic stimulation, which was inhibited by silencing sgk and enhanced by overexpressing sgk (fig. a, b) , indicating that the hypotonic-induced upregulation of foxo a phosphorylation was mediated by sgk . next, h o -induced foxo a translocation was evaluated using hoechst staining to label cell nuclei and an anti-foxo a antibody with a fluorescent secondary antibody to determine the location of foxo a. as shown in fig. c , d, the results showed that h o increased the localization of foxo a in the cell nucleus, suggesting increased transcription of apoptosisrelated proteins, and this was reversed by hypotonic challenge. this effect of hypotonic solution was alleviated and promoted by silencing and overexpressing sgk , respectively. a similar result was also obtained using western blotting (fig. s ), which showed that h o increased the translocation of foxo a from the cytosol to the cell nucleus and that this effect was reversed by hypotonic challenge. silencing and overexpressing sgk increased and decreased this translocation of foxo a, respectively. bim is a foxo a target that is related to mitochondria-dependent apoptosis. in this study, we found that the expression of bim was upregulated under treatment with h o . hypotonic challenge reduced bim expression, which was attenuated in cells transfected with sgk sirna and enhanced in cells transfected with sgk adenovirus (fig. ) . these results demonstrate that the activation of vrccs protects against h o -induced apoptosis via sgk , the inhibition of downstream foxo a translocation into the nucleus and bim transcription. in the present study, we investigated whether the protective effect of vrccs against h o -induced apoptosis is mediated by sgk in basmcs. the results show that the activation of vrccs bax, which was measured by western blotting, in different treatment conditions. densitometric analysis of the bcl- /bax ratio is shown in the bar graphs (*p < . vs. con in iso, # p < . vs. con in iso + h o , + p < . vs. si or ad in iso + h o , n = ) apoptosis were evaluated by detecting mmp loss, which was measured by jc- staining. a reduced ratio of red/green fluorescence represented the loss of mmp. densitometric analysis of the ratio of red/ green fluorescence intensity is shown in the bar graphs (*p < . vs. con in iso, # p < . vs. con in iso + h o , + p < . vs. si or ad in iso + h o , n = ). c, d sgk mediated the protective effect of hypotonic challenge against h o -induced cyt-c release from the mitochondria (mito) into the cytosol (cyto). the effects of hypotonic challenge plus sgk silencing (c) or overexpression (d) on h o -induced apoptosis were evaluated by detecting the expression of cyt-c in the cyto and mito, which was measured by western blotting. densitometric analysis of cyto-c in the cyto and mito is shown in the bar graphs (*p < . vs. con in iso, # p < . vs. con in iso + h o , + p < . vs. si or ad in iso + h o , n = ) protects against h o -induced mitochondria-dependent apoptosis via sgk , which inhibits foxo a translocation from the cytosol into the nucleus and subsequent bim transcription. regulatory volume decrease is the mechanism by which cells maintains a constant volume when hypotonic challenge favors the influx of free water, which is related to vrcc activation [ ] . it has been proven that the opening of vrccs protects against h o -induced apoptosis via a mitochondria-dependent pathway in vascular smooth muscle cells (vsmcs) [ , ] and by inhibiting oxidative stress in pc cells [ ] . ischemia/reperfusion-induced apoptosis is also inhibited by the activation of vrccs in the myocardium [ ] . moreover, vrccs act as dual sensors of both hypoosmolarity and low ph o and reduce neuronal injury during ischemia stimulation and n-methyl-d-aspartate-induced apoptosis the effects of hypotonic challenge plus sgk silencing (a) or overexpression (b) on h o -induced apoptosis were evaluated by detecting the expression and activation of caspase- (cas- ), caspase- (cas- ), and parp, which were measured by western blotting, in different treatment conditions. densitometric analysis of the expression of cleaved cas- and cleaved cas- is shown in the bar graphs (*p < . vs. con in iso, # p < . vs. con in iso + h o , + p < . vs. si or ad in iso + h o , n = ) cell nuclei were stained with hoechst (blue), and foxo a was stained with a fitc-labeled antibody (green) (n = ) [ ] . however, the mechanism by which vrccs regulate apoptosis needs further exploration. apoptosis relies on an intracellular proteolytic cascade, which includes two types of caspases, namely, initiator caspases (such as caspase- ) and executioner caspases (such as caspase- ). apoptosis is essentially mediated by two important pathways, the extrinsic pathway and the intrinsic (or mitochondrial) pathway [ ] . notably, the mitochondrial pathway can be activated following oxidative damage via ros, which induce outer mitochondrial membrane permeabilization followed by cyt-c release from the mitochondria. the translocation of cyt-c into the cytosol initiates the cleavage of caspase- and subsequently caspase- , resulting in the cleavage of downstream targets, such as parp, and induces apoptotic cell death [ ] . it is well established that sgk activation is critical to cell survival and is related to the onset and progression of several pathophysiological disorders [ ] . extensive studies have demonstrated that the inhibition of sgk remarkably triggers apoptosis in breast cancer cells [ ] , colon cancer cells [ ] , prostate cancer cells [ ] , intestinal epithelial cells [ ] , and human umbilical vein endothelial cells [ ] . in sgk -null animals, the absence of sgk results in embryonic and extraembryonic angiogenic defects and increased apoptosis of endothelial cells and vsmcs [ ] . in the present study, we observed that the silencing of sgk increased apoptosis, whereas the overexpression of sgk inhibited it, through the mitochondria-dependent intrinsic pathway in basmcs. these results are consistent with the results of previous studies. foxo a, also called forkhead transcription factor fkhrl , is an intracellular target of sgk [ ] . foxo a is phosphorylated by sgk , resulting in its translocation from the nucleus to the cytoplasm and its inability to stimulate its pro-apoptotic gene targets, such as p and bim [ , ] . it has been reported that wnt signaling inhibits foxo a-induced apoptosis by increasing sgk synthesis [ ] . however, the inhibition of sgk promotes autophagy-dependent apoptosis via the mtor-foxo a pathway [ ] . the present results revealed that the activation of sgk by hypotonic challenge increases foxo a phosphorylation levels, resulting in the inhibition of foxo a translocation into the cell nucleus and the suppression of bim expression. this result provides compelling evidence that sgk is an important regulator of mitochondria-dependent apoptosis via the regulation of foxo a phosphorylation. sgk activity is regulated by extracellular osmotic pressure. previously, the human isoform of sgk was identified as a cell volume-regulated gene [ ] . this observation was also proven by other research groups, which showed that hypotonic conditions increase sgk levels in distal nephritic cells of xenopus leavis [ , ] and canine pulmonary artery smooth muscle cells [ ] . however, other studies have demonstrated that hypertonic conditions also increase sgk levels in some other cells, such as hepatocytes [ ] , pancreatic acinar cells [ ] , mammary tumor cells [ ] , and renal collecting duct cells [ ] . these results indicate that sgk is involved in osmotic cell adaptation via the dual regulation of cell volume in a cell typedependent manner. our results demonstrated that hypotonic challenge remarkably increased sgk phosphorylation and that the phosphorylation of sgk in basmcs recovered to normal levels within several minutes. therefore, these data only provide evidence that acute alterations in the intracellular cl − concentration affects the phosphorylation of sgk and this effect is transient. however, we cannot exclude the possibility that downstream signaling, such as foxo a signaling, is not already stimulated. a similar result showing that the phosphorylation of sgk is increased after min of incubation with hypotonic culture medium and returns to the normal level within min in pulmonary artery smooth muscle cells has been reported [ ] . we speculate that the difference in time course between this study and our study was due to the different cell types. to achieve long-term regulation of sgk phosphorylation, we used sgk sirna and adenovirus to alter the expression of sgk in subsequent experiments. the results showed that increased sgk prevented h o -induced apoptosis in basmcs. therefore, it appears that the opening of vrccs induces the activation of sgk , resulting in a subsequent inhibitory effect on the apoptosis signaling cascade. regarding the mechanism of vrcc-induced sgk phosphorylation, we speculate that there are at least two possible mechanisms. the first is the phosphoinositide- -kinase (pi k) signaling pathway. it has been demonstrated that sgk is highly homologous to the akt kinase family, sharing similar upstream activators, such as pi k [ , ] . previous works have reported that silencing candidate basic components of vrccs, such as lrrc a [ , ] and clc- [ , ] , remarkably reduces the activation of pi k/akt signaling pathway [ , , ] , indicating that vrccs might regulate sgk activity via pi k. the second pathway is the wnk signaling pathway. wnk is a serine-threonine protein kinase with an atypically placed catalytic lysine, and the wnk family is sensitive to the [cl − ] i [ ] . wnk has been found to activate sgk directly to regulate the effects of hypotonic challenge plus sgk silencing (a) or overexpression (b) on h o -induced apoptosis were evaluated by detecting the expression of bim, which was measured by western blotting, in different treatment conditions. densitometric analysis of the expression of bim is shown in the bar graphs (*p < . vs. con in iso, # p < . vs. con in iso + h o , + p < . vs. si or ad in iso + h o , n = ) the action of epithelial sodium channels [ ] . in addition, hypotonic challenge can evoke wnk phosphorylation [ ] . therefore, wnk might be another link between vrccs and sgk . moreover, wnk activates sgk in a pi k-dependent manner, indicating an effect of crosstalk between pi k and wnk on sgk activity [ ] . however, these speculations need further exploration in the future. as of now, the functional significance of sgk is still far from understood. functional analysis of gene-targeted mice lacking sgk has provided insights into the sgk -dependent regulation of physiological functions. although sgk knockout does not lead to a severe phenotype, the multiple physiological deficits it induces, such as a decreased ability to retain salt, an enhanced ability to excrete k + [ , ] , resistance to hypertension [ ] , blunted intestinal glucose uptake and decreased uptake of glucose into peripheral tissues [ ] , point to the broad functional role of sgk [ ] . in this regard, targeting sgk might present a novel therapeutic strategy to benefit sgk -related diseases. in summary, our findings show that sgk mediates the protective effect of vrccs against h o -induced mitochondriadependent apoptosis in basmcs. this work highlights the role of sgk , which is downstream of vrccs, in h o -induced apoptosis in basmcs and sheds new light on the treatment of apoptosisassociated cardiovascular diseases, such as vascular remodeling, angiogenesis, and atherosclerosis. cl − channels in apoptosis biophysics and physiology of the volumeregulated anion channel (vrac)/volume-sensitive outwardly rectifying anion channel (vsor) identification of lrrc heteromers as an essential component of the volume-regulated anion channel vrac swell , a plasma membrane protein, is an essential component of volume-regulated anion channel bestrophin is indispensable for volume regulation in human retinal pigment epithelium cells regulation of intracellular cl − concentration through volume-regulated clc- chloride channels in a vascular smooth muscle cells molecular identification of a volume-regulated chloride channel clc- chloride channel prevents apoptosis induced by hydrogen peroxide in basilar artery smooth muscle cells through mitochondria dependent pathway pathophysiological significance of the serum-and glucocorticoid-inducible kinase isoforms characterization of sgk, a novel member of the serine/threonine protein kinase gene family which is transcriptionally induced by glucocorticoids and serum hypotonic induction of sgk and na+ transport in a cells intracellular calcium plays a role as the second messenger of hypotonic stress in gene regulation of sgk and enac in renal epithelial a cells hypotonic activation of volume-sensitive outwardly rectifying chloride channels in cultured pasmcs is modulated by sgk second akt: the rise of sgk in cancer signalling serum and glucocorticoidinducible kinase (sgk ) is necessary for vascular remodeling during angiogenesis foxo transcription factors directly activate bim gene expression and promote apoptosis in sympathetic neurons protein kinase sgk mediates survival signals by phosphorylating the forkhead transcription factor fkhrl (foxo a) cystic fibrosis transmembrane conductance regulator (cftr) prevents apoptosis induced by hydrogen peroxide in basilar artery smooth muscle cells wnk is required for proliferation induced by hypotonic challenge in rat vascular smooth muscle cells simvastatin ameliorates rat cerebrovascular remodeling during hypertension via inhibition of volumeregulated chloride channel tmem a contributes to endothelial dysfunction by facilitating nox nadph oxidase-derived reactive oxygen species generation in hypertension physiology of cell volume regulation in vertebrates tyrosine phosphorylation is required for clc- chloride channel activation in vascular smooth muscle cells involvement of volumeactivated chloride channels in h o preconditioning against oxidant-induced injury through modulating cell volume regulation mechanisms and membrane permeability in pc cells activation of volume regulated chloride channels protects myocardium from ischemia/reperfusion damage in second-window ischemic preconditioning the volumeregulated anion channel (lrrc ) in nodose neurons is sensitive to acidic ph cell death pathways: a novel therapeutic approach for neuroscientists oxidative stress: the mitochondria-dependent and mitochondria-independent pathways of apoptosis antiapoptotic effect of serum and glucocorticoidinducible protein kinase is mediated by novel mechanism activating i{kappa}b kinase wnt signaling inhibits forkhead box o a-induced transcription and apoptosis through upregulation of serum-and glucocorticoid-inducible kinase sgk inhibition induces autophagy-dependent apoptosis via the mtor-foxo a pathway sgk inhibits cellular apoptosis and promotes proliferation via the mek/erk/p pathway in colitis serum glucocorticoid inducible kinase (sgk)- protects endothelial cells against oxidative stress and apoptosis induced by hyperglycaemia cloning and characterization of a putative human serine/threonine protein kinase transcriptionally modified during anisotonic and isotonic alterations of cell volume genomic organization and chromosomal localization of the human sgk protein kinase gene deranged transcriptional regulation of cell-volume-sensitive kinase hsgk in diabetic nephropathy hyperosmotic stress stimulates promoter activity and regulates cellular utilization of the serumand glucocorticoid-inducible protein kinase (sgk) by a p mapk-dependent pathway effect of poria cocos on hypertonic stress-induced water channel expression and apoptosis in renal collecting duct cells silence of clc- chloride channel inhibits cell proliferation and the cell cycle via g/s phase arrest in rat basilar arterial smooth muscle cells swell is a regulator of adipocyte size, insulin signalling and glucose homeostasis the regulation of salt transport and blood pressure by the wnk-spak/osr signalling pathway wnk activates sgk to regulate the epithelial sodium channel wnk activates sgk by a phosphatidylinositol -kinase-dependent and non-catalytic mechanism renal tubular sgk deficiency causes impaired k + excretion via loss of regulation of nedd - /wnk and enac inducible kidney-specific sgk knockout mice show a salt-losing phenotype resistance of mice lacking the serum-and glucocorticoid-inducible kinase sgk against saltsensitive hypertension induced by a high-fat diet serum-and glucocorticoid-inducible kinase mediates salt sensitivity of glucose tolerance yyg designed the study; mmm and byc wrote the manuscript; byc, cch, xfl, hqz, yjz, and ls performed the experiments; byc, cch, xfl, and mmm analyzed the data; yyg and glw revised the manuscript; yyg and mmm are the guarantors of this work and, as such, had full access to all the data in this study and take responsibility for the integrity of the data and the accuracy of the data analysis. the online version of this article (https://doi.org/ . /s - - -y) contains supplementary material, which is available to authorized users.competing interests: the authors declare no competing interests. key: cord- -xnkqg ef authors: lin, fang; reid, paul f.; qin, zheng-hong title: cobrotoxin could be an effective therapeutic for covid- date: - - journal: acta pharmacol sin doi: . /s - - - sha: doc_id: cord_uid: xnkqg ef nan a novel coronavirus, severe acute respiratory syndrome coronavirus (sars-cov- ), which causes coronavirus disease (covid- ) , was declared a pandemic in . it is generally agreed that the inflammatory cytokine storm, which causes systemic inflammation leading to multiple organ failures, is an important pathogenic mechanism in severe cases of covid- [ ] . based on previous studies of cobra venom by the authors and other independent researchers, cobrotoxin, a short-chain αneurotoxin from naja naja atra venom (nnav), could be an alternative therapy for covid- . historically, cobra venom has been used as a traditional folk medicine for the treatment of multiple illnesses. cobrotoxin comprises amino acid residues with a molecular weight of about kda. cobrotoxin can bind to several subtypes of the nicotinic acetylcholine receptors (nachrs) and also a few other receptors and ion channels [ ] . the wide distribution of nachrs may be the main cause of the multiple pharmacodynamic actions of nnav and cobrotoxin, including analgesic, antiviral, antiinflammatory, immunoregulatory, and antitumor activities. it has been reported that nnav and α-neurotoxins have antiinflammatory and immune regulatory actions. in numerous animal models and human clinical studies, cobra venom has demonstrated useful anti-inflammatory activity. cobrotoxin can inhibit the inflammatory process in rat models of rheumatoid arthritis induced by adjuvant [ ] . cobratoxin, also an α-neurotoxin, has analgesic effects in rodent models of inflammatory pain induced by formalin and acetate [ ] . cobrotoxin also can inhibit the accumulation of lymphocytes and macrophages and the excretion of pro-inflammatory cytokines induced by lipopolysaccharides (lps) (wang sz, unpublished). nuclear factor-κb (nf-κb) is a transcription factor that regulates the expression of genes involved in inflammatory responses and cell survival. under nonstimulated conditions, nf-κb localizes to the cytoplasm through an interaction with inhibitory proteins of κb family (iκbs). under pro-inflammatory stimuli, iκbs are rapidly phosphorylated by iκb kinase (ikk) and degraded by the proteasome, which results in the release of free nf-κb dimers (p and rel a) that translocate to the nucleus, to regulate the expression of target genes. cobrotoxin binds to the kinase domain of ikk and inhibits its phosphorylation, preventing the release of free nf-κb from iκb. cobrotoxin also binds with the critical cysteine residue (cys- ) of p in the n-terminal region of its dna-binding domain to prevent nf-κb binding to dna, thereby reducing the transcription of inflammatory genes [ ] . we also found that cobrotoxin and cobra venom inhibit the nuclear location of nf-κb in several independent experiments [ , , ] . in addition to inflammation inhibitory effects, nnav also has immune regulatory effects. nnav can increase the activity of natural killer cells in normal mice, increase the secretion of interferon (ifn)-γ and interleukin (il)- , promote cd tlymphocyte differentiation into th and th subsets, and increase b-lymphocyte proliferation stimulated by lps, as well as antibody production in response to sheep red blood cells [ ] . nnav markedly decreases t-lymphocyte proliferation stimulated by concanavalin a, suppresses cd and cd t-lymphocyte division, and inhibits il- secretion and th (cd + cd + il- + t) cell differentiation. although nnav decreases the numbers of both cd and cd t cells, the decrease in cd t cells is much more robust than that of cd t cells, resulting in restoration of the ratio of cd /cd . nnav also prevents the decrease in serum immunoglobulin igg and igm concentrations in immunosuppressed mice [ ] . previous studies have reported that α-neurotoxins inhibited replication and toxicity of several virus in both in vitro and in vivo. α-neurotoxins have sequence homology to viral glycoproteins, including glycoprotein of rabies virus and gp of human immunodeficiency virus (hiv), which can specifically prevent the binding of the virus to nachrs on cells and inhibit rabies virus [ ] and hiv [ ] infection. cobra venom can also upregulate the gene transcription of type i ifns. viruses may have developed immune escape mechanisms based on selective inhibition of some ifn-α subtypes [ ] . therefore, the increase in type i ifn expression could be important for enhancing the immune system to prevent viral infection. cobra venom showed great efficacy in treating a patient with multidrug-resistant hiv. after subcutaneous injection of . ml cobra venom preparation daily (trade name bioven) for month, the viral load of the patient decreased from , , copies/ml to copies/ml and the cd count increased from /mm to /mm [ ] . the peptide derived from oxidative detoxification of α-neurotoxin could reduce herpes virus replication in infected brain tissues and increase survival time by % in mice [ ] . in addition, cobrotoxin has a certain similarity to ace according to sequence alignment results. ace is the receptor through which the spike protein of sar-cov- can bind to human cells [ ] . the sequence similarity may suggest a possibility that nnav or cobrotoxin could compete with sars-cov- for binding to ace . nnav and α-neurotoxins have potent therapeutic effects in inflammation-induced lung diseases. pulmonary fibrosis is a progressive and lethal lung disease characterized by the accumulation of extracellular matrix and the loss of pulmonary function. in the authors' early research on cobra venom, it was found that nnav can improve lung gas exchange functionality and attenuate fibrotic lesions in the lung. in pulmonary fibrosis models induced by liposaccharide or bleomycin, nnav showed the potential to decrease the levels of the inflammatory markers il- β and tumor necrosis factor (tnf)-α in the serum. it was also found that nnav inhibited the inflammatory process through inhibition of nf-κb in the lps-induced model and the transforming growth factor-β/smad pathway in the bleomycin-induced model [ ] . in addition, cobrotoxin was shown to attenuate lpsinduced pulmonary edema, decrease the number of hematological cd + t cells, inhibit immune cell accumulation in bronchoalveolar lavage fluid, and inhibit pro-inflammatory cytokine excretion in rat acute lung injury and acute respiratory distress syndrome [ ] . furthermore, it was also demonstrated that cobrotoxin can relieve chronic pulmonary fibrosis in rats (wang sz, unpublished). patients with covid- have persistently high levels of il- , tnfα, il- β, il- , il r, and cytotoxic peptides such as perforin and granulysin [ ] . the resulting inflammatory cytokine storm exacerbates lung damage in addition to other fatal complications. therefore, in addition to inhibition of viral replication, antiinflammatory therapy is an important approach for combating covid- morbidity. lymphocytopenia is one of the most prominent markers of covid- [ ] . lymphocytes are directly invaded by the virus or indirectly damaged by the subsequent cytokine storm. corticosteroids are strong anti-inflammatory medications, but they also hamper the elimination of the virus by the immune system and increase the risk of secondary infections, mortality, and pulmonary fibrosis in survivors [ ] . some biological agents only target certain proinflammatory cytokines, leaving others unchecked, and thus have a limited impact on the cytokine storm. in addition, some anti-inflammatory medications inhibit the production of ifn-α, which may impede viral clearance. therefore, drugs that have broad-spectrum anti-inflammatory and antiviral activities are valuable. at this time, it is believed that cobrotoxin has the potential to treat patients with covid- or to inhibit sars-cov- infection. this conclusion is based on the following: . anti-inflammatory activity: nnav and α-neurotoxins have strong inhibitory effects on inflammation; thus, they could inhibit the cytokine storm caused by sars-cov infection. in addition, the symptoms of patients with covid- infection include deep vein thrombosis in hospitalized patients, which leads to serious adverse outcomes such as heart attack and stroke [ ] . inflammation and other factors contribute to a hypercoagulable state in covid- . nnav and cobrotoxin can inhibit inflammation and possibly inhibit thrombosis caused by inflammation in both arteries and veins. . immunoprotective activity: the numbers of white blood cells and lymphocytes are significantly reduced in the peripheral blood samples of patients infected with sars-cov- . the cellular immune responses triggered by covid- also develops through the overexpression of cd and hyperactivation of cytotoxic t lymphocytes [ ] . nnav and cobrotoxin inhibit the proliferation of cd t cells more than that of cd t cells and thus restore the cd /cd ratio. nnav also increases the concentration of serum igg and igm in mice with dexamethasone-induced immunosuppression, suggesting that nnav or cobrotoxin could have the potential to restore the immune balance in patients with covid- . . lung protection properties: three stages of covid- have been identified as viral infection, pulmonary inflammation, and fibrosis [ ] . as pulmonary fibrosis causes irreversible fatal respiratory failure, antifibrotic therapies that prevent pulmonary fibrosis should be considered [ ] . nnav and cobrotoxin can inhibit lung inflammation, improve lung gas exchange function, and attenuate the development of fibrotic lesions in the lung. . analgesic effect: some patients with covid- experience muscle pain and headache. nnav and cobrotoxin are effective analgesics [ ] that provide relief to patients with muscle pain and headache. . antiviral activity: nnav and α-neurotoxins have broadspectrum antiviral activities, although no direct evidence is available that nnav or cobrotoxin could inhibit covid- . . safety: cobra venoms have a long history of clinical use as an analgesic and were first approved by the us food and drug administration in the s. cobratide, an αneurotoxin from cobra venom, was approved as an analgesic in china and has been used safely since . the ld of nnav administered intragastrically in mice is mg/kg and the ld of subcutaneously injected cobrotoxin in mice is ~ µg/kg. furthermore, subcutaneous injection of ~ µg/kg cobrotoxin has good anti-inflammatory effects in mice. chronic toxicity data show that intragastric administration of ~ µg/kg nnav for months is safe in mice. in pilot trials for clinical pain treatment, it was suggested that ~ µg cobrotoxin by subcutaneous injection and µg nnav by mouth produced satisfactory effects. nnav and cobrotoxin are safe and may be effective in preventing and alleviating the symptoms caused by covid- . clinical features of patients infected with novel coronavirus in wuhan the structural loop ii of cobrotoxin is the main binding region for nachr and epitope in the region is conformation-dependent cobrotoxin extracted from naja atra venom relieves arthritis symptoms through anti-inflammation and immunosuppression effects in rat arthritis model involvement of cholinergic system in suppression of formalin-induced inflammatory pain by cobratoxin cobrotoxin inhibits nfkappa b activation and target gene expression through reaction with nf-kappa b signal molecules naja naja atra venom ameliorates pulmonary fibrosis by inhibiting inflammatory response and oxidative stress differential effects of naja naja atra venom on immune activity rabies virus infection of imr- human neuroblastoma cells and effect of neurochemical and other agents peptide derived from cobra venom inhibits hiv infection modulation of type i interferon system by african swine fever virus snake venom preparation for drug-resistant human immunodeficiency virus inhibition of herpes simplex virus replication by cobra alpha-neurotoxoid structure of the sars-cov- spike receptor-binding domain bound to the ace receptor pathological findings of covid- associated with acute respiratory distress syndrome clinical and immunological features of severe and moderate coronavirus disease clinical evidence does not support corticosteroid treatment for -ncov lung injury deep vein thrombosis in hospitalized patients with coronavirus disease (covid- ) in wuhan, china: prevalence, risk factors, and outcome increased expression of cd marker on t-cells in covid- patients a systematic review of pathological findings in covid- : a pathophysiological timeline and possible mechanisms of disease progression pulmonary fibrosis and covid- : the potential role for antifibrotic therapy a long-form alphaneurotoxin from cobra venom produces potent opioid-independent analgesia conflict of interest: all authors declare no conflict interest. key: cord- -i yhaagz authors: zhang, zhi-hao; he, jun-qiu; zhao, ying-yong; chen, hua-chao; tan, ning-hua title: asiatic acid prevents renal fibrosis in uuo rats via promoting the production of d-pgj , an endogenous ligand of ppar-γ date: - - journal: acta pharmacol sin doi: . /s - - - sha: doc_id: cord_uid: i yhaagz renal fibrosis is an inevitable outcome of all kinds of progressive chronic kidney disease (ckd). recently, asiatic acid (aa), a triterpenoid compound from chinese medicine centella asiatica, has been found to attenuate renal fibrosis. in the current study, we explored the mechanisms underlying antifibrotic effect of aa on uuo model. sd rats and icr mice were subjected to unilateral ureteral occlusion (uuo) surgery. prior the surgery, rats were administered aa ( mg·kg(− ) per day, ig) for days, whereas the mice received aa ( mg·kg(− ) per day, ig) for days. uuo group displayed significant degree of renal dysfunction, interstitial fibrosis, oxidative stress, and activation of the tgf-β/smad and wnt/β-catenin signaling pathway in the kidney, these pathological changes were greatly ameliorated by pretreatment with aa. in addition, we found that co-treatment with gw , a selective ppar-γ antagonist ( mg·kg(− ) per day, ip) for days, abolished the protective effects of aa. we further revealed that aa pretreatment did not significantly change the expression levels of ppar-γ in the kidney, but markedly increase the plasma levels of d-pgj , an endogenous ligand of ppar-γ. in uuo mice, pretreatment with d-pgj ( μg·kg(− ) per day, ip, for days) produced similar protective effect as aa. moreover, aa pretreatment upregulated the expression levels of active, nuclear-localized srebp- (nsrebp- ), whereas fatostatin, a specific inhibitor of srebp- , decreased the expression of nsrebp- , as well as the level of d-pgj . these results provide new insight into the antifibrotic mechanism of aa and endogenous metabolites might become a new clue for investigation of drug mechanism. chronic kidney disease (ckd) affects nearly % of adults in the united states, and the incidence and prevalence of this disease are increasing worldwide [ ] . ckd progresses to end-stage renal disease as renal function declines gradually when a critical number of functional nephrons are lost. treatment options for ckd are limited and only offer partial protection against ckd progression [ , ] . the development of more effective drugs to halt ckd progression is therefore a critical challenge for public health. natural small molecules remain promising drug sources. asiatic acid (aa), a triterpenoid compound, is isolated from centella asiatica, which is a traditional chinese medicine. previous studies have shown that aa has a variety of pharmacological effects, including anti-inflammation [ ] , antioxidation [ ] , and antitumor effects [ , ] . few studies have evaluated the mechanism of aa for the treatment of renal fibrosis. although previous studies have demonstrated that aa produces a significant effect on the inhibition of renal fibrosis by regulating tgf-β/smad signaling [ , ] , the details remain elusive. it is unclear whether aa regulates key proteins in the tgf-β/smad signaling pathway by direct binding or through other mediators. it has been reported that thiazolidinediones, peroxisome proliferator-activated receptor-γ (ppar-γ) agonists, attenuate renal interstitial fibrosis, and inflammation through a reduction in tgf-β [ ] . we hypothesize that aa attenuates renal injury and fibrosis by activating ppar-γ. to test this hypothesis, we investigated whether the selective and irreversible ppar-γ antagonist gw counteracts the protective effects against renal injury and fibrosis afforded by aa treatment in animals. our results showed that aa upregulated the expression of nuclear-localized sterol regulatory element-binding proteins- (nsrebp- ), enhanced d-pgj , activated ppar-γ, and consequentially attenuated renal damage in unilateral ureteral occlusion (uuo) models. chemicals and reagents lc-grade methanol and chloroform were purchased from merck (darmstadt, germany). internal standards (myristic acid), n, o-bis (trimethylsilyl) trifluoroacetamide (bstfa), methoxyamine hydrochloride, and pyridine were supplied by sigma-aldrich (st. louis, mo, usa). -deoxy-Δ , -prostaglandin j ( d-pgj ) was supplied by cayman chemical (ann arbor, mi, usa). aa was obtained from feiyu biotech ltd co (nantong, china), and gw was purchased from abmol bioscience (houston, tx, usa). fatostatin was supplied by targetmol (shanghai, china). the chemicals used in this study were of analytical grade, and their purity was above . %. all animal experiments were approved by the ethics committee for animal experiments of china pharmaceutical university. after acclimatization for week, rats and mice were used in the experiments. male sprague-dawley rats (weight - g) and male icr mice (weight - g) were given free access to water and regular chow. an animal model of uuo was established as described previously [ ] . in brief, under chloral hydrate anesthesia, the left ureter was double-ligated with a - silk suture following a midline abdominal incision. sham-operated animals underwent the same surgical intervention except for ureteral ligation. to investigate the effect of aa, the rats were randomly separated into the following four groups: the uuo group (n = ), the sham-operated group (control group, n = ), the uuo + aa group (n = ) (rats that were orally administered aa ( mg·kg − per day, once a day continuously for days) h prior to uuo surgery), and the uuo + aa + gw group (n = ) (rats that were administered gw ( mg·kg − per day, ip, once a day continuously for days) a half hour prior to the oral administration of aa). all rats were operated in the morning and sacrificed at : a.m. days later. for the verification test, male icr mice were used and randomly separated into the following four groups: the uuo group (n = ), the control group (n = ), the uuo + aa group (n = ) (uuo mice that were given aa ( mg·kg − per day, once a day continuously for days)), and the control + aa group (n = ) (sham-operated mice that were given aa ( mg·kg − per day, once a day continuously for days)). all mice were operated on in the morning and sacrificed at : a.m. days later. to verify the role of the alterative metabolite d-pgj in renal injury, we also used a mouse model, and mice were randomly separated into the uuo group (n = ), the control group (n = ), the uuo + d-pgj group (n = ) (mice that were administered d-pgj ( μg·kg − per day, ip, once a day continuously for days)), and the uuo + d-pgj + gw group (n = ) (mice were administered gw ( mg·kg − per day, ip, once a day continuously for days) a half hour prior to the administration of d-pgj ). all mice were operated on in the morning and sacrificed at : a.m. days later. to investigate the relationship between aa and the generation of d-pgj , a mouse model was used, and male icr mice were randomly separated into the uuo group (n = ), the control group (n = ), the uuo + aa group (n = ) (mice that were given aa ( mg·kg − per day, once a day continuously for days)), and the uuo + aa + fatostatin group (n = ) (mice that were given fatostatin ( mg·kg − per day, ip, once a day continuously for days) a half hour prior to the administration of aa). all mice were operated on in the morning and sacrificed at : a.m. days later. under chloral hydrate anesthesia, plasma was collected for biochemical detection or gc/ms analysis, and the left kidney was harvested and stored at − °c until analysis. blood parameters, histology, and western blotting blood analyses were performed on an olympus au automatic analyzer. the histological protocol was described in detail previously [ ] . the western blot protocol was described previously [ ] . the following primary antibodies were used: anti-collagen i (ab , abcam, cambridge, uk), anti-fibronectin gc-ms sample preparation internal standard solutions ( µl of myristic acid in methanol, mg/ml) were added to μl of plasma. the mixed solution was extracted with µl of methanol and chloroform ( : , v/v) and vortexed for s. the mixture was stored at room temperature for min and centrifuged at × g for min at °c. the resulting μl of supernatant was transferred to a sample vial for vacuum drying at room temperature. the residue was redissolved in μl of a methoxyamine solution ( mg/ml in pyridine) and vortexed for min. an oximation reaction was performed at °c for . h. then, μl of bstfa (containing % tmcs) was added to the solution, and the solution was vortexed for s. the sample was kept at °c for h and vortexed for s. the supernatant was transferred to a sample vial for gc-ms analysis. the samples were analyzed using an agilent chromatograph coupled with a b ms system (agilent technologies, santa clara, ca, usa). separation was achieved on a db- ms capillary column coated with % dimethyl % diphenyl polysiloxane ( m × . mm i.d., . -µm film). the initial gc oven temperature was set at °c for min, followed by a °c/ min oven temperature ramp to °c, which was maintained for min. the temperature of the inlet, transfer line, and ion source was set to , , and °c, respectively. the injection volume was μl with a split ratio of : . helium was used as the carrier gas with a constant flow rate of . ml/min. measurements were made with electron impact ionization ( ev) in full scan mode (m/z − ). the raw gc-ms data were converted and prepared for multivariate analysis according to the previously published methods [ ] . the peak area was normalized to the internal standard. pca analysis was used to visualize general clustering, trends and outliers among the observations. opls-da was used to validate the pca model and identify the differential metabolites. fold changes of the arithmetic mean values (each group/the control group) were calculated. student's t test was used to analyze the statistical significance of the results. the p values obtained from student's t test were further adjusted by a false discovery rate method based on the hochberg-benjamini method. differential metabolites were identified by a library search (nist and fiehn) and confirmed by available references. representative photographs of the left kidneys of the rats are shown in fig. a . as shown in fig. b , the uuo rats exhibited significant increases in serum creatinine (scr), blood urea nitrogen asiatic acid ameliorates renal fibrosis via ppar-γ zh zhang et al. ). *p < . , **p < . , ***p < . (compared with control group); # p < . , ## p < . (compared with uuo group) (bun), and triglyceride (tg) levels, suggesting a significant degree of renal dysfunction. treatment with aa group significantly attenuated the increase in bun and scr levels. however, the attenuation of bun and scr levels observed after aa treatment was reversed by the administration of gw , an antagonist of ppar-γ (fig. b) . histological analysis suggested that kidney tissues from the uuo rats exhibited severe tubular dilatation, tubular atrophy, and widened interstitial space with severe inflammatory cell infiltration (fig. c) . the oral administration of aa attenuated the interstitial injury, whereas the attenuation of interstitial damage was reversed by gw (fig. d, e) . uuo resulted in a significant increase in collagen i (col i), fibronectin (fn), and alpha-smooth muscle actin (α-sma) levels, as well as collagen iii levels, suggesting interstitial fibrosis (fig. a, b) . the treatment of rats with aa produced a significant attenuation of the uuo-mediated increase in col i, fn, and α-sma levels, indicating improvement in interstitial fibrosis (fig. b) . the administration of gw in aa-treated rats produced an increase in the expression of the abovementioned proteins similar to that in uuo rats and thus abolished the protective effect mediated by aa (fig. a, b) . wnt/β-catenin signaling is closely associated with renal fibrosis [ ] as well as the classic tgf-β/smad signaling axis. we next examined the effects of gw on the wnt/β-catenin and tgf-β/ smad signaling pathways in uuo rats treated with aa. uuo caused a significant increase in dvl , dvl , and β-catenin expression, which indicated the activation of wnt/β-catenin signaling. treatment with aa mediated a significant attenuation of the uuo-induced increase in dvl , dvl , and β-catenin expression, while the administration of gw abolished the protective effect of aa (fig. c) . meanwhile, we found that uuo caused a significant increase in tgf-β , smad , and smad expression and a decrease in smad expression, which indicated the activation of tgf-β/smad signaling (fig. d ). after treatment with aa, the upregulation of tgf-β , smad , and smad expression was significantly reduced; the downregulation of smad expression was significantly increased (fig. d) . the administration of gw in aa-treated rats produced a marked increase in tgf-β , smad , and smad expression as well as a marked decrease in smad expression, suggesting that it abolished the improvement in the expression of these proteins mediated by aa (fig. d) . in addition to fibrosis-related proteins and pathways, aa also affected oxidative stress in uuo rats. as shown in fig. f , the expression levels of reactive oxygen species (ros)-generating molecules including nox , rac , and -lox were significantly increased in uuo rats. treatment with aa produced a significant attenuation of the uuo-mediated increase in nox , rac , and -lox expression, and the administration of gw abolished the antioxidant effect mediated by aa (fig. f) . since an antagonist of ppar-γ, gw , can reverse the renal protective effects of aa, we hypothesized that aa may ameliorate renal fibrosis via activating ppar-γ. therefore, we investigated the expression of ppar-γ in kidneys from different group of rats. uuo rats induced a significant increase in the expression of ppar-γ compared with that in sham-operated rats (fig. e) . when compared with that in uuo rats, the expression of ppar-γ did not significantly change in rat kidneys after treatment with aa or gw (fig. e) , which indicates that aa activates ppar-γ through other processes instead of directly increasing its protein expression. untargeted gc-ms-based metabolomics identified important differential plasma metabolites typical total ion chromatograms of the representative samples obtained are presented in supplementary fig. s . a pca score plot of the plasma metabolites from the control and uuo groups is shown in fig. a . there was a clear separation observed in the plot of the plasma samples from the control and uuo groups, and a supervised method, opls-da, was also used to show the metabolic distinction between the two groups (fig. b) . the opls-da model parameters r y and q (cum) were . and . , suggesting good fitness and predictive ability of the opls-da model. pca and opls-da score plots of plasma metabolites in the control, uuo and uuo + aa groups are shown in fig. c , d. the uuo + aa group was more similar to the control groups than the uuo group was. twenty-two plasma metabolites were identified ( table ). out of the metabolites, were differentially expressed in uuo rats. then, a heatmap was created to visualize the relative levels of the differential metabolites in each group (supplementary fig. s ) . notably, of these identified metabolites, there were two ppar-γ ligands, namely, d-pgj and linoleic acid. both of them were significantly increased in aa-treated rats compared with uuo rats (fig. e) . targeted gc-ms-based metabolomics validated the production of d-pgj to validate whether aa can enhance the production of d-pgj , the plasma level of d-pgj in mice was measured by gc-ms. mice treated with aa showed a higher level of d-pgj than that exhibited by controls. in addition, uuo mice treated with aa showed an enhanced level of d-pgj compared with that shown by uuo mice (fig. f) . these results demonstrate that aa indeed increases the plasma level of d-pgj . d-pgj mediated similar therapeutic effects as those of aa in uuo mice to prove our hypothesis that aa ameliorates renal fibrosis via enhanced formation of d-pgj , we administered d-pgj to uuo mice to test whether d-pgj shows similar therapeutic effect as those of aa. uuo mice exhibited significant increases in bun and scr levels, suggesting a significant reduction in renal function. treatment with d-pgj significantly attenuated the increase in bun and scr levels. however, the attenuation of bun and scr levels observed after d-pgj treatment was reversed by the administration of gw (fig. a, b) . kidney tissues from uuo mice showed severe interstitial fibrosis and inflammatory cell infiltration. the administration of d-pgj significantly attenuated the interstitial damage, whereas the attenuation was reversed by gw ( figs. c and a ). in addition, uuo mediated a significant increase in the expression of type i collagen (col i), fn, and α-sma, which are marker proteins in fibrosis (fig. b) . the treatment of mice with d-pgj produced a significant attenuation of the uuo-induced increase in col i, fn, and α-sma expression, indicating an improvement in interstitial fibrosis, while the administration of gw abolished the protective effect mediated by d-pgj (fig. b) . similar to aa, d-pgj inhibited the activation of wnt/βcatenin and tgf-β/smad signaling pathways, and the effect was blocked by gw (fig. c, d) . taken together, these data suggest that d-pgj shows similar therapeutic effects as those of aa in the uuo animal model and that the renoprotective effects of d-pgj can be abolished by gw . aa regulated the formation of endogenous d-pgj through srebp- it was demonstrated that aa activates ppar-γ by promoting the production of endogenous d-pgj , the natural ligand of ppar-γ. to investigate how aa regulates d-pgj , we measured the expression of srebp- , which is of importance in the synthesis of fatty acids since d-pgj contains a long-chain fatty acid structure. we found that the expression of active nsrebp- was significantly increased in uuo rats treated with aa compared with model rats, while the expression of full-length srebp- was slightly decreased (fig. a) , and total srebp- mrna levels quantitative measurements of protein expression in kidneys from each group as indicated. *p < . , **p < . , or ***p < . remained unchanged between the two groups (fig. b) , suggesting that aa mediated srebp- activity at the posttranscriptional level. to validate whether srebp- regulates the formation of d-pgj , we gave uuo + aa mice fatostatin to test whether the level of d-pgj was decreased. the treatment of mice with aa significantly increased the expression of nsrebp- compared with that in uuo mice, while the administration of fatostatin, which is an inhibitor of srebp- , in aa-treated mice produced a decrease in the expression of nsrebp- (figs. c and s ) . the plasma level of d-pgj in the mice was measured by gc-ms. uuo mice treated with aa showed a higher level of d-pgj compared with that in the uuo group, while fatostatin significantly decreased the level of d-pgj compared with that in uuo mice treated with aa (fig. d) , which was consistent with the expression of nsrebp- . moreover, the expression of fibrotic proteins was decreased in aa-treated mice and upregulated after the administration of fatostatin (supplementary fig. s ). these results demonstrated that aa might regulate the level of endogenous d-pgj in mice through srebp- and play an antifibrotic role. in this study, we demonstrated that the upregulation of nsrebp- mediated by aa enhances the level of endogenous d-pgj , which activates ppar-γ and protects against renal damage and renal fibrosis (fig. e) . our major novel findings include the following: ( ) aa attenuates renal injury, oxidative stress, and fibrosis induced by the activation of ppar-γ through increasing its fig. a pca scores plot from control and uuo groups. b opls-da scores plot from control and uuo groups. c pca scores plot from control, uuo, and uuo + aa groups. d opls-da scores plot from control, uuo, and uuo + aa groups. e alteration of the level of d-pgj and linoleic acid in plasma samples of rats using gc-ms. asterisk denotes uuo + aa vs uuo, **p < . . f the plasma level of d-pgj in mice treated with aa. *p < . , **p < . natural ligand d-pgj in uuo rats; ( ) d-pgj attenuates interstitial fibrosis in uuo mice; and ( ) aa regulates the production of endogenous d-pgj , which may be through srebp- . this study demonstrates for the first time that the administration of the selective ppar-γ antagonist gw abolishes the renoprotective effects observed upon treatment with aa in rats. this result indicates that the renoprotective effects of aa are closely associated with ppar-γ. ppar-γ, which is expressed in many organs, including the kidney [ ] , is a member of the ligand-activated transcription factor superfamily. ppar-γ is involved in the expression of many genes and regulates glucose metabolism, insulin sensitivity, lipid metabolism, fibrosis, inflammation, and oxidative stress [ , ] . it has been reported that the ppar-γ agonist pioglitazone reduces proteinuria and preserves renal structure and function in passive heymann nephritis rats [ ] . in addition, a ppar-γ agonist can inhibit the generation of ros and the oxidative stress response [ , ] , and oxidative stress plays an important role in the pathogenesis of ckd and its complications [ , ] . in our study, aa attenuated oxidative stress and fibrosis via the activation of ppar-γ in uuo rats. ppar-γ expression was upregulated in uuo rats compared to control rats, which is consistent with previous studies of a renal ischemia reperfusion injury model [ , ] . compared with that in uuo rats, the expression of ppar-γ did not change upon treatment with aa, implying that the renoprotective effects afforded by aa may be due to the overproduction of endogenous ppar-γ agonists instead of the upregulation of the protein expression of ppar-γ. to verify whether endogenous ppar-γ ligands are overproduced, we applied untargeted gc-ms-based metabolomics to investigate plasma samples from the control, uuo, and uuo + aa groups. twenty-two plasma metabolites were identified and are shown in table ; we found two ligands of ppar-γ, namely, d-pgj , and linoleic acid. they were significantly increased in aatreated rats compared with uuo rats (fig. e) . endogenous d-pgj produced by the nonenzymatic dehydration of pgd has an affinity for ppar-γ of approximately μm [ ] . this metabolite is the most potent natural ligand of ppar-γ. ppar-γ is also activated by a variety of long-chain polyunsaturated fatty acids. linoleic acid has been reported to bind to and activate ppar-γ with relatively scale bar, µm; magnification, × ). *p < . , **p < . , ***p < . (compared with control group); # p < . , ## p < . (compared with uuo group) low affinity (k d = - μm) [ ] . then, we further validated that aa indeed enhances the plasma level of d-pgj in mice using targeted gc-ms-based metabolomics (fig. f) . moreover, the administration of d-pgj attenuated ( ) renal dysfunction (reduced the increase in scr and bun levels, fig. a, b) ; ( ) interstitial damage (h&e staining, fig. c); ( ) the expression of fibrotic proteins (fig. a, b) ; and ( ) the disorder of tgf-β/smad and wnt/β-catenin signaling pathways (fig. c, d) . overall, d-pgj showed similar therapeutic effects as those of aa in uuo animal model, and their renoprotective effects were abolished by gw . these data indicate that aa ameliorates renal fibrosis, at least in part via enhanced formation of endogenous d-pgj in a uuo animal model. srebps play a central role in cell metabolism by controlling the synthesis of fatty acids, tgs, and cholesterol [ ] . srebp- is one of the most important srebps, which are synthesized as inactive precursors bound to the er [ , ] . when sterols are depleted in the er membrane, srebp- is transported to the golgi, proteolytically released by two proteases and then shuttled to the nucleus to induce the expression of genes involved in fatty d western blots showing protein expression in the tgf-β/smad signaling pathway in kidneys from all groups. quantitative measurements of protein expression in kidneys from each group as indicated. *p < . , **p < . , or ***p < . acid synthesis. a previous study reported that the expression of an activated form of srebp- causes cells to produce ligand(s) of ppar-γ [ ] . however, the ligand that is produced is still unknown. our results show that uuo animals treated with aa exhibit significant increases in the levels of activated srebp- and d-pgj compared to those in the uuo group (figs. e and a) , while the administration of fatostatin in aa-treated mice produces a decrease in nsrebp- and d-pgj levels (fig. c, d) . the data presented here indicate clearly that nsrebp- triggers the generation of endogenous d-pgj . in summary, we demonstrated that aa increases the expression of activated srebp- , upregulates nsrebp- , enhances the level of endogenous d-pgj , which activates ppar-γ, attenuates oxidative stress, rebalances the disorder of the tgf-β/smad and wnt/β-catenin signaling pathways, and ameliorates renal fibrosis (fig. e) . our study, for the first time, reveals, in part, the mechanism by which aa can treat renal fibrosis through regulating endogenous metabolites. this study also provides a paradigm for mechanistic studies of molecules with similar characteristics as those of aa. fig. the protein (a) and mrna expression (b) of srebp- in kidneys from control, uuo, and uuo + aa rats. c western blots showing nsrebp- expressions in kidneys from control, uuo, uuo + aa, and uuo + aa + fatostatin mice. quantitative measurements of protein expression in kidneys from each group as indicated. *p < . , **p < . . d alteration of the plasma level of d-pgj in mice using gc-ms. *p < . , **p < . . e suggested antifibrotic mechanism of asiatic acid team. the authors would like to thank zhe wang for the discussion of the manuscript. zhz, hcc and nht designed the experiments. zhz and jqh collected and analyzed the data. yyz and nht contributed analytical tools. zhz wrote the manuscript. nht revised the paper. prevalence of chronic kidney disease in the united states effects of losartan on renal and cardiovascular outcomes in patients with type diabetes and nephropathy unmet need in renal protection-do we need a more comprehensive approach? inhibition of lps-induced no and pge production by asiatic acid via nf-kappa b inactivation in raw . macrophages: possible involvement of the ikk and mapk pathways antioxidant and cytotoxic activities of centella asiatica (l) urb asiatic acid, a triterpene, induces apoptosis through intracellular ca + release and enhanced expression of p in hepg human hepatoma cells asiatic acid induces apoptosis in sk-mel- human melanoma cells asiatic acid ameliorates tubulointerstitial fibrosis in mice with ureteral obstruction treatment of renal fibrosis by rebalancing tgf-beta/smad signaling with the combination of asiatic acid and naringenin ppar-gamma agonist attenuates renal interstitial fibrosis and inflammation through reduction of tgf-beta renal fibrosis is attenuated by targeted disruption of kca . potassium channels an integrated lipidomics and metabolomics reveal nephroprotective effect and biochemical mechanism of rheum officinale in chronic renal failure metabolomics analysis reveals the association between lipid abnormalities and oxidative stress. inflammation, fibrosis, and nrf dysfunction in aristolochic acid-induced nephropathy biomarkers of obstructive nephropathy using a metabolomics approach in rat matrix metalloproteinase- as a surrogate marker predicts renal wnt/β-catenin activity in ckd peroxisome proliferator-activated receptors (ppars): novel therapeutic targets in renal disease thermodynamics in gliomas: interactions between the canonical wnt/beta-catenin pathway and ppar gamma the role of ppars in lung fibrosis transcriptional regulation of nephrin gene by peroxisome proliferator-activated receptorgamma agonist: molecular mechanism of the antiproteinuric effect of pioglitazone rosiglitazone, a ppar-gamma ligand, protects against burn-induced oxidative injury of remote organs the metabolic effects of troglitazone in patients with diabetes and end-stage renal disease bardoxolone methyl and kidney function in ckd with type diabetes targeting the transcription factor nrf to ameliorate oxidative stress and inflammation in chronic kidney disease study of peroxisome proliferator-activated receptor (ppar)-gamma in renal ischemia-reperfusion injury the selective ppar-gamma antagonist gw reverses the protection of lps in a model of renal ischemia-reperfusion a prostaglandin j metabolite binds peroxisome proliferator-activated receptor gamma and promotes adipocyte differentiation fatty acids, eicosanoids, and hypolipidemic agents identified as ligands of peroxisome proliferator-activated receptors by coactivator-dependent receptor ligand assay protein sensors for membrane sterols srebps: activators of the complete program of cholesterol and fatty acid synthesis in the liver add /srebp activates ppargamma through the production of endogenous ligand this work was financially supported by the national natural science foundation of china (grant ) and the program for jiangsu province innovative research the online version of this article (https://doi.org/ . /s - - - ) contains supplementary material, which is available to authorized users.competing interests: the authors declare no competing interests. key: cord- - goljir authors: yuan, meng; song, zi-han; ying, mei-dan; zhu, hong; he, qiao-jun; yang, bo; cao, ji title: n-myristoylation: from cell biology to translational medicine date: - - journal: acta pharmacol sin doi: . /s - - - sha: doc_id: cord_uid: goljir various lipids and lipid metabolites are bound to and modify the proteins in eukaryotic cells, which are known as ‘protein lipidation’. there are four major types of the protein lipidation, i.e. myristoylation, palmitoylation, prenylation, and glycosylphosphatidylinositol anchor. n-myristoylation refers to the attachment of -carbon fatty acid myristates to the n-terminal glycine of proteins by n-myristoyltransferases (nmt) and affects their physiology such as plasma targeting, subcellular tracking and localization, thereby influencing the function of proteins. with more novel pathogenic n-myristoylated proteins are identified, the n-myristoylation will attract great attentions in various human diseases including infectious diseases, parasitic diseases, and cancers. in this review, we summarize the current understanding of n-myristoylation in physiological processes and discuss the hitherto implication of crosstalk between n-myristoylation and other protein modification. furthermore, we mention several well-studied nmt inhibitors mainly in infectious diseases and cancers and generalize the relation of nmt and cancer progression by browsing the clinic database. this review also aims to highlight the further investigation into the dynamic crosstalk of n-myristoylation in physiological processes as well as the potential application of protein n-myristoylation in translational medicine. lipids are one of the principal components that structure the cell membrane and provide the barrier required for cells to survive. in addition, various lipids and lipid metabolites are also generated for modifying proteins in eukaryotic cells in a process known as 'protein lipidation'. generally, there are four major types of protein lipidation: myristoylation, palmitoylation, prenylation, and glycosylphosphatidylinositol (gpi) anchoring. these lipid modifications have been defined by different functional properties that are classified according to the characteristics of lipid attachment, the covalent bond, the specific sequence on the protein and the enzymes involved [ , ] . these lipidation distinctions impact the charge, hydrophobicity, and other aspects of targeted-protein chemistry, resulting in marked differences in the physiology of the targeted protein, such as its conformation, trafficking, localization, and binding affinity for cofactors. therefore, due to the deregulated lipid metabolism that occurs, protein lipidation may contribute to various diseases. this review primarily summarizes the current knowledge of nmyristoylation with updated study results and discusses the strategy of using n-myristoylation in the treatment of diseases. n-myristoylation consists of the addition of the -carbon fatty acid, myristate, to the n-terminal glycine residue of a protein via a covalent amide bond. in rare cases, including those of ras gtpases and tnf [ , ] , myristic acid is attached to a lysine residue [ ] through an amide bound, a process named lysine myristoylation. previously, it was observed that the myristate attaches to the nascent peptide in the first min of translation during protein biosynthesis. therefore, n-myristoylation is considered a cotranslational modification with the most accurate step occurring after the removal of the methionine initiator by methionine aminopeptidase (metap) (fig. a, b) . furthermore, it is recognized that nmyristoylation can also occur posttranslationally on an internal glycine exposed by caspase cleavage during apoptosis (fig. c) . for many protein signaling systems, the n-myristoyl moiety represents an essential feature and contributes to numerous effects, including changing protein stability, influencing protein-protein interactions, enhancing subcellular targeting to organelles or the plasma membrane, and so on [ ] [ ] [ ] [ ] [ ] . two nmt isozymes during the multiple enzymatic steps of n-myristoylation, the process of myristate transfer is completed by nmt, which is classified as a member of the gcn -related n-acetyltransferase (gnat) superfamily. some well-studied structural and biochemical investigations of yeast and human nmts revealed an ordered bi bi kinase mechanism (fig. d ) that involves a structural transformation of nmt during nmt catalysis. in fact, nmt is ubiquitous in eukaryotes while no protozoans possessed it. typically, lower eukaryotes (e.g., s. cerevisiae and drosophila) have only one isoform of nmt. however, for some mammalian species, including humans and mice, two isozymes have been identified, referred to as nmt and nmt . these isozymes are encoded by distinct genes but share approximately % peptide sequence identity with unique substrate specificities in the n-terminus, suggesting a distinct physiologic role of each isozyme in mammals. additionally, each isozyme has a conserved sequence in the catalytic domain of divergent species, implicating an essential role for each gene family throughout evolution. in humans, there are four isoforms of nmt and two isoforms of nmt , which are translated from splice variants of mrna with different reading frames. the main differences among the nmt isoforms are found in the n-termini. although the n-terminal structure of these nmts does not contribute to the construct of the kinase pocket, some investigations have characterized an nterminal truncation that increases kinase activity without affecting enzyme stability [ ] . in addition, the isoforms of these nmts may have specific effects on their intracellular localization and substrate selectivity [ , ] , suggesting that nmts are involved in diverse physiological processes. physiological roles of nmts unequivocally, nmts play essential roles in the survival and cell proliferation of diverse species [ , ] . some evidence suggests a principal role of nmt in the embryonic development of mice. in normal mice, the expression level of nmt is similar to that of nmt in a wide variety of tissues, but it is higher than the nmt levels in embryos. knocking out nmt severely impaired the differentiation ability of embryonic stem cells. homozygous nmt −/− -knockout mice were not born alive, and induction of nmt activity alone was unable to elicit the survival of heterozygous nmt +/− mice. in addition, it was reported that the subcellular localization and catalytic activity of both nmt and nmt were altered during apoptosis. nmt was transported to the cytosol from ribosomes and membranes following caspase- -and caspase- -mediated nmt cleavage, and % of the nmt activity was eliminated h after the induction of apoptosis. however, the relocalization of the cytosolic fraction to the membrane and reduced activity of nmt were also found under the same conditions. in addition, the depletion of nmt caused a . -fold increase in the apoptosis rate compared to the apoptosis rate upon depletion of nmt [ ] . this evidence led to the speculation that nmt may be responsible for ribosome-based cotranslational n-myristoylation, while nmt may be the major contributor to apoptosis-related posttranslational n-myristoylation. in summary, the widespread and conservative presence of nmt in different species emphasizes its importance in basic physiological processes, such as embryonic development, while nmt appears only in higher-level organisms, suggesting that it is necessary for sophisticated physiological processes. in the future, in-depth biochemical and pharmacological research is expected to improve the understanding of the unique roles of nmt and nmt as they apply to the clinic. protein demyristoylation some reports have revealed the demyristoylation ability of some proteins. human sirtuin (sirt ), which is a member of the lysine deacetylase sirtuin protein family, was found to exhibit more efficient demyristoylase activity than deacetylase activity [ ] . the crystal structure implied that, in complex with a thiomyristoyl peptide, sirt has a dominant hydrophobic pocket that can adopt a myristoyl group. the hydrophobic acyl pocket of sirt resembles that of sirt , which has been previously demonstrated to possess efficient fatty acid deacylase activity [ ] . in addition, the two pockets are different in certain aspects, suggesting differential adoption of fatty acid chains. moreover, the other homologs, sirt and sirt , each have a hydrophobic acyl pocket very similar to that of sirt , hinting at analogous demyristoylation activities among them. the shigella virulence factor ipaj was identified as an irreversible demyristoylase [ ] that cleaves the peptide bond between nmyristoylated gly- and asn- in some n-myristoylated proteins such as human adp-ribosylation factor p (arf ) and c-src. this irreversible demyristoylation mechanism provides a new approach to exploring the functional effects of n-myristoylated proteins in human health and diseases. in most cases, n-myristoylation on a protein is irreversible, indicating that the myristoyl motif may orient the protein toward a specific destiny, as if it is pressing a button that will irrevocably affect the dynamics of the protein and its subsequent pathway. therefore, it is reasonable to study the interactions among the factors of n-myristoylation and those of biological signaling pathways to understand the significant role of n-myristoylation. although n-myristoylation is irreversible, it cannot shield the myristoylated protein from cross talk. in contrast, cross talk is regarded as a means of interfering with n-myristoylation functions. it has been proposed that one protein modification might initiate the signaling that leads to the addition or removal of a second protein modification or the binding of another protein, suggesting that cross talk between protein modification components may serve as an important bypass of regulating protein functions. for example, both methylation and phosphorylation are able to trigger acetylation of histones [ ] . here, while introducing the physiological functions of nmyristoylation, we also delineate the cross talk of nmyristoylation components with signaling constituents in light of well-established studies to explore the robust role of nmyristoylation in cell biology. dynamic structural changes in membrane anchoring and intracellular trafficking one of the major functions of n-myristoylation is to facilitate protein binding in membranes. in fact, peitzsch and mclaughlin established a tenet stating that the myristoyl motif is insufficient for the stable anchorage of a protein to a lipid bilayer [ ] . therefore, a second signal, comprising a group of hydrophobic residues, positively charged amino acids or another lipid moiety, is required for stable membrane attachment. in one scenario called the 'ligand-dependent switch' (fig. a) , the conformation of a protein is changed upon ligand binding, exposing the myristoyl motif that attaches to a component in the lipid bilayer. for example, the gtp-myristoyl switch facilitates the membrane interaction of arf [ , ] . the exposed myristoyl motif and the basic hydrophobic residues in the n-terminus facilitate the interaction of arf -gtp with the membrane. the second scenario refers to a cluster of positively charged amino acids that are associated with a cofactor, such as calcium (fig. b) , or are phosphorylated (fig. c) ; the former cluster accumulates a positive charge to strengthen membrane binding, while the latter attenuates the positive charge to weaken membrane binding and cause membrane dissociation. the binding of two calcium ions to ef-hand motifs in the recoverin protein facilitates the exposure of a myristoyl group from a hydrophobic cavity to solvent (fig. b ) [ ] . another example is the myristoylated alanine-rich c kinase substrate (marcks) protein. the phosphorylation of serine residues contributes to its membrane dissociation, since the phosphate moiety reduces the positive charge ( fig. c ) [ ] . ece c. gaffarogullari et al. [ ] proposed a novel myristoyl/phosphorylation switch in a pka-c model of membrane attachment. pka-c maintains conformational equilibrium between a myr-in state and a myr-out state, which refer to the myristoyl group tucked into the hydrophobic pocket of the pka-c or extruded from the hydrophobic pocket, respectively. in the myr-out sate, the exposed myristoyl group inserts into the lipid bilayer and facilitates pka-c binding to the membrane. therefore, it was proposed that a large population of pka-c maintains the myr-in state distal to the membrane and that pka-c shifts to the myr-out state in the proximity of the plasma membrane or when phosphorylated at ser independent of the regulatory subunit. the palmitoyl moiety, which consists of a -carbon fatty chain, also induces signal transduction (fig. d) . it was observed that h-ras requires both a myristoyl group and palmitoyl group to target the membrane and stimulate kinase activity, whereas only n-myristoylated h-ras acts a substrate for palmitoyltransferases and therefore is palmitoylated [ , ] . the n-myristoylationnegative mutants of cdpk [ ] and fibroblast growth factor receptor substrate α (frs α) [ ] do not incorporate palmitate and therefore do not attach to the plasma membrane. moreover, proteins with myristoyl moieties show a tendency for inclusion in the membrane fraction. for example, it was reported that both n-myristoylation and palmitoylation appear to have opposing roles and different membrane lipid microdomain preferences for the g protein-membrane interactions i (gαi ) monomer, which are likely due to the conformational differences in the presence of different fatty acids [ ] . the gαi protein that is n-myristoylated but not palmitoylated preferentially anchors to lamellar-prone membrane domains with a net negative charge. in contrast, the gαi protein that is both n-myristoylated and palmitoylated preferentially localizes to raft-like lamellar membranes without negative charges. n-myristoylated akt (myrakt ) exhibits a distinct substrate preference that is not exhibited by the cytosolic or the membrane fractions that do not include lipid rafts, indicating that akt for which oncogenicity is conferred by nmyristoylation is enriched in lipid rafts [ ] . in addition, the evidence that simvastatin or cholesterol depletion preferentially ablates the phosphorylation of myrakt in lipid rafts suggests that myrakt is modified in a cholesterol-sensitive manner (fig. e ). it is well known that the most organelles in cells, such as the endoplasmic reticulum (er), mitochondrion and endosome, are bound to the plasma membrane and that these membrane interactions produce different effects. in addition, some proteins must undergo n-myristoylation for subcellular trafficking and localization. some mitochondria-related proteins, such as tomm , samm [ ] and, clpabp [ ] , were demonstrated to require the function of myristoylated proteins to bind to the mitochondrial outer membrane. in one mechanism, the hydrophobic myristoyl group motif increases dependence of the protein to reduce cytoplasmic shuttling. for example, in mice, binding domain-containing protein (stbd ) was observed to be a transmembrane resident protein, and nonmyristoylated stbd was shuttled with ease between the er and mitochondria [ ] . ring finger protein (rnf ) colocalizes with both early and recycling endosomes. while rnf requires n-myristoylation and spalmitoylation for membrane binding, n-myristoylation plays a greater role [ ] . the removal of myristoylate results in protein diffusion. in another mechanism, the hydrophobicity of the myristoyl moiety increases protein flexibility, facilitating its shuttling through hydrophobic areas. in a yeast model [ ] , the proteasome subunit rpt is cotranslationally n-myristoylated. loss of rpt n-myristoylation causes abnormal dynamic nucleuscytoplasm translocation of proteasomes and the aberrant accumulation of ubiquitinated protein levels (fig. f) . requirements for protein assembly and protein-protein interaction many proteins require assembly for maturity and function, and some evidence indicates that n-myristoylation drives the aggregation of proteins in some cases [ ] . spassov et al. reported that src dimerization is mediated by the myristoylated n-terminal region of one partner interacting with the hydrophobic kinase domain of its counterpart [ , ] . both y autophosphorylation and dimerization are codependent and activate src kinases (fig. a) . the functions of src kinases are disrupted by interfering dimerization, emphasizing the importance of n-myristoylation to src activity. in addition, the myristoyl-group-driven aggregation in lipid bilayers is also observed for h-ras [ ] . some protein-protein interactions require a myristoyl group. the brain-specific protein cap- /nap binds calmodulin (cam) with high affinity even though it lacks any canonical cam-binding domain. a crystal structure of ca + -cam-cap- /nap -cam complex showed that the myristoyl group of cap- /nap passes through a hydrophobic tunnel created by two hydrophobic components exclusively in the pockets of the terminus of cam, implying the direct involvement of the myristoyl group in this interaction. further, the interaction of calmodulin induces the phosphorylation of cap- /nap- [ ] . in hiv infections, both nef and gag are myristoylated by the host nmt cell to execute proper functions. in the assembly of hiv, nef and gag proteins are essential for infection. nef has many virulence factors that attenuate the immune system recognition of infected cells and enhance infectivity and viral replication [ ] , and gag is a precursor protein for structural components of the viral capsid. because this virus lacks nmt proteins, both nef and gag are myristoylated by the host nmt cell, and the gag-gag interaction triggers an entropic switch toward a myristoyl-exposed conformation, providing the impetus for protein assembly (fig. b) . in contrast, in beta-retroviruses, such as mouse mammary tumor virus (mmtv), the oligomerization of the matrix (ma) domain, which contains the n-terminal residue in gag, adopts a myristoylsequestering conformation [ , ] . moreover, the results from screens of the interaction of the host factors with the hiv- ma domain showed that heme oxygenase (ho- ) specifically binds the myristoyl moiety of gag via a hydrophobic channel adjacent to its heme-binding pocket, which inhibits virus production. in addition, ho- binds n-myristoylated tram, an adaptor protein for toll-like receptor (tlr ) [ ] , which inhibits the tramdependent lipopolysaccharide(lps)-tlr pathway. these findings suggest ho- is a novel cellular n-myristoylated protein that negatively regulates both virus replication and host inflammatory responses [ ] . recent studies have revealed that a glycine positioned in the nterminus can act as a potent degron, indicating that nmyristoylation may contribute to the removal of proteolytic cleavage products. richard t. timms et al. [ ] identified two cul cullin-ring e ubiquitin ligase complexes called cul zyg b and cul zer , both of which target n-myristoylated proteins for proteasomal degradation by recognizing n-terminal glycine degrons, which presumably play important roles in the quality control of protein n-myristoylation. nonmyristoylated c-src has enhanced stability compared to that of soluble n-myristoylated c-src [ ] . borja belda-palazon et al. [ ] found that rglg , an e ligase in arabidopsis, is n-myristoylated by nmt . n-myristoylation facilitates the attachment of rglg to the plasma membrane. the phytohormone abscisic acid (aba) induces the degradation of pp ca, which is predominantly localized in the nucleus, through rglg / e ligases. this degradation mechanism was found by aba downregulation of nmt , which hindered the nmyristoylation of rglg and promoted its translocation to the nucleus, where it interacted with pp ca, increasing pp ca degradation. in addition, in aspergillus nidulans [ ] , the swof gene was found to encode an nmt. the enhanced activity of the s proteasome and the accumulation of ubiquitinated substrates in the n-myristoylate-deficient swof mutant, compared with the activity and accumulation in wild-type strain, resulted in impaired cell morphogenesis. alterations to signaling pathways n-myristoylation influences downstream kinase activity directly or indirectly, usually by the mechanisms described above, through ras and src. c-abl is a member of the src family of tyrosine kinases. a 'myristoyl/phosphotyrosine' switch has been identified in the regulation of the kinase activity of c-abl [ ] . nmyristoylation locks the protein into an autoinhibitory conformation when the sh domain docks to the kinase domain. in contrast, myristoylation leads to an unpredicted function: c-src is induced into a conformation optimal for kinase activity. a series of studies have unequivocally demonstrated that n-myristoylation of the β-subunit is a prerequisite for the initiation of ampk signaling in response to amp [ , , ] . therefore, in the case of nmt deficiency or upon cell treatment with an nmt inhibitor, suppressed n-myristoylation diminished the extent of α-thr phosphorylation of amp and abolished its activation, thereby causing multiple morbid physiological outcomes. moreover, the myristoyl switch regulated by amp may affect the selectivity of the substrates and serve as a gatekeeper for transducing signals of metabolic stress [ ] . recently, it was suggested that pka-c n-myristoylation may account for the enriched kinase activity at the membrane and the preferential phosphorylation of membrane substrates. these findings indicate an important role for n-myristoylation in synaptic function normality and plasticity during normal pka regulation [ , ] . in another pattern of results from recent studies, several proteins were found to phosphorylate nmt, resulting in the activation of nmt. rajala et al. [ ] verified that lyn and fyn are nmyristoylated by nmt before they phosphorylate nmt. in addition, it has been hypothesized that the interaction between nmt and the lyn tyrosine kinase proceeds in a phosphorylationdependent manner [ ] . manipulation of substrates during apoptosis apoptosis, or programmed cell death, is the physiologically and tightly controlled process for eliminating unnecessary or redundant cells in eukaryotic organisms. in addition, dysfunctional apoptosis is one of the characteristics of malignant cells. apoptosis is orchestrated by a signaling cascade of proteases called caspases (cysteine-containing aspases), which leads to the exposure of novel n-termini susceptible to various posttranslational modifications. since the initial discovery of myristoylated bid, it has been revealed that multiple proteins are modified posttranslationally by n-myristoylation upon caspase cleavage. using a myristoylated protein-labeling method in jurkat t cells and mcf- cells, at least and posttranslationally nmyristoylated proteins were discovered, respectively, during apoptosis [ ] . further characterization of these proteins revealed that caspase truncated (ct)-gelsolin and ct-pkc have antiapoptotic roles while ct-bid and ct-pak have proapoptotic roles upon posttranslational n-myristoylation, suggesting that posttranslational n-myristoylation plays an important role in maintaining a balance between cell death and survival. moreover, it has been found that nmt and nmt are substrates of different caspases. furthermore, n-myristoylation may be involved in sphingolipid biosynthesis pathway, which includes ceramide synthesis and is involved in the induction of apoptosis. in the last step of ceramide biosynthesis, a trans △ -double bond in the carbon chain of dihydroceramide is specifically catalyzed by dihydroceramide △ desaturase (des ), which is n-myristoylated by nmt [ ] . myristic acid upregulates novel des activity, which differs from that of saturated fatty acids, possibly because of the accumulation of n-myristoylated des . n-myristoylation causes a proportion of des to be translocated from the er to the mitochondrial outer membrane, leading to an increase in ceramide levels. since the production of ceramide can induce apoptosis by inducing mitochondrial dysfunction [ ] , the myristic acid-associated increase in des activity can enhance apoptosis induction in cells, which shows the importance of n-myristoylation in the process of apoptosis. n-myristoylated lancl , which belongs to the eukaryotic lanc-like protein family, is known as the testisspecific adriamycin sensitivity protein (tasp). one biochemical study identified a phosphatidylinositol phosphate (pip)-binding site in the n-terminus of lancl . in addition, both nmyristoylation and pip binding are crucial for lanc binding to the membrane. it is possible that the overexpression of lancl increases cell sensitivity to adriamycin, which is dependent on both n-myristoylation and membrane association [ ] . in summary, we can conclude that a myristoyl group as a hydrophobic motif always stably binds to substrates to change the conformation and increase the hydrophobicity of the protein. further, it affects protein localization and the ease with which a protein binds to substrates. however, insufficient hydrophobicity of the -carbon myristoyl group leads to it being influenced by its surroundings. the hydrophilic group can ease protein binding, and the hydrophobic group can stabilize the state of the n-myristoylated protein. it seems that n-myristoylation triggers consequential functions of protein necessarily but insufficiently. accumulating evidence has demonstrated that n-myristoylation is an evolutionarily conserved lipidation that is essential for cell viability in different organisms. currently, interventions of protein n-myristoylation are principally achieved through nmt inhibitors. therefore, nmt inhibitors are very suitable for use against diseases caused by proliferating cells or pathogens, such as infectious diseases caused by various pathogens and malignancies. in fact, broadly classified inhibitors for each process in nmt-induced disease are designated as antifungal and antiviral agents. typically, these inhibitors can be divided into three categories [ ] : ( ) myristoyl-coa and myristate derivatives. although the myristoyl-coa binding sites in human nmt and fungal nmt are highly conserved, their peptide-binding sites are divergent, which provides an explanation for the selectivity of inhibitors that do not induce adverse toxicity in humans. ( ) histidine analogs. in most conserved regions in the catalytic domain eeveh ( - ), histidine is critical for the myristoyl-coa transfer [ ] . ( ) myristoyl peptide derivatives. as previously mentioned, lipid metabolism disorders can affect protein lipidation [ ] . various saturated and unsaturated fatty acids have been evaluated as potent inhibitors of human nmt . indeed, the development of nmt inhibitors is not limited to proteins involved in infectious diseases because they are great prospects as immunodeficiency and cancer treatments. as mentioned previously, nmt has been characterized as the principal enzyme for the early development of embryogenesis. further investigation was focused on the role of nmt in myelopoiesis through which blood cell types are matured. bone marrow-derived macrophages (bmdms) from nmt +/− -deficient mice have defective morphology compared with that of mature bmdms in wild-type mice. during bmdm maturation, the nmt activity increased during the initial period and then decreased for the remaining time due to differential nmt expression. although the nmt activity in the bmdms of nmt +/− -deficient mice followed a trend similar to that of the bmdms in the wild-type mice, the nmt expression levels were reduced to approximately one-half in the mutant mice. this report addressed an essential role of nmt during monocytic/macrophage differentiation. of course, studies are continuously uncovering valuable evidence not only for nmt but also for nmt in both normal and malignant hematopoiesis. a bioinformatic database of gene expression in different cancer cell lines revealed decreased nmt expression and preserved nmt expression in some hematological cancer cell lines, such as burkitt lymphoma, diffuse large b-cell lymphoma and acute myeloid leukemia (aml) cell lines. a study performed by ryan stubbins et al. suggested a correlation between nmt and aml. nmt and nmt protein levels in the marrow aspirates or peripheral blood of aml patients were assayed by fluorescence-activated cell counting with intracytoplasmic staining, expressed as the mean fluorescent intensity (mfi). the evidence showed that the nmt mfi was higher in the lymphocytes and lower in the monocytes, suggesting that the regulation of nmt protein levels may influence early lymphoid/myeloid lineage commitment. further, the overall trend revealed by a survival analysis showed higher nmt mfi values, portending a worse prognosis for aml patients and suggesting a role for nmt as a novel prognostic biomarker for intermediate-risk aml [ ] . in terms of adaptive immunity, it has been reported that nmyristoylation is indispensable for t cell development [ , ] . a comparative analysis of thymuses showed that mice with deficient nmt or nmt levels had reduced medullary volume, which was % and % lower, respectively, than it was in wild-type mice, and mice with double nmt and nmt deficiency had a much greater medullary volume reduction ( %). these findings suggest nonredundant roles for both nmts in maintaining the development of thymocytes, since the thymus has high nmt activity levels [ ] . in agreement with these results, it was demonstrated that n-myristoylation and its potential applications m yuan et al. t-cell receptor (tcr) signaling is disrupted in mice with t-cell lineages characterized by specific nmt and nmt activity deficiencies and results in retarded thymocyte development. these outcomes may be attributable to the mislocalization of nmyristoylation-deficient src family tyrosine kinases, such as lck or fyn [ ] . for example, n-myristoylation contributes to the cytomembrane targeting of fyn and facilitates its binding with the z chain of tcr. non-n-myristoylated lck is in the cytoplasm and unable to facilitate tcr signaling. however, the regulation of n-myristoylation or nmt activity in these processes during adaptive immunity has not been well established to date. to gain in-depth understanding of the regulation of immune responses, further investigation into the specific roles of nmyristoylation, which may involve nmt as a potential target of immune modulation, is warranted. parasitic and other infectious diseases it is known that some n-myristoylated proteins in small rna viruses and retroviruses are essential for virus assembly during viral replication or production of infectious viral particles, suggesting that n-myristoylation may be related to the survival and propagation of pathogens. in addition, some pathogens need to utilize host cellular machinery to replicate within host cells due to deficiency in viral nmts. in immunosuppressed patients, cryptococcus neoformans can easily cause chronic meningitis; however, it is unable to survive at °c if it harbors mutant nmt with reduced activity. some disease-causing parasitic protozoa such as falciparum (malaria), leishmania donovani (leishmaniasis), and trypanosoma brucei (african sleeping sickness) retain the nmt necessary for their survival. given that the peptide-binding pocket of nmt is not strictly conserved across species, the search for species-specific nmt inhibitors focused on the binding pocket is worthwhile [ ] . notwithstanding, current anti-infectious agents have some drawbacks, such as drug resistance, poor oral bioavailability, and renal toxicity. a group of studies have suggested nmt as a novel target for use in anti-infective drugs. theoretically, according to the respective skeletal structures, nmt inhibitors are classified into four categories (table ) . among these inhibitors, benzofuran and, benzothiazole have high species selectivity [ ] . anti-infective drugs targeting nmt have certain advantages. first, myristoyl-coa is found at very low accumulation levels, at approximately nm, in mammalian cells. in addition, the strict hydrophobic structure of the fatty carbon chains reduces the likelihood that incompatible fatty acid groups will be recognized by the nmt, even in cases where the palmitic acid concentration is higher than the myristic acid in vitro [ ] . these physiological phenomena suggest that a small amount of inhibitor can have a good inhibitory effect. second, nmt inhibitors can be designed for high selectivity because of the significant differences in substrate specificity in the human and parasitic organisms. moreover, many fungal and parasites must use the nmt of the host to synthesize essential proteins for their own reproduction. potential targets of cancer treatments given that altered nmt expression is observed in many types of cancer tissues and because many n-myristoylated proteins are involved in signaling processes that regulate cell proliferation, growth and death, it has been proposed that n-myristoylation or nmts can be considered as therapeutic targets for cancer. the premise for their use is based on a thorough understanding of the abnormal regulation of n-myristoylation in carcinogenesis. for instance, given that n-myristoylation can facilitate src-mediated prostate tumorigenesis, the myristoyl-coa analog b and its derivative lcl [ , ] have been identified as inhibitors of nmt enzymatic activity and blockers of src n-myristoylation; their actions are based on competing for myristoyl-coa binding site, which provides a promising approach to inhibit src family kinase-mediated oncogenic activity, and offers preclinical support for the use of protein n-myristoylation inhibitors in treating cancer [ ] . an organopalladium compound, tris(dibenzylideneacetone) dipalladium (tris dba) was identified as a novel human nmt inhibitor that not only blocks the kinase activity of nmt but also reduces its expression. it showed potent antiproliferative activity against melanoma cells by inhibiting several proliferation-related signaling pathway proteins including mapk, akt, and stat- [ ] . the most successful examples of myristoylation-related anticancer inhibitors are allosteric abl tkis such as abl [ ] [ ] [ ] , gnf and gnf [ ] (table ) . these allosteric inhibitors selectively target the myristoyl-binding pocket in the c-lobe of the abl kinase domain and not the atp-binding pocket. moreover, these inhibitors increase the sensitivity of a bcr-abl (t i) mutant to atp-competitive tkis. a series of phase i clinical trials of abl and tkis (nilotinib, imatinib, and dasatinib) therapy have been ongoing for cml and ph+all patients (https://clinicaltrials.gov/ ct /show/nct ). some studies have shown the potential targets of nmt in cancer cells. the cancer genome atlas (tcga) reports a group of genomic alterations induced by nmt and nmt in several cancers. although the occurrence of somatic mutations is more common than the occurrence of genomic amplification in nmt and nmt , the roles of these mutations in regulating pathological mechanisms remain to be determined (https://www.cbioportal. org/). moreover, patients with diseases such as liver cancer, cervical cancer, and lung cancer and high expression levels of nmt or nmt are more likely to have a poor prognosis, as indicated consistently in some reports. in addition, it is noteworthy that a cohort with renal cell carcinoma with either high expression of nmt or low expression of nmt was found to have worse overall survival, suggesting the unique roles of these proteins in kidney cancer. however, no evidence showed that their catalytic activity is linked to tumor progression in kidney cancer (http:// kmplot.com/analysis/). in addition, several nmt activators that enhance nmt enzyme activity have been identified. l-histidine and d-histidine can activate human nmt activity in a concentration-dependent manner. this finding suggests that two analogs may be involved in myristoyl-coa transfer by interacting with his- of nmt. naf was identified as an nmt activator factor in bovine brain [ ] . the reconstitution of naf and nmt likely resulted in an open conformation where the active site is expanded. a novel stratagem of nmt activation is enhanced nmt specificity for specific myristoyl-coa substrates. on the basis that nmt can transfer abundant palmitoyl-coa but can use only the rare myristoyl-coa for acylation of a substrate protein, eric soupene et al. [ ] determined that the acyl-coa-binding protein domain (acbd ) stimulates the activity of nmt . acbd can block the access of palmitoyl-coa to the nmt -binding site and enhance its catalytic activity, which requires interaction between nmt and acbd . a mutant acbd unable to interact with nmt or with deficient ligand binding at its own n-terminus does not stimulate nmt activity. in this review article, we outlined advanced studies of nmyristoylation, focusing on the role of protein n-myristoylation in physiology and pathology. the important role of nmyristoylation underlies the early stages of protein maturation. after the protein is folded in the golgi apparatus or the er, cotranslationally n-myristoylation is likely to influence the transport and localization of the protein, which can affect the biofunction of protein. the insufficient nmt kinase activity in pathogens and the variations in different species emphasize the selective lethality of nmt inhibitors for infectious diseases for which either no valid drug is qualified or for which available drugs induce drug resistance. the breakthroughs of nmt inhibitors will likely be as tumor treatments. abl is being pioneered continuously for cml and ph+all patients in phase i trials, which suggests a strategy focused on the n-myristoylation of oncoproteins. furthermore, posttranslational n-myristoylation in the apoptotic process suggests the participation of nmts, specifically nmt , in cell death. the function of the n-myristoylated protein in the apoptotic process, whether pathogenically or physiologically normal, can further indicate the orientation of the treatment strategy for targeting nmt. the knowledge of other protein modifications, such as ubiquitination or palmitoylation, which involves a multimember kinase family, can inform many specific enzyme-substrate studies and the design of selective inhibitors. in contrast, scientists need more precise approaches for analyses of nmts and nmyristoylation. nevertheless, traditional methods based on radioactively labeled probes to detect n-myristoylation are insensitive and time consuming, and the technical difficulties in obtaining three-dimensional structures of myristate-attached proteins need to be overcome. further, bioinformatics on n-myristoylation and nmts is still in its infancy; however, since databases are integrated, these data provide many clues linked to other biological fields. regardless of the technical difficulties, it is worth exploiting novel nmt inhibitors as single agents and exploring the potential drug synergies that might improve multiple clinical applications and enhance therapeutic efficacy, reverse drug resistance, or extend the therapeutic index for drugs already used in the clinic. it is likely that two approaches [ ] can be used to reveal the prospects of nmt inhibitors for oncology therapy in the future: ( ) identify currently unknown sensitivities of certain cancer types by widely screening cancer cell line panels and ( ) discover the essential sensitivity or resistance mechanisms in resistant cell lines and wild-type cell lines by proteomic analyses of nmt substrate profiles and proteomic changes. as our knowledge of the biochemistry and cell biology of n-myristoylation continues to grow, more substrate proteins will be identified, and scientists will continue to deduce the effects of this lipid attachment on protein structure and function. protein lipidation in cell signaling and diseases: function, regulation, and therapeutic opportunities protein lipidation sirt and lysine fatty acylation regulate the transforming activity of k-ras a hdac regulates type i interferon signaling through defatty-acylation of shmt protein lysine acylation and cysteine succination by intermediates of energy metabolism protein myristoylation in health and disease cholesterol sensitivity of endogenous and myristoylated akt myristoylated naked antagonizes wnt-beta-catenin activity by degrading dishevelled- at the plasma membrane myristoylation of the dual-specificity phosphatase c-jun n-terminal kinase (jnk) stimulatory phosphatase is necessary for its activation of jnk signaling and apoptosis inhibition of enterovirus vp myristoylation is a potential antiviral strategy for hand, foot and mouth disease n-terminal region of the catalytic domain of human nmyristoyltransferase acts as an inhibitory module hiv- production is specifically associated with human nmt long form in human human n-myristoyltransferase amino-terminal domain involved in targeting the enzyme to the ribosomal subcellular fraction fatty acids regulate germline sex determination through acs- -dependent myristoylation comparison of myristoyl-coa: protein n-myristoyltransferases from three pathogenic fungi: cryptococcus neoformans, histoplasma capsulatum, and candida albicans two n-myristoyltransferase isozymes play unique roles in protein myristoylation, proliferation, and apoptosis efficient demyristoylase activity of sirt revealed by kinetic and structural studies sirt regulates ras-related protein r-ras by lysine defatty-acylation nmyristoyltransferase is essential in early mouse development protein kinase c coordinates histone h phosphorylation and acetylation molecular determinants of the myristoyl-electrostatic switch of marcks structural basis for activation of arf gtpase: mechanisms of guanine nucleotide exchange and gtp-myristoyl switching structure and membrane interaction of myristoylated arf sequestration of the membranetargeting myristoyl group of recoverin in the calcium-free state myristoylation-dependent n-terminal cleavage of the myristoylated alanine-rich c kinase substrate (marcks) by cellular extracts a myristoyl/phosphoserine switch controls camp-dependent protein kinase association to membranes c-terminal kda fragment of cytoskeletal actin is posttranslationally n-myristoylated upon caspase-mediated cleavage and targeted to mitochondria n-terminally myristoylated ras proteins require palmitoylation or a polybasic domain for plasmamembrane localization membrane localization of a rice calcium-dependent protein kinase (cdpk) is mediated by myristoylation and palmitoylation myristoylationdependent palmitoylation of the receptor tyrosine kinase adaptor frs alpha g proteinmembrane interactions i: galphai myristoyl and palmitoyl modifications in protein-lipid interactions and its implications in membrane microdomain localization identification and characterization of protein n-myristoylation occurring on four human mitochondrial proteins, samm , tomm , mic , and mic role of n-myristoylation in stability and subcellular localization of the clpabp protein mouse stbd is n-myristoylated and affects er-mitochondria association and mitochondrial morphology multiple modification and protein interaction signals drive the ring finger protein (rnf ) e ligase to the endosomal compartment n-myristoylation of the rpt subunit of the yeast s proteasome is implicated in the subcellular compartment-specific protein quality control system aggregation of lipid-anchored full-length h-ras in lipid bilayers: simulations with the martini force field a dimerization function in the intrinsically disordered n-terminal region of src a myristoyl-binding site in the sh domain modulates c-src membrane anchoring crystal structure of a myristoylated cap- /nap- n-terminal domain complexed with ca +/calmodulin a myristoyl switch regulates membrane binding of hiv- gag myristoylation drives dimerization of matrix protein from mouse mammary tumor virus the myristoylation of trif-related adaptor molecule is essential for toll-like receptor signal transduction heme oxygenase binds myristate to regulate retrovirus assembly and tlr signaling a glycine-specific ndegron pathway mediates the quality control of protein n-myristoylation myristoylation and membrane binding regulate c-src stability and kinase activity aba inhibits myristoylation and induces shuttling of the rglg e ligase to promote nuclear degradation of pp ca a novel interaction between n-myristoylation and the s proteasome during cell morphogenesis a myristoyl/phosphotyrosine switch regulates c-abl beta-subunit myristoylation is the gatekeeper for initiating metabolic stress sensing by ampactivated protein kinase (ampk) n-myristoyltransferase deficiency impairs activation of kinase ampk and promotes synovial tissue inflammation exploring protein myristoylation in toxoplasma gondii liberated pka catalytic subunits associate with the membrane via myristoylation to preferentially phosphorylate membrane substrates myristoylated alanine-rich c-kinase substrate effector domain phosphorylation regulates the growth and radiation sensitization of glioblastoma phosphorylation of human n-myristoyltransferase by n-myristoylated src family tyrosine kinase members potential role of n-myristoyltransferase in cancer rapid detection, discovery, and identification of post-translationally myristoylated proteins during apoptosis using a bio-orthogonal azidomyristate analog myristic acid increases the activity of dihydroceramide delta -desaturase through its nterminal myristoylation monounsaturated fatty acid modification of wnt protein: its role in wnt secretion myristoylation of human lanc-like protein (lancl ) is essential for the interaction with the plasma membrane and the increase in cellular sensitivity to adriamycin effects of l-histidine and its structural analogues on human n-myristoyltransferase activity and importance of eeveh amino acid sequence for enzyme activity expression and activity of n-myristoyltransferase in lung inflammation of cattle and its role in neutrophil apoptosis deficiency of n-myristoylation reveals calcineurin activity as regulator of ifn-gammaproducing gamma delta t cells myristoylation: an important protein modification in the immune response n-myristoylation of ampk controls t cell inflammatory function the residue at position of the n-terminal region of src and fyn modulates their myristoylation, palmitoylation, and membrane interactions vhy, a novel myristoylated testis-restricted dual specificity protein phosphatase related to vhx novel analogs of d-e-mapp and b . part : signature effects on bioactive sphingolipids role of long-chain fatty acyl-coa esters in the regulation of metabolism and in cell signalling novel analogs of de-mapp and b . part : synthesis and evaluation as potential anticancer agents blocking myristoylation of src inhibits its kinase activity and suppresses prostate cancer progression tris (dibenzylideneacetone) dipalladium, a n-myristoyltransferase- inhibitor, is effective against melanoma growth in vitro and in vivo discovery of asciminib (abl ), an allosteric inhibitor of the tyrosine kinase activity of bcr-abl allosteric inhibitors of bcr-abl: towards novel myristate-pocket binders the allosteric inhibitor abl enables dual targeting of bcr-abl inhibitors of the abl kinase directed at either the atp-or myristate-binding site differential activation of bovine brain n-myristoyltransferase(s) by a cytosolic activator acbd protein controls acyl chain availability and specificity of the n-myristoylation modification of proteins n-myristoyltransferase inhibition induces er-stress, cell cycle arrest, and apoptosis in cancer cells this work was supported by grants from the national natural science foundation of china ( to j.c.), zhejiang provincial natural science foundation (y h to j.c.), and the talent project of zhejiang association for science and technology (no. ycgc to j.c.). my and jc conceived and designed the review article. my, zhs, and jc collected the related research articles contributed to the paper. my, mdy, hz, by, qjh, and jc made amendments to the paper. competing interests: the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as potential competing interests. key: cord- - odny f authors: lai, qiong; yuan, guang-ying; wang, hao; liu, ze-liang; kou, jun-ping; yu, bo-yang; li, fang title: exploring the protective effects of schizandrol a in acute myocardial ischemia mice by comprehensive metabolomics profiling integrated with molecular mechanism studies date: - - journal: acta pharmacol sin doi: . /s - - - sha: doc_id: cord_uid: odny f schizandrol a (sa) is an bioactive component isolated from the schisandra chinensis (turcz.) baill., which has been used as a remedy to prevent oxidative injury. however, whether the cardioprotective effect of sa is associated with regulating endogenous metabolites remains unclear, thus we performed comprehensive metabolomics profiling in acute myocardial ischemia (ami) mice following sa treatment. ami was induced in icr mice by coronary artery ligation, then sa ( mg·kg(− )·d(− ), ip) was administered. sa treatment significantly decreased the infarct size, preserved the cardiac function, and improved the biochemical indicators and cardiac pathological alterations. moreover, sa ( , m) significantly decreased the apoptotic index in ogd-treated h c cardiomycytes in vitro. by using hplc-q-tof/ms, we conducted metabonomics analysis to screen the significantly changed endogenous metabolites and construct the network in both serum and urine. the results revealed that sa regulated the pathways of glycine, serine and threonine metabolism, lysine biosynthesis, pyrimidine metabolism, arginine and proline metabolism, cysteine and methionine metabolism, valine, leucine and isoleucine biosynthesis under the pathological conditions of ami. furthermore, we selected the regulatory enzymes related to heart disease, including ecto- ’-nucleotidase (nt e), guanidinoacetate n-methyltransferase (gamt), platelet-derived endothelial cell growth factor (pd-ecgf) and methionine synthase (mtr), for validation. in addition, sa was found to facilitate pi k/akt activation and inhibit the expression of nox in ami mice and ogd-treated h c cells. in conclusion, we have elucidated sa-regulated endogenous metabolic pathways and constructed a regulatory metabolic network map. furthermore, we have validated the new potential therapeutic targets and underlying molecular mechanisms of sa against ami, which might provide a reference for its future application in cardiovascular diseases. cardiovascular disease (cvd) remains the leading cause of mortality and morbidity worldwide, and approximately % of cvd cases are associated with ischemic heart disease [ ] . acute myocardial ischemia (ami) is a severe cardiovascular disease that is due to a sudden decrease in blood flow and oxygen supply to the heart, consequently leading to cardiac dysfunction, cardiac metabolic disorder, myocardial infarction, and even death [ ] . it is widely acknowledged that the molecular mechanisms of ami are complex and involve multiple pathways. this complexity offers the opportunity for metabolomics studies to uncover new insights into the progression of ami. currently, β-blockers, statins and angiotensin-converting enzyme inhibitors are the mainstays of current treatment [ ] . however, these treatments provide limited symptomatic relief, and the attempts to treat ami thus far have been far from sufficient. schizandrol a (sa) is a bioactive component isolated from schisandra chinensis (turcz.) baill. and is also one of the major effective ingredients of the well-known tcm formula shengmai preparations, which acts as a remedy to prevent oxidative injury and treat coronary heart diseases [ , ] . previous studies have reported that sa exerts protective effects against h/r-induced vascular endothelial injury and glutamate-induced neuronal excitotoxicity [ ] [ ] [ ] and that sa also exhibits anti-inflammatory activity [ ] . in our previous study, a new drug combination comprising ginsenoside rb , ruscogenin and sa (grs; : . : ratio) was shown to be effective in mitigating myocardial ischemia/reperfusion injury by suppressing mitochondrial-mediated apoptosis [ ] . as a component of this drug combination, sa showed cardioprotective effects against acute myocardial infarction [ ] . however, the molecular mechanism of the functions of sa against ami remains unclear, and further studies are still needed to elucidate this mechanism. currently, metabolomics, which provides comprehensive profiling of small molecular substances in biological systems, is being increasingly applied to elucidate disease mechanisms and understand potential new drug targets [ ] . recently, there has been an increasing number of metabolomics investigations of cardiovascular diseases. recent studies have identified panels of specific biomarkers useful for the diagnosis of coronary artery disease based on metabolomics analysis of plasma samples from patients from independent centers [ ] . moreover, in a further study, a functional metabolomics strategy was employed to characterize metabolite signatures associated with coronary artery disease. neu ac was identified as a key metabolite, and its regulatory enzyme neuraminidase- may serve as a new avenue for therapeutic intervention for myocardial ischemia injury [ ] . li et al. conducted comprehensive profiling of the rat plasma and heart in response to ami and danqi tongmai tablets via lc-msbased metabolomics and lipidomics [ ] . however, there have been few studies on the cardioprotective effects of sa from the perspective of metabonomics. therefore, in our present study, metabonomics technology was employed to clarify the specific metabolic pathways modulated by sa to induce improvements in ami. furthermore, endogenous metabolites differentially regulated by sa were identified, and a regulatory metabolic network map was constructed. several novel potential therapeutic targets closely related to cardiovascular diseases were selected, and the effects of sa on these targets were verified. we hope this work will help us understand more about the mechanisms of sa from the perspective of endogenous metabolites and provide some references for the investigation of sa in the future. reagents and materials acetonitrile, methanol and formic acid were obtained from merck (darmstadt, germany). ultrapure water was obtained from a milli-q purification system (milford, ma, usa). schizandrol a (sa) was purchased from chengdu pufei de biotech co., ltd. (chengdu, china; purity: hplc ≥ %, lot number: ). metoprolol tartrate tablets were obtained from astrazeneca (taizhou, china; lot number: a ). creatine kinase (ck) and lactate dehydrogenase (ldh) assay kits were purchased from nanjing jiancheng bioengineering institute (nanjing, china). c-reactive protein (crp), tumor necrosis factor-α (tnf-α), and cardiac troponin i (ctn-i) assay kits were obtained from nanjing jin yibai biological technology co., ltd (nanjing jin, china). antibodies against gapdh were purchased from shanghai kangchen biotech inc. (shanghai, china). antibodies against pd-ecgf, mtr, and nt e were obtained from proteintech (wuhan, china). antibodies against β-actin and gamt were purchased from bioworld technology (st. louis park, mn, usa). antibodies against pi k, akt, p-akt, and nox were purchased from cell signaling technology (boston, ma, usa). animals and the ami injury model icr male mice ( weeks old, - g) were purchased from the experimental animal center of yangzhou university (yangzhou, china; certificate no scxk - ). all animals were housed individually in cages under hygienic conditions and placed in an environment with controlled temperature ( ± °c), humidity ( %- %) and light ( -h light/dark cycle) with commercial standard solid rodent chow and water provided ad libitum. all animal welfare and experimental procedures were in accordance with the national institutes of health guide for the care and use of laboratory animals, and the protocols used were approved by the animal ethics committee of china pharmaceutical university, china pharmaceutical university, nanjing, china. the ami model was produced as previously described [ ] , and the rats were anesthetized with sodium pentobarbital ( mg/kg) intraperitoneally (i.p.). the adequacy of anesthesia was controlled by monitoring the lack of response to toe pinch. then, the chest wall was shaved, and left thoracotomy was performed. a - silk suture was tied around the left anterior descending coronary artery approximately - mm from its origin. sham-operated mice were subjected to the same surgical procedures without ligation of the left anterior descending coronary artery. successful myocardial ischemia injury by complete ligation of the left ascending (lad) coronary artery was confirmed by the occurrence of st-segment elevation on an electrocardiogram monitor. the mice were randomly divided into four groups (n = /group): ( ) the sham group (normal saline, i.p.); ( ) the model group (normal saline, i.p.); ( ) the sa group (sa, i.p.); ( ) and the metoprolol (positive control drug) group (met, i.g.). the dosage of sa in this experiment was mg·kg − ·day − , which was based on the effective dose identified in our previous study [ ] . the clinically used dosage of met was converted for use in our experiment, and the dosage was determined to be . mg·kg − ·day − . all drugs were administered intraperitoneally after min of ischemia. ttc staining after h of ligation, the mice were euthanized and perfused with pbs by lv puncture. one-millimeter short-axis sections of the heart were incubated in % ttc solution at °c for min. the infarcted area was analyzed using imagej software (bethesda md, usa). blood sample analysis at the end of the experiment, blood samples were obtained. serum samples were collected by centrifuging the blood samples at rpm for min. then, the supernatant was obtained and immediately stored at − °c until analysis. the activities of ck and ldh and the content of crp, tnf-α, and ctn-i in the serum were determined with commercial kits according to the manufacturer's instructions. after h of ligation, cardiac function was evaluated using an echocardiographic system consisting of a vevo imaging system (visual sonics, toronto, canada) equipped with a mhz transducer. the mice were anesthetized with isoflurane in o gas. after anesthetization, each mouse was placed on a heated imaging platform. the following parameters were calculated: left ventricular (lv) ejection fraction (ef) ((lvedv-lvesv)/lvedv), lv fractional shortening (fs) ((lvdd-lvds)/lvdd), and stroke volume (sv) (lv vold-lv vols). hematoxylin-eosin staining of the heart heart samples from each group were fixed in % phosphatebuffered formalin for h. next, the samples were embedded in paraffin wax, and -µm to -µm histological sections of the paraffin-embedded tissues were stained with hematoxylin-eosin. the sections were imaged under a light microscope (dx , olympus microsystems ltd., japan) [ ] . masson's trichrome staining paraffin-embedded heart sections ( -µm to -µm thick) were stained with masson's trichrome. normal tissue appeared red, and collagen tissue appeared blue. the heart sections were imaged at high pixel resolution with an olympus dp digital camera. cell preparation and culture the rat cardiomyocyte cell line h c was purchased from shanghai institute of cell biology, chinese academy of sciences (shanghai, china). the h c cells were maintained in dmem supplemented with % fbs at °c in a humidified atmosphere of % co . the cells were subcultured or subjected to experimental procedures at %- % confluence. oxygen glucose deprivation (ogd)-treated cell model and drug treatment to mimic ischemic injury in vitro, the ogd model was employed. ogd injury was induced by incubating cells with glucose-free dmem and exposing them to a hypoxic environment of % n , % co , and % o for h. the cells were treated with sa ( - μm) during hypoxia. cell viability and ldh assays cell viability was detected using the mtt assay. after different treatments, cells were incubated with mtt at a final concentration of . mg/ml for h. then, the medium was discarded, and μl of dmso was added to dissolve the formazan crystals. the absorbance was read at nm with a reference wavelength of nm, and cell viability was calculated as the percentage of absorbance relative to the control value. in addition, the release of ldh was also determined to measure the extent of cell injury. at the end of the incubation, the culture supernatant was collected, and ldh was detected at nm. tunel staining apoptotic cells were detected using the tunel apoptosis assay kit according to the manufacturer's instructions as previously described [ ] . five fields from each slice were randomly analyzed under a confocal scanning microscope (lsm , zeiss, usa). the extent of cell apoptosis was expressed as the ratio of tunelpositive nuclei. flow cytometry analysis fitc-conjugated annexin v and pi were used to detect apoptotic cells. h c cardiomyocytes were harvested, washed with pbs, and resuspended in binding buffer, and then fitc-annexin-v and pi were added at a final concentration of ng/ml. the mixture was incubated for min in the dark and analyzed with a flow cytometer. untargeted metabonomics analysis sample pretreatment. serum and urine were collected after ligation for h. serum samples were obtained by centrifuging the blood samples at rpm for min at °c. urine samples were collected at °c. all samples were kept at − °c until analysis. for untargeted extraction, μl aliquots of serum and urine were precipitated by adding μl of methanol and mixed for s. the precipitated proteins were subsequently removed by centrifugation ( , rpm, min) at °c. then, μl of the supernatants were collected and dried under a gentle stream of nitrogen at room temperature. finally, the supernatants were reconstituted with μl of methanol for further analysis. hplc-q-tof ms analysis. metabolite separation was performed using an agilent technologies accurate-mass q-tof lc/ms system (usa). the mobile phase consisted of water with . % formic acid (eluent a) and acetonitrile with . % formic acid (eluent b). serum analyses were archived on a synergi tm fusion-rp c column ( mm × mm i.d., . μm). the gradient program began with % b at - min, %- % b at - min, %- % b at - min, %- % b at - min, %- % b at - min, %- % b at - min, and %- % b at - min and then returned to the initial conditions with min for equilibration. the flow rate was . ml/min. urine analyses were performed on a tsk-gel amide- column ( mm × . mm i.d., μm). the gradient program began with % b at - min, %- % b at - min, %- % b at - min, and %- % b at - min and then returned to the initial conditions with min for equilibration. the flow rate was . ml/min. the sample injection volumes were μl. mass spectrometry was performed with an electrospray ionization ion source in the positive (esi + ) and negative (esi − ) ion modes. the ms parameters were set as follows: a fragmental voltage of v, a nebulizer gas of psig, a capillary voltage of v, a drying gas flow rate of l/min, and a temperature of °c. the data were collected in centroid and profile modes with a mass range of - m/z using the highresolution mode ( ghz). method validation. the repeatability and robustness of the analytical procedure were validated by using the pooled quality control (qc) sample. the qc sample was prepared by mixing µl of each test sample, and the pretreatment method of the qc samples was the same as that of the test samples. the qc sample was divided into five aliquots, which were randomly injected throughout the sequence list. unsupervised pca analysis showed good repeatability and robustness of the analytical method, as indicated by the tight grouping of the qc samples in the score plot ( supplementary fig. s ). meanwhile, we overlapped the total ion chromatograms (tics) of the qc samples, as shown in supplementary fig. s , which showed that the response intensity and retention time of each chromatographic peak basically overlapped. these data all demonstrated the high reproducibility of the method and the stability of the instrument during the experiment. data processing. the raw data (.d) acquired from q-tof lc/ms esi + and esi − analysis were transformed to data format (.mzdata) files by masshunter workstation software (version b. . , agilent technologies). using r foundation for statistical computing, data pretreatment procedures such as nonlinear retention time alignment, peak discrimination, filtering, alignment, and matching were performed with the xcms package (http://metlin.scripps. edu/download/). ion features that were present in less than % of the samples were screened out. the intensities of each peak detected were generated by virtue of the retention times and the m/z data pairs for each ion. after being normalized to total peak intensity, log-transformed and pareto scaled, the processed data were imported into simca-p . (umetrics, sweden) and metaboanalyst (https://www.metaboanalyst.ca/), where they were subjected to multivariate data analyses, including pareto-scaled principal component analysis (pca) and orthogonal partial leastsquares discriminant analysis (opls-da). metabolites with a vip value > were further subjected to student's t-test at the univariate level to measure the significance of each metabolite, and p-values less than . were considered statistically significant. western blot analysis. as reported [ ] , cells were lysed in icecold ripa buffer supplemented with mm pmsf. the infarct areas of heart tissues were homogenized in ripa buffer. the protein concentrations were quantified with a bca assay kit following the manufacturer's instructions. equal amounts of proteins ( μg) were electrophoresed on sds-page gels, transferred to pvdf membranes, and blocked with % bsa solution. the blots were probed with primary antibodies overnight at °c and then incubated with the appropriate secondary antibodies at room temperature for h. the ecl plus system was used to detect the protein signal. the band densities were visualized by the chemidoc™ mp system (bio-rad), and the relative values were expressed relative to the signal of gapdh or β-actin. immunohistochemistry. twenty-four hours after ligation, the hearts were collected and processed for immunohistochemistry to analyze the expression of ecto- '-nucleotidase (nt e) and methionine synthase (mtr). the heart tissues were fixed in % paraformaldehyde and embedded in paraffin. the heart tissues were sectioned at a thickness of µm, deparaffinized, rehydrated in pbs, and incubated with % hydrogen peroxide to block endogenous peroxidase activity. then, the sections were incubated for h at °c with blocking liquid (beyotime, shanghai, china). primary antibodies against mtr and nt e ( : ) were added dropwise to the sections, and the sections were incubated at °c for h. after washing, the sections were incubated with the hrp-conjugated secondary antibody ( : , biogot technology, nanjing, china) at °c for h. after incubation with dab and counterstaining with hematoxylin, a light microscope (dx , olympus microsystems ltd., japan) was used to observe the dehydrated sections, and the sections were imaged at × magnification. immunofluorescence staining. slices of heart tissues were washed with ice-cold pbs, fixed in % paraformaldehyde, permeabilized with . % triton x- in pbs for min, blocked with % bsa, and incubated with a rabbit pd-ecgf antibody at a : dilution overnight at °c followed by an alexa fluor -conjugated donkey anti-rabbit antibody at a : dilution for h. nuclei were visualized using dapi. fluorescence was observed with a confocal laser scanning microscope. statistical analysis. all values in the text and figures are expressed as the mean ± sem. statistical analysis was carried out using student's two-tailed t-test for comparisons between two groups and one-way analysis of variance (anova) followed by dunnett's test for comparisons between three or more groups. p < . was considered significant. mice were treated with sa by intraperitoneal administration after h of myocardial ischemia induced by coronary artery ligation. the myocardial infarct size and some serum biochemical indicators were measured to evaluate the effect of sa on ami. as illustrated in fig. , h of ischemia resulted in myocardial injury, as evidenced by an increased infarct size, increased ck and ldh activities, and increased levels of ctn-i, crp, and tnf-α. however, sa and met treatment significantly inhibited the increase in infarct size (fig. a, b) . simultaneously, sa and met markedly reduced the activities of ck and ldh (fig. c, d) , as well as the levels of ctn-i, crp, and tnf-α ( fig. e-g) . furthermore, echocardiography was performed to detect the effects of sa on cardiac performance. as shown in fig. , compared to sham surgery, ami significantly impaired the lvef, lvfs, and sv. in contrast, sa and met markedly attenuated the ami-induced impairment of cardiac function (fig. a-d) . in addition, histopathological examination of ami mouse heart tissues revealed widespread myocardial structural disarray, a large number of inflammatory cells infiltrating the myocardial tissue and increased left ventricular wall fibrosis. the histological features were normalized or became milder, showing marked improvement, after sa treatment ( fig. e-h) . sa inhibited myocardial apoptosis in ami mice and ogd-induced cardiomyocyte apoptosis as shown in fig. , the exposure of h c cardiomyocytes to ogd led to a decrease in cell viability, whereas treatment with sa maintained cell viability (fig. a) . as ldh release is a recognized marker of cell injury, the release of ldh into the culture medium was also investigated. compared to that in the control group, ldh release significantly increased after ogd injury, while treatment with sa markedly inhibited the release of ldh (fig. b) . myocardial apoptosis is an essential element associated with infarct size expansion and cardiac dysfunction after ischemic injury [ ] . therefore, flow cytometry analysis and tunel staining were employed to explore whether sa prevented ami-induced myocardial apoptosis. quantitative analysis using flow cytometry confirmed that the apoptotic index was markedly increased compared with that of the control group. however, the apoptotic index was significantly decreased by treatment with sa (fig. c, d) . compared to the shamtreated mice, the ami mice exhibited obvious tunel-positive (apoptosis) cells in the ischemic myocardium following ami. in contrast, a markedly lower proportion of apoptotic cells was observed in heart slices from sa-treated mice (fig. e, f) . principal component and orthogonal partial least squares discriminant analyses of serum and urine samples principal component analysis (pca) was used to perform unsupervised data analysis on the sham, model and sa groups. most of these groups could be easily distinguished from each other. supplementary fig. s shows that the farthest distance was however, the sa-treated samples were closer to the sham operation group than the model group. these results further indicated that sa improved myocardial ischemia, as myocardial ischemia mice were increasingly similar to healthy mice after sa administration. we further used opls-da for supervised data analysis to elucidate the metabolic variations. all data acquired from the urine and serum were analyzed using simca-p software for the discrimination and selection of significant variables. the opls-da model was constructed to determine the distinction of metabolic patterns between the model group and sa group in both the positive and negative ion modes. in theory, the r y and q values should be close to , which indicates a high predictive ability [ ] . as shown in table , the parameters of model quality reflected the good fitness and predictability of opls-da. moreover, as illustrated in fig. a -d, the metabolic profiles of both urine and serum samples from the model group were distinctly different from those of urine and serum samples from the sa group. next, an s-plot was employed to identify altered metabolites that markedly contributed to the differences between the model and sa groups (fig. e-h) . the metabolites with vip values over and p-values lower than . , which are listed in tables , , were considered potential biomarkers [ ] . in addition, a hierarchical clustering heatmap was applied to view the data more intuitively. the heatmap indicated that the concentrations of metabolic biomarkers in the sa group differed from those in the model group (fig. ). to reveal the metabolic processes of the metabolites, the metabolites that were significantly changed in the serum and urine were entered into metaboanalyst . (https://www. metaboanalyst.ca/) for pathway enrichment analysis. twenty associated pathways in total were identified. according to the enrichment fold of the pathways, the major modulated pathways involved were glycine, serine and threonine metabolism, glycerolipid metabolism, pyrimidine metabolism, spermidine and spermine biosynthesis, and methionine metabolism (fig. a-c) . furthermore, pathway and enrichment analysis of related regulatory enzymes was performed by string (https://string-db. org/) and metascape (https://www.metascape.org/). a protein interaction network was constructed and is shown in fig. d . the results of go enrichment analysis showed that the functions of the regulatory enzymes mainly included cellular amino acid metabolic processes, drug metabolic processes, the metabolism of nucleotides, and nucleobase catabolic processes (fig. e) . our results suggested that regulatory enzymes affected by sa were mainly involved in drug metabolism, pyrimidine metabolism, pantothenate and coa biosynthesis pathways, as identified by kegg enrichment analysis (table ). according to these results, a schematic diagram of the altered metabolic pathways in both the serum and urine, which is shown in fig. , was constructed; sa may affect these pathways to ameliorate ami. validation of the potential therapeutic targets of sa according to the results of kegg enrichment analysis of regulatory enzymes of differential metabolic markers, the regulatory enzymes involved in the top pathways were selected. the regulatory enzymes related to heart disease, including ecto- '-nucleotidase (nt e), guanidinoacetate n-methyltransferase (gamt), plateletderived endothelial cell growth factor (pd-ecgf) and methionine synthase (mtr), were selected for further validation. as illustrated in fig. , ami resulted in a noticeable decrease in nt e, gamt, and mtr expression and a significant increase in pd-ecgf expression. however, sa markedly promoted the expression of nt e, pd-ecgf, and mtr and exerted no effect on gamt expression ( fig. a-d) . in addition, immunohistochemical analysis showed that sa significantly increased the expression of nt e and mtr (fig. e , f) and further promoted pd-ecgf expression, as evidenced by immunofluorescence staining (fig. g ). sa ameliorated ami by regulating the pi k-akt-nox signaling pathway as shown in fig. , the western blotting results showed that the expression of pi k, p-akt, and nox was upregulated in ami mice and ogd-induced cardiomyocytes. sa treatment significantly metabolomics and mechanism of schizandrol a on ami q lai et al. further increased the expression of pi k and p-akt and downregulated the expression of nox . in the present study, we confirmed the efficacy of sa in amiinjured mice and ogd-injured cardiomyocytes, as evidenced by the decrease in infarct size and the most common biomarkers for myocardial ischemia, such as ldh, ck, ctn-i, crp, and tnf-α. in addition, echocardiography is clinically applied to evaluate cardiac function and structure [ ] . lvef and lvfs measurements are used as key markers of contractile capabilities both in basic and clinical studies. the main function of the heart in the circulatory system is to pump blood to meet the needs of metabolism. thus, sv is also a basic indicator used to evaluate cardiac function [ ] . our present results demonstrated that treatment with sa preserved cardiac function and improved cardiac tissue damage and fibrosis in mice subjected to ami injury. these positive effects metabolomics and mechanism of schizandrol a on ami q lai et al. of sa were associated with the suppression of cardiomyocyte apoptosis. accumulating pharmacological studies have suggested that the loss of cardiomyocytes as a result of apoptosis is essential in various heart diseases and inevitably contributes to heart failure [ ] . intervening in the apoptotic process could attenuate the loss of cardiomyocytes, alleviate cardiac injury and ultimately inhibit the occurrence and development of ami injury. in addition, the hplc-q-tof/ms metabolomics approach was selected to explore metabolic changes in mice with ami and uncover the treatment mechanism of sa. forty-eight differentially regulated metabolites were found between the model group and sa group. sa markedly increased the levels of fructoselysine, -methylguanosine, n-acetylhistamine, creatinine and l-isoleucine, d-glutamine, and citric acid compared to those in ami mice. some studies have shown that later-stage or advanced-stage glycation processes involve the degradation of fructoselysine and the glycation of proteins by methylglyoxal to form advanced glycation end products, which contribute to the pathogenesis of vascular complications of diabetes [ ] . additionally, -methylguanosine was identified as a marker that predicts the development of nephropathy in t dm patients [ ] . meanwhile, in the general population, moderately elevated plasma creatinine and l-isoleucine levels are correlated with an increased risk of myocardial infarction, ischemic heart disease and early death [ , ] . in addition, glutamine has been found to maintain cardiac function perioperatively and promote glycogen metabolism by enhancing mitochondrial function and stimulating mtorc [ ] . citric acid is an essential metabolite involved in the tricarboxylic acid cycle (tca) cycle. the tca cycle is the ultimate metabolic pathway of the three major nutrients (carbohydrates, lipids, amino acids), and it is also an important hub of the metabolic link between carbohydrates, lipids and amino acids [ ] . the levels of xanthurenic acid, d-serine and thymine in the sa group were lower than those in the ami group. xanthurenic acid is a biomarker related to the kynurenine pathway. in patients with suspected stable angina pectoris, elevated levels of plasma kynurenines predict an increased risk of acute myocardial infarction, and risk estimates are generally stronger in subgroups with evidence of impaired glucose homeostasis [ ] . furthermore, atherosclerosis is the underlying cause of most myocardial infarction and ischemic stroke episodes. recent studies have suggested that there is a direct relationship between d-serine and intima media thickness, which is an early sign of atherosclerosis [ ] . moreover, larger cytosine-thymine-guanine expansions have been reported to be associated with a higher rate of conduction disease progression and a trend toward an increased risk of cardiac events [ ] . subsequently, the pathways of differential metabolites and regulatory enzymes were further analyzed, and we found that sa mainly regulated metabolic pathways, including the glycine, serine and threonine metabolism, lysine biosynthesis, pyrimidine metabolism, arginine and proline metabolism, cysteine and methionine metabolism, and valine, leucine and isoleucine biosynthesis pathways. the glycine, serine and threonine metabolism pathway previous studies have shown that glycine and serine can reduce myocardial inflammation by activating glycine receptors and indirectly inhibit collagen production in cardiac fibroblasts, ultimately inhibiting myocardial fibrosis [ ] . in addition, impairments in cardiac energy metabolism and mitochondrial function are intricately correlated with cardiac dysfunction. mitochondrial dysfunction contributes to increased oxidative stress, cell death and myocardial remodeling. recent developments suggest that mitochondrial protein lysine biosynthesis modulates the sensitivity of the heart to stress and hence the propensity for heart failure [ ] . furthermore, during arginine and proline metabolism, creatine, which reacts with atp during the catalysis of creatine kinase, is broken down into phosphocreatine and creatinine. under conditions of ami, the level of creatine is depressed significantly. cardiac ischemia and reperfusion seem to shift arginine metabolism from forming nitric oxide to forming polyamine, which may limit functional recovery after reperfusion [ ] . in addition, the cysteine and methionine metabolic pathways are also widely associated with the pathogenesis of myocardial infarction, atherosclerosis and other cardiovascular diseases [ ] [ ] [ ] . to further explore the potential targets of sa in inducing cardioprotection, nt e, gamt, pd-ecgf, and mtr were identified and primarily verified in the present study. nt e is the key enzyme that controls the degradation of adenine nucleotides into adenosine via the enzymatic dephosphorylation of amp in the ischemic myocardium. adenosine, a degradation substance of atp, exerts an essential role in regulating cardiac contractility, coronary vascular tone, and cardiac substrate utilization [ ] [ ] [ ] . recent studies have demonstrated that oxidative stress may cause the inactivation of nt e and decrease the concentration of adenosine in the rat heart [ ] . our results indicated that ami inhibited the expression of nt e while sa markedly reversed this suppression. moreover, creatine is mainly produced through two essential chemical reactions and a second step catalyzed by gamt. gamt knockout mice show increased susceptibility to ischemia/reperfusion injury, which is reflected by changes in high-energy phosphate metabolism [ ] . our results indicated that sa had no effect on the promotion of gamt expression. in addition, previous studies have documented an increase in the expression of the angiogenic protein pd-ecgf in a rat model of mi [ ] . in our present study, sa further promoted pd-ecgf and mtr (f) was detected using immunohistochemistry. the expression of pd-ecgf was measured by immunofluorescence staining (g). the results are presented as the mean ± sem. ## p < . vs. the control group, *p < . , **p < . vs. the model group expression, which is also a potent peptide with anti-apoptotic activity. simultaneously, sa promoted the expression of mtr, which is one of the key enzymes in folate metabolism. several studies have suggested that mtr deficiency can increase the risk of congenital heart disease [ ] . based on the above results, it can be reasonably speculated that sa might attenuate amiinduced cardiomyocyte apoptosis through the promotion of nt e, pd-ecgf, and mtr expression. furthermore, we explored the potential pathways regulated by sa. previous investigations have demonstrated that nt e regulates the production of adenosine and that adenosine modulates vascular tone via pi k/akt signaling cascade-mediated enos activation and no production [ ] . in our present study, we found that sa promoted the expression of nt e, while sa was found to facilitate pi k/akt activation and inhibit the expression of nox . these findings suggest that the cardioprotective effect of sa is at least partially mediated by the pi k/akt-nox signaling pathway. in summary, for the first time we revealed the endogenous metabolic small molecules and metabolic pathways regulated by sa in ami through nontargeted metabonomics and the construction of a regulatory metabolic network map. furthermore, several potential new targets of sa, such as nt e, pd-ecgf, and mtr, were identified and verified, and sa was shown to exert cardioprotective effects against ami, at least in part through the pi k/akt-nox signaling pathway. fig. the effect of sa on the pi k-akt-nox signaling pathway in ami mice and ogd-induced cardiomyocytes. the expression of pi k (a), akt, p-akt (b), and nox (c) in ami mice was detected using western blot analysis. the expression of pi k (d), akt, p-akt (e) and nox (f) in ogdinduced cardiomyocytes was determined using western blot analysis. # p < . , ## p < . vs. the control group, *p < . , **p < . vs. the model group global, regional, and national burden of cardiovascular diseases for causes myocardial ischemia: current concepts and future perspectives cell biology of ischemia/reperfusion injury identification of multiple constituents in the traditional chinese medicine formula sheng-mai san and rat plasma after oral administration by hplc-dad-ms/ms brain oxidative stress as basic target of antioxidant traditional oriental medicines identification of schisandrin as a vascular endothelium protective component in yiqifumai powder injection using huvecs binding and hplc-dad-q-tof-ms/ms analysis schizandrin protects primary cultures of rat cortical cells from glutamate-induced excitotoxicity schizandrin, an antioxidant lignan from schisandra chinensis, ameliorates aβ - -induced memory impairment in mice upregulation of heme oxygenase- via pi k/akt and nrf- signaling pathways mediates the antiinflammatory activity of schisandrin in porphyromonas gingivalis lps-stimulated macrophages cardioprotection by combination of three compounds from shengmai preparations in mice with myocardial ischemia/reperfusion injury through ampk activation-mediated mitochondrial fission a strategy for optimizing the combination of active components based on chinese medicinal formula sheng-mai-san for myocardial ischemia metabolomics and metabolic diseases: where do we stand? comprehensive metabolomic characterization of coronary artery diseases functional metabolomics characterizes a key role for n-acetylneuraminic acid in coronary artery diseases exploring the protective effects of danqi tongmai tablet on acute myocardial ischemia rats by comprehensive metabolomics profiling a novel and efficient model of coronary artery ligation and myocardial infarction in the mouse enhanced anti-tumor activity and reduced toxicity by combination andrographolide and bleomycin in ascitic tumor-bearing mice the nuclear melatonin receptor ror α is a novel endogenous defender against myocardial ischemia/reperfusion injury a tutorial review: metabolomics and partial least squares-discriminant analysis-a marriage of convenience or a shotgun wedding a consensus orthogonal partial least squares discriminant analysis (opls-da) strategy for multiblock omics data fusion mr as a diagnostic tool in heart disease: what is the future? low altitude simulation without hypoxia improves left ventricular function after myocardial infarction by reducing ventricular afterload inhibition of myocardial apoptosis reduces infarct size and improves regional contractile dysfunction during reperfusion hidden complexities in the measurement of fructosyllysine and advanced glycation end products for risk prediction of vascular complications of diabetes identification of urinary metabolite biomarkers of type diabetes nephropathy using an untargeted metabolomic approach creatinine, egfr and association with myocardial infarction, ischemic heart disease and early death in the general population blood metabolomic fingerprint is distinct in healthy coronary and in stenosing or microvascular ischemic heart disease glutamine regulates cardiac progenitor cell metabolism and proliferation citric acid cycle intermediates in cardioprotection associations of plasma kynurenines with risk of acute myocardial infarction in patients with stable angina pectoris peripheral biomarker for vascular disorders does cytosine-thymineguanine (ctg) expansion size predict cardiac events and electrocardiographic progression in myotonic dystrophy? a serine elastase inhibitor reduces inflammation and fibrosis and preserves cardiac function after experimentally-induced murine myocarditis the growing landscape of lysine acetylation links metabolism and cell signalling mechanism and consequences of the shift in cardiac arginine metabolism following ischaemia and reperfusion in rats plasma total cysteine and total homocysteine and risk of myocardial infarction in women: a prospective study plasma methionine and risk of acute myocardial infarction: effect modification by established risk factors cysteine proteases in atherosclerosis nt e mutations and arterial calcifications cardiac metabolism and its interactions with contraction, growth, and survival of cardiomyocytes adenosine and its receptors in the heart: regulation, retaliation and adaptation oxidative stress inactivates ecto- '-nucleotidase by inhibiting protein kinase c in rat hearts in vivo reduced inotropic reserve and increased susceptibility to cardiac ischemia/reperfusion injury in phosphocreatine-deficient guanidinoacetate-n-methyltransferaseknockout mice myocardial expression of pdecgf is associated with extracellular matrix remodeling in experimental myocardial infarction in rats genetic variants reducing mtr gene expression increase the risk of congenital heart disease in han chinese populations a b adenosine receptor contributes to penile erection via pi k/akt signaling cascade-mediated enos activation this research work was supported by the national natural science foundation of china (no. , , , and ), the natural science foundation of jiangsu province (bk ), the china postdoctoral science foundation ( m and t ), and the "double first-class" university project (cpu gf and cpu gf ). fl and byy conceived the study. ql and gyy performed the metabolomic analysis. ql, hw, and zll established the animal model and performed the in vivo experiments. gyy and hw collected the samples. ql, gyy, and hw interpreted the data. ql, jpk, and fl aided in the data analysis and wrote the paper. all authors read and approved the final paper. the online version of this article (https://doi.org/ . /s - - - ) contains supplementary material, which is available to authorized users. the authors declare no competing interests. key: cord- -clbqckj authors: yan, you-you; shi, ke-yu; teng, fei; chen, jing; che, jin-xin; dong, xiao-wu; lin, neng-ming; zhang, bo title: a novel derivative of valepotriate inhibits the pi k/akt pathway and causes noxa-dependent apoptosis in human pancreatic cancer cells date: - - journal: acta pharmacol sin doi: . /s - - - sha: doc_id: cord_uid: clbqckj natural compound valepotriate exhibits inhibitory activity against a number of cancers, but the effect of valepotriate against pancreatic cancer is unclear, and the structure–activity relationship of valepotriate has not been characterized. in this study, we performed a structure-based similarity search and found hit compounds. among the hits, ( s, s, r)- -(acetyloxy)- -[( -methylbutanoyl)oxy]- a, , , a-tetrahydro- h-spiro[cyclopenta[c]pyran- , ’-oxiran]- -ylmethyl -methylbutanoate (denoted as amcp) exhibited superior anticancer activity against human pancreatic cancer bxpc- and sw cells. the anti-proliferation activity of amcp was validated in human pancreatic cancer bxpc- and sw cells in vitro. amcp more effectively induced apoptosis in bxpc- and sw cells than gemcitabine. at a concentration of μm, amcp significantly suppressed the pi k/akt pathway and disrupted the mitochondrial membrane equilibrium through modulation of noxa and mcl- balance in both cell lines. meanwhile, knockdown of noxa substantially attenuated amcp-induced reduction of cell viability and anti-apoptotic protein mcl- level in bxpc- cells. in addition, amcp showed synergistic anticancer effects when combined with gemcitabine in bxpc- cells. to conclude, this work not only suggests that amcp possesses a dual-inhibitory activity towards pi k/akt pathway and mcl- , but also enlightens further development of bioactive valepotriate derivatives. pancreatic ductal adenocarcinoma (pdac) is an aggressive malignancy and is predicted to be the second leading cause of cancer-related mortality in the near future [ ] . systemic chemotherapy is an important part of treatment for pdac patients, especially those who are diagnosed at advanced stages. although great efforts have been made to explore efficacious compounds for treating pdac in both preclinical and clinical studies, the -year survival rate remains at~ % and has not changed for the past years [ ] . gemcitabine has been a key component of gold standard chemotherapy regimens for pancreatic cancer, but the actual clinical response rate to gemcitabine has been unsatisfactory. it was reported that the phosphoinositide -kinase (pi k)/akt signaling pathway was aberrantly activated in most pancreatic cancer patients [ ] and the concomitant inhibition of pi k/akt and mcl- was considered to be a promising strategy to inhibit the progression of pancreatic cancer [ ] [ ] [ ] . in addition, noxa is a bh- domain-containing protein that is capable of binding and sequestering pro-survival bcl- family proteins such as mcl- and bcl- , and therefore the downregulation of mcl- via noxa activation is considered to be an efficacious therapeutic strategy for a variety of cancers [ , ] . unfortunately, most clinical tests of revised chemotherapy regimens have failed, probably due to the complexity of pancreatic cancer [ ] . therefore, novel chemotherapeutic agents are still urgently needed to combat this aggressive neoplasm. valeriana is a natural medicinal herb, for which the root extracts have been extensively studied in many aspects as part of anticancer, anti-bacterial, and anti-anxiety investigations [ ] [ ] [ ] . valepotriate is the key bioactive component extracted from valeriana and is reported to exert anti-proliferation cytotoxic effects by regulating the redox balance or suppressing the mitogen-activated protein kinase pathway in cancer cells [ , ] . however, the anticancer activity of valepotriate against pancreatic cancer has been rarely shown in the last few decades. in addition, it remains unclear whether the structure-activity relationship of valepotriate is unique. therefore, extensive work and deeper exploration are required to fully investigate valepotriate and its derivatives as anticancer agents. herein, we obtained a series of valepotriate derivatives and aimed to explore candidate compounds with potent anticancer activity against pancreatic cancer cells. by using a structure-based similarity search, we identified compounds that had similar backbones but differed in terms of their substitution groups. among these compounds, ( s, s, r)- -(acetyloxy)- -[( -methylbutanoyl)oxy]- a, , , a-tetrahydro- h-spiro [cyclopenta[c]pyran- , 'oxiran]- -ylmethyl -methylbutanoate (denoted as amcp) was chosen for the following experiments and its mechanisms of action were validated. cell culture rpmi- medium and fetal bovine serum (fbs) were purchased from gibco (grand island, ny, usa). dapi ( ′, -diamidino- phenylindole) was purchased from wuhan goodbio technology co., ltd (wuhan, china). the annexin v-fitc apoptosis kit was purchased from bestbio (shanghai, china). the mitochondrial membrane potential assay kit was purchased from signalway antibody (college park, md, usa). the primary antibodies against poly(adp-ribose) polymerase (parp), caspase- , cleaved caspase- , pi k-p ɑ, p-akt (ser ), akt, mammalian target of rapamycin (mtor), p-mtor (s ), p-p s , p s , p-s ( / ), p-s ( / ), s , and bcl- were purchased from cell signaling technology (beverly, ma, usa). the primary antibodies against x-linked inhibitor of apoptosis protein (xiap), bax, noxa, bcl-xl, mcl- , and β-actin were purchased from abcam, inc. (cambridge, ma, usa). the human pancreatic cancer cell lines bxpc- (catalog number tchu ) and sw (catalog number tchu ) were purchased from the chinese academy of sciences (shanghai, china). cells were cultured in rpmi- medium containing % fbs and u/ml penicillin/streptomycin at °c in % co in a humidified atmosphere. amcp was dissolved in dimethyl sulfoxide (dmso) at a concentration of mm. gemcitabine was dissolved in dmso at a concentration of mm. the structure-based similarity search was performed using the structure search plugin on the molport website (https://www. molport.com/shop/find-chemicals). ( r)- , , , a-tetrahydrospiro [indene- , '-oxirane] was used as the initial structure. the similarity value was set to . . after searching, compounds were chosen and purchased from targetmol (shanghai, china). cell viability assay cell proliferation was measured by using a cell counting kit- (cck- ) assay (bestbio, shanghai, china). cells were cultured in -well plates at a concentration of × - × /well. cells were cultured for h and treated with . , . , . , , and μm amcp. after , , or h of treatment, the supernatant was completely removed and μl of cck- solution was added to each well, and the cells were cultured for another h at °c. cell viability was quantified by a spectramax m e instrument (molecular devices, san jose, ca, usa) at nm. cell viability was calculated for each well using the formula od treated cells/ od control cells × %. assays were performed in three independent experiments. dapi staining bxpc- or sw cells ( × cells/well) were cultured in -well plates. after exposure to amcp or gemcitabine for h, the cells were fixed with % paraformaldehyde for min and stained with dapi for min. after washing with phosphate-buffered saline (pbs), the cells were observed under a fluorescence microscope (nikon, ti-e, japan). apoptosis assay cells in the exponential growth phase were seeded in six-well plates ( × /well) and cultured overnight in a % co atmosphere at °c. after treatment with amcp or gemcitabine for h, the cells were collected and washed with pbs. then, cells were stained with an annexin v-fitc apoptosis kit according to the manufacturer's instructions and analyzed by flow cytometry (becton dickinson, franklin lakes, nj, usa). assays were performed in three independent experiments. detection of the mitochondrial membrane potential the mitochondrial membrane potential was visualized by , ', , 'tetrachloro- , ', , ' tetraethyl-imidacarbocyanine iodide (jc- ) staining. cells were seeded into six-well plates at a density of × /well and cultured for h. after h of treatment, the cells were collected, washed with pbs, and incubated with jc- for min at °c. after washing off the dye, the cells were immediately analyzed using flow cytometry (becton dickinson, franklin lakes, nj, usa). assays were performed in three independent experiments. noxa transient sirna knockdown small interfering rnas (sirnas) targeting noxa [noxa-homo (sirna- ), noxa-homo (sirna- ), noxa-homo (sirna- )], and control sirna (sicontrol) were purchased from guannan co. ltd. (hangzhou, china). the target sequences were as follows: transient transfection of bxpc- cells was performed using µm of each sirna with lipofectamine (invitrogen, carlsbad, ca, usa). knockdown of nova was verified by realtime quantitative reverse-transcriptase pcr. western blotting analysis after cells were treated with different concentrations of amcp or gemcitabine, the total proteins were extracted using ripa lysis buffer. a total of μg of protein was subjected to % sdspolyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membranes (bio-rad, hercules, ca, usa). the membranes were blocked with % non-fat milk at room temperature for h and then incubated with specific primary antibodies ( : - : dilution) overnight at °c. after washing with tris-buffered saline and tween , the membranes were incubated with secondary antibodies ( : dilution) at room temperature for another h. the protein bands were visualized by adding the ecl reagent wbkls (emd millipore, billerica, ma, usa) and analyzed using bio-rad laboratories quantity one software (bio-rad, hercules, ca, usa). statistical analysis was performed in graphpad prism . a twosided student's t-test or one-way analysis of variance was used to analyze the differences among groups. the results are expressed as the mean ± sd of at least three independent experiments. a pvalue < . was considered statistically significant. amcp induced apoptosis in bxpc- and sw cells a similarity search was employed to find derivatives of valepotriate that were potentially active against cancer cells and the results identified hit compounds that were purchased for bioactivity tests. examples of five of the hit compounds are shown in fig. in both bxpc- and sw cells. to verify the anti-proliferation effects of amcp, both bxpc- and sw cells were incubated with the indicated concentrations of amcp for the indicated times, as shown in fig. b . as a result, amcp produced a concentrationand time-dependent inhibitory effect against both bxpc- and sw cells. in addition, gemcitabine, which is considered the standard single chemotherapeutic agent in the clinic, was used as a positive control to verify the effects against pancreatic cancer cells. by using dapi staining, shrunken cell nuclei and reduced cell numbers were observed after treatment with increasing concentrations of amcp. at a concentration of µm, amcp was more efficacious in inhibiting the proliferation of pancreatic cancer cells than gemcitabine (fig. c) . to demonstrate that the anti-proliferation effect of amcp was attributed to the induction of apoptosis, cells were stained with annexin v/propidium iodide and analyzed by flow cytometry. as shown in fig. a , amcp dose-dependently induced both early and late apoptosis in bxpc- and sw cells. at a concentration of µm, amcp significantly increased the number of apoptotic cells compared with gemcitabine in both cell lines (fig. b ). in addition, western blotting was applied to determine the proteins involved in the regulation of the occurrence of apoptosis. an increase in cleaved caspase- and the appearance of cleaved parp after treatment with µm amcp suggested the occurrence of caspase-dependent apoptosis (fig. c) . moreover, bxpc- and sw cells were pretreated with the caspase paninhibitor z-vad-fmk for h before culturing them with the indicated concentrations of amcp (fig. a) . the addition of z-vad-fmk substantially reversed the inhibitory effects of amcp on bxpc- and sw cells, and reduced the downregulation of caspase- , indicating that caspase-dependent apoptosis played a critical role in the anti-proliferation effects of amcp (fig. b) . amcp selectively suppressed the activation of the pi k/akt pathway and disrupted the mitochondrial membrane potential aberrant activation of pi k pathways is commonly found in many pancreatic cancer patients, indicating a need for the development of novel compounds targeting these key regulators of the pi k pathway [ ] . we therefore intended to explore the effects of amcp on the pi k pathway. amcp substantially suppressed the phosphorylation of akt and mtor, and slightly affected their total protein levels (fig. c) . in contrast, treatment with gemcitabine scarcely affected the phosphorylation of proteins in the pi k/akt pathway, thus highlighting the unique mechanisms of action of amcp. as the disruption of the pi k/akt pathway causes mitochondrial instability [ ] , we detected the mitochondrial membrane potential after exposure to amcp. as expected, cells treated with amcp exhibited a substantial decrease in the mitochondrial membrane potential, which was significantly more severe than that in cells treated with gemcitabine (fig. a, b) . in addition, proteins that play major roles in regulating the mitochondrial membrane potential were analyzed by western blotting (fig. c) . it is worth mentioning that the anti-apoptotic protein mcl- was obviously downregulated in both cell lines after amcp treatment, whereas noxa was apparently upregulated by µm amcp treatment. noxa-dependent mcl- downregulation was critical for amcpinduced apoptosis it has been reported that noxa can act as an e ligase and mediate the degradation of mcl- under certain circumstances [ ] . therefore, we aimed to determine the role of noxa in amcpinduced apoptosis. noxa was successfully knocked down by customized sirna interference in bxpc- cells (fig. d) and sirna # was used for the following experiments. after knocking down noxa, the number of viable cells significantly increased from~ % to % during exposure to µm amcp (fig. e ). in addition, amcp-induced apoptosis was apparently attenuated after noxa knockdown, indicating increased levels of caspase- and parp (fig. f) . meanwhile, the decrease in the protein levels of both mcl- and bcl- caused by amcp was obviously reversed by knocking down noxa, suggesting that noxa is a key protein involved in regulating mcl- . in addition, cycloheximide was used to pretreat cells for . h to explore the degradation dynamics of mcl- in cells with or without amcp. interestingly, amcp obviously accelerated the degradation of mcl- after or h of treatment (fig. g) . synergistic effects of amcp with gemcitabine in bxpc- cells it was shown that amcp exhibited potent anticancer activity against pancreatic cancer cells as a single agent. in addition, amcp displayed a synergistic effect when used in combinatorial treatment with gemcitabine in bxpc- cells. both . and . µm amcp significantly enhanced the cytotoxicity of gemcitabine (fig. a) . combinatorial treatment with amcp and gemcitabine synergistically induced apoptosis in bxpc- cells (fig. b, d) and this effect could be partially overcome by the addition of the pancaspase inhibitor z-vad-fmk (fig. c) . furthermore, depolarization of the mitochondrial membrane was found to be significantly increased after combination treatment with amcp and gemcitabine, suggesting that mitochondrion-dependent apoptosis might play a pivotal role in this event (fig. e) . iridoids isolated from valeriana have recently been reported to possess various biological activities, including anticancer effects [ ] [ ] [ ] . however, large numbers of bioactive iridoids remain to be identified and their structure-activity relationships still need to be validated. in this study, we identified a series of valepotriate derivatives through a similarity search. during the initial antiproliferative activity screening, amcp exhibited a potent anticancer activity against pancreatic cancer cells in vitro and was therefore chosen for the following experiments. as caspases are frequently cleaved into active components once they are activated by various cellular stimuli, we examined the protein expression of both proand cleaved caspase- , and the proteolytic cleavage of parp [ , ] . as shown in fig. , the expression of activated caspase- was significantly upregulated and the expression of parp was diminished after amcp exposure, suggesting that amcp induced caspase- -mediated apoptosis in a concentration-dependent manner [ ] . in addition, the protein expression of xiap was gradually decreased and active caspase- could be detected (fig. c) . it was reported that xiap is an inhibitor of apoptosis that represses the activation of caspase- and ; on the other hand, xiap activity could be attenuated by caspase- during the inhibition of caspase- [ , ] . we also found that pretreatment with the pancaspase inhibitor z-vad-fmk significantly reversed amcp-induced proliferation suppression in both bxpc- and sw cells (fig. d) . taken together, the results showed that the activation of caspase- played an essential role in amcpinduced apoptosis in pancreatic cancer cells. the pi k/akt signaling pathway is commonly activated in many human cancers, including pancreatic cancer, and s . c cells were treated with the indicated concentrations of amcp or gemcitabine for h before western blotting assays. the protein bands were visualized by an ecl system and analyzed using bio-rad laboratories quantity one software. *p < . , **p < . . c cells were treated with amcp or gemcitabine for h before western blotting assays. three independent experiments were performed and the data were presented as the mean ± sd. *p < . , **p < . . fig. amcp disrupted the mitochondrial membrane potential and affected mitochondrial proteins. a bxpc- and sw cells were seeded into six-well plates at a density of × /well and were cultured for h. after treatment with amcp for h, jc- staining was used to observe the depolarized mitochondrial membrane potential. cells were analyzed by flow cytometry. b quantitative analysis of cells with a depolarized mitochondria membrane. c cells were treated with the indicated concentrations of amcp or gemcitabine (gem) for h before western blotting assays. *p < . . d the protein level of noxa was dramatically reduced by noxa sirna knockdown in bxpc- cells. e bxpc- cells were exposed to amcp for h with or without noxa knockdown. cell viability was determined by cck- assays. f noxa was knocked down by sirna in bxpc- cells before treatment and cells were exposed to the indicated concentrations of amcp or gemcitabine for h before western blotting analysis. g bxpc- cells were pretreated with µg/ml cycloheximide (chx) for . h and incubated with µm amcp for , , or h. the mcl- protein level was examined and quantitative analysis was performed using imagej . q software (national institute of health, usa). *p < . . (fig. ) . therefore, amcp was capable of reducing the protein level of mcl- by increasing its degradation. in this study, we identified a novel derivative of valepotriate (denoted as amcp) via a similarity search. amcp showed potent anticancer activity against pancreatic cancer cells through the inhibition of the mcl- and pi k/akt pathways, thereby stimulating caspase-dependent apoptosis. moreover, amcp had a synergistic effect when combined with gemcitabine. therefore, our work not only suggests that amcp is a promising dual-inhibitory anticancer agent but also highlights the importance of the further development of bioactive valepotriate derivatives. the combination of amcp and gemcitabine synergistically induced apoptosis in bxpc- cells. a bxpc- cells were treated with . , . , . , or µm gemcitabine (gem) alone or in combination with . or . µm amcp for h before the cck- assay. b dapi staining was used to visualize the cell nucleus after treatment with gemcitabine, amcp, or a combination of both for h. c bxpc- cells were pretreated with z-vad-fmk for h before exposure to gemcitabine, amcp, or a combination of both. cell viability was determined by cck- assays after h of treatment. d bxpc- cells were treated with gemcitabine, amcp, or a combination of both for h before collection. cells were then stained with annexin v/pi and analyzed by flow cytometry. e after treatment with gemcitabine, amcp, or a combination of both for h, jc- was used to measure the depolarization of the mitochondria membrane and bxpc- cells were then analyzed by flow cytometry. scale bar = µm. *p < . , **p < . . pancreatic cancer genomes: implications for clinical management and therapeutic development pancreatic cancer chemoresistance to gemcitabine preclinical validation of -phosphoinositide-dependent protein kinase inhibition in pancreatic cancer mesothelin confers pancreatic cancer cell resistance to tnf-alpha-induced apoptosis through akt/pi k/nf-kappab activation and il- /mcl- overexpression cdk inhibitor downregulates mcl- and sensitizes pancreatic cancer cell lines to navitoclax enhanced fgfr signalling predisposes pancreatic cancer to the effect of a potent fgfr inhibitor in preclinical models structure-guided design of pyridoclax derivatives based on noxa/mcl- interaction mode mcl- phosphorylation without degradation mediates sensitivity to hdac inhibitors by liberating bh -only proteins therapeutic developments in pancreatic cancer: current and future perspectives new iridoids from the roots of valeriana dioscoridis sm cytotoxic and antibacterial activities of iridoids and sesquiterpenoids from valeriana jatamansi iridoids and sesquiterpenoids of valeriana stenoptera and their effects on ngf-induced neurite outgrowth in pc cells anti-invasion and antimetastasis effects of valjatrate e via reduction of matrix metalloproteinases expression and suppression of mapk/erk signaling pathway jatamanvaltrate p induces cell cycle arrest, apoptosis and autophagy in human breast cancer cells in vitro and in vivo combating pancreatic cancer with pi k pathway inhibitors in the era of personalised medicine puma and bim are required for oncogene inactivation-induced apoptosis found in translation: how preclinical research is guiding the clinical development of the bcl -selective inhibitor venetoclax iridoids from valeriana jatamansi induce autophagy-associated cell death via the pdk /akt/mtor pathway in hct human colorectal carcinoma cells chemical constituents from valeriana polystachya smith and evaluation of their effects on the acetylcholinesterase and prolyl oligopeptidase activities valepotriates from the roots and rhizomes of valeriana jatamansi jones as novel n-type calcium channel antagonists ampelopsin inhibits human glioma through inducing apoptosis and autophagy dependent on ros generation and jnk pathway hydrogen sulfide donating ent-kaurane and spirolactone-type , -seco-ent-kaurane derivatives: design, synthesis and antiproliferative properties l-serine protects mouse hippocampal neuronal ht cells against oxidative stress-mediated mitochondrial damage and apoptotic cell death caspase attenuates xiap (x-linked inhibitor of apoptosis protein)-mediated inhibition of caspase the novel microrna hsa-mir-cha regulates cell proliferation and apoptosis in human lung cancer by targeting xiap lamb mediates apoptotic, proliferative, invasive, and metastatic behaviors in pancreatic cancer by regulating the pi k/akt signaling pathway cudc- displays potent antitumor activity against human pancreatic adenocarcinoma in vitro and in vivo through inhibition of hdac to downregulate c-myc expression low-molecular-mass hyaluronan induces pulmonary inflammation by up-regulation of mcl- to inhibit neutrophil apoptosis via pi k/ akt pathway bcr signaling inhibitors differ in their ability to overcome mcl- -mediated resistance of cll b cells to abt- targeted inhibition of pi kalpha/delta is synergistic with bcl- blockade in genetically defined subtypes of dlbcl ursolic acid facilitates apoptosis in rheumatoid arthritis synovial fibroblasts by inducing sp -mediated noxa expression and proteasomal degradation of mcl- conflict of interest: the authors declare no conflicts of interest. key: cord- -wsjob p authors: wang, yan-hang; lv, hai-ning; cui, qing-hua; tu, peng-fei; jiang, yong; zeng, ke-wu title: isosibiricin inhibits microglial activation by targeting the dopamine d /d receptor-dependent nlrp /caspase- inflammasome pathway date: - - journal: acta pharmacol sin doi: . /s - - - sha: doc_id: cord_uid: wsjob p microglia-mediated neuroinflammation is a crucial risk factor for neurological disorders. recently, dopamine receptors have been found to be involved in multiple immunopathological processes and considered as valuable therapeutic targets for inflammation-associated neurologic diseases. in this study we investigated the anti-neuroinflammation effect of isosibiricin, a natural coumarin compound isolated from medicinal plant murraya exotica. we showed that isosibiricin ( − μm) dose-dependently inhibited lipopolysaccharide (lps)-induced bv- microglia activation, evidenced by the decreased expression of inflammatory mediators, including nitrite oxide (no), tumour necrosis factor-α (tnf-α), interleukin- (il- ), interleukin- β (il- β) and interleukin- (il- ). by using transcriptomics coupled with bioinformatics analysis, we revealed that isosibiricin treatment mainly affect dopamine receptor signalling pathway. we further demonstrated that isosibiricin upregulated the expression of dopamine d / receptors in lps-treated bv- cells, resulting in inhibitory effect on nucleotide binding domain-like receptor protein (nlrp )/caspase- inflammasome pathway. treatment with dopamine d / receptor antagonists sch ( μm) or sultopride ( μm) could reverse the inhibitory effects of isosibiricin on nlrp expression as well as the cleavages of caspase- and il- β. collectively, this study demonstrates a promising therapeutic strategy for neuroinflammation by targeting dopamine d / receptors. dopamine, as a crucial neurotransmitter, can transmit neuronal signals to regulate neurological function by acting on specific dopamine receptors [ , ] . currently, the dopamine receptor family is considered to play a key role in various advanced neural functions, including emotion, reward and autonomic movement [ ] . in addition, increasing attention has been paid to the role of the dopamine signal system in immunomodulation and inflammation control [ , ] . previous studies have shown that the genetic and pharmacologic regulation of dopamine receptors can have potential anti-inflammatory effects. for instance, dopamine d receptor (drd ) activation is widely involved in the alleviation of inflammatory bowel diseases [ , ] , pancreatic inflammation [ ] and arthritis [ ] . specifically, drd /d have been found to be widely expressed not only in neurons and t cells [ ] but also in microglia [ ] , indicating that drd /d might also be potential therapeutic targets for neuroinflammation in the central nervous system (cns). recently, the pharmacologic activation of drd was shown to exert an obvious inhibitory effect on neuroinflammation induced by intracerebral haemorrhage, by regulating the nuclear factor-κb (nf-κb) signalling pathway [ ] . moreover, new therapies based on drd /d agonists have been developed for experimental autoimmune encephalomyelitis [ ] . therefore, treating the microglia-derived inflammatory response by targeting dopamine receptors appears to be a promising therapeutic strategy for neuroinflammation. in particular, dopamine receptors have been found to be highly associated with the inflammasome signalling pathway. some previous research has suggested that spinal cord injury induces inflammatory cytokine production by activating the nucleotide-binding domain-like receptor protein inflammasome pathway, which is significantly suppressed by drd agonists [ ] . moreover, drd can also block nlrp inflammasome activation in a β-arrestin-dependent manner [ ] . collectively, these findings provide a potential therapeutic strategy for treating the neuroinflammatory response by targeting the drd-mediated inflammasome pathway. murraya exotica l. (rutaceae) has traditionally been used in china for the treatment of infectious diseases and pains [ ] . modern pharmacological studies have suggested that murraya koenigii and m. exotica possess a wide range of biological activities, including anti-inflammatory, antinociceptive, antioxidative stress, anti-mutagenic stress, and chondroprotective and anticancer metastasis effects [ , ] . isosibiricin is a natural bioactive coumarin compound isolated from m. exotica. several studies have indicated that the coumarin derivatives isofraxidin, osthole and urolithin can exert obvious anti-inflammatory activities both in vitro and in vivo [ ] [ ] [ ] . therefore, in this study, we explored the mechanism of the anti-neuroinflammatory effects of isosibiricin in a bv- microglial model and highlighted that isosibiricin can significantly inhibit the production of multiple inflammatory mediators induced by bacterial lipopolysaccharide stimulation via targeting the drd /d -dependent inflammasome pathway, providing a potential therapeutic strategy for inflammation-related neurological disorders. mouse strains balb/c mice ( - weeks old) were purchased from the vital river laboratories (beijing, china). all mice were raised in a specific pathogen-free environment. the bv- cell line was purchased from the cell bank of peking union medical college in china. the cells were cultured in dulbecco's modified eagle's medium containing % fetal bovine serum, u/ml penicillin and mg/ml streptomycin. the cell culture conditions were maintained at °c in a % co incubator under % absolute humidity. nitrite oxide analysis bv- cells were treated with isosibiricin ( , , and μm) with or without lps for h. then, the supernatants were used to analyse the production of nitrite oxide (no) with a nitric oxide analysis kit (jiancheng bioengineering institute, nanjing, china) enzyme-linked immunosorbent assay bv- cells were treated with isosibiricin ( , , and μm) with or without lps. after treatment for h, μl of the cell supernatants was centrifuged at × g for min to evaluate the accumulation of tumour necrosis factor-α (tnf-α). in addition, μl of the cell supernatants was also centrifuged at × g for min to evaluate il- levels h after treatment. the two experiments were carried out with enzyme-linked immunosorbent assay (elisa) kits (excell bio company, shanghai, china) following the manufacturer's protocol. the cells were collected and rna was extracted by the rnaprep pure cell/bacteria kit (tiangen biotech, beijing, china). target analyses were carried out by the yuanquan yike company (beijing, china). amino allyl-labelled antisense rna was prepared and purified prior to hybridization. purified, coupled antisense rna was quantified using nanodrop nd- to explore the targets affected by isosibiricin. transcriptome analysis pathway analysis [ ] on isosibiricin was also performed by the yuanquan yike company. after the targets of isosibiricin were determined by amino allyl-labelled antisense rna hybridization, kyoto encyclopedia of genes and genomes (kegg) pathway enrichment was used to perform the pathway analysis. signal pathways significantly influenced by isosibiricin compared with lps were selected. western blotting after treatment, the cells were lysed in ice-cold ripa buffer containing % protease inhibitors (macgene, beijing, china). whole-cell proteins were collected by centrifugation at × g for min. the concentration of the total proteins was detected by a protein assay kit (transgen, beijing, china). protein solutions of equal concentration were separated by %- % sds-polyacrylamide gel electrophoresis and transferred to pvdf membranes. next, the pvdf membranes were blocked with % skim milk at °c for min. subsequently, the membranes were incubated with primary antibodies at °c for h. after incubation with a second antibody for h, the membranes were developed with supersignal west femto maximum sensitivity substrate. the protein bands were imaged by a tanon imaging analysis system (tanon, shanghai, china). relative densitometry analysis was carried out by imagej software. reverse-transcription pcr the cells were collected and total rna was extracted using the rnaprep pure cell/bacteria kit (tiangen biotech, beijing, china). the mrna was reverse transcribed using a cdna synthesis kit (transgen, beijing, china). reverse-transcription pcr (rt-pcr) was performed using a rt-pcr superkit (transgen, beijing, china) on an agilent technologies stratagene mx p system. a single cycle was carried out at °c for s, °c for s and °c for s, and this cycle was repeated times. the relative transcriptional levels of drd and drd normalized to those of gapdh was calculated by the comparative −ΔΔct method [ ] . dopamine receptor agonist analysis hek cells stably expressing drd /gα were cultured in -well plates for h. after the supernatant was removed, fluo- / am, a calcium fluorescence probe, was added at °c for min in an incubator under % absolute humidity. the fluo- /am was removed and a calcium buffer solution was added. flexstation ii was used to measure the levels of gα protein activation induced by isosibiricin ( , . nm, . nm, nm, nm, nm, μm and μm) at nm. dopamine was used as a positive control. the ec was analysed by graphpad prism. all experiments were carried out by the national center for drug screening (shanghai, china). nine balb/c mice were randomly divided into the control, lps and isosibiricin groups. the isosibiricin group was treated with isosibiricin ( mg/kg body weight). after h, both the lps group and the isosibiricin group were injected intraperitoneally with lps ( mg/kg body weight). then, the brain tissues were sectioned and stained with specific antibodies. cycle adenosine monophosphate analysis bv- cells were treated with μm isosibiricin with or without lps for h. then, the cell lysates were used to analyse the level of cycle adenosine monophosphate (camp) with a camp direct immunoassay kit (bioway, beijing, china) all experiments were carried out at least three times in triplicate. statistical analyses were performed by using one-way analysis of variance with graphpad prism . software. multiple comparisons were performed by comparing the mean of each column with the mean of every other column. student's t-test was used without correcting for multiple comparisons. all data were expressed as the means ± s.d. a p-value < . was considered to be significant. in this study, the anti-inflammatory effect of isosibiricin (fig. a) was measured by using an no release assay. as shown in fig. b , lps obviously stimulated no release, which was significantly inhibited by isosibiricin ( , and μm) in a concentrationdependent manner. to evaluate the effect of isosibiricin on other inflammatory factors, the levels of tnf-α and il- were further analysed by elisa. as shown in fig. c , d, tnf-α and il- expression was significantly increased upon lps treatment; however, isosibiricin ( , and μm) inhibited tnf-α and il- production in a concentration-dependent manner. moreover, cox- and inos expression was markedly upregulated in lpstreated bv- cells, and isosibiricin reduced cox- and inos expression in a concentration-dependent manner (fig. e) . taken together, these findings indicated that isosibiricin can attenuate neuroinflammation by inhibiting the expression of multiple inflammatory mediators in lps-treated bv- cells. isosibiricin upregulates drd /d expression in lps-treated bv- cells and balb/c mice to investigate the potential mechanism of the anti-inflammatory effect of isosibiricin, transcriptomics coupled with bioinformatics analysis was performed to explore the anti-inflammatory signalling pathway of isosibiricin in lps-treated bv- cells. the results suggested that genes, including upregulated genes and downregulated genes ( genes related to inflammation and genes related to the synthesis, storage, release, termination of dopamine and the expression of drds) were significantly changed by isosibiricin compared with lps. notably, dopamine receptors were most strongly related to isosibiricin treatment (fig. a) . moreover, kegg pathway analysis was carried out to explore the pathway affected by isosibiricin. as shown in fig. b , dopamine activation of the neurological reward system was strongly influenced by isosibiricin. therefore, we speculated that isosibiricin might exert an anti-inflammatory effect by targeting the dopamine receptor signalling pathway. nevertheless, isosibiricin did not directly target or regulate the functions of dopamine receptors (supplementary table ). thus, we hypothesized that isosibiricin can regulate the expression of dopamine receptors. as shown in fig. c , d, lps downregulated the mrna expression of drd and drd , and this was significantly reversed by isosibiricin. in addition, isosibiricin also upregulated drd /d expression in the ca region and cortex of lps-treated balb/c mice (supplementary fig. s a) . collectively, these observations revealed that isosibiricin can effectively upregulate drd and drd expression in vitro and in vivo, which might represent a crucial antiinflammatory mechanism. isosibiricin inhibits the nlrp /caspase- inflammasome pathway in lps-or nigericin-treated bv- cells and lps-treated balb/c mice it has been reported that the expression of the pro-inflammatory mediator il- β significantly increases in drd -null mice compared with wild-type mice [ ] . in addition, drd agonists are able to inactivate the nlrp inflammasome pathway [ ] . thus, we detected the effect of isosibiricin on the nlrp inflammasome pathway. as shown in fig. a , isosibiricin significantly reduced nlrp , caspase- , il- β and il- expression in a concentrationdependent manner, indicating that isosibiricin exerts an inhibitory effect on the nlrp /caspase- inflammasome pathway. moreover, the nlrp inflammasome inducer nigericin was used to activate bv- cells and we found that nigericin significantly increased nlrp , caspase- , il- β and il- expression, which was the predicted pathways (a) and targets (b) of isosibiricin inferred by the differential expressed gene-based method. c, d isosibiricin upregulated dopamine d receptors (c) and dopamine d receptor (d) expressions in lps-induced bv- cells. ## p < . vs. control group. **p < . vs. lps group effectively inhibited by isosibiricin (fig. b) [ ] . animal experiments were also carried out to evaluate the anti-inflammatory effect of isosibiricin. as shown in fig. c , isosibiricin significantly inhibited inflammasome-related il- β and il- induced by lps in the ca region and cortex of balb/c mice. these results suggested that isosibiricin exerts an anti-inflammatory effect via the inhibition of the nlrp inflammasome pathway. drd /d inhibitors reverse the isosibiricin-mediated inactivation of nlrp /caspase- inflammasome pathway considering the effect of isosibiricin on dopamine receptors (fig. c, d) and the inflammasome pathway (fig. a, b) , we hypothesized that isosibiricin might inhibit the inflammasome pathway through dopamine receptors. therefore, drd and drd inhibitors, sch and sultopride were used to investigate whether drd /d are involved in isosibiricinmediated anti-inflammatory effects. we found that the anti-inflammatory effect of isosibiricin was significantly reversed upon treatment with sch and sultopride. nlrp , caspase- , il- β and il- expression was markedly increased by sch and sultopride (fig. a) . furthermore, sch and sultopride were also used in nigericin-treated bv- cells. similar to what was observed in the lps-induced experiment, nlrp , caspase- , il- β and il- expression was reversed in the sch -and sultopride-treated groups compared with the isosibiricin-treated group (fig. d) . taken together, the results suggested that drd and drd inhibitors are able to reverse the isosibiricin-mediated inhibition of the nlrp /caspase- inflammasome pathway. dopamine agonists (das), such as dopamine, act on dopamine receptors to regulate a great diversity of biological functions [ ] . fig. isosibiricin inhibits nlrp /caspase- inflammasome pathway in vitro and in vivo. a isosibiricin inhibited inflammasome-related nlrp , pro-caspase- , caspase- , pro-il- β, il- β, pro-il- and il- expressions in lps-induced bv- cells. quantification for caspase- , il- β and il- expressions were relative to gapdh in lps-induced bv- cells. b isosibiricin inhibited inflammasome-related nlrp , pro-caspase- , caspase- , pro-il- β, il- β, pro-il- and il- expressions in nigericin-induced bv- cells. quantification for caspase- , il- β and il- expressions were relative to gapdh in nigericin-induced bv- cells. c isosibiricin inhibited inflammasome-related il- β and il- in the ca region and cortex stained with the specific antibodies il- β and il- . arrows indicate the activated inflammatory factors. ## p < . vs. control group. **p < . vs. lps group or nigericin group in the past decade, das have been mainly used clinically for the treatment of parkinson's disease and, recently, the development and clinical application of das for various other neurological diseases have begun to draw attention from researchers [ , ] . neuroinflammation is an inflammatory response in the cns characterized by increased microglial over-activation [ , ] . thus, the development of anti-neuroinflammatory agents is of great importance for the treatment of inflammation-related neurological disorders, such as motor paralysis, amyotrophic lateral sclerosis and dementia [ ] [ ] [ ] . in the current study, we reported that isosibiricin exerts a significant anti-neuroinflammatory effect on lps-treated bv- cells. lps is a common inflammation inducer that is used to investigate different types of inflammatory pathology, such as tissue inflammation [ ] , vascular inflammation [ ] and myocarditis [ ] . dopaminergic neurons play an important role in the pathological process of parkinson's disease [ ] , and lps is widely used to induce neuroinflammation to establish models of parkinson's disease. in addition, some references have indicated that dopamine receptors are also involved in the regulation of the classical lps signalling pathway [ ] [ ] [ ] . therefore, lps was used to induce neuroinflammation and damage dopamine receptors in the substantia nigra [ ] . the potential mechanism of isosibiricin may involve the upregulation of drd /d expression and the further inactivation of the nlrp /caspase- inflammasome pathway, which results in the blockage of the production of multiple inflammatory mediators. in addition, we used pipeline pilot to measure the solubility, intestinal absorption and blood-brain barrier (bbb) level of isosibiricin. isosibiricin exhibited good solubility in water at °c and was easily absorbed by the intestine. the bbb level was , which means that isosibiricin can pass through the bbb. thus, our study provides an attractive therapeutic methodology for inflammation-associated neurological disorders, as well as an anti-neuroinflammatory lead compound that may become a clinical candidate. dopamine receptors were recently discovered as key therapeutic targets involved in immunoregulation and the inflammatory response [ ] . previously reported active compounds were shown to mainly activate or inhibit dopamine receptors [ , ] ; however, we found that isosibiricin did not directly activate drd /d but significantly upregulated the expression of drd /d mrna, further promoting drd /d -dependent inflammasome signalling pathway inactivation and blocking inflammatory mediator production. in addition, the level of camp was regulated by drd /d , but we detected that isosibiricin did not affect the level of camp ( supplementary fig. s b ). these mechanisms are quite different from those of current small molecular regulators, which directly activate drd /d [ ] . however, the molecular mechanism by which drd mrna expression is regulated still needs to be explored. we speculate that some transcription factors, such as nf-κb, nrf and creb, might be involved in this process. dopamine receptors, including five subtypes from drd to drd , are seven-transmembrane-spanning g-protein-coupled receptors [ , ] . based on their structures and pharmacological characteristics, dopamine receptors are divided into two major categories: dopamine d -like receptors (drd and drd ) and d like receptors (drd , drd , and drd ) [ ] . notably, the specific drd inhibitors sch (for drd ) and sultopride (for drd ) significantly reversed the inhibitory effect of isosibiricin on the nlrp signalling pathway, further supporting the observation that isosibiricin suppresses neuroinflammation by blocking the nlrp / caspase- inflammasome pathway. it is worth mentioning that the regulatory effect of drd inhibitors on the isosibiricin-dependent inhibition of il- β production was more significant in lps-induced bv- cells than in nigericin-induced bv- cells. a possible explanation may be that the mechanism by which lps induces inflammasome signal activation differs from that of nigericin. fig. dopamine d / receptor inhibitors reverse isosibiricin-mediated inhibition of nlrp /caspase- inflammasome pathway. a sch and sultopride reversed isosibiricin-mediated inhibition of nlrp , pro-caspase- , caspase- , pro-il- β, il- β, pro-il- and il- expressions in lps-induced bv- cells. quantification for caspase- , il- β and il- expressions was relative to gapdh in lps-induced bv- cells. b sch and sultopride reversed isosibiricin-mediated inhibition of nlrp , pro-caspase- , caspase- , pro-il- β, il- β, pro-il- and il- expressions in nigericin-induced bv- cells. quantification for caspase- , il- β and il- expressions was relative to gapdh in nigericin-induced bv- cells. ## p < . vs. control group. $$ p < . vs. lps group or nigericin group. **p < . vs. the isosibiricin group isosibiricin regulates drd / to inhibit neuroinflammation yh wang et al. meanwhile, isosibiricin-mediated anti-neuroinflammation was highly associated with the canonical lps-induced neuroinflammation pathway. of course, further studies are needed to elucidate this speculation. in conclusion, our observations demonstrated that isosibiricin exerts a marked anti-neuroinflammatory effect via targeting the drd /d -dependent nlrp /caspase- inflammasome pathway. this finding indicates that isosibiricin may a promising candidate molecule for that treatment of neuroinflammation, and that drd /d may be a key target for inflammatory programmes in neurological diseases. dopamine promotes ascorbate release from retinal neurons: role of d receptors and the exchange protein directly activated by camp type (epac ) modulatory effect of dopamine receptor on the neurosecretory dahlgren cells of the olive flounder, paralichthys olivaceus a concise review of the conflicting roles of dopamine- versus dopamine- receptors in wound healing exercise decreases oxidative stress and inflammation and restores renal dopamine d receptor function in old rats dopamine d -like receptor antagonist attenuates th -mediated immune response and ovalbumin antigen-induced neutrophilic airway inflammation role of dopamine and d dopamine receptor in the pathogenesis of inflammatory bowel disease dopamine d receptor polymorphisms in inflammatory bowel disease and the refractory response to treatment dopamine d receptor signalling controls inflammation in acute pancreatitis via a pp a-dependent akt/ nf-κb signalling pathway dopamine d receptor is involved in alleviation of type ii collagen-induced arthritis in mice dopamine, t cells and multiple sclerosis (ms) microglial dopamine receptor elimination defines sex-specific nucleus accumbens development and social behavior in adolescent rats activation of dopamine d receptor suppresses neuroinflammation through αb-crystalline by inhibition of nf-κb nuclear translocation in experimental ich mice model dopamine d receptor agonist a- inhibits nlrp inflammasome activation, controls inflammation, and alleviates histopathology in a rat model of spinal cord injury α-synuclein disrupts the antiinflammatory role of drd via interfering β-arrestin -tab interaction in astrocytes selective activation of nociceptor trpv channel and reversal of inflammatory pain in mice by a novel coumarin derivative muralatin l from murraya alata anti-inflammatory, analgesic and antiulcerogenic effect of total alkaloidal extract from murraya koenigii leaves in animal models evaluation of anti-inflammatory and antinociceptive activities of murraya exotica isofraxidin inhibits interleukin- β induced inflammatory response in human osteoarthritis chondrocytes osthole protects against acute lung injury by suppressing nf-κb-dependent inflammation urolithin a attenuates proinflammatory mediator production by suppressing pi -k/akt/nf-κb and jnk/ ap- signaling pathways in lipopolysaccharide-stimulated raw macrophages: possible involvement of nadph oxidase-derived reactive oxygen species transcriptome analysis of dairy goat mammary gland tissues from different lactation stages an integrated proteomics and bioinformatics approach reveals the anti-inflammatory mechanism of carnosic acid suppression of neuroinflammation by astrocytic dopamine d receptors via αb-crystallin the inflammasome drives gsdmd-independent secondary pyroptosis and il- release in the absence of caspase- protease activity behavioral and cellular dopamine d and d receptor-mediated synergy: implications for l-dopa-induced dyskinesia a randomised, open-label, crossover study of the dopamine agonist, pramipexole, in patients with sleep bruxism efficacy and safety of low-and high-dose cariprazine in acute and mixed mania associated with bipolar i disorder: a double-blind, placebo-controlled study formononetin inhibits neuroinflammation and increases estrogen receptor β (erβ) protein expression in bv microglia neurotropin inhibits neuroinflammation via suppressing nf-κb and mapks signaling pathways in lipopolysaccharide-stimulated bv cells (-)-β-caryophyllene, a cb receptor-selective phytocannabinoid, suppresses motor paralysis and neuroinflammation in a murine model of multiple sclerosis disease origin and progression in amyotrophic lateral sclerosis: an immunology perspective nstearoylethanolamine protects the brain and improves memory of mice treated with lipopolysaccharide or immunized with the extracellular domain of α nicotinic acetylcholine receptor suppressive effects of pelargonidin on lipopolysaccharide-induced inflammatory responses emodin attenuates lipopolysaccharide-induced injury via down-regulation of mir- in h c cells tubeimoside i protects dopaminergic neurons against inflammation-mediated damage in lipopolysaccharide (lps)-evoked model of parkinson's disease in rats spinal dopaminergic projections control the transition to pathological pain plasticity via a d /d -mediated mechanism dopamine induces il- -dependent il- production via d -like receptor on cd naive t cells and d -like receptor antagonist sch- inhibits cartilage destruction in a human rheumatoid arthritis/scid mouse chimera model effects of antiglaucoma drugs glc , a novel dopamine d agonist and d antagonist, and timolol on endotoxininduced tnf-alpha release in serum of rats modulation of m /m polarization by capsaicin contributes to the survival of dopaminergic neurons in the lipopolysaccharide-lesioned substantia nigra in vivo stimulation of dopamine d receptors in the nucleus accumbens inhibits inflammatory pain long-term treatment with dopamine d receptor agonists induces a behavioral switch that can be rescued by blocking the dopamine d receptor antagonism of the d dopamine receptor enhances tremor but reduces voluntary muscle activation in humans ck oppositely modulates l-dopa-induced dyskinesia via striatal projection neurons expressing d or d receptors dopamine receptor subtypes differentially regulate autophagy predicting subtype selectivity of dopamine receptor ligands with three-dimensional biologically relevant spectrum characterization of an invertebrate-type dopamine receptor of the american cockroach, periplaneta americana this work was financially supported by the national natural science foundation of china (numbers , and ) and the drug innovation major project (number zx - - ). kwz, pft, and yj designed the research. yhw and hnl conducted majority of the experiments. qhc coordinated the experiments. kwz and yhw wrote the manuscript. kwz provided suggestions for the experimental design and manuscript. the online version of this article (https://doi.org/ . /s - - - ) contains supplementary material, which is available to authorized users.competing interests: the authors declare no competing interests. key: cord- -ewc awv authors: bian, yu; li, xin; pang, ping; hu, xue-ling; yu, shu-ting; liu, yi-ning; li, xin; wang, ning; wang, jin-hui; xiao, wei; du, wei-jie; yang, bao-feng title: kanglexin, a novel anthraquinone compound, protects against myocardial ischemic injury in mice by suppressing nlrp and pyroptosis date: - - journal: acta pharmacol sin doi: . /s - - - sha: doc_id: cord_uid: ewc awv pyroptosis is a form of inflammatory cell death that could be driven by the nucleotide-binding oligomerization domain-like receptor family pyrin domain-containing (nlrp ) inflammasome activation following myocardial infarction (mi). emerging evidence suggests the therapeutic potential for ameliorating mi-induced myocardial damages by targeting nlrp and pyroptosis. in this study, we investigated the myocardial protection effect of a novel anthraquinone compound ( , -dihydroxy- -methyl- , -anthraquinone- -ethyl succinate) named kanglexin (klx) in vivo and in vitro. male c bl/ mice were pre-treated either with klx ( , mg· kg(− )per day, intragastric gavage) or vehicle for consecutive days prior to ligation of coronary artery to induce permanent mi. klx administration dose-dependently reduced myocardial infarct size and lactate dehydrogenase release and improved cardiac function as compared to vehicle-treated mice h after mi. we found that mi triggered nlrp inflammasome activation leading to conversion of interleukin- β (il- β) and il- into their active mature forms in the heart, which could expand the infarct size and drive cardiac dysfunction. we also showed that mi induced pyroptosis, as evidenced by increased dna fragmentation, mitochondrial swelling, and cell membrane rupture, as well as increased levels of pyroptosis-related proteins, including gasdermin d, n-terminal gsdmd, and cleaved caspase- . all these detrimental alterations were prevented by klx. in hypoxia- or lipopolysaccharide (lps)-treated neonatal mouse ventricular cardiomyocytes, we showed that klx ( μm) decreased the elevated levels of terminal deoxynucleotidyl transferase dutp nick end labeling- and propidium iodide-positive cells, and pyroptosis-related proteins. we conclude that klx prevents mi-induced cardiac damages and cardiac dysfunction at least partly through attenuating nlrp and subsequent cardiomyocyte pyroptosis, and it is worthy of more rigorous investigations for its potential for alleviating ischemic heart disease. myocardial infarction (mi) is one of the leading causes of hospitalization and death globally. myocardial injury due to mi is largely due to the loss of damaged cardiomyocytes, which results in reduced cardiac contractility and cardiac dilatation, eventually leading to heart failure (hf) [ ] . the current treatment for mi is percutaneous coronary intervention, which dramatically improves the survival of patients. however, the reperfusion of blood can further accelerate injury to the ischemic heart, and surviving patients are still at risk for developing hf [ ] . therefore, it is of critical importance to develop novel pharmacological therapeutic strategies to prevent cardiomyocyte death and thus limit myocardial injury in patients with mi. programmed cell death and necrosis are two major forms of myocardial injury after mi. in contrast to necrosis, programmed cell death mainly occurs in the infarct border zone and has been identified as an important determinant of the severity of cardiomyocyte injury and the subsequent outcome of mi patients [ ] . recently, a new form of inflammatory programmed cell death known as pyroptosis was recognized. pyroptosis, which shares the morphological characteristics of apoptosis (typical programmed cell death) and necrosis, including dna fragmentation, cell swelling, an impairment of membrane integrity, and the release of proinflammatory cytokines such as interleukin- β (il- β) and il- , is initiated by caspase- [ ] . specifically, caspase- activation cleaves gasdermin d (gsdmd) and generates n-terminal fragment oligomers within the cell membrane, leading to membrane rupture and pyroptotic cell death through the formation of large pores [ ] . several studies have confirmed the involvement of pyroptosis in response to different pathological stresses, particularly in the post-mi heart [ , ] . the nucleotide-binding oligomerization domain-like receptor family pyrin domain-containing (nlrp ) inflammasome is activated upon mi and acts as an initiator of pyroptosis [ ] . the activated nlrp inflammasome aggravates myocardial injury by directly activating caspase- -mediated cell pyroptosis and indirectly through the release of pro-inflammatory cytokines [ ] . in contrast, the inhibition of the nlrp inflammasome signaling pathway reduces infarct size and preserves cardiac function via attenuating cardiomyocyte pyroptosis in post-mi mice [ ] [ ] [ ] . these findings suggest that targeting nlrp inflammasome activation and subsequent pyroptosis may be a promising therapeutic strategy for suppressing mi-induced myocardial injuries. there is increasing evidence that monomeric compounds with various pharmacological properties can have beneficial effects on cardiovascular and cerebrovascular diseases. in particular, anthraquinones are a crucial class of new and synthetic compounds that possess a broad range of applications. numerous studies have documented that several anthraquinone compounds provide protection from a variety of cardiac disorders [ ] [ ] [ ] [ ] [ ] . in this study, we chemically designed and synthesized a novel anthraquinone compound ( , -dihydroxy- -methyl- , anthraquinone- -ethyl succinate) called kanglexin (klx). we aimed to investigate the potential cytoprotective effects of this novel compound against ischemic injury in a mouse model of mi and the modification of the nlrp inflammasome and pyroptosis as underlying cellular and molecular mechanisms. animal experiments were performed in accordance with the nih guide for the care and use of laboratory animals and were approved by the animal ethical committee of harbin medical university. male c bl/ mice (~ weeks old, weighing ± g) were obtained from changsheng biotechnology company (liaoning, china). the mice were randomly divided into four groups of equal numbers (fig. b) : the sham group, the mi + vehicle group, the mi + klx-low (klx-l; mg· kg − ) group, and the mi + klx-high (klx-h; mg· kg − ) group. the mice were administered or mg· kg − per day klx by intragastric gavage, and the sham and mi groups received an equivalent volume of solvent for days prior to mi surgery. the mouse model of mi was generated using previously described procedures [ ] . briefly, the mice were anesthetized with avertine ( . g ·kg − ; sigma-aldrich corporation, usa), and they underwent open-chest surgery to expose the hearts. mi was established by ligation of the left anterior descending coronary artery mm below the left auricle with a / nylon suture for h. the surgical procedures for the sham control mice were identical to those for the mi model, but did not include coronary artery ligation. cardiac function was determined by echocardiographic measurements after anesthesia, and blood and heart tissue were stored at − °c for later use. echocardiographic measurements twenty-four hours after surgery, echocardiography was performed using an echocardiographic system with a vevo ultrasound machine (visualsonics, toronto, canada) at a probe frequency of mhz. the mice were anesthetized with avertine throughout the entire surgery period for noninvasive examination. two-dimensional images were obtained at the papillary muscles. the ejection fraction (ef) and fractional shortening (fs) were measured on the m-mode the experimental protocol for the preventative klx study in mi mice. c representative lv sections stained with ttc and evans blue (upper panel) and statistical data of the lv infarct size. the infarct size (%) is expressed as the percentage of infarct area relative to the total lv area. the blue area represents the nonischemic region, the area at risk is stained red, and the infarct region is stained white. scale bar = μm; n = . klx-l: mg ·kg − ; klx-h: mg· kg − . d, e echocardiographic analysis of the lv ef% and fs% in the mice; n = - . f serum ldh level in the mice; n = . g representative h&e staining of paraffin sections of the lv from the mice (× ). scale bar = μm. the data are presented as the mean ± sem; *p < . , **p < . , ***p < . vs. sham; # p < . , ## p < . , ### p < . vs. mi tracings and calculated by the machine as the average of three cardiac cycles. the mice were injected through the abdominal aorta with evans blue ( %; solarbio, china) to stain the non-infarcted areas. the hearts were cut transversely into -mm sections and stained with % triphenyltetrazolium chloride (ttc; solarbio) at °c for min. images were captured with a microscope (zeiss, germany). the infarct size was calculated as the percentage of infarct area relative to the total area of the left ventricle. electron microscopic examination cardiac slices were fixed in . % glutaraldehyde and rinsed with . mol· l − phosphate-buffered saline (pbs). the samples were placed in % osmium tetroxide at °c. after fixation for h, the samples were stained with % uranyl acetate dehydrated in ethanol and embedded in epoxy resin, and stained with uranyl acetate and lead citrate. the samples were examined under a jem- electron microscope (fei, usa). lactate dehydrogenase release assay blood samples were collected before the mice were sacrificed, and the serum was separated. the concentration of lactate dehydrogenase (ldh) in the serum was measured according to the manufacturer's experimental procedure (nanjing jiancheng, jiangsu, china). hematoxylin and eosin staining middle-transverse sections of the left ventricular (lv) wall were fixed in % paraformaldehyde for embedding in paraffin. the tissues were cut into -μm-thick sections and stained with hematoxylin and eosin (h&e). images were captured by microscopy (fv ). culture and treatment of neonatal mouse ventricular cardiomyocytes one-to three-day-old neonatal c bl/ mice were subjected to open-chest surgery to expose the hearts. the heart tissues were digested into single cardiomyocytes with . % trypsin (solarbio, china). the cells were seeded into culture dishes at × cells per well and then exposed to various treatments. for lps treatment, cardiomyocytes were treated with μg· ml − lps for h. for hypoxia treatment, cardiomyocytes were placed in an anoxic chamber with % co and % n for h and cotreated with klx or . % dimethyl sulfoxide (dmso) as a vehicle control. tunel and hoechst/pi staining cardiomyocytes were cultured in -well plates at a density of × cells per well to evaluate pyroptosis after drug treatment. the cardiomyocytes were fixed and permeabilized in % paraformaldehyde and . % triton x- . the cells were incubated with terminal deoxynucleotidyl transferase dutp nick end labeling (tunel) staining agents at °c in the dark for h and dapi ( ′, diamidino- -phenylindole) for min. the cardiomyocytes were then incubated with hoechst and propidium iodide (pi) (solarbio, china) in the dark at °c for min. the fluorescence was detected by confocal laser scanning microscopy (fv , olympus, japan). western blot analysis protein samples were extracted using lysis buffer containing % protease inhibitor (roche, switzerland). protein concentrations were measured by a bca protein kit (beyotime institute of biotechnology, shanghai, china). the proteins were separated by % sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred onto polyvinylidene difluoride membranes (pall, usa). the membranes were incubated with the antibodies against nlrp ( : ; #ab , abcam, cambridge, uk), gsdmd ( : ; #ab , abcam), il- β ( : ; #ab ), caspase- ( : ; #wl a, wanleibio, china), il- ( : ; #a , abclonal, wuhan, china), and β-actin ( : ; #bs- r, bioss, boston, ma, usa) at °c overnight and then with a fluorescence-conjugated anti-rabbit igg secondary antibody ( : , ; li-cor bioscience, lincoln, ne, usa) for min in the dark. the gray value of each protein was quantified by an infrared fluorescence imaging detector (li-cor bioscience) and normalized to β-actin as an internal control. rna isolation and real-time pcr total rna was extracted from myocardial tissues or neonatal mouse ventricular cardiomyocytes (nmvcs) using trizol reagent (invitrogen, carlsbad, ca, usa) according to the manufacturer's instructions. a total of μg of rna was reverse transcribed into complementary dna under the conditions of °c for min, °c for min, and °c using a reverse transcription kit (toyobo, osaka, japan). sybr green (toyobo) was used for real-time pcr to quantify messenger rna (mrna) level with β-actin as an internal control on a fast real-time pcr system (abi, waltham, ma, usa). the normalized level of nlrp (forward primer: ′-caacctcacgtcacactgct- ′; reverse primer: ′-tttcagacaaccccaggttc- ′) mrna expression was calculated according to the −ΔΔct method. the data are presented as the mean ± sem. the results were analyzed using student's t test for two-group comparisons and one-way analysis of variance followed by tukey's post hoc correction multiple-group comparisons. a p value of < . was considered statistically significant. graphpad prism was used for the statistical analysis. to determine whether klx (fig. a) possesses cytoprotective effects against mi damage and associated cardiac dysfunction, we employed a mouse model of mi. the experimental protocol for the in vivo study is depicted in fig. b . evans blue/ttc double staining was performed to measure the infarct size h after mi. as shown in fig. c , the infarct size was significantly reduced by treatment with klx-h at a dosage of mg· kg − per day. echocardiographic measurements of lv systolic function showed that, compared to sham operation, mi leds to dramatic decreases in the ef percentage (ef%) and fs percentage (fs%), which were remarkably reversed by klx-h treatment (fig. d, e) . klx-h failed to alter cardiac function, indicating that this dose of klx had no toxic effect on the heart (supplementary fig. ) . furthermore, klx-h remarkedly diminished the release of ldh, an indicator of cardiac damage, from the infarcted myocardium into the serum (fig. f) . by comparison, klx group at a dosage of mg· kg − per day did not elicit any significant beneficial effects (fig. c-f ). h&e staining of lv sections showed a significant reduction in the infiltration of inflammatory cells in the mice treated with klx-h compared with untreated mi mice (fig. g) . klx effectively inhibits pyroptosis processes in the mouse hearts with mi pyroptosis is a new form of inflammatory programmed cell death, which has been shown to contribute to cardiomyocyte loss in the heart following mi [ , ] . we sought to explore whether the protective effects of klx on ischemic hearts occur through the attenuation of pyroptosis. tunel staining and electron microscopy were used to evaluate the morphological characteristics of pyroptotic cell death in our experimental model. as shown in fig. a representative electron micrographs (× , ) of lv sections. the red arrowheads indicate membrane pores, the yellow arrowheads indicate swollen mitochondria, and the blue arrowheads indicate inflammatory cells. c western blot analysis of cleaved caspase- , n-gsdmd, and gsdmd in mouse hearts. the relative protein levels of d cleaved caspase- (n = ), e gsdmd, and f n-gsdmd (n = ). g, h the relative protein levels of mature il- β (n = ) and il- (n = ) in mouse hearts. protein expressions were normalized to β-actin. the data are presented as the mean ± sem. *p < . , **p < . , ***p < . vs. sham; # p < . , ## p < . vs. mi. d-f, h one-way anova followed by tukey's post hoc analysis formation, and inflammatory cell infiltration, which were prevented by klx-h pretreatment. the protein levels of cleaved caspase- , gsdmd, and n-terminal gsdmd (n-gsdmd) were markedly elevated in mi, and these effects were significantly reversed by klx-h (fig. c-f ). caspase- activation causes the transformation of il- β and il- into their active forms, which in turn triggers further myocardial damage in mice subjected to mi. the release of cytokines into the blood is a hallmark of pyroptosis [ ] . thus, we further examined the expression of these two pro-inflammatory cytokines. the mi-induced elevation of the protein levels of intracardiac il- β and il- in the lv was significantly repressed by klx-h treatment compared with vehicle treatment (fig. g, h) . these results indicated that klx suppresses pyroptosis in mi mice. klx attenuates the activation of the nlrp inflammasome upon mi in mouse hearts the nlrp inflammasome, a pro-inflammatory protein complex, has been shown to be activated and to contribute to cell death via pyroptosis in a caspase- -dependent manner [ ] . the inhibition of the nlrp inflammasome offers cardioprotective benefits by reducing cardiomyocyte pyroptosis [ ] . to determine whether the anti-pyroptotic effects of klx are attributable to the suppression of the nlrp inflammasome, we measured the nlrp protein level. consistent with a published study [ ] , our western blot analysis showed that mi resulted in the upregulation of nlrp h after mi, and similar expression patterns of proteins downstream of nlrp , including mature il- β, n-gsdmd, and gsdmd, were observed, supporting the notion that nlrp inflammasome activation initiates downstream effectors associated with pyroptosis (fig. a, b) . moreover, klx significantly inhibited the elevation of nlrp mrna and protein levels induced by mi (fig. c, d) . hypoxia is a critical event in the setting of mi and is well recognized to simulate in vivo ischemic conditions in cells [ , ] . in addition, hypoxia has been shown to promote pyroptosis klx-h: μm) and exposed to hypoxic conditions for h. the nuclei were stained with dapi (blue), and cells with chromosomal dna fragmentation were stained with tunel (green). scale bar = μm; n = . b representative images of hoechst /pi staining (left) and the quantitative analysis of pi-positive nmvcs (right). the nuclei were stained with hoechst , and cells with a ruptured membrane were stained with pi (red). scale bar = μm; n = . c, d western blot analysis of the protein levels of nlrp (n = ), cleaved caspase- (n = ), gsdmd (n = ), n-gsdmd (n = ), mature il- β (n = ), and il- (n = ) in nmvcs treated with μm klx and exposed to hypoxic conditions for h. the data are presented as the mean ± sem. *p < . , **p < . , ***p < . vs. control; # p < . , ## p < . , ### p < . vs. hypoxia. a-d one-way anova followed by tukey's post hoc analysis kanglexin attenuates myocardial infarction y bian et al. through nlrp inflammasome activation [ ] . to determine whether klx can prevent cardiomyocytes from pyroptosis, we cultured nmvcs under hypoxic conditions. cell membrane rupture and chromosomal dna fragmentation are morphological features of pyroptosis [ ] . the numbers of tunel-positive cells and pipositive cells were both significantly increased in the hypoxia group compared with the normoxia group, and treatment with and μm klx, but not μm klx, significantly blunted these deleterious changes in cardiomyocytes (fig. a, b) . moreover, hypoxia markedly elevated the protein expressions of nlrp , cleaved caspase- , gsdmd, n-gsdmd, il- β, and il- , and klx reversed these alterations (fig. c, d) . these results indicated that klx protects against cardiomyocyte pyroptotic cell death in response to hypoxia. lipopolysaccharide (lps) has been shown to cause cardiomyocyte pyroptosis and simulate inflammatory injury resembling mi [ ] . to corroborate our in vitro observations, we investigated the effects of klx on lps-induced changes in nmvcs. lps stimulation significantly induced cardiomyocyte pyroptosis, as indicated by the increased numbers of tunel-and pi-positive cells, which were remarkably suppressed by treatment with and μm klx (fig. a, b) . in accordance with our observations under hypoxic conditions, lps also elicited profound increases in the expression levels of nlrp and the pyroptosis-related proteins mature il- β and il- , and these increases were significantly abrogated by μm klx (fig. c, d) . here, we report the beneficial effects of a novel chemically modified monomeric compound, klx, on cardiac damage, its function in a mouse model of mi and the underlying mechanisms. our results showed that klx significantly reduces the infarct size and improves cardiac dysfunction induced by mi. moreover, klx significantly suppressed the expression of the nlrp inflammasome, the release of the pro-inflammatory cytokines il- β and il- and pyroptotic cell death in both an in vivo model of mi and an in vitro model of ischemic injury by hypoxia or lps (fig. ) . mi-induced cardiac ischemia remains one of the leading causes of mortality worldwide, despite the use of advanced therapeutic interventions and drugs. the major reason for myocardial injury upon mi is cardiomyocyte death, which is also a therapeutic target for preventing mi and subsequent consequences. however, commonly used drugs are limited by their therapeutic efficacy and side effects [ ] [ ] [ ] . accumulating evidence has shown that traditional chinese medicine offers huge beneficial effects in the treatment of heart diseases [ , ] . in the current study, we found that klx had beneficial effects on reducing the myocardial infarct size and ldh release by affecting the nlrp inflammasome and pyroptosis. pyroptosis is a newly identified form of inflammatory cell death triggered by caspase- , and it has been reported to be casually linked to a variety of diseases, particularly to cardiomyocyte loss following mi and ischemia/reperfusion [ , , ] . gsdmd acts as the executor of pyroptosis by producing an n-terminal fragment, which oligomerizes in the cell wall and causes pyroptotic cell death by forming membrane pores [ ] . moreover, the inhibition of caspase- decreases the infarct size and improves cardiac function by attenuating cardiomyocyte pyroptosis [ ] . our study showed that klx significantly suppressed the cardiac production of several pyroptosis-related proteins, including gsdmd, n-gsdmd, and cleaved caspase- . the morphological features of pyroptosis are nuclear condensation, dna fragmentation, and the destruction of cell membrane integrity. as a result, the intracellular ion balance is destroyed, cell swells, and excess inflammatory reaction is triggered after the release of the cellular contents [ ] . in agreement with this view, our results indicated that mi induces pyroptosis in the heart, as evidenced by increased dna fragmentation and mitochondrial swelling and cell membrane rupture, and notably, these detrimental alterations were markedly reversed by klx treatment. these results suggest that the protective effects of klx on the ischemic heart can likely be ascribed to the attenuation of pyroptosis. mi initiates an intensive inflammatory response that is essential for cardiac wound healing, but it can also aggravate cardiac damage and dysfunction [ ] . nlrp is a member of the inflammasome, which is a large multimeric protein complex, and the nlrp inflammasome can readily be activated upon tissue injury [ ] . mi triggers nlrp inflammasome activation, leading to the conversion of il- β and il- to their active mature forms in a caspase- -dependent manner [ ] . il- β and il- are early and strong pro-inflammatory cytokines that expand the infarct size and drive cardiac dysfunction [ ] . the pharmacological inhibition of il- β and il- with a neutralizing antibody significantly reduces myocardial infarct size and ameliorates cardiac dysfunction in mi mice [ , ] . additionally, the nlrp inflammasome promotes ischemic myocardial injury by causing pyroptosis via activating caspase- [ ] , whereas the inhibition of the nlrp inflammasome reduces the myocardial infarct size and preserves normal cardiac function post mi [ , , ] . in line with the reported finding that nlrp is activated in acute mi [ ] , our results showed that nlrp inflammasome expression was increased, reaching a significant difference at h in the infarcted myocardium of mice. furthermore, klx significantly attenuated the elevation of nlrp , il- β, and il- protein levels. h&e staining analysis showed reduced inflammatory cells in mi mice treated with klx. collectively, these data suggest that klx reduces cardiac damage by attenuating the pro-inflammatory response and reducing subsequent pyroptosis. hypoxia and lps are potent stimuli for cardiomyocyte injury and the secretion of inflammatory factors, which can in part mimic the conditions of cardiac damage in the setting of mi [ , ] . recent studies have found that hypoxia and lps induce cell death by activating the nlrp inflammasome and pyroptosis in cardiomyocytes [ , ] . our results showed that the levels of il- β, il- , nlrp and pyroptosis-related proteins were diminished in nmvcs treated with klx. our tunel and hoechst /pi staining results further demonstrated that klx improved dna damage and cell membrane rupture of cardiomyocytes induced by hypoxia or lps treatment, indicating that it suppressed pyroptotic cell death in cardiomyocytes. there was no significant difference in the protective effect between the μm and μm klx groups, indicating that the cardioprotective effect of klx was efficacious at μm in our model. in conclusion, our study provides the first evidence that klx exerts myocardial protection by inhibiting nlrp inflammasome activation and subsequent pyroptosis in mi hearts and in cardiomyocytes treated with hypoxia or lps as a cellular model of mi injury. our results suggest that klx merits more rigorous and detailed studies regarding its potential for the treatment of mi. in this regard, it is known that inflammasomes and caspase- / pyroptosis are important parts of the innate immune system. it has also been suggested that agents targeting innate immunity may exert acute cardioprotective effects [ ] . our finding that klx acts cardiac remodeling-concepts and clinical implications: a consensus paper from an international forum on cardiac remodeling. behalf of an international forum on cardiac remodeling task force on the management of st-segment elevation acute myocardial infarction of the european society of cardiology (esc) mir- promotes cardiomyocyte apoptosis by inhibiting foxo a expression -hydroxycholesterol induces both p x -dependent pyroptosis and caspase-dependent apoptosis in human skin model: new insights into degenerative pathways inflammasome-activated gasdermin d causes pyroptosis by forming membrane pores pyroptosis: host cell death and inflammation the inflammasome promotes adverse cardiac remodeling following acute myocardial infarction in the mouse the nlrp inflammasome in acute myocardial infarction nlrp inflammasome as a novel player in myocardial infarction nf-kappab-gasdermin d (gsdmd) axis couples oxidative stress and nacht, lrr and pyd domains-containing protein (nlrp ) inflammasome-mediated cardiomyocyte pyroptosis following myocardial infarction caspase- inhibition by vx- administered at reperfusion in p y receptor antagonisttreated rats provides long-term reduction in myocardial infarct size and preservation of ventricular function liraglutide attenuates nlrp inflammasome-dependent pyroptosis via regulating sirt /nox /ros pathway in h c cells aloe-emodin attenuates myocardial infarction and apoptosis via up-regulating mir- expression emodin protects h c cells against hypoxiainduced injury via regulation of mir- a/survivin and the jak /stat pathway emodin attenuates lipopolysaccharide-induced injury via down-regulation of mir- in h c cells rhein protects the myocardiac cells against hypoxia/reoxygention-induced injury by suppressing gsk beta activity aloe-emodin relieves high-fat diet induced qt prolongation via mir- inhibition and ik up-regulation in rats thymosin beta activates integrin-linked kinase and promotes cardiac cell migration, survival and cardiac repair role of nlrp (cryopyrin) in acute myocardial infarction inhibition of the nlrp inflammasome limits the inflammatory injury following myocardial ischemia-reperfusion in the mouse lncrna zfas as a serca a inhibitor to cause intracellular ca + overload and contractile dysfunction in a mouse model of myocardial infarction pedf inhibits the activation of nlrp inflammasome in hypoxia cardiomyocytes through pedf receptor/phospholipase a pyroptosis: gasdermin-mediated programmed necrotic cell death trimetazidine attenuates cardiac dysfunction in endotoxemia and sepsis by promoting neutrophil migration beyond medication prescription as performance measures: optimal secondary prevention medication dosing after acute myocardial infarction electrocardiographic changes associated with beta-blocker toxicity intracerebral haemorrhage after dermal nitrate application dihydromyricetin protects against diabetic cardiomyopathy in streptozotocin-induced diabetic mice salvianolic acid b and tanshinone iia attenuate myocardial ischemia injury in mice by no production through multiple pathways cytoprotective activated protein c averts nlrp inflammasome-induced ischemia-reperfusion injury via mtorc inhibition inhibition of nlrp inflammasome prevents lps-induced inflammatory hyperalgesia in mice: contribution of nf-kappab, caspase- / , asc, nox, and nos isoforms molecular definitions of cell death subroutines: recommendations of the nomenclature committee on cell death the selective nlrp -inflammasome inhibitor mcc reduces infarct size and preserves cardiac function in a pig model of myocardial infarction could nlrp -inflammasome be a cardiovascular risk biomarker in acute myocardial infarction patients? cardiac cytokine expression is upregulated in the acute phase after myocardial infarction. experimental studies in rats interleukin- beta modulation using a genetically engineered antibody prevents adverse cardiac remodelling following acute myocardial infarction in the mouse neutralization of interleukin- ameliorates ischemia/reperfusion-induced myocardial injury nlrp plays no role in acute cardiac infarction due to low cardiac expression p x receptor inhibition attenuated sympathetic nerve sprouting after myocardial infarction via the nlrp /il- beta pathway innate immunity as a target for acute cardioprotection this work was supported in part by the national natural science foundation of china (grants number: , , , and ), the scientific research starting foundation for returned overseas chinese scholars of heilongjiang province, the heilongjiang natural science foundation (grants number: h ), and the china postdoctoral science foundation ( m ). on the components of innate immunity (i.e., inflammasomes and caspase- /pyroptosis) encourages further studies on this process. bfy, wx, wjd, nw, and yb designed the present study; yb, xl, pp, xlh, sty, and ynl performed the experiments; jhw conducted the synthesis of klx; yb, xl, pp, and xl analyzed the data; yb, wjd, and bfy wrote the paper; wjd and nw revised paper. the online version of this article (https://doi.org/ . /s - - - ) contains supplementary material, which is available to authorized users.competing interests: the authors declare no competing interests. key: cord- -qzu gz d authors: zhang, xiao-lei; li, zhuo-ming; ye, jian-tao; lu, jing; ye, lingyu linda; zhang, chun-xiang; liu, pei-qing; duan, dayue d title: pharmacological and cardiovascular perspectives on the treatment of covid- with chloroquine derivatives date: - - journal: acta pharmacol sin doi: . /s - - -x sha: doc_id: cord_uid: qzu gz d the novel severe acute respiratory syndrome coronavirus- (sars-cov- ) causes coronavirus disease (covid- ) and an ongoing severe pandemic. curative drugs specific for covid- are currently lacking. chloroquine phosphate and its derivative hydroxychloroquine, which have been used in the treatment and prevention of malaria and autoimmune diseases for decades, were found to inhibit sars-cov- infection with high potency in vitro and have shown clinical and virologic benefits in covid- patients. therefore, chloroquine phosphate was first used in the treatment of covid- in china. later, under a limited emergency-use authorization from the fda, hydroxychloroquine in combination with azithromycin was used to treat covid- patients in the usa, although the mechanisms of the anti-covid- effects remain unclear. preliminary outcomes from clinical trials in several countries have generated controversial results. the desperation to control the pandemic overrode the concerns regarding the serious adverse effects of chloroquine derivatives and combination drugs, including lethal arrhythmias and cardiomyopathy. the risks of these treatments have become more complex as a result of findings that covid- is actually a multisystem disease. while respiratory symptoms are the major clinical manifestations, cardiovascular abnormalities, including arrhythmias, myocarditis, heart failure, and ischemic stroke, have been reported in a significant number of covid- patients. patients with preexisting cardiovascular conditions (hypertension, arrhythmias, etc.) are at increased risk of severe covid- and death. from pharmacological and cardiovascular perspectives, therefore, the treatment of covid- with chloroquine and its derivatives should be systematically evaluated, and patients should be routinely monitored for cardiovascular conditions to prevent lethal adverse events. coronavirus disease is caused by infection with severe acute respiratory syndrome coronavirus- (sars-cov- ), which has spread around the globe and is causing an ongoing severe pandemic with , , confirmed cases and , deaths worldwide that have been reported to the world health organization (who) as of july , (https://www.who.int/ emergencies/diseases/novel-coronavirus- ). to date, there is still no known approved curative drug therapy specific for covid- , especially for patients with the severe and critical forms [ ] . however, chloroquine phosphate and its derivative hydroxychloroquine, which have been used for decades in the treatment and prevention of malaria and chronic inflammatory diseases such as rheumatoid arthritis and systemic lupus erythematosus, were discovered to have a high inhibitory potency against sars-cov- infection in vitro [ ] [ ] [ ] [ ] and favorable clinical and virologic benefits in covid- patients [ ] [ ] [ ] [ ] [ ] , and they have emerged as important therapies for covid- in several countries, including china, france, usa, and india, although the mechanisms of their anti-covid- effects remain unclear. the desperation to control the pandemic has overridden the concerns regarding the serious adverse effects of chloroquine derivatives and azithromycin, including lethal arrhythmias and cardiomyopathy. clinical trials in several countries have provided controversial results. the findings that covid- is a multisystem disease involving injuries to many organs, including the lung, heart, and vasculature, have made the risks of using these drugs even more complex. covid- patients with preexisting cardiovascular conditions (hypertension, arrhythmias, heart failure, etc.) are at higher risk of severe covid- and death (table ) . here, we provide pharmacological and cardiovascular perspectives on the application of chloroquine derivatives in the treatment of covid- . systematic evaluations of their efficacy and safety, especially of the potential cardiovascular toxicity of chloroquine and hydroxychloroquine and combination therapies with other drugs in the treatment of covid- , and genetic variability in the metabolism of these drugs in patients are required to prevent lethal cardiovascular adverse events. in several in vitro studies, chloroquine [ , ] and hydroxychloroquine [ , ] were found to be able to inhibit sars-cov- infection and show clinical and virologic benefits in covid- patients [ ] [ ] [ ] [ ] [ ] . the two drugs inhibited sars-cov- at low-micromolar concentrations with % cytotoxic concentration (cc ) values over μm, implying the efficacy and safety of these drugs [ ] [ ] [ ] [ ] . although these in vitro and anecdotal data are still limited and the mechanisms of the anti-covid- effects remain unclear, the apparent efficacy and safety of chloroquine phosphate or hydroxychloroquine have attracted much attention for application of these drugs as potential therapies for covid- . chloroquine phosphate was therefore recommended in the guidelines for the prevention, diagnosis, and treatment of covid- by the national health commission (nhc) of china. an open-label nonrandomized study by gautret et al. [ ] that used hydroxychloroquine in combination in some patients with azithromycin, an azalide antibiotic with putative antiviral properties, has garnered unusual attention. hydroxychloroquine in combination with azithromycin was granted limited emergency-use authorization by the usa fda to treat covid- patients. preliminary clinical data showed that chloroquine or hydroxychloroquine prevented the exacerbation of pneumonia, promoted viral clearance and shortened the disease course in sars-cov- -infected patients [ ] [ ] [ ] [ ] . until may th, , a total of clinical trials proposed using hydroxychloroquine, and studies that proposed using chloroquine in the treatment of covid- were registered with the fda (tables and ). for patients diagnosed with mild, moderate and severe cases of sars-cov- pneumonia, mg chloroquine phosphate tablets were given twice a day for days according to the sixth and seventh editions of the guidelines for the prevention, diagnosis, and treatment of pneumonia caused by covid- recommended by the nhc of china. in several other countries, including the united states, hydroxychloroquine has been administered to hospitalized covid- patients due to its higher in vitro activity against sars-cov- and wider availability in these countries compared with chloroquine phosphate. chloroquine and hydroxychloroquine are oral prescription drugs that have been used for several decades in the treatment of malaria and autoimmune diseases. currently, it is not clear why they can inhibit sars-cov- and have therapeutic effects on covid- . since both of these drugs contain an amino group attached to a quinoline ring (fig. ) , they are weak diprotic bases that probably accumulate within intracellular acidic compartments such as lysosomes [ ] . they increase the ph of lysosomes and lead to the expansion, vacuolization and dysfunction of lysosomes [ ] . as a result, these drugs interfere with the fusion process and prevent rna release by neutrophil-to-lymphocyte ratio . ( . - . ) . [ ] the viruses, thus inhibiting the growth of these intracellular pathogens [ , , ] . in addition, these drugs can alter the terminal glycosylation of ace , the cellular receptor of the coronaviruses sars-cov and sars-cov- , preventing virus-receptor binding and abrogating the infection [ ] . this might help to explain the broadspectrum antiviral effects of chloroquine and hydroxychloroquine. indeed, both drugs have been previously demonstrated to have profound antiviral activity against various viruses, including sars-cov [ ] , dengue virus [ , ] , hiv- [ , ] , influenza a [ ] , ebola virus [ ] , and human coronavirus oc [ ] . anti-inflammatory and immunomodulatory effects chloroquine and hydroxychloroquine are regarded as potent antiinflammatory agents and immunomodulators. these drugs have been widely used for many years in the treatment of malaria and autoimmune diseases such as rheumatoid arthritis, systemic lupus erythematosus and sjögren's syndrome. both drugs impair the production of proinflammatory cytokines, including tumor necrosis factor α (tnfα) [ ] [ ] [ ] , interleukin (il)- [ , ] , il- [ , , ] , and interferon-γ [ ] . additionally, they prevent innate immune activation by blocking the interaction between cytosolic dna and the nucleic acid sensor cyclic gmp-amp (cgamp) synthase (cgas) [ ] and the interaction of toll-like receptors (tlrs) with nucleic acid ligands [ ] [ ] [ ] . considering that inflammatory reactions and cytokine storms occur commonly in severe covid- patients, the anti-inflammatory properties of chloroquine and hydroxychloroquine might provide important clinical benefits in the treatment of covid- as well. adverse effects although chloroquine and hydroxychloroquine are generally well tolerated and usually considered safe even during pregnancy, we should be aware of their serious adverse effects, even with a short duration of treatment. these adverse effects include gastrointestinal and cutaneous manifestations, hypoglycemia, neuropsychiatric effects, drug-drug interactions, idiosyncratic hypersensitivity reactions, myopathy, peripheral neuropathy, and cardiac toxicity [ ] . according to a systematic review of case series concerning cardiac complications attributed to chloroquine and hydroxychloroquine, conduction disorders are the main reported side effect, affecting % of patients; other nonspecific adverse cardiac events include ventricular hypertrophy ( %), heart failure ( . %), hypokinesia ( . %), pulmonary arterial hypertension ( . %), and valvular dysfunction ( . %) [ ] . for patients reported to have been withdrawn from treatment, only . % recovered normal heart function, whereas . % died and . % reported irreversible damage [ ] . it is not currently known, however, whether heart is more susceptible to chloroquine treatment than other organs. chloroquine-or hydroxychloroquine-related cardiac disorders, such as prolongation of the qtc interval, are rare but severe and life-threatening. both chloroquine and hydroxychloroquine interfere with ventricular repolarization and may lead to prolongation of the qt interval and increase the risk of torsades de pointes. the effect is dose-dependent: after receiving a dose of mg, the mean qtc increases by . ms, and after a dose of mg, the mean qtc increases by ms [ , ] . however, the effect varies among individuals. among children given short courses of chloroquine for malaria, experienced an increase in the qtc interval of ms after just day of treatment [ ] . potential mechanisms of the adverse effects blockade of k + channels. the activities of k + channels are the major determinants of the repolarization of cardiac myocytes. chloroquine and hydroxychloroquine have been reported to inhibit multiple k + currents, including the inward rectifier k + (kir) current (i k ) [ ] [ ] [ ] [ ] , the rapidly activating delayed rectifier k + current (i kr ) associated with the human eether-a-go-go-go related there are total studies as may th, (searching key word "covid- " and "chloroquine"). gene (herg) [ , ] , the fast transient outward k + current (i to ) [ ] , and the atp-sensitive inward rectifier k + current (k atp ) [ , ] . ( ) i k : chloroquine, hydroxychloroquine and other quinines inhibit the cardiac i k current and can induce lethal ventricular arrhythmias. chloroquine causes a dose-and voltage-dependent reduction in the i k current magnitude, action potential (ap) duration and effective refractory period [ ] [ ] [ ] [ ] . the peak outward current elicited by the ventricular ap is inhibited by chloroquine at a half-maximal inhibitory concentration (ic ) of~ . - μm [ , , , , ] . chloroquine and related compounds can inhibit kir channels by multiple potential mechanisms. first, chloroquine can bind to the cytoplasmic pore domain and block the cytoplasmic conduction pathway, which is stabilized by negatively charged and aromatic amino acids within a central pocket [ ] . unlike most ion channel blockers, chloroquine does not bind within the transmembrane pore and thus can reach its binding site even while polyamines remain deeper within the channel vestibule [ ] . these binding features of chloroquine could explain its relatively low affinity for kir channels but high effectiveness in blocking the i k current [ ] . second, chloroquine shows allosteric effects on channel gating. comparative molecular modeling and ligand docking of chloroquine in the intracellular domains of kir . , kir . and kir . suggest that chloroquine blocks k + flow by interacting with negatively charged amino acids facing the ion permeation vestibule of the channels [ , ] . in addition, chloroquine abrogates the cardiac protective effect of the i k channel activator zacopride, exacerbating myocardial infarction (mi) and post-mi cardiac remodeling [ , ] . ( ) i herg : chloroquine inhibits i herg in a concentration-and time-dependent manner at a relatively low ic of . μm, implying that it is a potent blocker of i herg [ , ] . inhibition of the herg current (i herg ) can lead to a prolongation of the qt interval, which may, under certain circumstances, lead to torsade de pointes [ ] . however, not all herg blockers have a high risk of torsade de pointes. there are drugs that block herg and prolong the heart rate-corrected qt (qtc) interval but have a low risk of torsade de pointes (e.g., ranolazine, verapamil, and amiodarone) because they block other inward currents such as late na + and/or l-type ca + currents [ ] . blocking these inward currents has antiarrhythmic effects by preventing early afterdepolarizations [ ] . thus, these drugs prolong qtc without heart ratecorrected j-t peak (j-t peak c) prolongation and are considered to be balanced ion channel blockers [ ] . to further evaluate the torsade risk of chloroquine, a comprehensive in vitro proarrhythmia assay (cipa) was conducted. chloroquine prolongs both the qtc interval and the j-t peak c interval, suggesting that chloroquine is a strong blocker of i herg with a high torsade risk [ ] . ( ) i to : chloroquine also blocks another important outward k + current, the transient outward k + current i to , at an ic of . ± . mm [ ] . chloroquine accelerates the apparent inactivation time constant of i to . exposure to mm chloroquine results in a rightward shift of the steady-state inactivation constant, whereas recovery from inactivation is only mildly affected. these data suggest that chloroquine is an open-channel blocker of i to and has potential clinical proarrhythmic effects [ ] . ( ) k atp : chloroquine inhibits the k atp current via a fast-onset effect and a slow-onset voltage-independent effect. the fast-onset effect of chloroquine involves direct channel blocking by binding to the channel pore from the cytoplasmic side. in contrast, the slow-onset effect is regulated by the disruption of interactions between k atp and phosphatidylinositol , -bisphosphate [ ] . inhibition of these k + currents leads to delayed repolarization of cardiac myocytes, prolongation of the cardiac action potential duration (apd) and the qt interval of the electrocardiogram, which is a sensitive but nonspecific risk marker for the development of torsade de pointes -a potentially lethal polymorphic ventricular tachyarrhythmia [ ] . therefore, the inhibition of multiple k + currents strongly suggests the proarrhythmic potential of chloroquine. the prominent clinical features of cardiac conduction disorders caused by chloroquine and hydroxychloroquine are prolongation of the qt interval, which increases the risk of sudden death due to the development of torsade de pointes, especially if the drug is coprescribed with other k + channel blockers, such as azithromycin, or used in patients with preexisting cardiac diseases. inhibition of the autophagy-lysosome pathway. as an evolutionarily conserved cellular housekeeping and quality control mechanism essential for homeostasis and survival, autophagy is responsible for the removal of superfluous, aging, or damaged cytoplasmic proteins and organelles from cells and tissues [ ] [ ] [ ] [ ] [ ] [ ] [ ] . the substrates of the autophagic response involve dedicated double-membrane organelles and are commonly known as autophagosomes. autophagosomes are ultimately delivered to the lysosome for degradation [ ] . since chloroquine interferes with acidic hydrolases in lysosomes, it acts as an inhibitor of the autophagy-lysosome pathway [ ] . since dysregulation of autophagy is linked to the occurrence and development of chronic diseases, including cardiovascular diseases, impairing autophagic flux with chloroquine may play a role in the development of various cardiovascular diseases. chloroquine at a high dose ( mg·kg − ·d − , for weeks) exacerbated pressure overload hypertrophy and impaired cardiac contractility in rats [ ] . ultrastructurally, chloroquine accentuates mitochondrial fragmentation and cristae destruction by producing a plethora of autophagosomes containing collapsed mitochondria and lysosomal lamellar bodies, suggesting the impairment of the autophagy-lysosome pathway [ ] . in addition, high-dose chloroquine significantly impairs the mitochondrial antioxidant buffering capacity and accentuates oxidative stress and mitochondrial dysfunction. these observations highlight the risk of chloroquine administration in patients under high-oxidative stress conditions, such as pathological myocardial hypertrophy or heart failure [ ] . during myocardial infarction, cardiomyocyte necroptosis results in the loss of functional cardiac cells, subsequently leading to left ventricular remodeling, cardiac dysfunction and heart failure [ ] . by disrupting autophagic degradation, treatment with chloroquine aggravates cardiac myocyte necroptosis and cardiac dysfunction, contributing to adverse ventricular remodeling and progressive heart failure after myocardial infarction [ ] . although autophagy is regarded as a double-edged sword, it is well accepted that activation of autophagy promotes adaptation to stress and supports cellular viability in response to pathological stimulation [ , ] . thus, inhibition of autophagy generally accelerates the death of cells exposed to potentially lethal perturbations of homeostasis [ , ] . numerous studies have reported that the activation of autophagy might contribute to ameliorating myocardial infarction/reperfusion and myocardial ischemia, protecting cardiomyocytes against apoptosis and necroptosis, and preventing cardiac hypertrophy and cardiac fibrosis [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . however, treatment with chloroquine might reverse the cardioprotective effects by inhibiting autophagic flux [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . furthermore, chloroquine could influence ca + channels through repression of autophagy [ ] . blocking autophagy with chloroquine results in depolarization of the mitochondrial pharmacology of chloroquine in covid- xl zhang et al. membrane potential and increased production of mito-ros, finally facilitating the oxidation of ryanodine receptor (ryr ) [ , ] . ryr is a ca + -releasing channel located on the sarcoplasmic reticulum of cardiomyocytes. the oxidation and dysfunction of ryr enhance pro-arrhythmic spontaneous ca + release under βadrenergic stimulation and disturb intracellular ca + homeostasis, finally resulting in cardiac disorders such as arrhythmia, cardiac hypertrophy and heart failure [ , ] . immunologically mediated adverse reactions. although rare, several severe cutaneous adverse reactions, including stevens-johnson syndrome, toxic epidermal necrolysis [ ] , and drug reaction with eosinophilia and systemic symptoms (dress) [ ] , have been reported to be associated with the use of chloroquine and hydroxychloroquine. patients may manifest with clinical symptoms of new-onset fever and mucositis, particularly when presenting with new hematologic abnormalities (such as lymphopenia, eosinophilia or atypical lymphocytosis) or unexplained liver or kidney injury after the start of treatment with chloroquine or hydroxychloroquine. hydroxychloroquine-azithromycin combination: azithromycin is a macrolide antibiotic agent primarily used for the treatment of respiratory infections and some sexually transmitted infections. since azithromycin also showed antivirus [ , ] and antiinflammatory [ , ] activities and might have synergistic effects when combined with chloroquine or derivatives, a french group used the combination of hydroxychloroquine and azithromycin to treat covid- and observed clinical benefits [ , ] . however, it should be highlighted that the cardiovascular risk would probably be increased. in fact, the tendency of azithromycin to induce arrhythmia and the risk of prolonged cardiac repolarization and qt interval, leading to fatal torsade de pointes, have been well documented [ ] [ ] [ ] . recent data from some cohort studies and retrospective studies demonstrated that chloroquine, hydroxychloroquine and concurrent treatment with azithromycin significantly prolonged the qtc interval in a clinically relevant matter [ ] [ ] [ ] . more than % of patients receiving chloroquine, hydroxychloroquine or the hydroxychloroquine and azithromycin combination developed qtc interval prolongation of > ms, which is a known marker of a high risk of torsade de pointes [ ] [ ] [ ] . compared with monotherapy, the hydroxychloroquine/ azithromycin combination was associated with greater changes in qtc [ ] . the sporadic incidence of torsade de pointes has also been reported [ ] [ ] [ ] . thus, additional caution is advised when administering this combination in patients with cardiac dysfunction and renal and hepatic diseases. chloroquine-propranolol combination: propranolol is widely used for cardiovascular diseases, including angina, hypertension, tachyarrhythmia, cardiac hypertrophy, and prophylaxis of myocardial infarction. however, a study in anesthetized dogs reported that administration of propranolol potentiates the proarrhythmic effects of chloroquine on prolongation of ecg intervals and bradycardia [ ] , suggesting the increased proarrhythmic potential of the chloroquine-propranolol combination. in view of the common use of propranolol in cardiovascular patients, the therapeutic use of chloroquine or hydroxychloroquine in covid- patients under propranolol treatment should be carefully considered. chloroquine-diclofenac combination: although this combination is unlikely to be used in covid- patients, combined therapy with these two drugs for rheumatoid arthritis is not rare. the combination could shift the balance between pro-and antiapoptotic proteins in the left ventricule towards myocardial apoptosis, thus indicating a risk of myocardial damage [ ] . drug-drug interactions. both chloroquine and hydroxychloroquine are metabolized by the hepatic cytochrome p enzyme d (cyp d ), and they also competitively inhibit cyp d activity [ ] . this has the potential to influence the fate of other drugs reliant on cyp d for metabolism. azithromycin exhibits little inhibition of cytochrome p enzymes or drug-transport proteins such as p-glycoprotein. as such, azithromycin is unlikely to precipitate clinically important drug-drug interactions. the expression of cyp d varies among individuals as a result of genetic polymorphisms. genetic variability in the metabolism of these drugs is considerable and may also influence their safety and effectiveness when used in combination for covid- . overdose. overdoses of chloroquine and hydroxychloroquine are extremely toxic. rapid onset of central nervous system toxicity, such as seizures and coma, cardiovascular collapse, including qrs widening and qt interval prolongation, and hypokalemia resulting from intracellular changes may occur. since overdose is the major cause of life-threatening toxicity and adverse events, an appropriate therapeutic dosage within the safety window is extremely important. the strategies used for the treatment of covid- with chloroquine and hydroxychloroquine in clinical applications vary in different countries or hospitals. some of the currently available treatment strategies and the efficacy of chloroquine, hydroxychloroquine and combination therapy for treatment of covid- in clinical studies are summarized in table . we checked the guidelines from the who for drug therapy of covid- and failed to find any guidelines or suggestions on the usage of chloroquine or hydroxychloroquine for covid- . in fact, this is true for any drugs used for covid- . instead, who released advice for "offlabel" use. as stated by the who, "no pharmaceutical products have yet been shown to be safe and effective for the treatment of covid- . however, a number of medicines have been suggested as potential investigational therapies, many of which are now being or will soon be studied in clinical trials, including the solidarity trial cosponsored by who and participating countries. in many countries, doctors are giving covid- patients medicines that have not been approved for this disease. the use of licensed medicines for indications that have not been approved by a national medicine regulatory authority is considered "off-label" use. further, such prescribing should be done on a case-by-case basis." however, the nih released a new guideline for the usage of chloroquine and hydroxychloroquine for covid- in june . the panel recommends against the use of high-dose chloroquine ( mg twice daily for days) for the treatment of covid- (ai). high-dose chloroquine ( mg twice daily for days) has been associated with more severe toxicities than lower-dose chloroquine ( mg twice daily for day, followed by mg once daily for days). the steady-state plasma concentration of chloroquine might reach at least . μm in the treatment of viral infectious diseases [ ] . according to in vitro studies, the ec of chloroquine in inhibiting sar-cov- ranges from . to . μm, whereas that of hydroxychloroquine ranges from . to . μm [ , , ] . however, these concentrations exceed the ic of chloroquine used for blockade of kir ( . - μm) [ , , , ] and herg channels ( . μm) [ ] and might possibly result in increased proarrhythmic risk. at the recommended dosage of chloroquine phosphate in china, the steady-state plasma concentration can reach the therapeutic concentration ( . μm), which also exceeds the ic necessary for kir and herg blockade and is sufficient to result in adverse effects and cause myocardial dysfunction. the safety margin of chloroquine and hydroxychloroquine is narrow. thus, attention should be paid to the proarrhythmic risk of the use of chloroquine or hydroxychloroquine, especially in patients with hepatic or renal dysfunction, which might cause accumulation of drugs. patients with chloroquine or hydroxychloroquine administration should undergo electrocardiogram monitoring. a large number of patients with a history of cardiovascular disease or cardiovascular risk factors appeared to have heightened vulnerability to developing covid- with more severe symptoms and worse clinical outcomes. in some confirmed sars-cov- infection cases, cardiovascular symptoms were one of the most important presentations. clinical data reported that~ %- % of covid- patients have hypertension, and~ . %- % have coronary heart disease [ ] [ ] [ ] [ ] . in a report of , cases from china's cdc, covid- patients who required admission to an intensive care unit were more likely to have comorbidities, the majority of which were cardiovascular disease [ ] . according to the nhc, among the patients who died from covid- , . % had substantial heart damage with elevated troponin i levels or cardiac arrest during hospitalization. another large-scale analysis of , confirmed cases indicated an increased mortality risk for those with diabetes ( . %), hypertension ( %), and cardiovascular disease ( . %), although the mortality rate remains highly variable depending on the region (currently less than % in germany and china and more than % in france and italy) [ ] [ ] [ ] [ ] [ ] . the case fatality rate for underlying cardiovascular disease ( . %) is larger than that for patients with underlying chronic respiratory disease ( . %) [ ] [ ] [ ] [ ] [ ] . for covid- patients in weak cardiac condition, increased oxygen and energy demand imposed by viral illnesses is poorly tolerated. therefore, cardiac involvement is an important feature of covid- and is associated with a poor prognosis. additional evidence that aids in understanding the interplay between covid- and cardiovascular disease from clinical studies may help to attenuate the risk for patients with preexisting cardiovascular diseases or cardiovascular risk factors. covid- patients suffer from cardiovascular injury, and it is necessary to revisit the potential mechanisms underlying these cardiac adverse effects to reexamine the possible drug-drug interactions that might increase cardiovascular risk and to reassess the therapeutic dosage of chloroquine or hydroxychloroquine in the treatment of covid- . with the increasing number of confirmed cases and the accumulating clinical data, it has become clearer that many covid- patients suffer from cardiovascular injury, which is closely associated with worse disease progression in addition to the typical respiratory symptoms caused by sars-cov- infection. the cardiovascular complications of covid- have drawn more attention from both clinicians and investigators, although the exact mechanism for the myocardial injuries caused by sars-cov- is not completely understood [ ] [ ] [ ] . recently, new clinical syndromes associated with coagulopathy and vasculopathy have emerged as a cause of sudden death and other serious clinical manifestations in younger covid- patients [ ] . angiotensinconverting enzyme (ace ), the receptor for sars-cov- and other coronaviruses, is a transmembrane protein expressed by lung alveolar epithelial cells, enterocytes, and vascular endothelial cells whose physiological role is to induce the maturation of angiotensin i to generate angiotensin - , a peptide hormone that controls vasoconstriction and blood pressure [ , ] . the common cardiac manifestations of covid- include acute myocardial injury and arrhythmias. the overall incidence of acute cardiac injury has been variable, but~ %- % of positive cases are known to develop significant elevation of ctni [ ] [ ] [ ] . both tachy-and brady-arrhythmias have occurred in covid- patients. the incidence was much higher ( . %) in patients who required intensive care unit (icu) admission than in those who did not require icu admission ( . %) [ ] . an estimated . % of covid- deaths in china were associated with substantial heart damage with elevated levels of ctni or cardiac arrest during hospitalization, although the deceased patients had no pre-existing cardiovascular disease [ ] . in another case study of patients with covid- , . % of patients had myocardial injury that caused cardiac dysfunction and arrhythmias. myocardial injury is significantly associated with a fatal outcome for covid- [ ] . the prognosis of patients with underlying cardiovascular disease but without myocardial injury was relatively improved [ ] . another recent study found that . % of the hospitalized patients with covid- had arrhythmias, and . % of them had acute myocardial injury [ ] . collectively, covid- patients with severe symptoms often have complications involving acute myocardial injury. cardiac injury had an increased prevalence among covid- patients requiring icu admission. myocardial injury-associated biomarker levels were significantly higher in patients requiring icu admission than those in patients not treated in the icu. therefore, a full understanding of the cardiovascular complications of covid- may help in making more precise and effective therapeutic decisions and improve outcomes by reducing mortality. although covid- has been spreading rapidly and has caused a global pandemic for several months, it is still too early to predict the long-term outcomes for patients who recover from this illness. however, there are reports of complications that occurred soon after the resolution of the acute symptoms [ ] [ ] [ ] [ ] [ ] . case reports from italy showed fulminant myocarditis in a convalescent patient one week after respiratory symptoms were resolved [ ] . moreover, sudden cardiac death (scd) was found to be likely to occur in many nonhospitalized patients with mild symptoms, who were found dead at their home during self-quarantine in the epicenter of the covid- outbreak in italy [ ] . insidious cardiovascular injury might be a potential trigger of scd. therefore, biomarkers for myocardial injury (ctni and nt-probnp) and coagulation (d-dimer) should be pharmacology of chloroquine in covid- xl zhang et al. evaluated early after hospitalization in all covid- patients to attenuate risk and promote intervention. the concern is that background inflammation may persist and evolve silently and manifest later in an insidious manner. there may be chronic sequelae even after an apparent "complete recovery". even after hospital discharge, myocardial injury might result in ventricular fibrosis, which may lead to cardiac arrhythmias and scd. important lessons should be learned from previous experiences with sars, a disease caused by sars-cov that shares considerable similarity with sars-cov- . many survivors from the epidemic develop avascular necrosis, pulmonary fibrosis, and dyslipidemia [ ] [ ] [ ] . thus, cardiovascular involvement may persist long after the resolution of the acute illness. these latter manifestations are particularly important because they represent cardiovascular risk factors that may lead to further cardiovascular injury. therefore, careful followup of cured covid- patients would be critical for understanding the long-term impact of this illness and the protection of these patients from future cardiovascular disease. potential mechanisms for sars-cov- -induced cardiovascular injuries understanding the effects of sars-cov- infection on the cardiovascular system is essential to provide comprehensive medical care for cardiac patients. several studies have shown that high expression of ace is key for sars-cov- to enter into human lung, heart, kidney, and liver cells. single-cell rna-seq analysis of the ace receptor in major human physiological systems revealed that the lung and heart are vulnerable to sars-cov- infection [ ] . it was reported that the blood concentration of ang ii in covid- patients is higher than that in healthy controls and positively correlated with sars-cov- virus loads [ ] . downregulation of ace by sars-cov- infection may cause an increase in ang ii and vascular endothelial injury, increased blood pressure, and reduced contraction function. clinical evidence has also shown that increased viral loads are linked to an increase in cardiac enzymes such as hs-tni and ck-mb and myocardial injury [ ] . pathological manifestations also included interstitial mononuclear inflammatory infiltrates in the hearts of covid- patients [ ] . the cytokine storm caused by sars-cov- infection may lead to further cardiovascular injury. covid- patients had increased blood concentrations of interleukin (il)- , il- , granulocyte-colony stimulating factor (g-csf), interferon-γinducible protein (ip- ), monocyte chemoattractant protein (mcp ), macrophage inflammatory protein -α (mip- α), and tumor necrosis factor-α (tnf-α) [ , ] . unbalanced reactions of th and th cells, increases in ccr + th cells and decreases in cd + /cd + t cells are also observed in covid- patients [ , ] . a similar cytokine profile was found to be associated with the severity of covid- disease. increased inflammatory reactions and cytokine storms lead to tissue swelling, ischemia-reperfusion injury, and heart failure in covid- patients. severe hypoxia from acute respiratory damage caused by sars-cov- infection may impair the myocardial oxygen demand-supply relationship and result in myocardial injury from increased myocardial oxygen demand in the presence of severe hypoxia due to acute lung injury. in addition to the altered myocardial demand-supply ratio, increased coronary blood flow due to systemic inflammation and increased shear stress may lead to plaque rupture and result in acute myocardial infarction. the prothrombotic milieu associated with exaggerated systemic inflammation may further aggravate the risk. cerebrovascular disease and ischemic stroke associated with covid- were reviewed, and it seems that sars-cov- infection was more likely to induce ischemic stroke than influenza a infection [ ] [ ] [ ] . therefore, as shown in fig. , the potential mechanisms underlying sars-cov- -induced cardiovascular effects may be complex, with the involvement of ( ) direct viral infection of cardiac myocytes by sars-cov- ; ( ) systemic inflammation and immune overreactions, including cytokine storm and imbalances in t cells, helper t cells and regulatory t cells; ( ) acute cardiac stress, including an altered myocardial demand-supply ratio, plaque rupture and coronary thrombosis due to respiratory failure and hypoxemia; and ( ) endothelial cell involvement across vascular beds of different organs in covid- and direct viral infection of endothelial cells and diffuse endothelial inflammation (endotheliitis), which could explain the systemic impaired microcirculatory function in different vascular beds and the resulting clinical sequelae in patients with covid- [ ] . from both the pharmacological and cardiovascular points of view, it is urgent to perform a systematic evaluation of the efficacy and safety of the current therapy of covid- with chloroquine and hydroxychloroquine and associated combination therapies. it is extremely important to show caution and to closely monitor cardiovascular function with electrocardiography because the adverse effects of these drugs on the cardiovascular system, especially in covid- patients with cardiovascular conditions and injury as well as kidney and liver diseases, these effects are potentially fatal. covid- treatment: close to a cure? -a rapid review of pharmacotherapies for the novel coronavirus hydroxychloroquine, a less toxic derivative of chloroquine, is effective in inhibiting sars-cov- infection in vitro chloroquine is a potent inhibitor of sars coronavirus infection and spread in vitro antiviral activity and projection of optimized dosing design of hydroxychloroquine for the treatment of severe acute respiratory syndrome coronavirus (sars-cov- ) remdesivir and chloroquine effectively inhibit the recently emerged novel coronavirus ( -ncov) in vitro janus sword actions of chloroquine and hydroxychloroquine against covid- hydroxychloroquine and azithromycin as a treatment of covid- : results of an openlabel non-randomized clinical trial clinical and microbiological effect of a combination of hydroxychloroquine and azithromycin in covid- patients with at least a six-day follow up: a pilot observational study update on use of chloroquine/hydroxychloroquine to treat coronavirus disease (covid- ) breakthrough: chloroquine phosphate has shown apparent efficacy in treatment of covid- associated pneumonia in clinical studies lysosomal sequestration of amine-containing drugs: analysis and therapeutic implications induction of lysosomal dilatation, arrested autophagy, and cell death by chloroquine in cultured arpe- cells targeting endosomal acidification by chloroquine analogs as a promising strategy for the treatment of emerging viral diseases chloroquine could be used for the treatment of filoviral infections and other viral infections that emerge or emerged from viruses requiring an acidic ph for infectivity antiviral activity of chloroquine against dengue virus type replication in aotus monkeys hydroxychloroquineinhibited dengue virus is associated with host defense machinery chloroquine and hydroxychloroquine as inhibitors of human immunodeficiency virus (hiv- ) activity the anti-hiv- activity of chloroquine chloroquine for influenza prevention: a randomised, double-blind, placebo controlled trial chloroquine inhibited ebola virus replication in vitro but failed to protect against infection and disease in the in vivo guinea pig model antiviral activity of chloroquine against human coronavirus oc infection in newborn mice chloroquine inhibits production of tnfalpha, il- beta and il- from lipopolysaccharide-stimulated human monocytes/ macrophages by different modes chloroquine inhibits tumor necrosis factor production by human macrophages in vitro chloroquine and hydroxychloroquine equally affect tumor necrosis factor-alpha, interleukin , and interferon-gamma production by peripheral blood mononuclear cells selective regulation of cytokine secretion by hydroxychloroquine: inhibition of interleukin alpha (il- -alpha) and il- in human monocytes and t cells enhancement of adeno-associated virus-mediated gene therapy using hydroxychloroquine in murine and human tissues chloroquine protects mice from challenge with cpg odn and lps by decreasing proinflammatory cytokine release targeting toll-like receptors by chloroquine protects mice from experimental cerebral malaria chloroquine and inhibition of toll-like receptor protect from sepsis-induced acute kidney injury current and future use of chloroquine and hydroxychloroquine in infectious, immune, neoplastic, and neurological diseases: a mini-review cardiac complications attributed to chloroquine and hydroxychloroquine: a systematic review of the literature pharmacokinetic interactions between primaquine and chloroquine randomized dose-ranging controlled trial of aq- , a candidate antimalarial, and chloroquine in healthy volunteers high-dose chloroquine for uncomplicated plasmodium falciparum malaria is well tolerated and causes similar qt interval prolongation as standard-dose chloroquine in children electrophysiological and pharmacological characterization of human inwardly rectifying kir . channels on an automated patch-clamp platform action potential clamp and chloroquine sensitivity of mutant kir . channels responsible for variant short qt syndrome the molecular basis of chloroquine block of the inward rectifier kir . channel specific residues of the cytoplasmic domains of cardiac inward rectifier potassium channels are effective antifibrillatory targets assessment of multi-ion channel block in a phase i randomized study design: results of the cipa phase i ecg biomarker validation study inhibition of herg k + currents by antimalarial drugs in stably transfected hek cells open channel block of the fast transient outward k + current by primaquine and chloroquine in rat left ventricular cardiomyocytes molecular mechanisms of chloroquine inhibition of heterologously expressed kir . /sur a channels structural bases for the different anti-fibrillatory effects of chloroquine and quinidine chloroquine blocks a mutant kir . channel responsible for short qt syndrome and normalizes repolarization properties in silico activation of i k channel by zacopride attenuates left ventricular remodeling in rats with myocardial infarction i k channel agonist zacopride alleviates cardiac hypertrophy and failure via alterations in calcium dyshomeostasis and electrical remodeling in rats in vitro cardiovascular effects of dihydroartemisin-piperaquine combination compared with other antimalarials molecular and cellular mechanisms of cardiac arrhythmias aging and autophagy in the heart autophagy: a lysosome-dependent process with implications in cellular redox homeostasis and human disease autophagy in cardiovascular health and disease autophagy as an emerging target for covid- : lessons from an old friend, chloroquine autophagy as a regulator of cardiovascular redox homeostasis autophagy and mitophagy in the myocardium: therapeutic potential and concerns autophagy and mitophagy in diabetic cardiomyopathy high-dose chloroquine is metabolically cardiotoxic by inducing lysosomes and mitochondria dysfunction in a rat model of pressure overload hypertrophy necroptosis mediated by impaired autophagy flux contributes to adverse ventricular remodeling after myocardial infarction autophagic dysregulation in doxorubicin cardiomyopathy gastrodin pretreatment alleviates myocardial ischemia/reperfusion injury through promoting autophagic flux autophagy activation attenuates angiotensin ii-induced cardiac fibrosis the cardioprotective compound cloxyquin uncouples mitochondria and induces autophagy tetramethylprazine attenuates myocardial ischemia/reperfusion injury through modulation of autophagy sevoflurane postconditioning protects against myocardial ischemia/reperfusion injury by restoring autophagic flux via an no-dependent mechanism spermidine-enhanced autophagic flux improves cardiac dysfunction following myocardial infarction by targeting the ampk/mtor signalling pathway dexmedetomidine prevents septic myocardial dysfunction in rats via activation of alpha nachr and pi k/ akt-mediated autophagy ischemic postconditioning regulates cardiomyocyte autophagic activity following ischemia/reperfusion injury nobiletin attenuates adverse cardiac remodeling after acute myocardial infarction in rats via restoring autophagy flux enhancing autophagy diminishes aberrant ca + homeostasis and arrhythmogenesis in aging rabbit hearts hydroxychloroquine-induced fatal toxic epidermal necrolysis complicated by angioinvasive rhizopus hydroxychloroquine-induced dress syndrome azithromycin protects against zika virus infection by upregulating virus-induced type i and iii interferon responses azithromycin, a -membered macrolide antibiotic, inhibits influenza a(h n ) pdm virus infection by interfering with virus internalization process the anti-inflammatory effects of erythromycin, clarithromycin, azithromycin and roxithromycin on histamine-induced otitis media with effusion in guinea pigs role of prophylactic azithromycin to reduce airway inflammation and mortality in a rsv mouse infection model azithromycin and the risk of cardiovascular death electrophysiologic studies on the risks and potential mechanism underlying the proarrhythmic nature of azithromycin azithromycin and the risk of cardiovascular complications chloroquine-induced qtc prolongation in covid- patients risk of qt interval prolongation associated with use of hydroxychloroquine with or without concomitant azithromycin among hospitalized patients testing positive for coronavirus disease (covid- ) the qt interval in patients with covid- treated with hydroxychloroquine and azithromycin the effect of chronic chloroquine toxicity on blood pressure of rats rho-kinase inhibitors ameliorate diclofenac-induced cardiotoxicity in chloroquine-treated adjuvant arthritic rats influence of hydroxychloroquine on the bioavailability of oral metoprolol clinical features of patients infected with novel coronavirus in wuhan covid- and the cardiovascular system clinical characteristics of hospitalized patients with novel coronavirus-infected pneumonia in wuhan characteristics of and important lessons from the coronavirus disease (covid- ) outbreak in china: summary of a report of cases from the chinese center for disease control and prevention clinical characteristics of coronavirus disease in china cardiovascular implications of the covid- pandemic: a global perspective vasculopathy and coagulopathy associated with sars-cov- infection covid- : are we dealing with a multisystem vasculopathy in disguise of a viral infection? the sars-cov- receptor, ace- , is expressed on many different cell types: implications for ace-inhibitor-and angiotensin ii receptor blocker-based cardiovascular therapies the ace expression in human heart indicates new potential mechanism of heart injury among patients infected with sars-cov- hypertension and its severity or mortality in coronavirus disease (covid- ): a pooled analysis sanchis-gomar f. cardiac troponin i in patients with coronavirus disease (covid- ): evidence from a meta-analysis laboratory abnormalities in patients with covid- infection cardiovascular implications of fatal outcomes of patients with coronavirus disease (covid- ) cardiac involvement in a patient with coronavirus disease (covid- ) altered lipid metabolism in recovered sars patients twelve years after infection thin-section ct in patients with severe acute respiratory syndrome following hospital discharge: preliminary experience avascular necrosis of bone in severe acute respiratory syndrome single-cell rna-seq data analysis on the receptor ace expression reveals the potential risk of different human organs vulnerable to -ncov infection clinical and biochemical indexes from -ncov infected patients linked to viral loads and lung injury covid- and decompressive hemicraniectomy for acute ischemic stroke cerebrovascular disease in patients with covid- : a review of the literature and case series covid- and stroke: casual or causal role? endothelial cell infection and endotheliitis in covid- predictors of mortality for patients with covid- pneumonia caused by sars-cov- : a prospective cohort study diabetes mellitus is associated with increased mortality and severity of disease in covid- pneumonia -a systematic review, meta-analysis, and meta-regression clinical course and risk factors for mortality of adult inpatients with covid- in wuhan, china: a retrospective cohort study lactate dehydrogenase levels predict coronavirus disease (covid- ) severity and mortality: a pooled analysis neutrophil-to-lymphocyte ratio as an independent risk factor for mortality in hospitalized patients with covid- efficacy of hydroxychloroquine in patients with covid- : results of a randomized clinical trial a pilot study of hydroxychloroquine in treatment of patients with moderate covid- no evidence of rapid antiviral clearance or clinical benefit with the combination of hydroxychloroquine and azithromycin in patients with severe covid- infection competing interests: the authors declare no competing interests. key: cord- -t zmvm authors: guo, chen-hong; cao, ting; zheng, long-tai; waddington, john l; zhen, xue-chu title: development and characterization of an inducible dicer conditional knockout mouse model of parkinson’s disease: validation of the antiparkinsonian effects of a sigma- receptor agonist and dihydromyricetin date: - - journal: acta pharmacol sin doi: . /s - - - sha: doc_id: cord_uid: t zmvm parkinson’s disease (pd) is a common neurodegenerative disease characterized by motor impairment and progressive loss of dopamine (da) neurons. at present, the acute application of neurotoxic drugs such as -methyl- -phenyl- , , , -tetrahydropyridine (mptp) and -hydroxydopamine ( -ohda) are commonly used to simulate the pathology of pd; however, it is difficult to induce the progressive pathogenesis of pd with these models. in this study, we employed dat promoter-mediated cre transgenic mice to establish tamoxifen-inducible dicer conditional knockout (cko) mice in an effort to mimic the progressive loss of da neurons and the development of pd-like behavioral phenotypes. the results showed that dicer cko mice exhibited progressive loss of da neurons in the substantia nigra (sn) following tamoxifen administration. significant da loss was observed weeks after tamoxifen administration; accordingly, progressive motor function impairment was also observed. we also found that a significant neuroinflammatory response, as evidenced by microglial proliferation, another hallmark of pd pathogenesis, accompanied the loss of da neurons. the acute application of levo-dopa (l-dopa) relieved the pd-like motor impairments in dicer cko mice to exert its antiparkinsonian action, indicating that the model can be used to evaluate the antiparkinsonian efficacy of pd drugs. to further elucidate the potential application of this novel pd animal model for pd drug development, we employed the powerful neuroprotective agent dihydromyricetin (dhm) ( mg/kg) and the selective sigma- receptor agonist pre- ( mg/kg), both of which were previously shown to produce antiparkinsonian effects. the results indicated that the chronic administration of either dhm or pre- attenuated the dicer cko-induced loss of da neurons and motor impairments, although the two drugs acted through different mechanisms. these data indicate that the dicer cko mouse model may be a useful model for investigating the pathological development of pd and intervention-mediated changes. in conclusion, this transgenic mouse model appears to simulate the progressive pathogenesis of pd and may be a potentially useful model for pd drug discovery. parkinson's disease (pd) is characterized by progressive damage to dopamine (da) neurons in the midbrain substantia nigra (sn) [ , ] . -methyl- -phenyl- , , , -tetrahydropyridine (mptp), hydroxydopamine ( -ohda), and other neurotoxic compounds are commonly used in the laboratory to establish pd models. these neurotoxins are selectively taken up by da neurons and are mainly targeted to the mitochondrial complex, eventually causing the death of da neurons, and they have been widely used for pd drug evaluation and pathological studies of diseases [ , ] . however, these neurotoxin-induced animal models cause acute death of da neurons and therefore do not simulate the progressive pathogenesis of pd. thus, there is an unmet need to develop novel animal models that resemble the chronic pathological development of pd. micrornas (mirnas) are single-stranded noncoding rnas composed of nucleotides that play an important role in neuronal development and function in both physiological and disease conditions [ ] [ ] [ ] . dicer is a key enzyme in the progression of mirna synthesis. dicer-specific knockout of hippocampal neurons using the calmodulin kinase (camk ii) promoter induces hippocampal neuronal death and neuroinflammation [ ] ; dicerspecific knockout in the cerebellum also causes purkinje cell death and behavioral ataxia in mice [ ] . moreover, kim and colleagues constructed a mouse line with dicer conditional knockout (cko) driven by the dat promoter using cre recombinase, revealing the key role of mirna in da neuronal survival for the first time. they found that the loss of da neurons began weeks after dicer deletion and that % da cell death was reached after weeks [ ] . in addition, pang et al. employed a different approach, using virus-mediated cre expression in adult floxed dicer knockin mice and found that the motor ability of mice was impaired due to the loss of da neurons in the sn, further confirming the importance of dicer for the survival of da neurons [ ] . these studies revealed that dicer depletion-induced da neuronal death is a progressive process that is distinct from the neurotoxin-induced acute death of da neurons and that may somehow resemble the progressive death in pd pathology. however, the detailed pathological changes related to dicer depletion and behavioral alterations remain to be addressed. moreover, the potential value of this model for pd drug evaluation requires further study. in addition, given that pd is an aging-related disease, inducible dicer depletion in da neurons in adult animals may be a better approach for mimicking the pathology of pd. for this purpose, we developed dat promoter-mediated cre transgenic mice to establish an inducible dicer cko mouse line to mimic progressive da neuron loss. we first characterized developmental changes in both behavior and pathology following dicer cko in da neurons. these results indicated that dicer cko in da neurons in response to tamoxifen administration in adult mice induced the progressive development of pd-like phenotypes. we further investigated the potential significance of the dicer cko mouse model for pd drug evaluation by comparing the effects of various compounds, such as levo-dopa (l-dopa), the neuroprotective agent dihydromyricetin (dhm) and a sigma- receptor agonist, which were previously shown to elicit promising anti-pd effects through different mechanisms. these results clearly indicated that the dicer cko mouse model can serve as an alternative experimental pd model for drug development with the advantage of mimicking the progressive development of pd pathology. inducible dicer cko mouse preparation floxed dicer mice (dicer f/f ) and dat-icreer transgene mice were donated by dr. xinyuan lv. (medical college of georgia, usa) and bred under spf conditions (temperature: ± °c, humidity: % ± %, -h light-dark cycle) with free access to food and water. all animal experiments were approved by the institutional animal care and use committee of soochow university and were in accordance with the guidelines for the care and use of laboratory animals. dicer f/f mice were crossed with dat-icreer mice to obtain dat-icreer;dicer f/+ mice, the latter were bred with dicer f/f mice to produce dat-icreer;dicer f/f mice; these mice were regarded as dicer cko mice, and their littermates were used as controls. the genotypes of the mice were determined by pcr using genomic dna from the toes of the mice and the following primer sequences: ( ) dicer: forward ′-cctgacagtgacggtccaaag- ′ reverse ′-catgactcttcaactcaaact- ′. dicer deletion was induced in -to -week-old mice. briefly, mg of tamoxifen (sigma-aldrich, usa, t ) was diluted in sunflower oil at a final concentration of mg/ml and injected intraperitoneally (i.p.) twice a day over five consecutive days. the animals then received different treatments: ( ) tamoxifen administration alone: the mice were used to observe pathological and behavioral changes at various times following dicer cko; ( ) the l-dopa treatment group: the mice were injected with mg/kg l-dopa (sigma-aldrich, usa, d ) combined with mg/kg benserazide (sigma-aldrich, usa, b ) weeks after tamoxifen administration to validate the antiparkinsonian effect of l-dopa through behavioral measurements; ( ) dhm treatment: the mice were administered tamoxifen followed by daily injection of mg/kg dhm (nature standard, china, st mg) for weeks to evaluate the neuroprotection and antiparkinsonian effects of the drug; and ( ) sigma- receptor agonist treatment: pre- (mce, usa, hy- a) was injected daily together with tamoxifen for consecutive weeks, and the antiparkinsonian effects were evaluated weekly after weeks of tamoxifen administration. pole test and rotarod test: the experiments were performed as previously reported by our group [ , ] . in brief, before the experiments, the mice were trained to crawl downward. during the experiment, the mice were placed head down at the top of a rough wooden pole ( cm length, cm in diameter), and the time required for the mice to descend from the top of the pole to the ground was recorded. the experiment was repeated three times for each mouse. for the rotarod test, the mice were first trained until they were able to remain on the rotarod for more than s at a speed of revolutions per minute (r/min). during the experiment, the speed of the rotarod was increased from to r/min within min. the mice were allowed to walk freely on the rotarod, and the latency time to fall from the rotarod was recorded. the measurement was repeated three times for each mouse. western blotting after the behavioral tests, the mice were sacrificed to collect brain tissues for biological tests. total protein isolated from mouse sn tissues were lysed in ripa buffer and denatured at °c for min. protein concentrations were determined using a bca protein assay kit. proteins ( μg) were loaded on sds-page gels and transferred to pvdf membranes for h at ma. the membranes were incubated with % nonfat milk for h at room temperature before incubation with the respective antibodies, including anti-th ( : ; millipore, usa), anti-α-tubulin ( : , ; sigma-aldrich, usa), and anti-β-tubulin ( : , ; sigma-aldrich, usa), at °c overnight. the membranes were washed three times with tbst. the membranes were incubated with respective mouse igg ( : , ; sigma-aldrich, usa) or rabbit igg ( : , ; sigma-aldrich, usa) secondary antibodies for h at room temperature. the results were analyzed using imagej software. immunofluorescence staining after the behavioral tests, some mice were anesthetized with % chloral hydrate and then perfused through the left ventricle with pbs (ph . ) followed by % paraformaldehyde in pbs. mouse brains were collected and postfixed in paraformaldehyde overnight at °c and then dehydrated in % sucrose at °c for days. the brain tissues were serially cut into -μm coronal sections using a freezing microtome. for immunofluorescence staining, the sections were washed three times with pbs and incubated in pbs containing % bsa and . % triton for h at room temperature. the sections were then incubated with an anti-th ( : , millipore, usa) or anti-iba ( : ; wako, japan) antibody for h at °c. then, the sections were washed in pbst (pbs containing . % triton) and incubated with alexa fluor conjugated goat anti-mouse igg ( : ; thermo fisher scientific, usa) or alexa fluor -conjugated goat anti-rabbit igg ( : ; thermo fisher scientific, usa) for h at room temperature in the dark. the sections were washed and then mounted using mounting medium. images were captured using a laser confocal fluorescence microscope. statistical analysis data analysis was performed by using graphpad prism . software. the results are expressed as the mean ± standard error of the mean (sem). one-way or two-way anova was used for multiple-group comparisons. *p < . was considered statistically significant. dicer cko mice showed a progressive decline in motor ability and damage to da neurons we first examined changes in the motor ability of the mice after tamoxifen administration to induce dicer cko in da neurons at different time points. as shown in fig. , impairments in motor activity were observed at week and worsened progressively with time. there was no difference in motor activity between male and female animals. in association with this observation, we also detected the progressive loss of da neurons in the sn, as measured by tyrosine hydroxylase (th) expression using western blotting. this was further confirmed by immunostaining with an anti-th antibody (fig. ) . these data confirmed that the induction of dicer cko in da neurons in adult mice resulted in progressive da neuronal loss and that the mice progressively developed a pdlike phenotype. these results indicated that these transgenic animals may represent a novel animal model for the study of pd, particularly the progressive development of pd-like behavioral and pathological phenotypes, that may be a useful tool for evaluating the protection of da neurons or for studying the mechanism of the effects of interventions or drugs. progressive neuroinflammation during pd-like pathological development in dicer cko mice neuroinflammation is believed to contribute to the development of pd. the modulation of neuroinflammation has become an important drug target for neurodegenerative diseases, including pd [ ] [ ] [ ] . to elucidate the inflammatory response during the development of pd-like phenotypes in the dicer cko mice, the animals were sacrificed and their brains were collected , , and weeks after tamoxifen administration. immunofluorescence staining for the microglial marker iba was performed (fig. ) . the results revealed a progressive increase in the number of iba immunopositive cells from week , which coincided with the onset of da neuronal death. the above results revealed that dicer knockout can cause progressive neuroinflammation in the sn, which is one of the hallmarks of pathology in pd. acute application of l-dopa to dicer cko mice relieved pd-like motor impairments we characterized the pd-like behavioral and pathological phenotypes in inducible dicer cko mice. we next tested the potential application of this novel model to study the efficacy of pd drugs. dicer cko mice, which steadily developed pd-like behaviors in response to tamoxifen induction, were administered l-dopa, and the results indicated that l-dopa treatment significantly relieved motor impairments, as evidenced by performance on the pole test and rotarod test (fig. ) , indicating that the dicer cko mouse line can be used as an alternative animal model for evaluating the efficacy of pd drugs. chronic application of the neuroprotective agent dhm or the sigma- receptor agonist pre- attenuated da neuronal damage and behavioral abnormalities in dicer cko mice to further evaluate the potential application of dicer cko mice in pd research, we investigated whether this animal model can be used to study the preventive effect of neuroprotective agents or antiparkinsonian agents on pd pathological development given the progressive nature of pd-like pathology in dicer cko mice. to this end, we employed dhm, a powerful neuroprotective agent that was previously shown to protect da neuron [ ] , and pre- , a selective sigma- receptor agonist, since the modulation of the sigma- receptor was recently identified an important target for pd drug discovery and treatment [ ] . dicer cko mice were administered dhm, pre- , or vehicle for consecutive weeks, and the drug treatments were started after tamoxifen induction. behavioral tests were performed every other week starting from week following tamoxifen administration. after the behavioral tests, the mice were sacrificed for biological tests (fig. a) . the results showed that, in comparison with tamoxifen alone, dhm treatment significantly attenuated dicer cko-induced impairments in motor functions (fig. b, c) . similarly, the chronic administration of the selective sigma- receptor agonist pre- also dramatically protected against impairments in motor functions (fig. d, e) . in support of these findings, the western blot results indicated that after dhm administration, the expression of th was significantly higher than that in the control (no drug treatment group) (fig. a, b) . meanwhile, we found that chronic dhm treatment significantly reduced the number of microglial cells, suggesting a decrease in neuroinflammation (fig. c-e) . in addition, treatment with the sigma- receptor agonist pre- also significantly restored th expression (fig. a, b) and fig. dicer cko mice exhibited a progressive impairment in motor activities. eight-to -week-old mice were injected with mg/ml tamoxifen (i.p.) twice a day for days. the basal motor activity was monitored before drug administration and after last drug injection. after last injection, mice were measured by a pole test and b rotarod test every other week. data were presented as mean ± sem. statistical analyses were performed by two-way anova. n = per group. (*p < . , **p < . ; tamoxifen♂ vs vehicle♂. # p < . , ## p < . ; tamoxifen♀ vs vehicle♀.^p < . ; tamoxifen♂, week vs time after tamoxifen administration. & p < . ; tamoxifen♀, week vs time after tamoxifen administration) suppressed reactive microglia proliferation, as evidenced by the decreased number of iba -positive cells (fig. c-e) . taken together, these results suggest that chronic dhm or pre- treatment can protect da neurons and reduce neuroinflammation in response to dicer cko. in this study, we established an inducible da neuron-specific dicer cko mouse line by crossing floxed dicer mice (dicer f/f ) with dat-icreer mice to obtain dat-icreer;dicer f/+ mice, the latter were bred with dicer f/f mice to produce dat-icreer;dicer f/f mice, which we called dicer cko mice. following tamoxifen induction, da neurons experienced progressive death, and a significant loss of da neurons was observed weeks after the depletion of dicer in the sn. accordingly, the behavioral tests revealed progressive pd-like impairment of motor functions. in addition, we also observed time-dependent inflammation in the sn during the development of pd-like phenotypes, and microglia-mediated neuroinflammation was regarded as the hallmark of pd pathogenesis. these data revealed that dicer cko mice develop pd-like pathological and behavioral phenotypes, specifically in a progressive manner. the acute administration of l-dopa to dicer cko mice significantly relieved the impairment of motor functions, indicating the potential of the dicer cko mouse model in evaluating pd drug efficacy. we further found that the chronic administration of dhm or a selective sigma- receptor agonist significantly attenuated the development of pd-like pathological and behavioral changes in dicer cko mice. although the mechanisms of the respective drugs are different, this model can also be used to investigate the pathological mechanism of pd and to validate the effects of various antiparkinsonian drugs. fig. dicer cko mice manifested a progressive reduction on th expression in sn. mice were sacrificed at indicated time points after tamoxifen administration. mice were subjected to tissue preparations for either dissecting for western assay or perfusion for immunostaining, respectively as described in methods. a the expression of th in sn were measured by western blot. b the immunofluorescence staining with th in sn. representative photomicrographs were shown at × magnification (scale bars, μm). c quantitative analyses of th expression and d counts of th-positive neurons. data were presented as mean ± sem. statistical analyses were performed by two-way anova. n = per group (**p < . ; tamoxifen vs vehicle) mirnas are single-stranded noncoding rnas that bind to targeted gene sequences to inhibit target gene translation. recently, many studies have revealed the important functional roles of mirnas in neuronal differentiation, development, and function [ , ] . moreover, alterations in mirnas have been widely reported to be associated with neuropsychiatric diseases [ , ] . for example, we recently reported that let- c and mirna- b are involved in microglial activation and contribute to neuronal survival and functional recovery in stroke [ , ] , indicating the importance of mirnas in neuroprotection and fig. . a double immunofluorescence staining with respective anti-th and anti-iba antibodies were performed in brain sections at various time points. iba -positive-cell numbers in sn were increased with time in response to the dicer cko. representative photomicrographs were shown at × magnification (scale bars, μm). b quantitative analyses of th-positive neurons and c ibal-positive neurons. data were presented as mean ± sem. statistical analyses were performed by one-way anova. n = per group (*p < . , **p < . ; tamoxifen vs vehicle) fig. l-dopa alleviated the motor disorder in dicer cko mice. eight weeks after tamoxifen or vehicle administration, mice were subjected to behavioral tests to monitor the motor impairments. mice were then injected with mg/kg l-dopa with mg/kg benserazide before the behavioral tests. a pole test and b rotarod were tested h later. data were presented as mean ± sem. statistical analyses were performed by two-way anova. n = per group (**p < . ; no l-dopa injection, vehicle vs tamoxifen. ## p < . ; dicer cko mice, no l-dopa vs l-dopa) neuroinflammation. in relation to the current study, alterations in mirnas in pd pathological development have been widely studied, and circulating mirnas have also been suggested to be potential novel biomarkers for pd. moreover, it has been suggested that targeting specific mirnas could be a potential therapeutic approach for pd. for instance, treatment with mir- agomir was shown to reduce the loss of da neurons in an mptptreated mouse model, inhibiting bim expression and thus suppressing bax translocation to mitochondria [ ] . dicer is a type iii rnase and is responsible for the processing of mirna precursors into functional mirnas, thus playing a crucial role in mirna regulation [ ] . dicer expression is essential not only for midbrain development in mice and da neuron maintenance and survival during early postnatal development, but also for da neuron survival in adult mice [ ] . recently, a reduction in dicer in the ventral midbrain and alterations in mirna expression profiles in laser-microdissected da neurons of aged mice have been demonstrated [ ] . moreover, mir- b was reported to be specifically deficient in midbrain da neurons from pd patient samples. it was further found that mir- b alters the survival of da neurons through negative feedback regulation with pitx [ ] . additionally, it was shown that the stimulation of mirna biosynthesis promotes the survival of cultured da neurons and reduces their vulnerability to thapsigargin-induced endoplasmic reticulum stress [ ] . in agreement with these observations, our adult dicer cko mice show progressive loss of da neurons in the sn in response to tamoxifen induction and consequently the presence of pd-like impairments in motor functions. we have characterized the detailed time-dependent changes and degeneration of da neurons in the sn in response to the specific deletion of dicer driven by dat-cre. a slightly different time course of pd-like phenotypes than that reported by a previous study that employed a different approach to delete dicer from da neurons, in which % da loss was observed at week . we found dramatic da loss at week following tamoxifen induction [ ] . the specific mechanism by which dicer cko induces da neuron loss remains to be further studied. in addition to the direct effect of dicer depletion on da neurons, we found that the number of iba -positive cells in the sn increased significantly weeks after tamoxifen injection, which was consistent with the time at which neurons began to die. the depletion of dicer induced the activation of microglia-mediated neuroinflammation. given that all mice were injected with mg/ml tamoxifen (i.p.) twice a day for days as described before. meanwhile, mice also received daily injection mg/kg of dhm, mg/kg of pre- or vehicle, respective for weeks. the behavioral test were conducted every other week started from week as described in methods. a flowchart of experimental procedures. b-e pole test and rotarod test were performed in mice at indicated times with or without dhm and pre- injection, respectively. data were presented as mean ± sem. statistical analyses were performed by two-way anova. n = per group (*p < . , **p < . , ***p < . ; tamoxifen vs vehicle. # p < . , ## p < . ; drugs vs tamoxifen) dicer conditional knockout parkinson's disease mouse model ch guo et al. neuroinflammation is another hallmark of pd pathology and that mirnas have been widely reported to be involved in the regulation of neuroinflammation [ ] , it is conceivable that dicer depletion-mediated microglial activation may also contribute to da neuron death. in addition to pathological development, the mice also exhibited progressive impairments in motor functions that resembled the pd-like phenotypes. it was also noted that we did not detect any difference between males and females. therefore, dicer cko mice may be an alternative pd animal model. indeed, the acute application of l-dopa significantly relieved motor impairments in response to dicer cko weeks after tamoxifen induction. furthermore, the chronic administration of neuroprotective agent dhm and the selective sigma- receptor agonist pre- significantly attenuated the development of pd-like behaviors and pathological phenotypes. all data indicate that dice cko mice may serve as a useful pd model. the chronic application of the neuroprotective agent dhm has been shown to produce powerful neuroprotective effects in models of different diseases, including pd and stroke [ , ] . as a traditional chinese medicine, it was initially used to treat cough and fever and later found to have many pharmacological effects, such as protective effects on the heart, diabetes, liver, and nervous system, possibly through reducing oxidative stress or antiinflammation [ , ] . the neuroprotective effects of dhm were further suggested to be associated with its modulation of the akt-gsk β signaling pathway [ ] . in this study, we confirmed that dhm administration for weeks reduced motor dysfunction, attenuated da neuron death, and reduced microglial activation in mice da neurons with dicer cko. the sigma- receptor is an important target for a few cns diseases [ , ] . the sigma- receptor agonist pre- can alleviate high spontaneous activity in mice and reduce da outflow and release induced by acute methamphetamine administration [ ] . in a chronic unpredicted mild stress model (cums), somcl- , a recently discovered sigma- allosteric modulator, was reported to elicit an antidepressant effect through the bdnf-gsk pathway [ ] . in addition, the administration of the low-dose sigma- receptor selective agonist pre- for consecutive weeks produced a good therapeutic effect in a -ohda-induced pd model [ ] . in agreement with these observations, we also found that fig. chronic dhm treatment alleviated da neuron loss in dicer cko mice. after last behavioral tests as described in fig. a , mice were sacrificed either for brain tissue dissection or for immunostaining as described in methods. a, b th expression was detected by anti-th antibody using western blot. c immunostaining was conducted with respective antibody of th and iba . representative photomicrographs were shown at × magnification (scale bars, μm). d quantitative analyses of th-positive neurons and e ibal-positive neurons. data were presented as mean ± sem. statistical analyses were performed by one-way anova. n = per group (*p < . , **p < . ; tamoxifen vs vehicle. # p < . , ## p < . ; dhm vs tamoxifen) dicer conditional knockout parkinson's disease mouse model ch guo et al. long-term administration of pre- improved motor ability, protected da neurons, and suppressed neuroinflammation, further confirming that our transgenic mouse model is valuable for evaluating the efficacy of pd drugs. in summary, the present study demonstrated that adult daticre;dicer f/f transgenic mice develop progressive da neuron loss and pd-like behaviors accompanied by progressive neuroinflammation following tamoxifen induction. the acute application of l-dopa can improve the impaired motor ability of dicer cko mice. therefore, we believe that this model could be used to simulate the progressive and irreversible pathogenesis of pd and to study the pathogenesis of pd. in addition, we evaluated dhm and pre- , two compounds that have been shown to elicit antiparkinsonian effects through different mechanisms, and found that the chronic application of either dhm or pre- attenuated the development of pd-like behavioral and pathological phenotypes, further implying that the use of this model is an alternative approach for pd drug discovery. fig. chronic pre- treatment alleviated da neuron loss in dicer cko mice. the experiments were conducted as described in fig. a . a, b th expression in sn. c th and iba immunostaining. representative photomicrographs were shown at × magnification (scale bars, μm). d quantitative analyses of th-positive neurons and e ibal-positive neurons. data were presented as mean ± sem. statistical analyses were performed by one-way anova. n = per group (*p < . , **p < . ; tamoxifen vs vehicle. # p < . , ## p < . ; pre- vs tamoxifen) parkinson's disease: a review parkinson's disease protocol for the mptp mouse model of parkinson's disease effect of intrastriatal -ohda lesions on extrastriatal brain structures in the mouse micrornas: genomics, biogenesis, mechanism, and function micrornas in neuronal function and dysfunction genetic ablation of dicer in adult forebrain neurons results in abnormal tau hyperphosphorylation and neurodegeneration age-dependent neuron loss is associated with impaired adult neurogenesis in forebrain neuron-specific dicer conditional knockout mice cerebellar neurodegeneration in the absence of micrornas a microrna feedback circuit in midbrain dopamine neurons dicer expression is essential for adult midbrain dopaminergic neuron maintenance and survival activation of ampk/ mtorc -mediated autophagy by metformin reverses clk deficiency-sensitized dopaminergic neuronal death dihydromyricetin protects neurons in an mptpinduced model of parkinson's disease by suppressing glycogen synthase kinase- beta activity clk deficiency promotes neuroinflammation and subsequent dopaminergic cell death through regulation of microglial metabolic reprogramming microglia function in the central nervous system during health and neurodegeneration neuroinflammation in parkinson's disease and its potential as therapeutic target pharmacological stimulation of sigma- receptors has neurorestorative effects in experimental parkinsonism micrornas in parkinson's disease conditional loss of dicer disrupts cellular and tissue morphogenesis in the cortex and hippocampus dysregulation of mirna and its potential therapeutic application in schizophrenia dicer deficiency differentially impacts microglia of the developing and adult brain microrna let- c- p protects against cerebral ischemia injury via mechanisms involving the inhibition of microglia activation mirna- b contributes to neuroinflammation following cerebral ischemia mir- regulates apoptosis and autophagy process in mptp model of parkinson's disease by targeting to bim jnk-mediated microglial dicer degradation potentiates inflammatory responses to induce dopaminergic neuron loss dicer and micrornas protect adult dopamine neurons dihydromyricetin protects against cerebral ischemia/reperfusion injury via suppressing microgliamediated neuroinflammation and activation of erk / -creb signaling pathway recent update on the pharmacological effects and mechanisms of dihydromyricetin dihydromyricetin ameliorates atherosclerosis in ldl receptor deficient mice sigma- receptor deficiency reduces mptp-induced parkinsonism and death of dopaminergic neurons allosteric modulation of sigma- receptors elicits rapid antidepressant activity the sigma- receptor modulates methamphetamine dysregulation of dopamine neurotransmission this work was supported by grants from the national natural science foundation of china (nos. , , and ) and the priority academic program development of the jiangsu higher education institutes (papd). support from the national center for international research ( b ) is also appreciated. author contributions chg, xcz, and jlw designed the research and wrote the paper; chg and tc performed the experiments; chg and ltz analyzed the data. competing interests: the authors declare no competing interests. key: cord- -x eyimug authors: hu, yue-huai; liu, jie; lu, jing; wang, pan-xia; chen, jian-xing; guo, ying; han, fang-hai; wang, jun-jian; li, wei; liu, pei-qing title: sfrp protects h c cardiac myoblasts from doxorubicin-induced apoptosis by inhibiting the wnt/pcp-jnk pathway date: - - journal: acta pharmacol sin doi: . /s - - -z sha: doc_id: cord_uid: x eyimug doxorubicin (dox) is an effective chemotherapy drug against a wide range of cancers, including both hematological and solid tumors. however, the serious cardiotoxic effect restricted its clinical application. we previously have illuminated the protective role of canonical wnt/β-catenin signaling in dox-induced cardiotoxicity. secreted frizzled-related protein (sfrp ) is one of the endogenous inhibitors of both canonical and noncanonical wnt signaling. in this study, we investigated the relationship between sfrp and noncanonical wnt/pcp-jnk (wnt/planar cell polarity-c-jun n-terminal kinase) pathway in dox-induced cardiotoxicity in vitro and in vivo. we showed that treatment of h c cardiac myoblasts with dox ( μm) time-dependently suppressed cell viability accompanied by significantly decreased sfrp protein level and increased wnt/pcp-jnk signaling. pretreatment with sp , the wnt/pcp-jnk signaling inhibitor, attenuated dox-induced apoptosis of h c cells. overexpression of sfrp protected h c cells from dox-induced apoptosis by inhibiting the wnt/pcp-jnk pathway. after intraperitoneal injection of a cumulative dose of mg/kg dox, rats displayed significant cardiac dysfunction; their heart showed inhibited wnt/β-catenin signaling and activated wnt/pcp-jnk signaling. these results suggest that sfrp may be a novel target for dox-induced cardiotoxicity. introduction doxorubicin (dox) is a potent antineoplastic compound with wide clinical applications in both hematological and solid tumors [ ] . however, the clinical use of dox is limited by the cardiac damage it causes [ ] . although the precise mechanisms responsible for dox cardiotoxicity are not well explained, various mechanisms have been suggested to be involved, including reactive oxygen species production, cell death, and inflammation [ , ] . doxinduced apoptosis is a programmed process of death characterized by cell shrinkage, chromatin condensation, and nuclear fragmentation [ ] . wnt signaling is an evolutionarily conserved pathway that plays an important role in a diverse range of cellular activities, including cell proliferation, calcium homeostasis, and cell polarity [ ] . the best understood wnt signaling pathway is the canonical wnt/βcatenin pathway [ ] . in our previous study, we illuminated the protective role of the wnt/β-catenin signaling pathway in doxinduced cardiotoxicity [ ] . to gain further understanding on the function of alternative wnt signaling pathway on dox-induced apoptosis, we focus on the noncanonical wnt/planar cell polarity (pcp) pathway in this study. the wnt/pcp pathway transduces signals by activating c-jun n-terminal kinase (jnk) [ ] , which leads to the expression of ptk , daam , vangl , and others [ , ] . the family of secreted frizzled-related proteins (sfrps) are reportedly antagonists of wnt signaling [ ] . sfrp , a member of the sfrp family, is highly expressed in the heart [ , ] and plays an important role in many cardiac diseases by regulating wnt signaling [ ] . the results from genetic and biochemical analyses revealed that sfrp regulates both the canonical wnt/β-catenin and noncanonical wnt/pcp-jnk pathways [ , ] . we explored the regulatory relationship between sfrp and wnt/β-catenin signaling in dox-induced cardiotoxicity in our previous study. whether the regulatory effect of sfrp on the wnt/pcp-jnk pathway is involved in dox-induced cell injury is the core issue to be resolved in this paper. chemical regents dox (purity of . %) was purchased from target molecule corp. (usa) and dissolved in sterile water to mm. sp (jnk inhibitor, purity of . %) was obtained from selleck chemicals (usa) and dissolved in dimethyl sulfoxide (dmso) to mm. anisomycin (jnk activator, purity of . %) was obtained from selleck chemicals (usa) and diluted in dmso to mm. all the prepared regents were stored at − °c. the experimental animal procedures were approved by the research ethics committee of sun yat-sen university and were conducted in accordance with the guide for the care and use of laboratory animals (nih publication no. - , revised ) . sprague-dawley (sd) rats (males weighing - g, spf grade, certification no. ) were obtained from the experimental animal center of sun yat-sen university (guangzhou, china). the animals were randomly separated into two groups. rats in the dox group were intraperitoneally injected with dox for a cumulative dose of mg/kg dox ( mg/kg/injection at days , , and ). the rats in the control group received an equal volume of normal saline (ns) in parallel. each group had six animals. cell culture and treatment h c cells were plated at a density of × cells per dish ( mm) filled with dulbecco's modified eagle's medium (dmem) (gibco) fig. dox induces apoptosis and respiratory defects in h c cells. h c cells were incubated with μm dox for the indicated time periods. a, b the expression of bax and cleaved caspase- was measured by western blot analysis. c cell viability was measured by the mts assay. d the cellular morphology was observed by direct visualization with a light microscope. scale bar: μm. e nuclear condensation and dna fragmentation were observed in the hoechst -stained cells. scale bar: μm. f the apoptosis rate was detected by flow cytometry. g, h seahorse assay was used to detect the respiratory function of the h c cells. the data are presented as the means ± sem. *p < . and **p < . vs. the control group; n = supplemented with % fetal bovine serum. the cells were grown at °c in humidified % co . h c cells were incubated with dox ( μm) for the indicated times. the cells were incubated with sp ( μm) and anisomycin ( μm) min before the dox treatment. adenovirus encoding sfrp was inoculated into the cells h before the dox treatment. crispr/cas -mediated knockdown of jnk (sgjnk) was carried out, and the following target sequence of sgrna was used: ′-ctcggtaggctcgcttagca- ′. h c cells were transfected with vectors encoding cas and sgjnk. to upregulate the expression of jnk, we transfected the h c cells with jnk plasmid by using lipofectamine transfection reagent (invitrogen corporation) according to the manufacturer's protocol. western blot analysis western blot analysis was performed as previously described [ ] . primary antibodies against bax (rabbit, diluted : ), poly(adpribose) polymerase (parp ) (rabbit, diluted : ), caspase- (rabbit, diluted : ), β-catenin (rabbit, diluted : ), phosphorylated (p)-jnk (rabbit, diluted : ), and total-jnk (t-jnk) (rabbit, diluted : ) were purchased from cell signaling technology. antibodies against sfrp (rabbit, diluted : ) were obtained from cloud-clone corp. (ccc, usa). primary antibodies against α-tubulin (diluted : , sigma, usa) and glyceraldehyde -phosphate dehydrogenase (diluted : , sigma, usa) served as loading controls for whole-cell lysates. the band intensity was analyzed by quantity one software (bio-rad, usa). cell viability assay cell viability was examined using the mts assay. briefly, h c cells were cultured in -well plates. after treatment, the medium was replaced with μl of fresh medium containing μl of mts reagent, and the cells were incubated in the dark for another h at °c. then, the optical density of each well was measured at nm by using a microplate reader (tecan, switzerland). determination of nuclear condensation and dna fragmentation nuclear condensation and dna fragmentation were examined by using hoechst . briefly, h c cells were cultured in -well plates. after dox treatment, μg/ml hoechst was added to the medium, and cells were incubated in the dark for another min at °c. then, the cell culture medium was replaced with dmem without phenol red, and the evidence of nuclear condensation and dna fragmentation was photographed using an evos fl auto cell imaging system (life technologies, bothell, wa, usa). the extent of cell apoptosis was assessed with an annexin v/propidium iodide (pi) apoptosis assay kit (bestbio, shanghai, china). briefly, h c cells were seeded in -well plates and treated with dox for h, harvested with trypsin without edta, and washed with pbs. annexin v and pi staining was performed using an annexin v/pi apoptosis assay kit (bestbio, shanghai, china) in accordance with the manufacturer's instructions. the stained cells were analyzed by flow cytometry (excitation at nm; emission at nm) with an epics xl instrument (beckman coulter, usa). oxygen consumption rate the oxygen consumption rate (ocr) was measured using a seahorse xfe extracellular analyzer (agilent, ca) according to the manufacturer's instructions. the ocr was examined using the seahorse xf cell mito stress test kit. h c cells were incubated in minimum essential media (mem) (ph . ) supplemented with glucose ( mm) and pyruvate ( mm). after basal measurements were taken, oligomycin ( μm), carbonylcyanide p-trifluoromethoxyphenylhydrazone ( . μm), and antimycin a/rotenone ( . μm) were autoinjected into the experimental wells sequentially, and the respiration rate was measured three times followed by each injection. each average basal or post-oligomycin treatment respiration rate was measured after the readings achieved a steady state. quantitative reverse transcription pcr quantitative reverse transcription pcr (rt-qpcr) was conducted as previously described [ ] . messenger rna levels of target genes were measured by qrt-pcr, and β-actin served as an internal control. the sequences of the rat-specific primers were as follows: ptk , ′-cattgtctgtcacccattcg- ′ and ′-gtttgataaggaggctac gg- ′; daam , ′-gcccatgaggtttgtaac- ′ and ′-agccaatga gggaggtat- ′; and β-actin, ′-tcgtgcgtgacattaaagag- ′ and ′-attgccgatagtgatgacct- ′. dox is an effective anticancer anthracycline antibiotic with limited application because it induces acute and serious cardiotoxic side effects [ ] . oxidative stress and apoptosis are the most important mechanisms of dox-induced cardiotoxicity [ ] . in our study, h c cells were treated with μm dox for the indicated times. the protein expression of bax and cleaved caspase- were upregulated with increased dox treatment times ( fig. a, b) . the results from the mts assay showed that cell viability was reduced as the dox incubation time increased (fig. c) . because cell injury was obvious at h, experimental measurements were taken after the cells were treated for h. morphological damage induced by dox was observed by light microscopy (fig. d) . hoechst staining of the h c cells showed that dox caused nuclear condensation and dna fragmentation (fig. e) . a flow cytometer was used to evaluate the extent of h c cell apoptosis, and the results implied that dox treatment increased both early and late apoptosis in the cells (fig. f ). in addition, results from the seahorse analysis showed serious respiratory defects in the h c cells. (fig. g, h) . changes in sfrp and wnt signaling during dox-induced apoptosis in our previous study [ ] , the protein level of sfrp was reduced in dox-treated neonatal rat cardiomyocytes (nrcms). in this study, reduced sfrp levels were also detected in the dox-treated h c cells (fig. a) . a recent study showed an association between the wnt/pcp-jnk pathway and apoptosis [ ] . to explore the involvement of the wnt/pcp-jnk pathway in dox-induced apoptosis, h c cells were incubated with dox, and the levels of p-jnk and t-jnk were detected. the level of p-jnk and the ratio of p-jnk/t-jnk were increased after cells were incubated with dox (fig. b ). in addition, canonical wnt/β-catenin signaling was suppressed by dox, as shown by the protein expression of β-catenin (fig. c) . inhibition of wnt/pcp-jnk signaling attenuates dox-induced apoptosis to further explore the role of wnt/pcp-jnk signaling in doxinduced apoptosis, h c cells were pretreated with sp ( μm), an inhibitor of the wnt/pcp-jnk pathway, for min followed by incubation with dox. it was obvious that the upregulated wnt/pcp-jnk signaling caused by dox was suppressed by sp (fig. a) . in addition, the inhibition of wnt/ pcp-jnk signaling led to the reduced levels of cleaved parp and caspase- (fig. b) . dox-induced morphological damage was diminished by sp , as observed in cells with a light microscope (fig. c) . nuclear condensation and dna fragmentation were ameliorated by sp , as revealed by hoechst staining (fig. d) . the results from the mts assay showed that the reduced cell viability caused by dox was reversed by sp (fig. e ). in addition, the expression of cleaved caspase- that had been induced by dox was reduced by crispr/cas system-mediated knockdown of jnk (fig. f) . the results described above indicated that the wnt/pcp-jnk signaling pathway was involved in dox-induced apoptosis of h c cells. the expression of cleaved parp and caspase- was determined by western blot analysis. c cellular morphology was observed by direct visualization with a light microscope. scale bar: μm. d nuclear condensation and dna fragmentation were observed in the hoechst -stained cells. scale bar: μm. e cell viability was measured by the mts assay. f the levels of jnk and cleaved caspase- were determined by western blot analysis. the data are presented as the means ± sem. *p < . and **p < . vs. the control or vector group. # p < . and ## p < . vs. the dox group or vector + dox group; n = sfrp protects h c cardiac myoblasts from doxorubicin-induced apoptosis yh hu et al. sfrp protects h c cells from dox-induced apoptosis the results described above showed that dox caused a reduction in sfrp level; therefore, a recombinant adenovirus vector encoding sfrp (ad-sfrp ) was used to investigate the effect of sfrp on dox-induced apoptosis. the results showed that overexpression of sfrp was successfully induced by the ad-sfrp vector (fig. a ). in addition, the results from the western blot analysis showed that the increased expression of cleaved caspase- induced by dox was suppressed by the overexpression of sfrp (fig. b) . the dox-induced morphological damage and nuclear condensation were diminished by ad-sfrp (fig. c, d) . the results from the mts assay showed that the reduced cell viability caused by dox was reversed by the overexpression of sfrp (fig. e) . the results described above indicated that sfrp played a protective role in the dox-induced apoptosis of h c cells. upregulation of sfrp inhibits the wnt/pcp-jnk signaling activity sfrp is reportedly the endogenous inhibitor of both the canonical wnt/β-catenin and noncanonical wnt/pcp-jnk pathways [ ] . in our previous study, we explored the regulatory relationship between sfrp and wnt/β-catenin in dox-induced cardiomyopathy [ ] . in this study, we focused on the regulatory relationship between sfrp and wnt/pcp-jnk pathway factors in dox-induced apoptosis of h c cells. the rt-qpcr results showed that dox promoted the transcription of ptk and daam , two wnt/pcp-jnk pathway target genes, but this effect was inhibited by the overexpression of sfrp (fig. a, b) . in addition, the upregulated p-jnk expressed induced by dox was suppressed by sfrp (fig. c) . anisomycin is an agonist of the jnk pathway. in our experiment, we found that the upregulated levels of p-jnk and cleaved caspase- that had been induced by dox were downregulated by the overexpression of sfrp , but these effects were diminished by anisomycin ( μm) treatment (fig. d) . overexpression of jnk exhibited a similar effect as that of anisomycin (fig. e ). in addition, anisomycin treatment also induced a significant increase in p-jnk and in cleaved parp and caspase- , but these effects were prevented in cells preinfected with ad-sfrp (fig. f, g) . the results described above indicated that sfrp protected h c cells from dox-induced apoptosis by inhibiting the wnt/pcp-jnk pathway. changes to the canonical and noncanonical wnt signaling pathways by dox-induced injury in vivo to further identify the changes in wnt signaling upon dox treatment, sd rats were intraperitoneally injected with a cumulative dose of mg/kg dox ( mg/kg/injection at days , , and ), and the control rats received the same volume of ns in parallel. echocardiography results revealed a decrease in the ejection fraction, fractional shortening, and cardiac output (co) (fig. a-d) . the hearts of the dox-treated rats were notably smaller than those of the control group (fig. e) . dox treatment also increased the disorganization of the myocytes and the apoptosis rate, as revealed by the hematoxylin-and eosin-stained cells (fig. f) and tunel (terminal deoxynucleotidyl transferase dutp nick end labeling) assay results (fig. g) . following dox treatment, the protein expression of p-jnk was increased (fig. h) , while the protein level of β-catenin was decreased (fig. i) . these results indicated that the canonical wnt/β-catenin pathway was inhibited and that the noncanonical wnt/pcp-jnk pathway was activated upon dox treatment. although dox is one of the most effective chemotherapeutic drugs for treating many cancers, the lethal nature of dox-induced the expression of cleaved caspase- was determined by western blot analysis. c cellular morphology was observed by direct visualization with a light microscope. scale bar: μm. d nuclear condensation and dna fragmentation were observed in the hoechst -stained cells. scale bar: μm. e cell viability was measured by the mts assay. the data are presented as the means ± sem. **p < . vs. the ad-gfp group; # p < . and ## p < . vs. the ad-gfp + dox group; n = cardiotoxicity is a serious problem. to date, strategies using pharmaceutical agents to prevent or reduce dox cardiotoxicity have been tested in many studies. dexrazoxane is the only compound to protect against dox-induced cardiotoxicity [ ] . diuretics, a low salt diet, β-blockers [ ] [ ] [ ] , and angiotensinconverting enzyme inhibitors [ ] are used in the clinic to relieve symptoms. however, to date, no single drug has been able to completely prevent dox-induced cardiotoxicity clinically. a better understanding of the cardiotoxic mechanism of dox may lead to the development of new therapeutic strategies. it is of great clinical significance to search for effective therapeutic targets. in our study, we demonstrated the important role of sfrp in dox-induced cardiotoxicity. we found that the level of sfrp was decreased in both the nrcms [ ] and h c cells (fig. a) . ectopic expression of sfrp protected h c cells from dox-induced apoptosis. in this study, we also studied the mechanism by which sfrp effectively protects cells from dox-induced apoptosis. numerous studies have shown that dox induces apoptosis through various pathways [ ] . it is thought that mitochondria play a significant role in dox-induced apoptosis by regulating the release of cytochrome c, which results in the activation of caspases and subsequent cell death [ , ] . several previous studies have suggested that sfrp plays an antiapoptotic role in cells exposed to ceramide [ ] , hypoxia [ ] , and/or inflammation [ ] . in these studies, the expression of cleaved caspase- was suppressed by sfrp . in our study, we obtained results consistent with the results of these previous studies. the wnt signaling network comprises several signaling pathways, of which the best studied are the canonical wnt/β-catenin pathway and noncanonical wnt/pcp-jnk pathway [ ] . these two pathways are primarily thought to act in a mutually repressive manner because they compete for common proteins, such as the scaffolding protein disheveled [ ] . therefore, it is reasonable that as the level of wnt/pcp-jnk signaling is upregulated, the level of wnt/β-catenin signaling is downregulated after dox treatment. our research showed that the inhibition of wnt/pcp-jnk signaling contributed to the protective effect of sfrp . in addition, anisomycin treatment induced the activation of wnt/pcp-jnk signaling and the apoptosis of the h c cells, and these effects were suppressed by sfrp . the results described above led to the conclusion that sfrp protected the h c cells from dox-induced apoptosis by inhibiting wnt/pcp-jnk signaling. in addition, in our d, e the levels of p-jnk, t-jnk and cleaved caspase- were determined by western blot analysis. g the expression of cleaved parp and caspase- was measured by western blot analysis. the data are presented as the means ± sem. *p < . and **p < . vs. the ad-gfp or vector group. ## p < . vs. the ad-gfp + dox group or ad-gfp + aniso group. δ p < . , δδ p < . vs. the ad-sfrp + dox group; n = previous study [ ] , we found that sfrp might regulate doxinduced apoptosis by interacting with other proteins, including those not in the wnt pathway. for example, it could attenuate apoptosis by inhibiting the activity of parp (which can be activated as a dna damage receptor after dna-damaged fragments are identified) in the nucleus. thus, it is believed that the protective effect of sfrp against dox-induced apoptosis is a comprehensive result, but more research is needed to reveal it completely. fig. changes in the canonical and noncanonical wnt signaling pathways in cells with dox-induced injury in vivo. sd rats were intraperitoneally injected with dox (cumulative dose was mg/kg) or sterile ns. a-c the echocardiographic parameters of ejection fraction (ef), fractional shortening (fs), and cardiac output (co) were detected. d-f echocardiogram of the gross heart and images of an he-stained transection of the left ventricle are shown. scale bar: μm. g apoptosis of rat heart cells was detected by tunel staining. scale bar: μm. h, i the expression of p-jnk and β-catenin was examined by immunofluorescence and immunohistochemistry, respectively. scale bar: μm. representative images of five independent experiments are presented. the data are presented as the means ± sem. **p < . vs. the ns group; n = the cardiotoxic mechanism of doxorubicin (dox) and pegylated liposomal dox in mice bearing c- colon carcinoma: a study focused on microrna role for toxicity assessment of new formulations anti-inflammatory agents and monoher protect against dox-induced cardiotoxicity and accumulation of cml in mice mechanisms and management of doxorubicin cardiotoxicity long-term effects of -monohydroxyethylrutoside (monoher) on dox-induced cardiotoxicity in mice wnt/beta-catenin signaling plays an ever-expanding role in stem cell self-renewal, tumorigenesis and cancer chemoresistance transsynaptic transmission of vesicular wnt signals through evi/wntless sfrp has a biphasic effect on doxorubicin-induced cardiotoxicity in a cellular location-dependent manner in nrcms and rats secreted frizzled-related protein (sfrp ) regulates the progression of renal fibrosis in a mouse model of obstructive nephropathy fzd drives in vitro aggressiveness in stem-a subtype of ovarian cancer via regulation of noncanonical wnt/pcp pathway the effect of maternal diabetes on the wnt-pcp pathway during embryogenesis as reflected in the developing mouse eye frza, a secreted frizzled related protein, induced angiogenic response secreted frizzled-related proteins: searching for relationships and patterns loss of secreted frizzled-related protein- leads to deterioration of cardiac function in mice and plays a role in human cardiomyopathy sfrp controls apicobasal polarity and oriented cell division in developing gut epithelium sfrp , sfrp , and sfrp regulate the wnt/beta-catenin and the planar cell polarity pathways during early trunk formation in mouse jmjd inhibition protects against isoproterenol-induced cardiac hypertrophy by suppressing beta-mhc expression the preventive effects of taurine on neural tube defects through the wnt/pcp-jnk-dependent pathway clinical practice guideline update summary: use of chemotherapy and radiation therapy protectants canadian cardiovascular society consensus conference guidelines on heart failure- update: best practices for the transition of care of heart failure patients, and the recognition, investigation and treatment of cardiomyopathies efficacy and safety of metoprolol in the treatment of doxorubicin-induced cardiomyopathy in pediatric patients carvedilol prevents doxorubicin-induced free radical release and apoptosis in cardiomyocytes in vitro ace inhibition and protection from doxorubicin-induced cardiotoxicity in the rat oxidative stress injury in doxorubicin-induced cardiotoxicity mitochondrial dysfunction is an early indicator of doxorubicin-induced apoptosis secreted frizzled-related protein (sfrp ) protects fibroblasts from ceramide-induced apoptosis secreted frizzled related protein protects h c cells from hypoxia/re-oxygenation injury by blocking the wnt signaling pathway secreted frizzled-related protein- improves postinfarction scar formation through a modulation of inflammatory response mechanisms of intercellular wnt transport. development dishevelled: the hub of wnt signaling yhh designed and performed the research; jie liu, jing lu, and fhh wrote the paper; pxw analyzed the data; jxc and yg prepared the reagents; and jjw, wl, and pql designed the research. competing interests: the authors declare no competing interests. key: cord- -s wf pix authors: prehn, jochen h m; jirström, elisabeth title: angiogenin and trna fragments in parkinson’s disease and neurodegeneration date: - - journal: acta pharmacol sin doi: . /s - - - sha: doc_id: cord_uid: s wf pix in this review, we summarise the evidence for a role of the ribonuclease angiogenin in the pathophysiology of neurodegenerative disorders, with a specific focus on parkinson’s disease (pd). angiogenin is a stress-induced, secreted ribonuclease with both nuclear and cytosolic activities. loss-of-function mutations in the angiogenin gene (ang) have been initially discovered in familial cases of amyotrophic lateral sclerosis (als), however, variants in ang have subsequently been identified in pd and alzheimer’s disease. delivery of angiogenin protein reduces neurodegeneration and delays disease progression in in vitro and in vivo models of als and in vitro models of pd. in the nucleus, angiogenin promotes ribosomal rna transcription. under stress conditions, angiogenin also translocates to the cytosol where it cleaves non-coding rna into rna fragments, in particular transfer rnas (trnas). stress-induced trna fragments have been proposed to have multiple cellular functions, including inhibition of ribosome biogenesis, inhibition of protein translation and inhibition of apoptosis. we will discuss recent evidence of trna fragment accumulation in pd, as well as their potential neuroprotective activities. acta pharmacologica sinica ( ) : - ; https://doi.org/ . /s - - - parkinson's disease (pd) is characterized by the progressive degeneration and loss of dopaminergic neurons in the substantia nigra pars compacta and catecholaminergic neurons in the locus coeruleus. loss of neurotrophic or neuroprotective support may therefore increase the likelihood of developing pd. angiogenin is a . kda protein that belongs to the superfamily of vertebrate secreted ribonucleases. other members of this family include rnase (pancreatic ribonuclease), rnase (eosinophil-derived neurotoxin), rnase (eosinophil cationic protein), rnase , rnase (k ), rnase and rnase [ ] . in contrast to most members of this family, angiogenin exhibits relatively low ribonuclease activity [ ] . angiogenin was initially discovered as a tumour-derived angiogenic factor in human colon adenocarcinoma cells [ ] . subsequent studies focused on its role in angiogenesis, cancer, ischaemia and infection (reviewed in [ ] ). as the name suggests, angiogenin stimulates endothelial cell proliferation and has been shown to be required for the angiogenic activity of vascular endothelial growth factor (vegf) and fibroblast growth factor- (fgf- ) [ ] . while vegf and fgf- signal through tyrosine kinase receptors to activate protein synthesis (via stimulation of mtor and s kinase pathways), angiogenin increases ribosomal rna (rrna) transcription in the nucleus [ ] . it thereby acts synergistically with vegf and fgf- to increase protein synthesis in endothelial cells and is required for their proliferation [ ] . angiogenin-induced rna transcription also requires the ribonuclease activity of angiogenin and occurs via an epigenetic activation of the ang promoter [ , ] . angiogenin has also been proposed to indirectly stimulate the pi -kinase and akt kinase pathways in endothelial cells and bladder cancer cells [ ] [ ] [ ] . additional cytosolic activities of angiogenin have recently emerged. under conditions of cellular stress such as oxidative stress, disruption of proteostasis or withdrawal of trophic factors, angiogenin accumulates in cytosolic stress granules [ , ] . stress granules are cytoplasmic foci containing untranslated mrna and rna-binding proteins that are formed in response to cellular stress and function to arrest protein translation [ ] [ ] [ ] . angiogenin is also a secreted factor that can be taken up by endocytosis into the cytoplasm of target cells [ ] [ ] [ ] where it may function in paracrine [ ] . as angiogenin is secreted from cells, several studies investigated potential angiogenin receptors and cellular uptake mechanisms. angiogenin has been shown to be a ligand for surface receptors of the plexin family. plexin-b was identified by yu et al. as receptor for angiogenin in endothelial, cancer, neuronal, and normal hematopoietic and leukaemic stem and progenitor cells [ ] . plexin-b is expressed in the postnatal and adult nervous system particularly in subventricular zone (szv)-derived neural stem cells [ ] . angiogenin may, however, also been taken up through additional mechanisms into other cell types. studies from our group demonstrated that neuronally-secreted or exogenous angiogenin protein is effectively taken up into astroglia [ ] and alters their secretome [ ] . indeed, astroglia are now considered an important contributor to neurodegenerative disorders including pd and amyotrophic lateral sclerosis (als) [ , ] . uptake and subsequent rna cleavage in astrocytes have been shown to require clathrin-mediated endocytosis (cme) and dependent on heparan sulfate proteoglycans [ ] . it is possible that other angiogenin-binding proteins responsible for angiogenin uptake and signalling will be identified going forward. the activity of angiogenin is critically regulated by an endogenous inhibitor, ribonuclease/angiogenin inhibitor (rnh ) [ ] . like angiogenin, rnh is expressed in endothelial cells and many other cell types, including neurons and glial cells [ ] [ ] [ ] . rnh inhibits the activity of angiogenin by binding to its catalytic triad residues lys- , his- and his- [ ] . under stress and anabolic conditions, rnh accumulates in the nucleus where it binds angiogenin and inhibits rrna transcription to save energy [ ] . angiogenin activity is therefore a process that is determined by the relative abundance and co-localisation of both proteins in cells. similar to other pro-angiogenic factors such as vegf, transcription of the ang gene is regulated by the transcription factor hypoxia-inducible factor- α (hif- α). through this mechanisms, increased ang expression stimulates angiogenesis in tissues that have insufficient oxygen supply [ ] . the hypoxia responsive element within the ang gene has been mapped to the consensus hif- α binding site ′-rcgtg- ′ [ , ] . hif- α has also been shown to be required for ang expression in neural cells in response to hypoxia [ ] . ang expression is also positively regulated by the transcription factor hepatic nuclear factor- α (hnf- α) [ ] . this transcription factor is involved in glucose and lipid metabolism in liver and pancreatic beta-cells. angiogenin became of interest to neuroscience with the initial discovery that ang gene variants were associated with familial and apparently sporadic cases of als in scottish, irish, english, swedish and northern american families [ ] . as part of this study, our laboratory identified that angiogenin is expressed and enriched in motor neurons. subsequent studies have identified ang variants in italian, french, german, dutch, belgian, hungarian, chinese and indian als patients ( table ) . several of the ang variants identified were predicted and subsequently validated to affect angiogenin's ribonuclease activity due to their proximity to the catalytic site of the protein (table ) . other angiogenin variants have been shown to inhibit the shuttling of angiogenin between nucleus and cytoplasm [ ] , or to reduce the stability of the protein [ ] . subsequent studies showed that angiogenin exerts neuroprotective activities in vitro in models of excitotoxic, hypoxic and trophic factor-withdrawal-induced injury to motor neurons and other neural cells, including dopaminergic sh-sy y neuroblastoma cells [ , , ] . many of the ang variants identified were shown to have reduced neuroprotective activity compared with wild-type ang when overexpressed at similar levels in neurons [ , ] . however, it is currently unknown which cell types in the nervous system (including endothelial cells) are susceptible to ang mutations. of note, subsequent studies also identified ang variants in familial forms of pd [ , ] . the two studies identified several non-synonymous ang variants in northern american, german, dutch and italian pd patients ( table ). the frequency of pd ang variants were highly similar in both studies ( . %/ . %) compared with controls ( . %/ %). furthermore, van es et al. reported similar frequency of als patients and pd patients carrying ang variants ( . %/ . % compared with . % in controls) [ ] . many of the reported pd ang variants were predicted to impair angiogenin protein function [ ] . in a more recent study, several of these variants were validated to have reduced levels of ribonuclease activity in comparison with wildtype angiogenin [ ] . interestingly, a recent study demonstrated ang mutations in familial cases of alzheimer's disease (ad) [ ] . collectively, these findings point to a general role of angiogenin as a protective factor for central nervous system neurons. of note, ang knock-out mice do not appear to develop reported by [ ] neurodegeneration and als-, pd-, or ad-like symptoms or neuropathology in their life span [ ] , highlighting that aging and/or additional disease processes are required to trigger neurodegeneration. ang can therefore be added to list of genes that regulate stress responses in neurons and are mutated and contribute to neurodegeneration in various neurological disorders (including pd), as seen in the case of tardbp mutations, fus mutations, c orf repeat expansions, and dj- mutations [ ] [ ] [ ] [ ] . the ang mutations that have been reported in als and pd patients suggested a direct involvement of angiogenin in pathways leading to motoneuron degeneration or degeneration of dopaminergic neurons. we demonstrated that angiogenin protects cultured primary mouse motor neurons against alsassociated, stress-induced cell death including excitotoxic injury by promoting and sustaining cell survival signalling through pi kinase/akt kinases [ ] . in further preclinical work, we demonstrated that daily, systemic (intraperitoneal) delivery of recombinant human angiogenin protein significantly increased life span and improved motor function in sod g a mice, an established mouse model of als [ ] . cell survival signalling in motoneurons was preserved in angiogenin-treated mice. importantly, the effect of angiogenin, when delivered post-symptom onset (from day onward) on life span and disease progression, was comparable to the effect of a pre-symptom angiogenin treatment (from day onward). these results suggested that angiogenin protein delivery may be beneficial in treating patients with newly diagnosed als. to further validate these findings, our group performed a sod g a mouse model study according to the preclinical guidelines for als animal studies set by the european als/ mnd group [ ] . in this study, we demonstrated that systemic delivery of human angiogenin three times per week postsymptom onset (from day onward) delayed motor dysfunction, significantly enhanced survival and protected against motoneuron loss and vascular network regression in the lumbar spinal cord [ ] . angiogenin may also be neuroprotective in pd. steidinger et al. demonstrated significantly decreased levels of endogenous angiogenin in an alpha-synuclein transgenic mouse model of pd and showed that recombinant human angiogenin protected against dopaminergic neuronal cell death and inhibited caspase- activation in neurotoxin-induced in vitro models of pd [ ] . a subsequent study by the same group found that virally-mediated overexpression of human angiogenin in the substantia nigra did not protect against dopaminergic cell loss in a neurotoxin-based mouse model of pd [ ] . these findings suggest that further in vivo studies are required to explore potential neuroprotective functions in animal models of pd, in particular in genetic models. during stress conditions, angiogenin accumulates in the cytosol where it cleaves non-coding rnas, including transfer rnas (trnas). cleavage of trnas by angiogenin generates fragments termed 'stress-induced trna fragments' or 'tirnas' [ , , ] . cleavage of trnas by angiogenin occurs in their anticodon loop, a process that is highly regulated by trna modifications [ ] [ ] [ ] [ ] [ ] , so that only specific subsets of trna fragments are generated [ ] . tirnas have been shown to inhibit ribosome assembly [ ] and to inhibit cap-dependent protein translation via interaction with the initiation factor eif f [ ] . both processes may facilitate the recovery of cells during stress conditions, so that resourceconsuming or error-sensitive cell functions are stalled during periods of stress. tirna generation has also been linked to the process of stem cell maintenance and inhibits the proliferation of hematopoietic stem/progenitor cells [ ] . due to their multiple mechanisms of action, lack of tirna production could be involved in the pathogenic effects of ang variants in human disease. trfs in general are now considered a new class on small noncoding rnas with multiple cellular functions, of which tirnas are only a subclass. trfs have been detected in various biological systems suggesting that trna cleavage by angiogenin and other ribonucleases is an evolutionary conserved process. other new functions of trfs beyond the regulation of protein translation have also been reported. 'shot-rnas', which are analogous to angiogenin-produced tirnas, were identified in hormone responsive cancer cells where they stimulate their proliferation [ ] . two studies in showed that trfs fragments are enriched in sperm cells and delivered to the zygote at fertilization where they modified gene expression by binding to the elements in the zygote's genome [ , ] . multiple ribonucleases other than angiogenin are able to generate such fingerprints, including the ribonucleases z and dicer [ ] [ ] [ ] [ ] [ ] . the identification of trf 'fingerprints' in different biological systems and disease conditions is still at its infancy, but interesting observations are now beginning to emerge. a study from our laboratory showed that specific trfs are associated with epilepsy [ ] . small rna sequencing analysis of plasma samples collected during video eeg monitoring of patients with focal epilepsy identified significant differences in three specific trfs fragments compared with healthy controls. interestingly, these trfs are different from angiogenin-generated fragments as cleavage did not occur in the anticodon loop. these fragments were elevated in the pre-seizure period, but lower in post-seizure samples, and may represent a novel class of biomarkers indicating seizure risk in epilepsy patients. a recent study performed in pd patients identified diseasespecific trfs in brain and biofluids [ ] . reanalyses of rnaseq data from three previous studies identified multiple differentially abundant trfs between pd patients and healthy controls in prefrontal cortex, cerebrospinal fluid and serum. of note, a subset of the identified trfs successfully distinguished pd patients from controls with high sensitivity and specificity in each sample collection. further research is required as to whether these fragments are generated by angiogenin or other ribonucleases. collectively, these findings suggested that trf signatures are promising candidates as non-invasive pd biomarkers. there is a significant body of evidence suggesting that angiogenin is a stress-induced survival factor for central nervous system neurons. while it has been shown that angiogenin is able to protect against dopaminergic neuron loss in vitro, further research is required to explore its role in animal models of pd. the arrival of new animal models of pd will likely accelerate this translation, as seen in als models. due to its pleiotropic mechanism of action, angiogenin may indeed be an interesting candidate for the treatment of neurodegenerative disorders. it stimulates angiogenesis in endothelial cells and promotes neuronal survival through akt signalling and possibly through the formation of tirnas, thereby facilitating the recovery of stressed neurons. moreover, trfs generated by angiogenin and other ribonucleases may deliver novel diagnostic or prognostic tools for the management of neurodegenerative disorders. studies are now required to explore the biological functions of these fragments in vitro and in vivo. rnase , a novel rnase a superfamily ribonuclease expressed uniquely in placenta characteristic ribonucleolytic activity of human angiogenin isolation and characterization of angiogenin, an angiogenic protein from human carcinoma cells three decades of research on angiogenin: a review and perspective endogenous angiogenin in endothelial cells is a general requirement for cell proliferation and angiogenesis the nuclear function of angiogenin in endothelial cells is related to rrna production angiogenin stimulates ribosomal rna transcription by epigenetic activation of the ribosomal dna promoter angiogenin-induced protein kinase b/akt activation is necessary for angiogenesis but is independent of nuclear translocation of angiogenin in huve cells angiogenin induces nitric oxide synthesis in endothelial cells through pi- and akt kinases angiogenin interacts with ribonuclease inhibitor regulating pi k/akt/mtor signaling pathway in bladder cancer cells angiogenin-induced trna-derived stress-induced rnas promote stress-induced stress granule assembly angiogenin-induced trna fragments inhibit translation initiation rna-binding proteins tia- and tiar link the phosphorylation of eif- alpha to the assembly of mammalian stress granules dynamic shuttling of tia- accompanies the recruitment of mrna to mammalian stress granules evidence that ternary complex (eif -gtp-trna(i)(met))-deficient preinitiation complexes are core constituents of mammalian stress granules motoneurons secrete angiogenin to induce rna cleavage in astroglia the cellular uptake of angiogenin, an angiogenic and neurotrophic factor is through multiple pathways and largely dynamin independent nuclear translocation of angiogenin in proliferating endothelial cells is essential to its angiogenic activity plexin-b mediates physiologic and pathologic functions of angiogenin plexin-b regulates the proliferation and migration of neuroblasts in the postnatal and adult subventricular zone angiogenin induces modifications in the astrocyte secretome: relevance to amyotrophic lateral sclerosis non-cell autonomous toxicity in neurodegenerative disorders: als and beyond the a astrocyte paradigm: new avenues for pharmacological intervention in neurodegeneration human placental ribonuclease inhibitor abolishes both angiogenic and ribonucleolytic activities of angiogenin ribonuclease inhibitor: structure and function proteomics. tissue-based map of the human proteome human protein atlas. version . rnh binding of placental ribonuclease inhibitor to the active site of angiogenin ribonuclease/ angiogenin inhibitor regulates stress-induced subcellular localization of angiogenin to control growth and survival regulation of angiogenin expression and epithelial-mesenchymal transition by hif- alpha signaling in hypoxic retinal pigment epithelial cells angiogenin protects motoneurons against hypoxic injury the mouse rnase and rnase /ang locus utilizes dual promoters for tissue-specific expression ang mutations segregate with familial and 'sporadic' amyotrophic lateral sclerosis angiogenin lossof-function mutations in amyotrophic lateral sclerosis characterization of human angiogenin variants implicated in amyotrophic lateral sclerosis control of motoneuron survival by angiogenin human angiogenin is a neuroprotective factor and amyotrophic lateral sclerosis associated angiogenin variants affect neurite extension/pathfinding and survival of motor neurons angiogenin variants in parkinson disease and amyotrophic lateral sclerosis angiogenin variation and parkinson disease structural insights into human angiogenin variants implicated in parkinson's disease and amyotrophic lateral sclerosis a novel nonsense angiogenin mutation is associated with alzheimer disease angiogenin promotes hematopoietic regeneration by dichotomously regulating quiescence of stem and progenitor cells decoding als: from genes to mechanism the p.a t tardbp gene mutation in sardinian patients affected by parkinson's disease and other degenerative parkinsonisms dj- mutations and parkinsonism-dementia-amyotrophic lateral sclerosis complex c orf repeat expansions are a rare genetic cause of parkinsonism guidelines for preclinical animal research in als/mnd: a consensus meeting pleiotropic activity of systemically delivered angiogenin in the sod (g a) mouse model a neuroprotective role for angiogenin in models of parkinson's disease angiogenin in parkinson disease models: role of akt phosphorylation and evaluation of aav-mediated angiogenin expression in mptp treated mice stress induces trna cleavage by angiogenin in mammalian cells angiogenin cleaves trna and promotes stress-induced translational repression rna methylation by dnmt protects transfer rnas against stress-induced cleavage queuosine modification protects cognate trnas against ribonuclease cleavage transfer rna demethylase alkbh promotes cancer progression via induction of trna-derived small rnas dnmt mediates intergenerational transmission of paternally acquired metabolic disorders through sperm small non-coding rnas aberrant methylation of trnas links cellular stress to neuro-developmental disorders angiogenin generates specific stressinduced trna halves and is not involved in trf- -mediated gene silencing shot-rnas: a novel class of trna-derived functional rnas expressed in hormone-dependent cancers sperm tsrnas contribute to intergenerational inheritance of an acquired metabolic disorder biogenesis and function of trna fragments during sperm maturation and fertilization in mammals filtering of deep sequencing data reveals the existence of abundant dicer-dependent small rnas derived from trnas mouse es cells express endogenous shrnas, sirnas, and other microprocessor-independent, dicerdependent small rnas extensive terminal and asymmetric processing of small rnas from rrnas, snornas, snrnas, and trnas a novel class of small rnas: trna-derived rna fragments (trfs) human trnaderived small rnas in the global regulation of rna silencing elevation in plasma trna fragments precede seizures in human epilepsy trna-derived fragments as sex-dependent circulating candidate biomarkers for parkinson's disease angiogenin mutations in hungarian patients with amyotrophic lateral sclerosis: clinical, genetic, computational, and functional analyses identification of new ang gene mutations in a large cohort of italian patients with amyotrophic lateral sclerosis identification of novel angiogenin (ang) gene missense variants in german patients with amyotrophic lateral sclerosis a case of als-ftd in a large fals pedigree with a k i ang mutation evidence for an oligogenic basis of amyotrophic lateral sclerosis accumulation of tdp- and alpha-actin in an amyotrophic lateral sclerosis patient with the k i ang mutation fus mutations in familial amyotrophic lateral sclerosis: genotype-phenotype correlations mutations of the ang gene in french patients with sporadic amyotrophic lateral sclerosis a novel angiogenin gene mutation in a sporadic patient with amyotrophic lateral sclerosis from southern italy identification of a novel missense mutation in angiogenin in a chinese amyotrophic lateral sclerosis cohort sod g d sporadic amyotrophic lateral sclerosis (sals) patient with rapid progression and concomitant novel ang variant identification and characterization of novel and rare susceptible variants in indian amyotrophic lateral sclerosis patients structural and molecular insights into the mechanism of action of human angiogenin-als variants in neurons jhmp and ej wrote the paper. competing interests: jhmp is a beneficiary of a patent relating to the use of angiogenin as a diagnostic and therapeutic for als and other neurodegenerative disorders. ej declares no competing interest. key: cord- -e js qrs authors: sun, qingxue; bai, xiangyang; sofias, alexandros marios; van der meel, roy; ruiz-hernandez, eduardo; storm, gert; hennink, wim e.; de geest, bruno; kiessling, fabian; yu, hai-jun; lammers, twan; shi, yang title: cancer nanomedicine meets immunotherapy: opportunities and challenges date: - - journal: acta pharmacol sin doi: . /s - - - sha: doc_id: cord_uid: e js qrs cancer nanomedicines have shown promise in combination immunotherapy, thus far mostly preclinically but also already in clinical trials. combining nanomedicines with immunotherapy aims to reinforce the cancer-immunity cycle, via potentiating key steps in the immune reaction cascade, namely antigen release, antigen processing, antigen presentation, and immune cell-mediated killing. combination nano-immunotherapy can be realized via three targeting strategies, i.e., by targeting cancer cells, targeting the tumor immune microenvironment, and targeting the peripheral immune system. the clinical potential of nano-immunotherapy has recently been demonstrated in a phase iii trial in which nano-albumin paclitaxel (abraxane®) was combined with atezolizumab (tecentriq®) for the treatment of patients suffering from advanced triple-negative breast cancer. in the present paper, besides strategies and initial (pre)clinical success stories, we also discuss several key challenges in nano-immunotherapy. taken together, nanomedicines combined with immunotherapy are gaining significant attention, and it is anticipated that they will play an increasingly important role in clinical cancer therapy. nanomedicines have been extensively investigated for tumortargeted drug delivery and reducing the toxicities/side effects of chemotherapeutic drugs [ , ] . tumor targeting by nanomedicines is mainly mediated by passive targeting (based on the enhanced permeability and retention effect [ , ] ) and/or active targeting [ ] . the field has witnessed the success of the first commercial liposomal nanomedicine in (liposomal doxorubicin; doxil), and afterwards, several more nanoformulations were approved by the fda and/or ema, including paclitaxel-loaded in albumin nanoparticles (abraxane) which is currently one of the best-selling cancer drugs on the market [ , ] . in recent years, the achievements of the nanomedicine field have been critically evaluated, mainly focusing on the average targeting efficiency and real clinical impact [ , ] . this has initiated intense discussions on the current clinical utilization and future directions of nanomedicines [ ] [ ] [ ] [ ] . among the main future ways forward is the combination of nanomedicines with immunotherapy, a therapeutic strategy that has been extensively studied preclinically [ ] [ ] [ ] [ ] [ ] [ ] and is also already being explored in the clinic [ ] . this combination approach has broadened the applicability of nanomedicines from solely targeting tumor tissues as monotherapies to targeting multiple other organs and cell types in combination modalities [ ] . in this context, a low degree of tumor accumulation for a specific nanoparticle or cancer type is not necessarily a disadvantage anymore, since nanomedicines targeting other cells and tissues may help to boost the therapeutic efficacy of combination immunotherapy, including that with checkpoint antibodies [ ] . as will be outlined in this paper, the ability of nanomedicines to activate cancer immunity and improve immunotherapeutic responses holds great potential, and there are already several pieces of evidence demonstrating that nano-immunotherapy has a bright clinical future. the interplay between cancer nanomedicine and immunotherapy has been demonstrated in multiple preclinical studies [ ] . to systematically summarize such combination therapies, we have utilized the concept of "cancer-immunity cycle" [ ] to showcase methods to improve immunotherapeutic outcomes [ ] . furthermore, we have proposed a simplified model which is composed of three immune targeting strategies for combination nanoimmunotherapy [ ] . integration of nanomedicines in the cancer-immunity cycle the cancer-immunity cycle is a model that describes the anticancer immune reaction cascade. as shown in fig. , it is composed of four sequentially connected steps. it starts with the release of antigens from cancer cells and the antigens are then taken up and processed by antigen-presenting cells (apcs). afterwards, they are presented to naive t cells to generate cytotoxic t cells. these cytotoxic t cells migrate via the circulation to find and kill cancer cells by releasing toxic molecules such as perforin and granzyme b. during this process of cancer cell death, more cancer antigens are released, and a new round of the immune reaction cascade can be triggered. cancer nanomedicines have been utilized to initiate the release of antigens from cancer cells by loading agents that are able to induce immunogenic cell death (icd) [ ] . examples of such agents include the classical chemotherapeutic drugs (anthracyclines, oxaliplatin, and cyclophosphamide) as well as molecules used for photodynamic/photothermal therapy [ ] . a number of studies have already shown that loading such agents into nanocarriers induces more potent icd compared with the free drug [ , ] . an early case was demonstrated with oxaliplatin-loaded poly(lactic-coglycolic acid) nanoparticles. in a mouse pancreatic carcinoma model, it was shown that the drug-loaded nanoparticles induced higher cell apoptosis than the free drug, and that immunoactivation effects such as enhanced effect or t cell infiltration in tumors, dendritic cell maturation, and interferon-gamma secretion were observed in mice treated with oxaliplatin-loaded nanoparticles. the therapeutic efficacy of oxaliplatin-loaded nps was found to be significantly higher than that of the free drug [ ] . in addition to inducing icd, nanomedicines can also be used to potentiate antigen uptake, processing, and presentation. a key aspect of these three processes is the delivery of antigens and adjuvating agents to immune cells. adjuvants, such as toll-like receptor (tlr) agonists, target pathways in immune cells that sense pathogen/danger-associated molecular patterns, which leads to the activation of apcs [ ] . such adjuvants have shown therapeutic effects, but they are unfortunately often associated with severe side effects [ ] . therefore, nanomedicines have on several occasions been used to target adjuvants to immune cells (mainly apcs) in the lymph nodes to avoid side effects and to enhance efficacy. for example, ph-sensitive nanogels have been designed to chemically conjugate the tlr / agonist imidazoquinoline. these nanogels were able to drain to lymph nodes of mice after subcutaneous injection, in which they were endocytosed by apcs and intracellularly degraded. the free polymer chains conjugated with imidazoquinoline were liberated, which triggered the tlr / pathway [ ] . furthermore, nanomedicines incorporating both adjuvants and tumor antigens have also been exploited as cancer vaccines to mount an adaptive immune response against overexpressed self-antigens or neo-antigens that arise from somatic mutations in the tumor [ ] . in addition to synthetic materials, cell-derived nanocarriers have shown their potential in delivering antigens for immunotherapy. for example, nanoparticles based on tumor cell membranes fused with erythrocyte membranes have been engineered, which displayed tumor antigens and resulted in efficient antigen responses in mice [ ] . the final step in the cascade is cancer cell recognition and killing by cytotoxic t lymphocytes at the tumor site. this process can be hampered by multiple phenomena, including the expression of t cell inhibitory cytokines and checkpoints. to maintain the expansion and effect or functions of t cells, liposomes loaded with two cytokines (interleukins il- and il- ) were fabricated with maleimide functional groups. the liposomes were able to chemically conjugate with the surface of t cells via thiol groups on the cells and slowly release cytokines to stimulate the t cells [ ] . the results showed that the liposome-treated t cells showed greater endurance in vivo in comparison to nontreated t cells, leading to improved therapeutic efficacy. analogously, the same group also developed liposomes binding to the thy . receptor of t cells, which were also functionalized with recombinant il- that binds to the il- receptor on activated t cells. these dualfunctional liposomes enabled the increased expansion of t cells in vivo and enhanced tumor eradication by t cells [ ] . targeting strategies in nano-immunotherapy depending on the targeting properties of nanomedicines, they can be utilized to boost cancer immunotherapy in three different ways [ ] (fig. ) . the first strategy is to target and kill cancer cells to induce specific forms of immune-activating cell death (i.e., icd). as discussed above, such nanomedicines typically incorporate icd-inducing chemotherapeutic drugs such as doxorubicin and oxaliplatin, and the added value of using nanomedicines in this regard is related to their ability to more efficiently deliver drugs to tumors (compared with the free drug) and at the same time to decrease accumulation and toxicity in healthy tissues. the tumor immune microenvironment (time) [ ] plays crucial roles in cancer development, metastasis, and resistance to (immuno)therapy. the time contains multiple types of suppressive immune cells, such as tumor-associated macrophages (tams), regulatory t cells and myeloid-derived suppressor cells. in addition, there are several classes of soluble inhibitors, such as indoleamine , -dioxygenase and transforming growth factor beta, which activate immunosuppressive pathways and thereby prohibit antitumor immunity. nanomedicines have been designed to target and modulate time. for example, nanomedicines such as iron oxide nanoparticles [ ] and caco nanoparticles [ ] have been repurposed to modulate the phenotype of tams, leading to increased ratios of anticancer m -like tams in tumor tissues. these tam-modulating nano-immunotherapy regimens improved the efficacy of checkpoint-inhibiting antibodies in multiple cancer models in mice [ ] . in addition to targeting cancer cells and modulating the time, nanomedicines can also target components of the peripheral immune system. such approaches primarily target apcs in secondary lymphoid organs as well as immune cells in the fig. integration of nanomedicines in the cancer-immunity cycle. nanomedicines can be used in each of the steps of the cancerimmunity cycle to potentiate antigen release from cancer cells, promote antigen uptake and processing by antigen-presenting cells, and support the presentation of cancer antigens to t cells to stimulate t cells to recognize and kill cancer cells [adapted with permission from ref. [ ] . copyright , the royal society of chemistry]. cancer nanomedicine meets immunotherapy q sun et al. circulation, including t cells. nanomedicines have been utilized for cancer vaccination by enabling the efficient delivery of antigens and/or adjuvants to peripheral immune organs and cells. for example, therapeutic nanovaccines based on mrnas encoding patient-specific antigens were fabricated with ionizable lipids. by tuning the physicochemical properties (size, surface charge, and stability), the nanovaccines accumulated in the spleen and transfected splenic apcs, leading to t cell responses to cancer antigens in mouse models and in patients [ ] . to target t cells in the circulation, nanomedicines carrying the leukemia-specific - bbz gene were designed to engineer peripheral t cells to express chimeric antigen receptor as a part of an alternative approach for car t therapy, which has the potential to reduce the high cost of adoptive t cell therapy [ ] . applications of nanomedicines in clinical immuno-oncology cancer nanomedicines have been applied in immunotherapy not only in preclinical studies but also in a number of clinical trials [ ] . to date, nanomedicines, including abraxane, doxil, and mrna nanovaccines, have been tested in~ clinical trials (fig. a) . most of these trials use chemotherapeutic nanomedicines (i.e., abraxane and doxil). the number of clinical trials involving nanomedicines in immuno-oncology has rapidly increased in the past years (fig. b) . the majority of current clinical trials are at stage i/ii; only a few trials with abraxane and doxil have proceeded to phase iii (fig. c ). fig. d shows that checkpointinhibiting antibodies are the most commonly used immunotherapeutics in combination with nanomedicines in clinics. importantly, abraxane combined with atezolizumab has been approved by the fda for the treatment of triple-negative breast cancer. the very positive outcome of this trial strongly supports the future development of combined nano-immunotherapy. there are multiple challenges that are encountered when aiming to translate immunomodulatory nanomedicines from the bench to the bedside. these are related not only to the material design but also to the clinical trial design. several key issues in this regard are discussed below. nanomedicine formulations are much more complex than small molecule drug formulations, and multiple physicochemical properties, such as size, charge, structure, composition, surface properties, and colloidal stability, all play important roles in determining their in vivo performance [ ] . the physicochemical properties of nanomedicines may significantly change after administration due to interactions with biological components (e.g., protein corona), which makes it difficult to predict in vivo performance [ ] . furthermore, because of their higher level of complexity, large scale production of nanomedicine formulations is more difficult than that of small molecule drugs. this not only affects nanomedicine production but also the reproducibility of preclinical studies involving nanomedicines. to address this issue, it might be useful to introduce standardized protocols for nanomedicine production, characterization, and results reporting [ ] . moreover, the scaling up of nanomedicine production can be difficult, especially for nanomedicines based on multiple different components. it is a significant challenge for complex nanomedicines to be produced at large scales for late-stage preclinical and clinical studies. therefore, to promote the clinical translation of immunomodulatory nanomedicines, nanomedicines should be designed based on components that are as simple as possible and on production protocols that are as scalable as possible [ ] . in addition, toxicities are also a critical issue that must be considered for the clinical translation of nanomedicines [ , ] . beyond issues related to the materials design and production, translation challenges in nanomedicine and nano-immunotherapy also entail clinical trial design. key issues in this regard are patient recruitment and patient stratification. patient recruitment is problematic because the numbers of chemo-immunotherapy and nano-chemo-immunotherapy trials have increased exponentially in the last - years [ ] . patient stratification is arguably one of the most important future challenges for cancer nanomedicine in general. nanomedicines are mostly applied nonselectively to patients in whom standard treatments fail. however, it is highly questionable whether this is the right strategy to explore their clinical therapeutic potential. as has been seen for molecularly targeted therapeutics and antibodies, patients have to be screened for certain biomarkers to be included in clinical trials. likewise, nanomedicines should only be used in the right patients, e.g., in those who show efficient accumulation of nanomedicines in tumors and/or metastases, to increase the chances of these patients showing good therapeutic responses [ ] . combination nano-immunotherapy, like other translational anticancer drug development and drug therapy fields, faces additional challenges because of gaps between preclinical and clinical studies. firstly, as already alluded to above, large numbers of patients are fig. targeting strategies in nano-immunotherapy. left: nanomedicines can be designed to target cancer cells and to elicit immunogenic cell death, thereby synergizing with immunotherapy. middle: nanomedicines targeting the tumor immune microenvironment are able to beneficially modulate the immunosuppressive local environment to promote immunoactivation. right: nanomedicines can also be developed to target the immune system outside of tumor tissues to potentiate antigen presentation in secondary lymphoid organs and to activate peripheral immune cells [adapted with permission from ref. [ ] . copyright , american chemical society]. cancer nanomedicine meets immunotherapy q sun et al. already being enrolled by a rapidly increasing number of immunooncology trials [ ] that mostly involve clinically established drugs together with novel immunotherapeutics. therefore, it is becoming increasingly challenging to recruit sufficiently high numbers of patients in a sufficiently short period of time for inclusion in nanoimmunotherapy trials. secondly, results from clinical trials on nanoimmunotherapy are not well in line with the outcomes of preclinical reports. for example, in preclinical studies, icd-inducing nanomedicines almost always strongly potentiate checkpoint inhibition therapy. clinically, however, various large trials involving nanomedicines have failed. the phase iii javelin ovarian trial, for instance, compared avelumab (bavencio®) plus doxil treatment to avelumab monotherapy in platinum-refractory ovarian cancer patients and failed to demonstrate a significant improvement of the therapeutic outcome by the combination treatment. one explanation for this could be that many patients who were included were not appropriate for combination treatment, as a retrospective analysis of the data showed a significant difference in response to the combination treatment between patients with positive or negative programmed death-ligand expression in their tumors [ ] . this exemplifies the crucial role of clinical trial design; especially in the case of nano-immunotherapy, it is critical to identify biomarkers for patient stratification and treatment outcome prediction. combining nanomedicines with immunotherapy will significantly expand their applicability in cancer therapy. nano-immunotherapy has demonstrated greatly improved therapeutic efficacy in preclinical study setups, and positive results have also already been reported for several clinical trials which have recently been completed or are currently ongoing. to further exploit the benefits of combining nanomedicines with immunotherapeutics, several challenges need to be overcome, including issues related to (the complexity of) nanomaterial design and clinical trial design. by carefully addressing these issues, nano-immunotherapy will become more broadly applied and appreciated, contributing to better treatment options for patients in need. cancer nanomedicine meets immunotherapy q sun et al. nanocarriers as an emerging platform for cancer therapy cancer nanomedicine: progress, challenges and opportunities a new concept for macromolecular therapeutics in cancer chemotherapy: mechanism of tumoritropic accumulation of proteins and the antitumor agent smancs microvascular permeability of normal and neoplastic tissues ligand-installed nanocarriers toward precision therapy the battle of "nano" paclitaxel survey of clinical translation of cancer nanomedicines-lessons learned from successes and failures to exploit the tumor microenvironment: since the epr effect fails in the clinic, what is the future of nanomedicine? analysis of nanoparticle delivery to tumours the role of liposomes in clinical nanomedicine development. what now? now what? smart cancer nanomedicine cancer nanomedicine: is targeting our target? evaluation of nanomedicines: stick to the basics nanomedicine and macroscale materials in immuno-oncology combining nanomedicine and immunotherapy cellular bioparticulates with therapeutics for cancer immunotherapy recent advances in nanomaterial-based synergistic combination cancer immunotherapy engineering nanoparticles to reprogram the tumor immune microenvironment for improved cancer immunotherapy disease immunotherapy: immunomodulatory nanosystems clinical translation of nanomedicine and biomaterials for cancer immunotherapy: progress and perspectives improving cancer immunotherapy using nanomedicines: progress, opportunities and challenges immunostimulatory nanomedicines synergize with checkpoint blockade immunotherapy to eradicate colorectal tumors oncology meets immunology: the cancer-immunity cycle immunogenic cell death in cancer therapy immunogenic cell death in cancer and infectious disease doxil synergizes with cancer immunotherapies to enhance antitumor responses in syngeneic mouse models inducing enhanced immunogenic cell death with nanocarrier-based drug delivery systems for pancreatic cancer therapy toll-like receptors and cancer rational design of small molecules as vaccine adjuvants ph-degradable imidazoquinoline-ligated nanogels for lymph node-focused immune activation peptide-tlr- / a conjugate vaccines chemically programmed for nanoparticle self-assembly enhance cd t-cell immunity to tumor antigens red blood cell-derived nanoerythrosome for antigen delivery with enhanced cancer immunotherapy therapeutic cell engineering with surface-conjugated synthetic nanoparticles in vivo targeting of adoptively transferred t-cells with antibody-and cytokine-conjugated liposomes understanding the tumor immune microenvironment (time) for effective therapy iron oxide nanoparticles inhibit tumour growth by inducing pro-inflammatory macrophage polarization in tumour tissues in situ sprayed bioresponsive immunotherapeutic gel for post-surgical cancer treatment systemic rna delivery to dendritic cells exploits antiviral defence for cancer immunotherapy in situ programming of leukaemia-specific t cells using synthetic dna nanocarriers physical chemistry of nanomedicine: understanding the complex behaviors of nanoparticles in vivo interactions of nanomaterials and biological systems: implications to personalized nanomedicine minimumin formation reporting in bio-nano experimental literature complexity and simplification in the development of nanomedicines nanotoxicology and nanomedicine: making hard decisions an overview of nanotoxicity and nanomedicine research: principles, progress and implications for cancer therapy the clinical trial landscape for pd /pdl immune checkpoint inhibitors competing interests: the authors declare no competing financial interests. key: cord- -h lrtecc authors: yu, hai-tao; zhen, juan; xu, jian-xiang; cai, lu; leng, ji-yan; ji, hong-lei; keller, bradley b title: zinc protects against cadmium-induced toxicity in neonatal murine engineered cardiac tissues via metallothionein-dependent and independent mechanisms date: - - journal: acta pharmacol sin doi: . /s - - -y sha: doc_id: cord_uid: h lrtecc cadmium (cd) is a nonessential heavy metal and a prevalent environmental toxin that has been shown to induce significant cardiomyocyte apoptosis in neonatal murine engineered cardiac tissues (ects). in contrast, zinc (zn) is a potent metallothionein (mt) inducer, which plays an important role in protection against cd toxicity. in this study, we investigated the protective effects of zn against cd toxicity in ects and explore the underlying mechanisms. ects were constructed from neonatal ventricular cells of wild-type (wt) mice and mice with global mt gene deletion (mt-ko). in wt-ects, cd ( − μm) caused a dose-dependent toxicity that was detected within h evidenced by suppressed beating, apoptosis, and ldh release; zn ( − μm) dose-dependently induced mt expression in ects without causing ect toxicity; co-treatment of ect with zn ( µm) prevented cd-induced toxicity. in mt-ko ects, cd toxicity was enhanced; but unexpectedly, cotreatment with zn provided partial protection against cd toxicity. furthermore, cd, but not zn, significantly activated nrf and its downstream targets, including ho- ; inhibition of ho- by a specific ho- inhibitor, znpp ( µm), significantly increased cd-induced toxicity, but did not inhibit zn protection against cd injury, suggesting that nrf -mediated ho- activation was not required for zn protective effect. finally, the ability of zn to reduce cd uptake provided an additional mt-independent mechanism for reducing cd toxicity. thus, zn exerts protective effects against cd toxicity for murine ects that are partially mt-mediated. further studies are required to translate these findings towards clinical trials. cadmium (cd) is a nonessential toxic heavy metal and a nondegradable environmental toxin. cd toxicity is associated with various cellular, metabolic, homeostatic, and repair mechanisms, including increased reactive oxygen species (ros) production and apoptosis [ ] . since cd mainly accumulates in the liver and kidneys, most research has focused on cd toxicity in these organs. however, cd is also toxic to other tissues including the heart, lungs, and adipose tissues [ ] . epidemiologic and preclinical studies show that low to moderate cd exposure is associated with cardiovascular (cv) dysfunction and disease including arrhythmias [ , ] , hypertension [ , ] , myocardial infarction [ , ] , and heart failure [ ] [ ] [ ] . urine cd levels are associated with increased cv disease incidence and mortality [ ] . cd toxicity to cardiomyocyte (cm) occurs at -fold lower dose than reported for hepatic and renal cell toxicity [ ] . pathological changes in the heart have been reported following cd exposure, and the underline mechanisms responsible for cd toxicity in cv disease [ ] and potential preventive strategies have not been fully characterized. by contrast to cd, zinc (zn) is an essential metal that supports a wide range of biological processes. the protective effect of zn on cd-induced injury has been studied in vitro and in vivo [ , ] , however, the underlying mechanisms remain undefined. since zn is a potent mt inducer via the metal-response element-binding transcription factor (mtf , [ ] [ ] [ ] [ ] ), it is possible that zninduced mt overexpression plays an important role in protection against cd toxicity [ ] [ ] [ ] [ ] [ ] . in contrast to zn-mediated mt induction, cd also results in mt induction as part of the cellular response to injury. attenuation of ros activation by zn also plays an important role [ ] . zn has been shown to be protective against cd toxicity in liver, kidney, and bone [ , , ] . our current study further explores underlying mechanisms for zn protection against cm cd toxicity. engineered cardiac tissues (ects) are now used globally as a robust platform technology that is well suited for tissue repair paradigms and for scalable in vitro drug screening and disease modeling [ ] [ ] [ ] [ ] [ ] [ ] . one of the strengths of the ect paradigm is that formulations can incorporate multiple cell lineages including cardiomyocytes, fibroblasts, endothelial cells, and mural cells [ ] . in general, ects cultured in vitro have not been shown to include immunologic cells. however, this ect paradigm has not been broadly applied to environmental toxicology related studies. while in vivo environmental toxicity models allow the validation of requisite pathways using transgenic over-expression and knockout strategies, these studies can require treatment for weeks to months to identify phenotypes with both on-target and off-target effects of compounds and countermeasures of interest while in vitro ect platforms can provide reproducible functional, biochemical, and molecular data within days [ ] . we have demonstrated that mouse-derived ects can serve as a robust model to study in vitro cd toxicity with respect to both ect dysfunction and cm injury, and ects derived from mt overexpressing mice display increased tolerance to cd toxicity [ ] . recognizing that zn has been described to reduce cd toxicity in other organs, we used our murine ect platform to address whether zn can protect ects from cd toxicity, in part via mt dependent pathways, as has been noted in animal models. we found that zn supplementation protected ects from cd toxicity. to determine the requirement for mt in zn-mediated protection from cd toxicity, we found that ects derived from mt-ko ventricular cells showed increased cd toxicity versus wt-ect and that zn could partially protect cd-induced cell death in mt-ko ects, likely due to zn-mediated, mt-independent, reduced cd uptake. we also found that although an ho- inhibitor enhanced cd toxicity, it had no impact on zn protection from cd toxicity. these findings are consistent with our hypothesis that both direct competition between zn and cd uptake and zn-mediated mt induction play important roles in zn protection from cm cd toxicity in murine ects. all mice were purchased from the jackson laboratory (bar harbor, me). mt-ko (mt −/− , both mt and mt gene knockout) and its wild-type s mice, as well as friend leukemia virus b (fvb) mice were housed in the university of louisville research resources center at • c with a -h light/dark cycle and were provided free access to standard rodent chow and tap water. all animal procedures were approved by the institutional animal case and use committee, which is certified by the american association for accreditation of laboratory animal care. we bred fvb to generate fvb pups for all experiments that did not relate the investigation of the role of mt knockout on the cd and zn responses. we bred s wt and s-mt −/− to generate s pups and mt −/− pups, respectively, in order to generate wt-and mt-ko ects. isolation of neonatal mouse ventricular cardiac cells neonatal mouse ventricular cardiac cells were isolated as previous described [ ] . briefly, hearts of -day-old pups were harvested and digested by trypsin and collagen. cm were enriched by preplating for min followed by rotating for h and then heart cells were collected and counted using a hemocytometer. ect construction and treatment ects were constructed as previously described [ , ] . approximately . × heart cells derived from four pup ventricles were mixed with collagen and matrigel, poured to form a cylinder construct within flexcell tissue train tm culture plates (flexcell international), and incubated for h ( °c, % co ) to form a cylindrical ect construct. following initial ect gelation, ml of mouse medium was added to each ect in the six-well culture dish. ects were maintained in vitro for days with media changes every other day. randomly selected ects were treated with μm cdcl for h starting on day , with µm zn for h starting on day , cotreated with zn and cdcl , or pretreated with ho- inhibitor (znpp µm) for h and then treated with μm cdcl or/and µm zn for another h starting on day . the concentrations for cdcl used in the zn cotreatment study were based on the dose-response curve shown in fig. and table . the zn dose used in the zn cotreatment study were based on the dose-response curve shown in fig. . ects were harvested from the tissue train tm plate on day and the mid-portion of the ect was washed times in pbs then placed in a tissue tek® cryomold (torrance, ca) with oct compound for embedding followed by snap freezing in liquid nitrogen. individual µm cryostat sections were mounted, fixed in acetone for - min, washed in % triton-x- /pbs for h at room temperature, then blocked with % triton-x- /pbs + % fbs for h. ect sections were stained with rabbit anti-nrf ( : dilution; abcam, cambridge, ma) as previously published [ , ] . other sections were mounted using slowfade™ gold antifade mountant to image dapi (thermo fisher scientific, eugene, or). nuclear nrf translocation was detected by fluorescence microscopy. protein extraction and western blotting protocols for ect western blot assays have previous described [ ] . briefly, ects were washed thoroughly in ice-cold pbs the rapidly homogenized in lysis buffer ( µl/ect) at °c for h. after measuring protein concentration, equal protein aliquots ( μg) were loaded for analysis. primary antibodies included cleaved caspase [ ] , ho- ( : , dilution; cell signaling technology, danvers, ma), and nrf ( : , dilution; abcam, cambridge, ma) using anti-rabbit secondary antibodies ( : , dilution; cell signal, danvers, ma). we report cleaved-caspase rather than total caspase because of the inability to detect both cleaved and total caspase in the same western (supplemental fig. ). mt expression was detected by a modified western blot protocol as previously described using an mt antibody ( : , dilution; dako, carpinteria, ca) [ ] . protein expression was normalized to gapdh ( : , dilution; abcam, cambridge, ma). rna extraction and real-time pcr quantification rna extraction and quantitative real-time pcr was performed as previously described [ , ] . tdt-mediated dutp nick-end labeling assay (tunel) we used the deadend™ fluorometric tunel system (promega, madison, wi) to determine the apoptotic cell proportion following the promega protocol [ ] . tunel positive cells were counted in fields per ect lactate dehydrogenase release in culture medium we used pierce™ ldh cytotoxicity assay kit (thermo fisher scientific, rockford, il) to determine the ldh release into the culture medium [ ] as an index of necrotic cell death [ ] . briefly, µl of each sample medium was transferred to a -well flat-bottom plate in duplicate wells, mixed with µl of reaction mixture, then incubated room temperature for min in the dark followed by the addition of µl of stop solution to each sample well. light absorbance at and nm was measured using a molecular devices sprectramax (molecular devices, sunnyvale, ca) to quantify signal ( nm) and noise ( nm) absorbance. to measure ect cd and zn, ects were removed, washed with milli-q water, then heated to dryness. ect supernatant was also collected on day during ect harvest. all samples were digested by ml of % nitric acid at °c for h, diluted into ml deionized water containing % nitric acid, then analyzed inductively coupled plasma mass spectrometry (icp-ms) (thermo zn protection from cd injury in engineered cardiac tissues ht yu et al. scientific, waltham, usa). concentrations are reported as nanograms of metal per ect or per µl medium (ng/ect or ng/ µl, [ ] ). data are presented as mean ± standard error of the mean (sem). statistical differences were determined using two-sided, unpaired student's t tests or two-way analysis of variance (anova) followed by tukey's multiple comparisons test. p value < . was considered statistically significant. we noted a dose-dependent increase of cleaved caspase , a surrogate for cell death, after ect cd treatment for h, as well as a time-dependent effect of cd toxicity measured by increasing induced cleaved caspase expression (fig. a) . as has been noted in previous ect studies, there is a low level of time-dependent cell death in the ect model [ , ] so that treatment effects need to be determined using groups of similar culture duration. cleaved caspase expression increased at h after µm and µm cd dosing, however, lower dose cd, µm, induced increased cleaved caspase expression only after and h of exposure (fig. a) . cumulative ldh release into the culture medium, an additional indicator of cell injury, also showed a dose-and timedependent response to cd treatment. because a µm cd dose was required to induce significantly increased cleaved caspase and ldh at h, we used this dose and duration for all subsequent experiments (fig. b) . the decrease of caspase cleavage after h incubation with µm and µm cd may be due to progressive cell loss resulting in fewer cells able to produce cleaved caspase (most of ects died at h time point, see table ). ect function also showed a dose-and time-dependent responses to cd toxicity with a progressive inhibition of ect beating with increased cd dose and duration (table ) . we defined cd-induced changes in ect function a global classification scheme: normal function (nl); increased ect deformation during shortening with regional dyssynchrony (a); increased ect deformation during shortening with regional dyssynchrony (b); (table ) with a progressive shift from normal, synchronous beating to increased deformation with regional dyssynchrony followed by global dyssynchrony and sensation of beating by h after treatment in wt ect, similar to our previously published data [ ] . tunel assay positive (green) cells increased after cd treatment compared with ctrl ects (fig. c) . zn and cd effect on mt expression both zn and cd increase mt expression in multiple cell types including cm [ ] . therefore, we determined the impact of zn and cd treatment on mt expression in murine ects. zn induced a significant dose-dependent increase in mt expression (fig. a) and noted increased ldh release as evidence for zn toxicity only at higher doses (fig. b) . we found that µm zn induced a significant increase of ldh release by h and no increase in ldh release was observed up to h after a single µm zn dose. therefore, we chose µm zn dosing for subsequent experiments. both cd ( µm) and zn ( µm) induced increased mt expression in murine ect, and cotreatment with cd and zn further increased mt expression (fig. ) . increased mt- gene expression paralleled the increase in mt protein expression noted after zn and/or cd ect treatment (fig. d) . zn impact on cd toxicity several studies have reported a therapeutic role of zn in reducing cd toxicity in vivo and in vitro [ , ] . we therefore tested the ability of zn to reduce cd-induced ect toxicity. as noted above, cd treatment increased cm apoptosis in ects indicated by increased cleaved caspase and zn reduced cd-induced cleaved zn protection from cd injury in engineered cardiac tissues ht yu et al. caspase (fig. a) . similar results were detected by ldh assay in culture medium (fig. b) . cd-induced ect toxicity increased the ect tunel positive cell ratio (fig. c) as well as some nonnuclear tunel staining consistent with cell death and nuclear rupture. consistent with the ability of zn to block cd-induced cell injury, we noted that zn prevented cd-induced ect functional impairment ( table ). the role of mt in zn protection from cd toxicity zn-induced mt overexpression has been reported to be partially responsible for the zn protective effect in several models [ ] [ ] [ ] . therefore, to further evaluate zn-dependent and independent roles for mt in ect protection from cd toxicity we generated ects from mt-ko cardiac cells in comparison with ects composed of control s cardiac cells. we confirmed that mt-ko derived ects lacked mt expression and that mt was not induced by zn in mt-ko ects (fig. a) . we noted no differences (gel compaction, beat rate, cellularity) in ect formulated from fvb versus s wt cells. we treated wt-ect and mt-ko ect with cd, zn, or zn + cd for h. ect function following cd exposure was digitally recorded for s at , , , and h starting at the time of cd treatment on day . ect function was then qualitatively classified as: normal function (nl); increased ect deformation during shortening with regional dyssynchrony (a); increased ect deformation during shortening with regional dyssynchrony (b); decreased beat rate with global dyssynchrony (c); and arrested beating (d). group sizes ranged from n = - . wt ects displayed dysfunction h after μm cd exposure with progressive dysfunction resulting in absent contractions by h after cd exposure. zn cotreatment with cd prevented cd induced ect dysfunction. n = - ects per group. zn protection from cd injury in engineered cardiac tissues ht yu et al. increased ldh release in culture medium (fig. b) and increased cleaved caspase expression (fig. c) . zn treatment ( µm × h) blocked increased cleaved caspase and ldh release in the zn + cd wt ect group. it is important to note that zn treatment partially suppressed cd-induced increased cleaved caspase and ldh release in mt-ko ect (fig. b, c ) consistent with an mt independent and as well as an mt requirement for zn protective effects. effect of cd and zn on nrf and downstream gene expression the nrf pathway has been shown to be upregulated during the antioxidative response to cellular stresses, including nrf induction following cd exposure [ ] and zn-mediated nrf induction [ , ] . following ect cd treatment, nrf was significantly activated (fig. a) as were nrf downstream genes ho- , noq- , and cat (fig. b) . this cd-mediated increase in nrf and downstream genes was reduced by zn cotreatment (fig. ) . of note, ect treatment with cd, zn, and zn + cd had no effect on sod (fig. b) . cd treatment increased nrf nuclear translocation that was partially reduced by zn cotreatment (fig. ). heme oxygenase- (ho- ), a stress-inducible enzyme that mediates antioxidative and cytoprotective effects to maintain cellular redox homeostasis and protects cells from oxidative stress. we noted a dose-and time-dependent increase in ho- expression after ect cd treatment (fig. a) that was reduced by zn cotreatment (fig. b) . we also found that ect pretreatment with the ho- inhibitor, znpp ( µm) enhanced cd toxicity indicated by increased cleaved caspase expression and increased ldh release (fig. a, b) . interestingly, ho- -inhibition had no impact on zn protection from cd toxicity (fig. a, b) suggesting that zn and ho- are both involved in the ect response to cd toxicity, but are not co-dependent. zn reduces ect cd uptake as an mt-independent protection from cd toxicity cd and zn are known to compete for cellular uptake via metal transporters and therefore zn inhibition of cd uptake could function as an mt-independent mechanism to reduce cd toxicity. we measured ect cd and zn uptake and found that zn cotreatment with cd significantly reduced ect cd accumulation with a complementary increase in culture medium cd content ( table ) . as would be expected, zn treatment increased ect zn content, though somewhat surprisingly, cd and zn cotreatment further increased ect zn content (table ) . our previous study established the basic paradigm of our murine ect model to study cd toxicity and noted that cm specific mt overexpression increased ect tolerance to cd toxicity. in the current study, we found that zn could induce mt overexpression in a dose-dependent manner (fig. a) , and zn cotreatment with cd could totally block the cytotoxicity of cd (fig. ) . we also defined that mt play a partial role in zn protection on cd toxicity by using mt-ko-derived ects (fig. b, c ). in addition, we found that nrf /ho- signaling pathway was involved in cd toxicity (figs. - , a, b), but it was not related to zn protection on cd toxicity (fig. a, b) . cd is a toxic heavy metal. in vitro and in vivo studies have proven the toxic effects of cd on a variety of tissues, including the liver [ ] , kidney [ ] , heart [ ] , lung [ ] , etc. upon uptake into the body, cd can complex with mt, a known heavy metal chelator, for detoxification [ ] . in wild-type and mt-null mice given increasing doses of cd, a remarkable acquired tolerance to cd lethality is evident in wild-type mice, with a sevenfold difference in ld values, however, such tolerance does not happen in mtnull mice [ ] . moreover, cd exposure in mt-null mice results in nephrotoxicity at one-tenth the nephrotoxic dose for control mice [ ] . conversely, mt overexpression mice are protected against acute cd lethality and hepatotoxicity [ ] . our current study used an established neonatal murine ect in vitro model to determine dose-and time-dependent cd toxicity with adverse effects noted on ect function and cell death and a protective effect of mt overexpression, as we have previously noted [ ] . we now show that cd exposure in mt-ko ects increased cell death with increased cleaved caspase- and ldh release and ect dysfunction confirming a critical role for mt in the response to cd exposure. the metal ion zn has been shown to be essential for normal biologic function in most living cells as a cofactor for many enzymes including the important zn-finger proteins. in addition to its structural and catalytic properties [ ] during normal homeostasis, zn has been also shown to support cellular antioxidant responses via several pathways including the inhibition of nadph oxidase, activation of superoxide dismutase (sod), induction of mt, and the upregulation of nrf and nrf downstream genes [ ] . it has been reported that the mechanism for zn-induced tolerance to cd toxicity results primarily from the induction of mt synthesis [ , ] . in renal cell lines from wt and mt- /mt- knockout (mt-ko) mice, pretreatment with zn increases mt expression and enhanced cd resistance in wt cells; however, zn pretreatment of mt-ko does not increase tolerance to cd [ ] . similar to previous in vivo studies, we noted that zn prevented cd induced-cell death in wt ects [ ] , however, zn treatment was only partially protective in mt-ko ects, likely via mt-independent inhibition of cd uptake. further studies are needed to determine the dose-responses of individual ect lineages (cm, ec, f) to cd toxicity and zn prevention of toxicity. many studies have investigated the role of oxidative stress and the balance between ros/reactive nitrogen species (rns) production in cd-mediated cellular toxicity [ ] . the level of cellular oxidative stress is dynamically determined by a biological system's ability to detoxify these reactive intermediates. nf-e related nuclear factor (nrf ) is rapidly upregulated early in the zn protection from cd injury in engineered cardiac tissues ht yu et al. response to oxidative stress and then translocates to the nucleus to increase the transcription of oxidative stress response genes as an adaptive mechanism. under normal conditions, nrf is sequestered by kelch-like ech associating protein (keap ) in the cytosol and degraded by proteasomes. in response to oxidative stimuli, nrf is released from keap , translocates into the nucleus, and induces its target genes by binding to antioxidant response elements (are) to regulate antioxidantmediated gene expression [ ] . we found that murine ect cd exposure increased nrf expression and enhanced the translocation of nrf into the nucleus, with an increase in nrf downstream gene expression, similar to the adaptive nrf response that has been noted in vitro [ , ] . zn treatment also facilitated the nrf adaptive response. we noted a reduced nrf induction following cd treatment in zn treated ects consistent with reduced injury and reduced injury response. additional studies using confocal imaging and lineage-specific staining may provide an additional refinement of the ect lineages (cm, ec, f, etc.) that show increased nrf expression and nuclear translocation in response to cd toxicity and electron microscopic imaging may provide additional insights into subcellular organelle injury following cd exposure. ects have multiple advantages over monolayer d culture methods including the formation of isotropic and aligned in vitro d myocardial tissues that electrically and mechanically function similar to in vivo myocardium. ects rapidly mature in vitro, begin to beat synchronously from culture day , and display altered beating rate and pattern in response to environmental stress (cd, [ ] . in the current study we noted that ect dysfunction within h of cd treatment and dose-dependent suspension of beating (tables , ). further studies are required to define the roles of altered calcium signaling [ , ] , mitochondrial function [ , ] , and changes in oxidative stress and ros as potential mechanisms for cd-mediated altered ect electrical and mechanical function. ects have one additional, essential advantage over in vivo models which is the ability to customize cell composition to include zn protection from cd injury in engineered cardiac tissues ht yu et al. multiple mesoderm lineages and/or genetically altered cm [ , ] . our current study uses ventricular myocardial cells from normal and transgenic newborn pups. this approach is highly adaptable to use transgenic models of interest to explore specific signaling pathways and to human stem-cell-derived mesodermal cells including cm, myofibroblasts, endothelial cells, and mural cells [ , ] . further studies are required to determine ect lineage-specific sensitivities to cd injury and potential differential effectiveness of zn to prevent lineage-specific cd related injury. ho- is one of the key downstream genes induced by nrf signaling pathway with important antioxidant and cytoprotective effects via a reduction in intracellular levels of the prooxidant heme and by the increased production of cytoprotective levels of carbon monoxide (co) and biliverdin (bv) [ ] . not surprisingly, we noted a significant dose-and time-dependent induction of ho- expression in response to ect cd exposure. we confirmed the adaptive role of ho- related to ect cd toxicity using an ho- inhibitor (znpp), which increased ect sensitivity to cd toxicity. however, ho- inhibition by znpp did not reduce znmediated protection from cd toxicity, confirming multiple cellular adaptive responses to cd toxicity. cd and zn possess similar chemical properties and have been shown to compete for uptake in a variety of tissues [ , [ ] [ ] [ ] and our results are consistent that one mechanism for znmediated reduction in cd toxicity is reduced cd accumulation in ects. further studies are required to identify the specific transporters and transporter efficiencies by which cd and zn compete for cellular uptake. candidate transporters for both cd and zn include zip [ ] , zip [ ] , dmt [ ] , and several calcium channels [ ] . specific transporter knockout mice may be helpful as novel sources for cardiac cells to further explore zn and cd transport and cd toxicity in myocardial tissues. since ros play fig. the ho- inhibitor znpp increases ect cd toxicity. a cleaved caspase following treatment with zn ( µm) and/or cd ( µm) and/or znpp ( µm) for h by wb. group sizes ranged from n = - per group. **p < . vs. corresponding group. b ldh release following treatment with zn ( µm) and/or cd ( µm) and/or znpp ( µm) for h. group sizes ranged from n = - per group. **p < . vs. corresponding group. as expected, cd content was only noted in treated ects. zn was present in ctrl ects and increased after zn treatment. ect cd content decreased and media cd content increased following zn and cd cotreatment. *p < . vs. corresponding µm zn group, **p < . vs. corresponding µm cd group. n = ects per group zn protection from cd injury in engineered cardiac tissues ht yu et al. an important role in cd toxicity and in zn protection, further studies are also required to quantify and explore modulation of ros in zn-mediated protection for cd toxicity. cadmium and cellular signaling cascades: interactions between cell death and survival pathways cadmium modulates adipocyte functions in metallothionein-null mice modelling cadmium-induced cardiotoxicity using human pluripotent stem cell-derived cardiomyocytes neonatal murine engineered cardiac tissue toxicology model: impact of metallothionein overexpression on cadmium-induced injury cadmium and hypertension in exposed workers: a meta-analysis environmental exposure to cadmium: health risk assessment and its associations with hypertension and impaired kidney function association of urinary cadmium and myocardial infarction interrelation of cadmium, smoking, and cardiovascular disease (from the national health and nutrition examination survey) role of cadmium and magnesium in pathogenesis of idiopathic dilated cardiomyopathy cadmium exposure and incidence of heart failure and atrial fibrillation: a population-based prospective cohort study cadmium exposure in association with history of stroke and heart failure cadmium exposure and incident cardiovascular disease cytotoxicity of cadmium and characteristics of its transport in cardiomyocytes the inhibitory effect of zinc on cadmium-induced cell apoptosis and reactive oxygen species (ros) production in cell cultures assessment of cadmium-induced hepatotoxicity and protective effects of zinc against it using an improved cellbased biosensor the transcription factor mtf- is essential for basal and heavy metal-induced metallothionein gene expression the transcription factor mtf- mediates metal regulation of the mouse znt gene mechanisms of mammalian zinc-regulated gene expression detection and manipulation of the stress response protein metallothionein basal and zinc-induced metallothionein in resistance to cadmium, cisplatin, zinc, and tertbutyl hydroperoxide: studies using mt knockout and antisense-downregulated mt in mammalian cells metallothionein: an intracellular protein to protect against cadmium toxicity metallothionein plays less of a protective role in cadmium-metallothionein-induced nephrotoxicity than in cadmium chloride-induced hepatotoxicity cytotoxicity and induction of protective mechanisms in hepg cells exposed to cadmium cadmium carcinogenesis zinc pretreatment prevents hepatic stellate cells from cadmium-produced oxidative damage beneficial effect of zinc supplementation on biomechanical properties of femoral distal end and femoral diaphysis of male rats chronically exposed to cadmium nephrotoxicity of cadmium-metallothionein: protection by zinc and role of glutathione threedimensional reconstitution of embryonic cms in a collagen matrix: a new heart muscle model system development of a drug screening platform based on engineered heart tissue neonatal mouse-derived engineered cardiac tissue: a novel model system for studying genetic heart disease engineered heart tissue model of diabetic myocardium terminal differentiation, advanced organotypic maturation, and modeling of hypertrophic growth in engineered heart tissue impact of cell composition and geometry on human induced pluripotent stem cells-derived engineered cardiac tissue zinc is essential for the transcription function of nrf in human renal tubule cells in vitro and mouse kidney in vivo under the diabetic condition metallothionein is downstream of nrf and partially mediates sulforaphane prevention of diabetic cardiomyopathy vegf inhibition alters neurotrophin signalling pathways and induces caspase- activation and autophagy in rabbit retina cardiac metallothionein induction plays the major role in the prevention of diabetic cardiomyopathy by zinc supplementation oxidative damage of cardiomyocytes is limited by extracellular regulated kinases / -mediated induction of cyclooxygenase- detection of necrosis by release of lactate dehydrogenase activity agency for toxic substances and disease registry (us) pdgf-bb protects cardiomyocytes from apoptosis and improves contractile function of engineered heart tissue zinc-metallothionein: a potential mediator of antioxidant defence mechanisms in response to dopamine-induced stress the zinc-metallothionein redox system reduces oxidative stress in retinal pigment epithelial cells renal improvement by zinc in diabetic mice is associated with glucose metabolism signaling mediated by metallothionein and akt, but not akt roles of ros, nrf , and autophagy in cadmium-carcinogenesis and its prevention by sulforaphane zinc might prevent heat-induced hepatic injury by activating the nrf -antioxidant in mice low-dose cadmium causes metabolic and genetic dysregulation associated with fatty liver disease in mice heavy metal poisoning: the effects of cadmium on the kidney effects of chronic exposure to lead, cadmium, and manganese mixtures on oxidative stress in rat liver and heart cadmium induces oxidative stress and apoptosis in lung epithelial cells a review of metallothionein isoforms and their role in pathophysiology protective effect of metallothionein against the toxicity of cadmium and other metals transgenic mice that overexpress metallothionein-i are protected from cadmium lethality and hepatotoxicity impact of labile zinc on heart function: from physiology to pathophysiology zinc: an antioxidant and anti-inflammatory agent: role of zinc in degenerative disorders of aging oxidative signaling response to cadmium exposure the keap -btb protein is an adaptor that bridges nrf to a cul -based e ligase: oxidative stress sensing by a cul -keap ligase degradation of transcription factor nrf via the ubiquitin-proteasome pathway and stabilization by cadmium human trpv and trpv : key players in cadmium and zinc toxicity cadmium inhibits the electron transfer chain and induces reactive oxygen species influence of mitochondrion-toxic agents on the cardiovascular system the myocardial regenerative potential of three-dimensional engineered cardiac tissues composed of multiple human ips cell-derived cardiovascular cell lineages a review on heme oxygenase- induction: is it a necessary evil role of zinc in protection against cadmium-induced toxicity in formation of embryonic chick bone in tissue culture dietary zinc reduces uptake but not metallothionein binding and elimination of cadmium in the springtail, orchesella cincta zinc supplementation protects against cadmium accumulation and cytotoxicity in madin-darby bovine kidney cells cd + versus zn + uptake by the zip hco -dependent symporter: kinetics, electrogenicity and trafficking slc a gene encodes zip , a metal/bicarbonate symporter: similarities to the zip transporter dmt : which metals does it transport? this work was supported by the kosair charities pediatric heart research fund to bk, the u.s-china pediatric research exchange training program to lc and bbk, and the national natural science foundation of china to hlj ( ). all personnel expenses and partial research-related expenses for hty and jz were provided by jilin university through a collaborative research agreement between the university of louisville and jilin university, changchun, china. basic science experiments were key: cord- -tgcrg db authors: liu, hao-chen; zhou, xiao-ting; zheng, yun-si; he, hua; liu, xiao-quan title: pk/pd modeling based on no-et homeostasis for improving management of sunitinib-induced hypertension in rats date: - - journal: acta pharmacol sin doi: . /s - - - sha: doc_id: cord_uid: tgcrg db sunitinib is an oral small molecule multitargeted tyrosine kinase inhibitor, which is currently used to treat severe cancers. clinical research has shown that patients treated with sunitinib develop hypertension. as soon as sunitinib-induced hypertension appears, it is usual to administer anti-hypertension agent. but this treatment may cause acute blood pressure fluctuation which may lead to additional cardiovascular risk. the aim of this study is to establish a mathematical model for managing sunitinib-induced hypertension and blood pressure fluctuation. a mechanism-based pk/pd model was developed based on animal experiments. then this model was used to perform simulations, thus to propose an anti-hypertension indication, according to which the anti-hypertension treatment might yield relative low-level auc and fluctuation of blood pressure. the simulation results suggest that the anti-hypertension agent may yield low-level auc and fluctuation of blood pressure when relative et- level ranges from − % to % and relative no level is more than % compared to control group. finally, animal experiments were conducted to verify the simulation results. macitentan ( mg/kg) was administered based on the above anti-hypertension indication. compared with the untreated group, the optimized treatment significantly reduced the auc of blood pressure; meanwhile the fluctuation of blood pressure in optimized treatment group was % less than that in immediate treatment group. this work provides a novel model with potential translational value for managing sunitinib-induced hypertension. sunitinib, an oral small-molecule multitargeted tyrosine kinase inhibitor, is currently approved for the treatment of metastatic renal cell carcinoma, imatinib-resistant gastrointestinal stromal tumor (gist), and pancreatic neuroendocrine tumor [ ] . previous clinical research has shown that patients treated with sunitinib develop hypertension [ ] . hypertension induced by sunitinib is associated with serious cardiovascular complications, such as coronary artery disease (cad) and heart failure, and the use of vascular endothelial growth factor (vegf) inhibition may be compromised in cancer patients who develop hypertension [ , ] . improving the management of hypertension caused by sunitinib may be helpful in preventing severe cardiotoxicity. however, randomized controlled trials on the treatment of angiogenesis inhibition-induced hypertension are not available, and no clear recommendation for a particular antihypertensive agent or class agents can be given [ ] . the clinical use of sunitinib may be compromised without a suitable antihypertension strategy. to address this challenge, preclinical studies on suitable antihypertensive strategies should be performed. previous preclinical studies have investigated the use of antihypertensive therapy for hypertension induced by sunitinib [ , [ ] [ ] [ ] . these studies identified treatments for inhibiting hypertension induced by sunitinib, but the fluctuation of blood pressure during antihypertensive treatment was ignored in these studies. in the previous studies, sunitinib and antihypertensive agents were administered simultaneously, which may have yielded dramatic fluctuations in blood pressure; e.g., the antihypertensive treatment proposed by witte et al. doubled blood pressure fluctuation levels compared to that in subjects that did not receive antihypertensive treatment. the treatment protocol reported by lankhorst et al. resulted in a similar fluctuation, but such a fluctuation was not observed in subjects that did not receive antihypertensive treatment [ , ] . previous studies have revealed that blood pressure variability increases cardiac, vascular, and renal damage [ , ] . therefore, the high blood pressure variability induced by antihypertensive treatment may limit the benefits provided by lowering blood pressure. the aim of this study was to develop a mechanismbased pharmacokinetics-pharmacodynamic (pk/pd) model for proposing an optimized antihypertensive management method that can lower both blood pressure and its fluctuation in rats. in this study, a mechanism-based pk-pd model was developed to optimize antihypertensive treatment. it is crucial to understand the pathophysiological mechanisms of hypertension induced by sunitinib to develop such a model. sunitinib acts by inhibiting vascular endothelial growth factor receptors (vegfr- , vegfr- , and vegfr- ), platelet-derived growth factor receptors (pdgfr-α and pdgfr-β), stem cell factor receptor (kit), fms-like tyrosine kinase- receptor (flt ), colony stimulating factor receptor type (csf- r), and the glial cell linederived neurotrophic factor receptor (ret) [ ] . among these targets of sunitinib, the vegf pathway is frequently associated with the development of severe hypertension [ ] . sunitinib can decrease no production by inhibiting the vegf pathway, which plays a key role in blood pressure regulation [ ] . moreover, previous studies have reported that sunitinib can elevate the plasma level of endothelin- (et- ), the most potent vasoconstrictor identified thus far [ , , ] . in summary, the mechanism of hypertension induced by sunitinib mainly involves two pathways. first, sunitinib inhibits vasodilation by decreasing no production. second, sunitinib induces vasoconstriction by elevating et- levels. in this study, based on the mechanisms mentioned above, a mechanism-based pk-pd model was developed. the present study was performed in four parts. first, animal experiments were performed to collect data for pk-pd modeling. second, a mechanism-based pk-pd that can provide insight into the relationship between the plasma concentration of sunitinib and blood pressure was developed. third, simulations based on the pk-pd model and progression analysis were performed according to the proposed hypothesis that the effect of antihypertensive treatment on blood pressure and its fluctuation are affected by variations in no-et homeostasis. the simulations demonstrated the different effects of antihypertensive treatment on blood pressure and its fluctuation under different states of no-et homeostasis. fourth, animal experiments were performed to verify the antihypertensive treatment proposed by the pk-pd model. our study provides a mechanism-based model to optimize an antihypertensive treatment for hypertension induced by sunitinib that not only inhibits hypertension but also reduces blood pressure fluctuations in rats. male wistar kyoto rats ( - g), obtained from super-b&k laboratory animal center (shanghai, china), were housed at ± °c on a -h dark/light cycle and given access to standard laboratory rat chow and water ad libitum. all animal experiments were approved by the animal ethics committee of china pharmaceutical university. all experiments were performed in accordance with relevant guidelines and regulations. in this study, an animal experiment was performed to obtain data for the development of a pk-pd model. in this experiment, rats were randomly administered sunitinib ( . mg/kg per day; n = ) or vehicle (n = ) by oral gavage for days. the dosage of sunitinib was based on previous experimental studies that investigated hypertension induced by sunitinib [ , , , ] . the protocol for systolic blood pressure (sbp) measurement was designed based on the method described by whitesall et al. and kubota et al. [ , ] . all rats were acclimated to restraint, tailcuff inflation and heating the day before the start of the experiment. the rats were placed in plastic restrainers with heating pads set to ~ °c. the instrument (alc-nibp, alcbio; shanghai, china) automatically took ten -s measurements. sbp values were recorded when more than five consecutive stable readings were available. the highest and lowest readings were discarded, and the remaining readings were averaged to obtain one data point. sbp was measured daily. blood samples were collected in tubes containing ethylene diamine tetraacetic acid (edta) daily. samples were collected at the same time on each sampling day. plasma was prepared by centrifugation at × g and °c for min. the prepared plasma samples were stored at − °c until analysis. the plasma concentration of sunitinib was determined by lc-ms/ms according to the method described in previous studies with slight modifications [ , ] . a total of μl of plasma was mixed with μl is working solution ( ng·ml − paliperidone in water). after vortex-mixing, ml of acetonitrile was added to the mixture to precipitate the proteins. then, the mixture was centrifuged for min at r/min at °c. a total of μl of the supernatant was diluted with μl of water for analysis. the prepared samples were injected into a shimadzu ods column ( . μm, mm × . mm). the mobile phase consisted of acetonitrile containing . % formic acid (phase a) and water containing . % formic acid (phase b). the flow rate of the mobile phase was . ml·min − , and gradient elution was used ( - . min, % phase b; . - . min, %- % phase b; . - . min, % phase b; . - . min, %- % phase b; . - . min, % b). the injection volume was μl. the column oven was set to + °c. a tsq quantum triple quadrupole mass spectrometer (thermo fisher, american) using selected reaction monitoring (srm) mode and an electrospray ionization source (esi) in positive ion mode was utilized to obtain mass spectra at a voltage of v. the sheath gas pressure and auxiliary gas pressure were maintained at l·min − and l·min − , respectively. the capillary temperature was maintained at °c throughout the run. the collision energy for the analyte and is was ev. the precursor to product ion transition (q to q ) for the quantitation (m/z) of sunitinib and is were programmed in the spectrometer as . to . and . to . , respectively. biomarker analysis plasma endothelin- (et- ) was measured using radioimmunoassay with a commercial kit obtained from beijing north institute of biological technology (beijing, china). plasma no levels are difficult to determine. therefore, the levels of the stable end products of no radicals, nitrite and nitrate, were determined to measure the production of no radicals based on the method described in a report of han moshage [ ] . fifty microliters of plasma was diluted with μl of distilled water. then, μl of nitrate reductase, μl of nadph, and μl of fad were added to the samples to yield final concentrations of u/l, μmol/l and μmol/l respectively. the samples were subsequently incubated for min at °c and then mixed with μl of lactate dehydrogenase at a final concentration of mg/l and μl of sodium pyruvate at a final concentration of mmol/l. the samples were further incubated for min at °c to oxidize nadph. ten microliters of zinc sulfate solution ( . g/ml) was mixed with the samples. after vortex-mixing, the mixture was centrifuged for min at r/min. then, μl of the supernatant and μl of griess reagent ( g/l sulfanilamide, g/l phosphoric acid, and . g/l n- -naphthylethylenediamine) were added to a microtiter plate well. after min of color development at room temperature, the absorbance was measured on a microplate reader at a wavelength of nm. creatinine was measured with a commercial kit obtained from nanjing jiancheng bioengineering institute (nanjing, china). the process of model development is shown in fig. . first, a mechanism-based pk-pd model was developed based on animal experiments. second, based on the pk-pd model, simulations were performed to investigate the potential appropriate antihypertensive methods. finally, the results of the simulations were verified by animal experiments. pk-pd model the pk parameters of sunitinib were estimated based on a single-dose pk profile reported by speed et al. [ ] . in this study, rats were administered sunitinib ( mg/kg or mg/kg) by oral gavage, and then the plasma concentration was determined , , , , , , and h after oral administration. then, the optimize anti-hypertension strategy by pk-pd model hc liu et al. sunitinib plasma concentration was simulated based on the above parameters after multi-dose administration. eq. was used to estimate the time course of sunitinib concentration in the plasma. k sunitinib a represents the absorption rate constant of sunitinib. v sunitinib represents the apparent volume of distribution. x represents the dose of sunitinib. k sunitinib out represents the elimination rate constant. the mechanism-based pharmacodynamics model was described in the system, which was composed of three linked turnover equations: the basal turnover of no was determined by a zero-order production rate (k no in ) and a first-order degradation rate (k no out ). in this system, the production of no may be affected by et- and sunitinib. on one hand, sunitinib may decrease no production by reducing the expression of endothelial nitric oxide synthase (enos) and neuronal nitric oxide synthase (nnos) [ , , ] . in this study, the inhibition of no production induced by sunitinib was assumed to be linear and concentration-dependent and was described by parameter k . on the other hand, et- may indirectly decrease the thick ascending limb of the loop of henle (talh) by activating etb receptors, increasing intracellular calcium concentration, and stimulating no release [ ] . in this study, the effect of et- on stimulating no release was assumed to be constant (e et ). the basal turnover of et- was determined by a zero-order production rate (k et in ) and a first-order degradation rate (k et out ). sunitinib may elevate et- [ ] . in this study, the sunitinib-induced increase in et- was assumed to be linear and concentration-dependent and was described by parameter k . the basal turnover of sbp was determined by a zero-order production rate (k sbp in ) and a first-order degradation rate (k sbp out ). both et- and no play important roles in blood pressure. et- may induce vasoconstriction and elevate blood pressure. the effect of et- on blood pressure was assumed to be linear and concentration-dependent and was described by parameter k . no may induce vasodilatation and reduce blood pressure. the effect of no on blood pressure was assumed to be linear and concentration-dependent and was described by parameter k . all the assumptions of the pk/pd model were proposed based on the experimental data used to acquire a parsimonious model with relatively good fitness. previous research has demonstrated that amlodipine and macitentan can help reduce blood pressure [ ] . in this study, amlodipine and macitentan were considered candidate agents. the simulations were performed based on the pk-pd model. the equation system used for the simulations contained three equations. the first two equations in this system were the same as eqs. , . as the anti-hypertension mechanisms of amlodipine and macitentan are different, the third equations for amlodipine and macitentan were different and were as follows: e macitentan ¼ c macitentan tr macitentan ð Þ nmacitentan n macitentan ! e Àtrmacitentant ( ) fig. the structure of the model-based research framework. optimize anti-hypertension strategy by pk-pd model hc liu et al. equations , were used to simulate the effect of amlodipine. e amlodipine represents the effect of amlodipine. c amlodipine is the plasma concentration of amlodipine. c amlodipine was obtained by simulating the multidose administration of amlodipine based on a first-order absorption one-compartment model whose parameters are estimated according to the data extracted from a previous experimental report [ ] . n amlodipine is the number of transit compartments of amlodipine. tr amlodipine stands for a transit rate constant between transit compartments of amlodipine. n amlodipine and tr amlodipine were estimated based on the data extracted from a previous experimental report [ ] . equations , were used to simulate the effect of macitentan. e macitentan represents the effect of amlodipine. c macitentan is the plasma concentration of macitentan. c macitentan was obtained by simulating the multidose administration of macitentan based on a first-order absorption one-compartment model whose parameters are estimated according to the data extracted from a previous experimental report [ ] . n macitentan is the number of transit compartments of amlodipine. tr macitentan stands for a transit rate constant between transit compartments of amlodipine. n macitentan and tr macitentan were estimated based on the data extracted from a previous experimental report [ ] . parameters n amlodipine , tr amlodipine , n macitentan , tr macitentan and the pharmacokinetic parameters of amlodipine and macitentan are listed in table . in the simulation, the antihypertensive effect was represented by the area under the curve (auc) of blood pressure, and the average real variability (arv) was used to measure the fluctuation of blood pressure (eq. ) [ ] . in eq. , n denotes the number of valid blood pressure (bp) measurements. to avoid bias based on background variation in the simulation, the percentage change in the arv compared to that of the control group (Δarv, eq. ) was used in the simulation. the simulation was performed based on three scenarios. the aim of scenario i was to choose a suitable antihypertensive agent. therefore, amlodipine and macitentan were administered at different time points (day to day , time points in total) after sunitinib was administered. then, the auc of blood pressure and Δarv yielded by amlodipine and macitentan treatment were compared to find a suitable antihypertensive agent. the dose of amlodipine was set to mg/kg (equivalent to the maximum tolerated dose in humans), and the dose of macitentan was set to mg/kg (equivalent to a high but well tolerated dose in humans) [ , ] . the aim of scenario ii was to find the appropriate dose of the selected antihypertensive agent. for this purpose, different doses of selected antihypertensive agents were administered. then, the auc of blood pressure and Δarv were calculated. the aim of scenario iii was to investigate the indication for antihypertensive treatment. for this purpose, the proposed antihypertensive agent was administered under different levels of et and no. then, the auc of blood pressure and Δarv were calculated. the relative levels of both no and et in the control group changed from − to %. a total of situations were simulated. the dose of sunitinib was . mg/kg, and the dose of macitentan was selected according to scenario ii. validation of modeling and simulation the pk model was validated by two pieces of data. first, it was validated by the plasma concentration after a single dose of sunitinib ( mg/kg) was administered [ ] . second, it was validated by the trough concentration after multiple doses of sunitinib ( . mg/kg and mg/kg) were administered. an animal experiment was performed as an experimental validation for the pk/pd model. in this experiment, rats were randomly administered low-dose sunitinib ( mg/kg; once daily; n = ) or vehicle (n = ) by oral gavage for days. the protocol of blood pressure measurement was the same as that used in the first experiment. blood samples were collected in tubes containing edta on days , , , , , and . the protocols for blood sample preparation and storage were the same as those used in the first experiment. the proposed antihypertensive method was also verified by another animal experiment. in this animal experiment, male wistar kyoto rats ( - g) were randomly divided into five groups. the first group was the control group (given vehicle; n = ). the second group (the sunitinib group) was orally administered sunitinib ( . mg/kg; n = ) once daily for days. the third group (the low-dose immediate treatment group; n = ) was orally and simultaneously administered sunitinib ( . mg/kg) and macitentan ( mg/kg). the fourth group (the high-dose immediate treatment group; n = ) was orally and simultaneously administered sunitinib ( . mg/kg) and macitentan ( mg/kg). the fifth group (the low-dose optimized treatment group; n = ) was orally administered sunitinib ( . mg/kg), and then macitentan ( mg/kg) was given until the no and et levels reached their critical values. the sixth group (the high-dose optimized treatment group; n = ) was orally administered sunitinib ( . mg/kg), and then macitentan ( mg/kg) was given until the no and et levels reached their critical values. the antihypertensive agent, the dosage of macitentan and the critical values of et and no levels were selected according to the simulation based on the pk-pd model. the detailed selection procedure are described in the "materials and methods" and "results" sections. the protocols for the measurements of sbp, no and et were the same as those used in the first experiment. macitentan was dissolved in vehicle containing . % methylcellulose aqueous solution and % tween . the results of the animal experiments are shown in fig. . the sbp level increased rapidly after sunitinib administration in the highdose group. the sbp level in the low-dose group was elevated day after sunitinib administration. the variation in the sbp level was dose-dependent. the level of no increased from day to day and decreased from day to day in the high-dose group. in the low-dose group, no was elevated at days. the et level increased continuously and dose-dependently in both the highdose group and the low-dose group. the pk-pd parameters were estimated based on the experiment that used high-dose sunitinib (the first experiment). in this table . pk-pd parameters of amlodipine and macitentan for simulation. experiment, rats were randomly administered sunitinib ( . mg/kg; once daily; n = ) or vehicle (n = ) by oral gavage for days. the estimated pk-pd model parameters are listed in table . random effects for parameters e et , k , k , and k sbp out were induced. the bootstrapping values of these parameters remained near the final parameter estimation with a relatively low coefficient of variance (cv). goodness-of-fit plots of the final model are shown in fig. a -c and suggest that the errors of both population and individual profiles were acceptable. the visual predictive check (vpc) for the pk-pd model is shown in fig. c . the vpc plots show that the observed average data fell within the % prediction confidence interval. moreover, a low-dose experiment was performed as an external verification of the pk-pd model. in this experiment, rats were randomly administered sunitinib ( mg/kg; once daily; n = ) or vehicle (n = ) by oral gavage for days. the experiment was used to simulate changes in no, et, and sbp to verify the pk-pd model, and the simulation results were compared with the experimental data. the results are shown in fig. a, b . the results suggest that the error was acceptable and that the pk-pd model fit the low-dose experiment data. furthermore, as sunitinib may induce renal damage, which may affect sbp and bias the pk-pd model, creatinine levels in the low-dose group, high-dose group and control group were measured to determine whether the model was biased by renal damage. the results are shown in fig. c . the results show that creatinine levels were not significantly different among these three groups at any of the time points (p > . , based on one-way anova), suggesting that no significant renal damage occurred during the experiment. therefore, the goodness of fit of the pk-pd model was satisfactory, and the model was not biased by the renal damage induced by sunitinib. the results of scenario i simulation are shown in fig. a, b . the simulation suggested that macitentan yielded similar blood pressure fluctuations but lower auc of blood pressure compared to those induced by amlodipine. therefore, macitentan was chosen as the antihypertensive agent. the result of scenario ii simulation is shown in fig. c . the simulation suggested that the blood pressure fluctuation decreased until the dose of macitentan increased to mg/kg and that if the dose of macitentan exceeded mg/kg, blood pressure fluctuation increased. the auc of blood pressure decreased as the dose of macitentan increased. thus, mg/kg macitentan should be selected for hypertension treatment. however, mg/kg macitentan may not be well tolerated (the maximum tolerated dose of macitentan is equivalent to mg/kg in rats.) [ ] . therefore, mg/kg macitentan rather than mg/kg macitentan was selected for hypertension treatment. the results of scenario iii simulation are shown in fig. a -c. we defined low-level auc as an auc of blood pressure in the treated group lower than % of that in untreated the group (auc treated / auc untreated ≤ %) and low-level Δarv as a fluctuation of blood pressure in the treated group lower than that in the untreated group (Δarv treated < Δarv untreated ). according to the response to the antihypertensive agent, there were four states of no-et homeostasis. the first state, termed the compensatory unbalanced state (fig. c) , was defined as an et level relative to that of the control group of less than − %. the second state, termed the weak balance state, was defined as a relative et level ranging from − % to % and a relative no level greater than %. the third state, termed the pathological balance state, was defined as a relative et level ranging from % to % and a relative no level greater than %. the fourth state, termed the pathological unbalanced state, was defined as a relative et level greater than %. if no-et homeostasis is in the compensatory unbalanced state, antihypertensive treatment may yield low-level auc and high-level Δarv. if no-et homeostasis is in the weak balance zone, antihypertensive treatment may yield low-level auc and low-level Δarv. if no-et homeostasis is in the pathological balance zone, antihypertensive treatment may yield high-level auc and low-level Δarv. if no-et homeostasis is in the pathological unbalanced zone, antihypertensive treatment may yield high-level auc and high-level Δarv. therefore, it is recommended to administer antihypertensive treatment in the weak balance state. the results of experimental verification of the antihypertensive method are shown in fig. . according to the treatment indication (fig. a) , macitentan should be given on the th day after sunitinib is administered. the high-and low-dose optimized treatment fig. the variation in sbp, no, and et. the variation in biomarkers is presented as the level relative to that of the control group. the black line in the figures represents the control group, which was normalized to . the red line represents the low-dose group. the blue line represents the high-dose group. optimize anti-hypertension strategy by pk-pd model hc liu et al. groups should be given mg/kg macitentan and mg/kg macitentan, respectively, at this time. the optimized treatment can lower auc and blood pressure fluctuation dose-dependently (fig. b) . compared with immediate antihypertensive treatment, optimized treatment can decrease % of blood pressure fluctuation. in this study, a mechanism-based pk/pd model was developed to propose a method for the management of hypertension induced by sunitinib. previous studies have proposed that the mechanism of hypertension-induced sunitinib involves variations in no-et homeostasis. the no system works in balance with the et system, but sunitinib may decease no, which makes no unable to balance the vasoconstrictor et [ ] [ ] [ ] . our study suggested that the balance between no and et may also affect the response of hypertension induced by sunitinib to antihypertensive agents. according to our experiment, from day to day after sunitinib administration, no-et homeostasis is in the compensatory unbalanced state. previous research has shown that sunitinib may decrease no, but our study showed that no increased from day to day and then decreased [ ] . the increase in et- may stimulate no release to balance the vasoconstrictive effect of et- [ ] . these results suggest that the system may still maintain no-et homeostasis in the compensatory unbalanced state. subjects in this state may be highly sensitive to antihypertensive treatment. our experiments and previous reports suggest that in the compensatory unbalanced state, antihypertensive treatment may lower blood pressure auc (auc treated /auc untreated ≤ %) and may cause acute blood pressure fluctuation (Δarv treated > Δarv untreated ) [ , [ ] [ ] [ ] . therefore, it is not recommended to administer any antihypertensive treatment in the compensatory unbalanced state because of the potential cardiovascular risk caused by blood pressure fluctuations. no-et homeostasis may enter the weak balance state on day . in this state, the elevated sunitinib plasma concentration may enhance the inhibition of no release, which may cause no to decrease and limit the ability of no-et homeostasis to maintain blood pressure. subjects in the weak balance state may be moderately sensitive to antihypertensive treatment. our simulations and experiments suggested that in this state, antihypertensive treatment can significantly lower auc and blood pressure fluctuations (p < . ). therefore, it is recommended to administer antihypertensive treatment in a weak balance state to reduce potential cardiovascular risk. from day to day after sunitinib administration, no-et homeostasis is in the pathological balance state. compared with that in the weak balance state, sensitivity to antihypertensive agents decreases in this state. our simulations showed that the effect of antihypertensive treatment in lowering blood pressure auc may be limited but that it is still helpful in managing blood pressure fluctuations. in the pathological unbalanced state, sbp and et- were markedly increased and no decreased continuously. in this state, hypertension may be related to multiple other mechanisms because both the simulations and the previous research showed that the effect of the et receptor antagonist macitentan is limited, which suggests that no-et homeostasis disorder may not be the sole causative factor of hypertension [ ] . apart from no-et homeostasis dysfunction, previous studies have proposed other hypotheses suggesting that decreased vegf function leads to remodeling of capillary beds and endothelial dysfunction [ , ] . as the mechanisms related to hypertension may be very complex, they remain to be further investigated. the potential translational value of the proposed model, after it is improved and validated by clinical data in the future, lies in providing a new method for optimizing the management of the hypertension induced by sunitinib. in this study, a mechanism-based pk/pd model was developed to investigate a management method for hypertension induced by sunitinib. our research suggested that it is recommended to use the state of the balance between et and no as an indicator for hypertension management. in this study, a novel mechanism- the results of sbp, sbp auc and sbp fluctuation (represented by relative arv (Δarv)). c a comparison of the effects of optimized treatment and immediate treatment on auc and the fluctuation of sbp (represented by relative arv (Δarv)). *p < . , **p < . . pkpd modeling of predictors for adverse effects and overall survival in sunitinib-treated patients with gist cardiovascular and renal toxicity during angiogenesis inhibition: clinical and mechanistic aspects treatment of hypertension and renal injury induced by the angiogenesis inhibitor sunitinib: preclinical study the use of -h ambulatory blood pressure monitoring (abpm) during the first cycle of sunitinib improves the diagnostic accuracy and management of hypertension in patients with advanced renal cancer hypertension during vascular endothelial growth factor inhibition: focus on nitric oxide, endothelin- , and oxidative stress rho kinase inhibition mitigates sunitinib-induced rise in arterial pressure and renal vascular resistance but not increased renal sodium reabsorption the vascular endothelial growth factor receptor inhibitor sunitinib causes a preeclampsia-like syndrome with activation of the endothelin system renal soluble guanylate cyclase is downregulated in sunitinib-induced hypertension maximum value of home blood pressure a novel indicator of target organ damage in hypertension assessment and management of bloodpressure variability pkpd modeling of vegf, svegfr- , svegfr- , and skit as predictors of tumor dynamics and overall survival following sunitinib treatment in gist hypertension induced by the tyrosine kinase inhibitor sunitinib is associated with increased circulating endothelin- levels greater sensitivity of blood pressure than renal toxicity to tyrosine kinase receptor inhibition with sunitinib impaired endothelium-dependent vasodilation does not initiate the development of sunitinib-associated hypertension comparison of simultaneous measurement of mouse systolic arterial blood pressure by radiotelemetry and tail-cuff methods evaluation of blood pressure measured by tail-cuff methods (without heating) in spontaneously hypertensive rats determination of sunitinib in human plasma using liquid chromatography coupled with mass spectrometry sunitinib lc-ms/ms assay in mouse plasma and brain tissue: application in cns distribution studies nitrite and nitrate determinations in plasma -a critical-evaluation pharmacokinetics, distribution, and metabolism of c- sunitinib in rats, monkeys, and humans sunitinib produces neuroprotective effect via inhibiting nitric oxide overproduction polymorphisms in endothelial nitric oxide synthase (enos) and vascular endothelial growth factor (vegf) predict sunitinib-induced hypertension endothelin inhibits thick ascending limb chloride flux via et(b) receptor-mediated no release negligible pharmacokinetic interaction between oral da- , a new erectogenic, and amlodipine in rats absorption, distribution, metabolism, and excretion of macitentan, a dual endothelin receptor antagonist, in humans a reliable index for the prognostic significance of blood pressure variability drug surveillance study of amlodipine in patients with hypertension not controlled with drug therapy: norcon study macitentan: entry-intohumans study with a new endothelin receptor antagonist receptor tyrosine kinase inhibition, hypertension, and proteinuria: is endothelin the smoking gun? role of endothelin- in clinical hypertension: years on systemic therapies for malignant pheochromocytoma and paraganglioma can exacerbate hypertension molecular basis of hypertension side effects induced by sunitinib this study was supported by the national natural science foundation of china (no. and no. ), "double first-class" university project (no. cpu gy ) and the fundamental research funds for the central universities (no. zd ). based model with potential translational value was developed for managing hypertension induced by sunitinib. hcl, xtz, xql, and hh designed the experiments. hcl, xtz, and ysz conducted the animal experiments. xtz analyzed the samples. hcl developed the mathematic model and wrote the manuscript. competing interests: the authors declare no competing interests. key: cord- - kzy rts authors: li, heng; yang, li; liu, fei-fei; ma, xin-na; he, pei-lan; tang, wei; tong, xian-kun; zuo, jian-ping title: overview of therapeutic drug research for covid- in china date: - - journal: acta pharmacol sin doi: . /s - - -y sha: doc_id: cord_uid: kzy rts since the outbreak of novel coronavirus pneumonia (covid- ) in december , more than , , people worldwide have been diagnosed with sars-cov- as of april . in response to this epidemic, china has issued seven trial versions of diagnosis and treatment protocol for covid- . according to the information that we have collected so far, this article provides an overview of potential therapeutic drugs and compounds with much attention, including favipiravir and hydroxychloroquine, as well as traditional chinese medicine, which have been reported with good clinical treatment effects. moreover, with further understanding of sars-cov- virus, new drugs targeting specific sars-cov- viral components arise and investigations on these novel anti-sars-cov- agents are also reviewed. in december , a new type of unexplained pneumonia appeared in the south china seafood wholesale market in wuhan, hubei province, but its source has not yet been found. to confirm the cause, whole-genome sequencing of samples from patients with this unexplained pneumonia revealed a betacoronavirus that had never been seen before, which was different from sars-cov and mers-cov [ ] . after the alert of this new pathogen, china quickly took measures to completely block off hubei province and implemented a quarantine policy nationwide to keep the outbreak under control. this efficient process was also recognized by the international who. this highly contagious new coronavirus was initially named -ncov. on feb , , the international committee on taxonomy of viruses introduced the name "severe acute respiratory syndrome coronavirus " (sars-cov- ) to refer to the virus, and the who named the related pneumonia coronavirus pneumonia . this article will go through basic virology of sars-cov- and review mainly the drugs used in the battle against covid- in china. by genome sequencing, it was found that sars-cov- has % sequence similarity with sars-cov [ , ] , and through phylogenetic tree analysis, it has % homology with bat coronaviruses at the genome level, for which reason bats were inferred to be the host for sars-cov- [ ] . the genome structure of sars-cov- is shown in fig. . sars-cov- is a single-stranded positive-sense rna virus and is classified in the order nidovirales, coronaviridae, and coronavirus [ ] . coronaviruses are a large class of viruses that can infect mammals and birds and can cause many diseases, such as respiratory, intestinal, liver, and nervous system diseases [ ] . coronaviruses are divided into four groups: alpha-, beta-, gamma-and delta-coronaviruses [ ] , and all known human pathogenic coronaviruses are beta-coronaviruses. the single-stranded rna genome of sars-cov- is nucleotides in size. the genes of sars-cov- from ′ to ′ are a ′ untranslated region, including ′ leader sequences; and open reading frame (orf) a/b; structural proteins, including the envelope glycoprotein spike (s), envelope (e), membrane (m) and nucleoprotein (n); accessory proteins, such as orf , , a, b, , and b; and a ′ untranslated region [ ] . orf a/b occupy two-thirds of the viral genome, and they encode two polyproteins, pp a and pp ab [ ] . these two polyproteins can be hydrolyzed into nonstructural proteins (nsps), including papain-like protease (plpro), c-like protease ( clpro), rna-dependent rna polymerase (rdrp), helicase, and exonuclease [ ] . the enveloped glycoprotein spike (s) forms a layer of glycoproteins protruding from the envelope and is closely related to virus infection of cells [ ] . there are multiple sequence segments in the receptor-binding domain of the s protein, which have high homology with sars-cov sequences, and some studies have confirmed that sars-cov- can bind to the ace receptor on the cell surface, similar to sars-cov [ , , ] . two additional transmembrane glycoproteins, envelope (e) and membrane (m), are incorporated into the virion. nucleoprotein encapsulates the viral (+) rna genome in the form of a spiral in the envelope to prevent degradation. accessory proteins do not participate in viral replication, but they interfere with the host innate immune response or have other unknown functions [ ] . the sars-cov- possible life cycle begins when the spike glycoprotein on the envelope binds to the ace receptor, and membrane fusion occurs either directly with the host cell membrane or with the endosome membrane. after membrane fusion, the viral rna genome is released into the cytoplasm, and the uncoated rna is translated into two polyproteins. the polyproteins are cleaved by the protease encoded by orf a into the nsps, which can form an rna replicase-transcriptase complex [ , ] . this complex drives the production of negative-sense rna, which is used as a template for the full-length (+) rna genome. during transcription, subgenomic rnas are produced by discontinuous transcription and then transcribed into subgenomic (+) mrna, which is translated into different structural proteins. the newly formed structural protein and viral genomic rna combine to form a nucleocapsid. the viral particles bud into the er-golgi intermediate compartment (ergic), and then the primary virion is released from the infected cells [ ] . a diagram of sars-cov- life cycle is shown in fig. . due to the sudden emergence of sars-cov- , no specific antiviral agents are available. however, many drugs under development are believed to have potential anti-sars-cov- activity due to common biological processes in the virus life cycle. currently, there are~ domestic clinical trials of covid- drugs from the chinese clinical trial registry (chictr) as of march, and most are for chemical drugs, chinese patent medicines, and combinations. all of these clinical trials were registered in , after the outbreak of covid- [ ] . among those covid- treatment-related clinical trials, a total of registered covid- treatment drug-related interventional studies were obtained from the chictr database (www.chictr.org. cn) with ethics committee approval and informed consent signed by march. statistic information of these registered trials are shown in fig. . the study phase of each trial was extracted, and nearly % of these clinical trials were registered as phase studies, which were stated as "postmarketing drugs" or "phase iv clinical trials" by the chictr definition. in phase four studies, many domestically approved chinese patent drugs and chemical agents are being used to treat covid- patients. with so many clinical trials registered in such a short period of time, we can see that china has responded to the epidemic in a timely manner and hopes to find sars-cov- -specific medicines to end the epidemic as soon as possible. this urgent need propelled china's drug research ability to its full power, and this motivation involved many academic institutions, hospitals, and companies. this approach has also led to concerns about this phenomenon. excessive research and drug trials can create the problem of squeezing each other for limited medical resources or even wasting them. this phenomenon and its impact on our medical system, as well as possible improvement in our medical administration, need thorough review and discussion at an appropriate time in the future. in these pooled clinical trials, a number of approved chemical and biomacromolecule drugs have been used in covid- treatment clinical trials for drug repurposing, most of which are nucleotide analogs and protease inhibitors against other viral pathogens, including influenza virus, hiv and hcv. comprehensive information about the chemical agents used in covid- drug clinical trials is shown in the table below (table ) . although the mortality rate of sars-cov- is not as high as that of sars-cov and mers-cov, sars-cov- is more contagious, and studies have shown that it can be transmitted during the incubation period [ ] . to date, there are no specific vaccines or antiviral drugs against covid- . for the treatment of patients, isolation and symptomatic supportive care are currently recommended, including oxygen therapy, water, and dielectric balance, nutritional interventions and fluid management to reduce symptoms and prevent end-organ dysfunction [ , ] . for the treatment of severe pneumonia, extracorporeal membrane oxygenation therapy is a new type of adjuvant treatment technology [ ] , and gas exchange through cardiopulmonary bypass technology can reduce ventilator-related damage and oxygen toxicity damage to promote early patient recovery. however, the needs for specific technical skill and high expense limit its usage. a number of nonspecific antiviral agents have been recommended in the latest th trial version of the "diagnosis and treatment protocol for novel coronavirus pneumonia". table shown below lists important information about currently available anti-sars-cov- drugs ( table ). type i interferons, including ifn-α and ifn-β, have broad-spectrum antiviral effects [ ] . ifn-α can directly inhibit virus replication or can achieve antiviral effects by activating innate or adaptive immunity [ ] . many studies have proven that ifn-α has an antiviral effect on sars-cov [ ] , and other experts discovered that ifn-α can prevent mers-cov infection and effectively inhibit the virus in the early stages of infection [ ] . because it is also a coronavirus and highly homologous to sars-cov, ifn-α is recommended for the treatment of sars-cov- infection. according to the latest new "diagnosis and treatment protocol for novel coronavirus pneumonia", ifn-α at a dose of~ million u or equivalent dose each time for adults is added to ml of sterile water, and it is aerosolized and inhaled twice a day [ ] . lopinavir/ritonavir lopinavir was first approved in the united states in for the treatment of hiv infection [ ] . it is a protease inhibitor and is usually used in combination with ritonavir to increase its half-life through the inhibition of cytochrome p [ ] . in vitro studies have shown that lopinavir/ritonavir can inhibit the replication of mers-cov and sars-cov and exert antiviral effects [ ] [ ] [ ] [ ] . at present, the drug has been used in the clinical treatment of covid- at a dose of mg/ mg for adults twice daily, and the course of treatment does not exceed days [ ] . however, this treatment has certain toxic and side effects on the treatment of covid- . therefore, its safety and effectiveness require further research. recently, some clinical studies have shown that lopinavir/ritonavir treatment has no significant effect [ , ] . ribavirin is a purine nucleoside analog with a broad-spectrum antiviral effect [ ] . it is used mainly to treat respiratory syncytial virus infection [ ] and in combination with interferon for hepatitis c [ ] . ribavirin was widely used in to treat sars-cov infection, but when used alone, it seemed to have no effect and caused significant hemolysis in many patients [ ] [ ] [ ] [ ] . when ribavirin was combined with ifn-β, it had good antiviral activity in in vitro studies [ ] . some studies have shown that in patients with severe mers-cov infection, ribavirin combined with ifn-α- a treatment can significantly improve patient -day survival [ ] . preliminary in vitro test results demonstrate that ribavirin can inhibit sars-cov- in a human cell line. in the latest "diagnosis and treatment protocol for novel coronavirus pneumonia", it is recommended to use ribavirin at a dose of mg each time for adults and in combination with interferon or lopinavir/ritonavir, with - intravenous infusions daily. the course of treatment does not exceed days [ ] . chloroquine phosphate chloroquine phosphate is an antimalarial drug that has been on the market for many years, and it also has a potential broadspectrum antiviral effect [ , ] . it can increase the ph of lysosomes to prevent virus fusion with the cell membrane and then block virus entry and infection [ ] . some studies have found that the spike glycoprotein on the virus envelope binds to the ace receptor to mediate sars-cov- infection [ , ] . chloroquine phosphate has been reported to interfere with the glycosylation of the ace receptor, thereby inhibiting the binding of sars-cov to cells and achieving therapeutic goals [ ] . therefore, chloroquine phosphate is used to treat covid- , and in vitro experiments show that chloroquine phosphate does inhibit sars-cov- and that its ec is . μm [ ] . apart from the above antiviral mechanism, chloroquine phosphate can continue to exert antiviral effects after sars-cov- invades cells, and it also has immunomodulatory activity to strengthen the antiviral effect [ ] . therefore, chloroquine phosphate has been included in the "diagnosis and treatment protocol for novel coronavirus pneumonia" and is undergoing clinical trials. chloroquine phosphate is used for the treatment of covid- in adults aged - years. those who weigh more than kg will receive mg twice daily for days, while those who weigh less than kg will receive mg twice daily on days and and mg once daily on days - [ ] . arbidol is a non-nucleoside broad-spectrum antiviral drug for upper respiratory tract infections caused by influenza a and b viruses, and it was first approved in russia [ ] . it can inhibit the adhesion of viruses to host cells and prevent them from invading human cells [ ] . at the same time, it can promote the synthesis of interferon, which can prevent influenza virus invasion and treat influenza virus infection [ ] . some studies have confirmed that arbidol has a good effect against sars-cov and mers-cov infections [ , ] . chinese scientists found in in vitro cell experiments that compared with a drug-untreated control group, - μm arbidol can effectively inhibit sars-cov- up to times and significantly inhibit the pathological effects of the virus on cells [ ] . as described in the latest "diagnosis and treatment protocol for novel coronavirus pneumonia", arbidol is used at a dose of mg for adults three times daily, and the course of treatment does not exceed days [ ] . it has been reported that some patients have improved symptoms after receiving arbidol [ ] . favipiravir is a nucleoside analog [ , ] with an ability to inhibit rna-dependent polymerase [ ] and was approved for marketing in japan in . it is used for antiviral treatment of influenza a and b [ ] and can effectively inhibit ebola virus, yellow fever virus [ ] , etc. in vitro experiments have shown that favipiravir is effective for covid- and that its ec is . μm [ ] . to date, some clinical trials of favipiravir in the treatment of covid- have been carried out in china. recent clinical studies have found that compared with the antiviral drug arbidol, the clinical effect of favipiravir is more significant. nucleic acid positive-to-negative time, mean antipyretic time and cough remission time were all better than those of the arbidol group [ ] . remdesivir(gs- ) remdesivir was first used to treat ebola virus, and it has completed phase clinical trials [ ] . as a nucleoside analog, it can interact with rdrp [ , ] , and the triphosphate form of remdesivir will compete with adenosine triphosphate, leading to delayed chain termination and inhibiting viral replication and transcription [ ] . a number of in vitro studies have shown that remdesivir has inhibitory effects on a variety of human and animal coronaviruses [ , ] . an in vivo study of remdesivir against sars-cov showed that remdesivir can reduce virus levels in the lungs of mice infected with sars-cov and reduce lung function damage caused by the virus [ ] . moreover, some studies have found that the antiviral effect of remdesivir against mers-cov is better than that of lopinavir/ritonavir combined with ifn-β [ ] . according to the above evidence, remdesivir has been used to treat sars-cov- , and in vitro experiments showed that remdesivir has a good inhibitory effect on sars-cov- , with an ec of . μm [ ] . recently, the first covid- patient in the united states was treated with remdesivir on the th day of hospitalization, and their clinical symptoms were improved significantly [ ] . at present, a phase three clinical trial of remdesivir for covid- has been officially launched in china, and a total of patients are planned to be enrolled in the study, which will be randomized, double-blind and placebo-controlled [ ] . as a derivative of chloroquine, hydroxychloroquine has similar efficacy and few adverse reactions. based on its characteristics of immunomodulation, antithrombotic activity, and improved inflammation, hydroxychloroquine has been used in the clinical treatment of systemic lupus erythematosus [ ] . hydroxychloroquine has been shown to have anti-sars-cov activity in vitro [ ] , and it is clinically safer than chloroquine [ , ] . some clinical studies have found that after treatment with hydroxychloroquine, the viral load significantly decreases or even disappears, and azithromycin can enhance the antiviral effect [ , ] . traditional chinese medicine has played an important role in the treatment of previous outbreaks of viral infectious diseases, so it has once again received attention during this epidemic of covid- [ , ] . in response to the outbreak, chinese medicine experts provided different chinese medicine prescriptions and proprietary chinese medicines for selection at different stages of the clinical treatment period of diagnosed patients according to the principle of tcm syndrome differentiation. for example, for patients with fatigue and fever during the medical observation period, jinhua qinggan granules, lianhua qingwen capsules (granules) and shufeng jiedu capsules (granules) can be used, while for patients with fatigue and gastrointestinal discomfort, the chinese patent medicine huoxiang zhengqi capsules (pills, liquid, and oral solution) can be used [ ] . lianhua qingwen capsules contain many chinese herbal ingredients with antiviral effects, such as licorice. glycyrrhizic acid, an extract of licorice, has been shown to inhibit virus replication and has been used clinically to treat hcv infection [ ] . glycyrrhizic acid has been reported to inhibit the replication of sars-cov in vitro [ ] . for the reasons above, lianhua qingwen capsules have been used in the clinic and have a certain effect on improving clinical symptoms because they can obviously alleviate the symptoms of cough, fever, and fatigue in patients with covid- , reduce the proportion of severe cases, and shorten fever time [ ] [ ] [ ] . in addition, lung cleansing and detoxifying decoction is also recommended for the treatment of confirmed cases in the latest "diagnosis and treatment protocol for novel coronavirus pneumonia" [ ] . in addition to traditional chinese medicine prescriptions, some traditional chinese medicine ingredients also have certain potential for the treatment of sars-cov- . baicalin is a flavonoid compound isolated from the root of scutellaria baicalensis and has been confirmed to inhibit sars-cov in vitro [ ] . ginsenoside improves human immunity and has a curative effect on viral infection [ ] . therefore, traditional chinese medicine can also be used as an option to treat covid- . many clinical trials have been launched to further analyze the safety and effectiveness of traditional chinese medicine, and the traditional chinese medicines mentioned above have been recommended in the latest "diagnosis and treatment protocol for novel coronavirus pneumonia" [ ] (table ) . chinese patent medicine chinese patent medicine is herbal medicines in traditional chinese medicine modernized into a ready-to-use form, such as tablets, oral solutions, or dry suspensions. chinese patent medicine has played very important roles in the domestic battle against covid- in china, and many clinical investigations have started to more precisely evaluate its effects on covid- patient treatment. chinese patent medicine, as well as many herbal medicines, has been very useful in improving symptoms such as coughing, weakness, and digestive system disorders, as well as alleviating anxiety. reports announced that up to % of covid- patients in china have been given chinese patent medicine or tcm prescriptions. a list of chinese patent medicine currently undergoing covid- clinical trials is shown below (table ) to present an overall message about chinese patent medicine in covid- treatment research (table ) . spike protein some studies have confirmed that sars-cov- infects cells through endocytosis via the ace receptor on at alveolar epithelial cells in the lungs [ , ] . at present, the protein structure of the s protein and ace interaction has been solved, providing reliable guidance for vaccine and drug design [ ] . aiming at the mutual use of the s protein and ace protein necessary for sars-cov- to enter host cells, multiple domestic research teams have discovered a variety of potential ace - papain-like protease (plpro) plpro is a multifunctional protein with protease and phosphatase activity that is involved in viral replication and ifn antagonism [ ] . plpro of sars-cov- and sars-cov have only % sequence similarity [ ] . however, the high-level structure of the protein that forms the active site is not altered in the two plpro proteins [ ] . there are already domestic teams working to find potential inhibitors of plpro through virtual screening, which needs to be confirmed by experiments. the clpro of sars-cov- and sars-cov have an amino acid sequence similarity of up to %, and the structure of clpro in sars-cov- has been solved [ ] . clpro is a key protein of the virus, and the virus needs to use it to replicate rna. therefore, finding sars-cov- clpro inhibitors can provide a more effective way to fight covid- . rna-dependent rna polymerase (rdrp) as an rna virus, sars-cov- -encoded rdrp plays a key role in the virus's rna replication. rdrp inhibitors can be used as broadspectrum antiviral drugs against rna viruses. the rdrp protein structure has a large and deep groove structural region as the active center of rna synthesis. the sequence similarity of the rdrp proteins of sars-cov- and sars-cov is as high as %, and structural differences exist outside the active center [ ] . therefore, high sequence conservation allows the development of rdrp inhibitors against sars-cov to be applied to the development of anti-sars-cov- drugs. tmprss (serine protease) a recent study showed that in addition to the use of the sars-cov receptor ace to enter the cell, sars-cov- has two other proteins, cathepsin b and l(catb/l) and tmprss , that activate the s protein, help sars-cov enter the cell, and play a key role in the process of invasion of normal cells by sars-cov- [ ] . when a tmprss inhibitor is used in combination with a catb/l inhibitor, they can completely inhibit the invasion of sars-cov- . therefore, tmprss may be a potential antiviral target. this article provides an overview of published information on domestic research and the development of coronavirus-related therapeutic agents. the drug-repurposing effort summarized in this article focused primarily on agents currently known to be active against other rna viruses, including sars-cov, mers-cov, influenza, hcv, and ebola, as well as anti-inflammatory drugs. this information provides a strong intellectual groundwork for support of current and future research and development for the discovery and development of therapeutic agents for the treatment of covid- and coronavirus-related diseases. however, there are still no officially approved specific antiviral drugs or vaccines for sars-cov- , and supportive care remains a key to treatment. tcm has accumulated thousands of years of experience in the treatment of pandemic and endemic diseases. complementary and alternative treatments are still urgently needed for the management of patients with sars-cov- infection, and experiences in tcm are certainly worth examining. many antiviral chinese patent medicines, such as shuanghuanglian oral solution and others, have been declared to have the effect of heat clearing and detoxifying, which could help clear viral respiratory pathogens and relieve symptoms according to tcm theory. chinese patent medicine has been used in many historic epidemics, such as the previous two coronavirus outbreaks (sars-cov in and mers-cov in ) and seasonal epidemics caused by influenza viruses and dengue virus. fighting against current epidemics also provides an opportunity to test the true value of tcm in treating emerging contagious diseases. it is encouraging that controlled clinical studies to evaluate the efficacy of tcm in the treatment of sars-cov were conducted and reported. we believe that specific and efficient antiviral drugs and therapy will arise from these ongoing and developing drugs, especially from the rich tradition of herbal medicines in china. competing interests: the authors declare no competing interests. evolution of the novel coronavirus from the ongoing wuhan outbreak and modeling of its spike protein for risk of human transmission recent advances in the detection of respiratory virus infection in humans genomic characterization of the novel human-pathogenic coronavirus isolated from a patient with atypical pneumonia after visiting wuhan discovery of a novel coronavirus associated with the recent pneumonia outbreak in humans and its potential bat origin coronavirus pathogenesis coronaviruses: an overview of their replication and pathogenesis sars and other coronaviruses as causes of pneumonia coronaviruses post-sars: update on replication and pathogenesis host factors in coronavirus replication sars and mers: recent insights into emerging coronaviruses a familial cluster of pneumonia associated with the novel coronavirus indicating person-toperson transmission: a study of a family cluster diagnosis and treatment protocol for novel coronavirus pneumonia (trial version ) advance in research on treatment of influenza viral pneumonia the yin and yang of viruses and interferons a rapid screening assay identifies monotherapy with interferon-ss and combination therapies with nucleoside analogs as effective inhibitors of ebola virus treatment of sars with human interferons treatment with lopinavir/ ritonavir or interferonbeta b improves outcome of mers-cov infection in a nonhuman primate model of common marmoset lopinavir/ritonavir: a review of its use in the management of hiv infection comparative therapeutic efficacy of remdesivir and combination lopinavir, ritonavir, and interferon beta against mers-cov role of lopinavir/ritonavir in the treatment of sars: initial virological and clinical findings combination therapy with lopinavir/ ritonavir, ribavirin and interferon-α for middle east respiratory syndrome small molecules targeting severe acute respiratory syndrome human coronavirus screening of an fda-approved compound library identifies four small-molecule inhibitors of middle east respiratory syndrome coronavirus replication in cell culture a trial of lopinavir-ritonavir in adults hospitalized with severe covid- favipiravir versus arbidol for covid- : a randomized clinical ribavirin: a drug active against many viruses with multiple effects on virus replication and propagation. molecular basis of ribavirin resistance comparative effectiveness of aerosolized versus oral ribavirin for the treatment of respiratory syncytial virus infections: a single-center retrospective cohort study and review of the literature ideal oral combinations to eradicate hcv: the role of ribavirin a major outbreak of severe acute respiratory syndrome in hong kong clinical features and short-term outcomes of patients with sars in the greater toronto area short term outcome and risk factors for adverse clinical outcomes in adults with severe acute respiratory syndrome (sars) severe acute respiratory syndrome: report of treatment and outcome after a major outbreak ribavirin and interferonbeta synergistically inhibit sars-associated coronavirus replication in animal and human cell lines ribavirin and interferon alfa- a for severe middle east respiratory syndrome coronavirus infection: a retrospective cohort study use of chloroquine in viral diseases effects of chloroquine on viral infections: an old drug against today's diseases? remdesivir and chloroquine effectively inhibit the recently emerged novel coronavirus ( -ncov) in vitro evidence for a common evolutionary origin of coronavirus spike protein receptor-binding subunits chloroquine is a potent inhibitor of sars coronavirus infection and spread progress in antiviral therapy of new coronavirus pneumonia arbidol as a broad-spectrum antiviral: an update arbidol: a broad-spectrum antiviral compound that blocks viral fusion the synthetic antiviral drug arbidol inhibits globally prevalent pathogenic viruses comparison of inhibitory effects of arbidol and lianhuaqingwen capsules on middle east respiratory syndrome coronavirus in vitro and in vivo clinical characteristics and therapeutic procedure for four cases with novel coronavirus pneumonia receiving combined chinese and western medicine treatment dancing with chemical formulae of antivirals: a personal account dancing with chemical formulae of antivirals: a panoramic view (part ) mechanism of action of t- against influenza virus. antimicrob agents chemother new nucleoside analogues for the treatment of hemorrhagic fever virus infections a randomized, controlled trial of ebola virus disease therapeutics nucleoside and nucleotide hiv reverse transcriptase inhibitors: years after zidovudine inhibitors of the hepatitis c virus polymerase; mode of action and resistance mechanism of inhibition of ebola virus rna-dependent rna polymerase by remdesivir coronavirus susceptibility to the antiviral remdesivir (gs- ) is mediated by the viral polymerase and the proofreading exoribonuclease. mbio broad spectrum antiviral remdesivir inhibits human endemic and zoonotic deltacoronaviruses with a highly divergent rna dependent rna polymerase broad-spectrum antiviral gs- inhibits both epidemic and zoonotic coronaviruses first case of novel coronavirus in the united states transport of ebolavirus nucleocapsids is dependent on actin polymerization: live-cell imaging analysis of ebolavirus-infected cells therapy and pharmacological properties of hydroxychloroquine and chloroquine in treatment of systemic lupus erythematosus, rheumatoid arthritis and related diseases design and synthesis of hydroxyferroquine derivatives with antimalarial and antiviral activities in vitro antiviral activity and projection of optimized dosing design of hydroxychloroquine for the treatment acute respiratory syndrome coronavirus (sars-cov- ) recommendations on screening for chloroquine and hydroxychloroquine retinopathy hydroxychloroquine and azithromycin as a treatment of covid- : results of an openlabel non-randomized clinical trial efficacy of hydroxychloroquine in patients with covid- : results of a randomized clinical trial unique synergistic antiviral effects of shufeng jiedu capsule and oseltamivir in influenza a viral-induced acute exacerbation of chronic obstructive pulmonary disease the chinese prescription lianhuaqingwen capsule exerts anti-influenza activity through the inhibition of viral propagation and impacts immune function antiviral activity of glycyrrhizin against hepatitis c virus in vitro glycyrrhizin, an active component of liquorice roots, and replication of sars-associated coronavirus clinical effectiveness and case analysis in ncp patients treated with lianhuaqingwen granules retrospective clinical analysis on treatment of novel coronavirus-infected pneumonia with traditional chinese medicine lianhua qingwen clinical observation on cases of suspected cases of new coronavirus pneumonia treated by chinese medicine lianhua qingwen in vitro susceptibility of clinical isolates of sars coronavirus to selected antiviral compounds combined adjuvant effect of ginseng stem-leaf saponins and selenium on immune responses to a live bivalent vaccine of newcastle disease virus and infectious bronchitis virus in chickens structure of the sars-cov- spike receptor-binding domain bound to the ace receptor structural basis of receptor recognition by sars-cov- cryo-em structure of the -ncov spike in the prefusion conformation the sars-coronavirus papain-like protease: structure, function and inhibition by designed antiviral compounds learning from the past: possible urgent prevention and treatment options for severe acute respiratory infections caused by -ncov nucleotide analogues as inhibitors of viral polymerases sars-cov- cell entry depends on ace and tmprss and is blocked by a clinically proven protease inhibitor key: cord- -e cai j authors: fusi, fabio; trezza, alfonso; sgaragli, giampietro; spiga, ottavia; saponara, simona; bova, sergio title: ritanserin blocks ca(v) . channels in rat artery smooth muscles: electrophysiological, functional, and computational studies date: - - journal: acta pharmacol sin doi: . /s - - - sha: doc_id: cord_uid: e cai j ca(v) . channel blockers or -ht( ) receptor antagonists constitute effective therapy for raynaud’s syndrome. a functional link between the inhibition of -ht( ) receptors and ca(v) . channel blockade in arterial smooth muscles has been hypothesized. therefore, the effects of ritanserin, a nonselective -ht( ) receptor antagonist, on vascular ca(v) . channels were investigated through electrophysiological, functional, and computational studies. ritanserin blocked ca(v) . channel currents (i(ca . )) in a concentration-dependent manner (k(r) = . µm); i(ca . ) inhibition was antagonized by bay k and partially reverted upon washout. conversely, the ritanserin analog ketanserin ( µm) inhibited i(ca . ) by ~ %. ritanserin concentration-dependently shifted the voltage dependence of the steady-state inactivation curve to more negative potentials (k(i) = . µm) without affecting the slope of inactivation and the activation curve, and decreased i(ca . ) progressively during repetitive ( hz) step depolarizations (use-dependent block). the addition of ritanserin caused the contraction of single myocytes not yet dialyzed with the conventional method. furthermore, in depolarized rings, ritanserin, and to a lesser extent, ketanserin, caused a concentration-dependent relaxation, which was antagonized by bay k . ritanserin and ketanserin were docked at a region of the ca(v) . α( c) subunit nearby that of bay k ; however, only ritanserin and bay k formed a hydrogen bond with key residue tyr- . in conclusion, ritanserin caused in vitro vasodilation, accomplished through the blockade of ca(v) . channels, which was achieved preferentially in the inactivated and/or resting state of the channel. this novel activity encourages the development of ritanserin derivatives for their potential use in the treatment of raynaud’s syndrome. ritanserin ( -[ -[ - [bis( -fluorophenyl)methylidene]piperidin- -yl] ethyl]- -methyl- [ , ] thiazolo[ , -a]pyrimidin- -one) is a potent, long acting, nonselective -ht receptor antagonist [ , ] and is classified as an antidepressant agent [ ] , although it has never been approved for clinical use. it has an additional intrinsic antidopaminergic effect, possibly underlying the reported improvement of negative symptoms in patients who have schizophrenia [ ] . moreover, a number of preclinical and clinical studies have examined the effectiveness of ritanserin at reducing cocaine cravings and/or cocaine use [ ] . the direct action of ritanserin on na + and ca + channels has been shown in canine purkinje fibers, where it produces significant depressant effects on transmembrane action potentials [ ] , which are credited for its antiarrhythmic activity. recently, evidence that ritanserin inhibits several lipids (e.g., diacylglycerol kinase-α, dgkα, a novel, potential therapeutic target in various cancers as well as in immunotherapy) [ ] [ ] [ ] and protein kinases (e.g., the feline encephalitis virus-related kinase fer) [ ] , as well as the rapidly accelerated fibrosarcoma kinase c-raf [ ] has been provided. the capacity of perturbing cellular signaling pathways important for cell survival and proliferation, through serotonin-independent mechanisms suggests that ritanserin may be a viable option for in vivo translation and a novel therapeutic tool with potential applications in various tumors. the serotoninergic antagonism of ritanserin, characterized by a long duration of action, has been shown in a variety of peripheral cardiovascular, gastrointestinal, and respiratory vascular tissues [ ] . in the rat tail artery, the interaction of ritanserin with an allosteric site near the -ht receptor was presumed, owing to ritanserin antagonism of the effects of -ht that are different from that of another -ht receptor antagonist, ketanserin ( -[ -[ -( fluorobenzoyl)piperidin- -yl]ethyl]- h-quinazoline- , -dione) [ ] . furthermore, ritanserin also antagonizes histaminergic (h ) and adrenergic (α ) responses in the rabbit femoral artery, though at concentrations one to three orders of magnitude higher than those needed to block -ht receptors. several studies have demonstrated the activity of this antagonist on vascular function. in vitro as well as in vivo, ritanserin potently inhibits the -ht-induced vasoconstriction and pressure responses of an isolated, perfused mesenteric artery preparation [ ] . furthermore, ritanserin is also an in vitro competitive antagonist of noradrenaline and decreases the mean arterial blood pressure of anaesthetized rats. ketanserin fails to antagonize -ht-induced contractions in rat aorta rings pretreated with verapamil, a ca v . channel blocker [ ] . this phenomenon was explained by the direct interaction of -ht receptor antagonists with the ca v . channel protein [ ] . finally, similarity in the pharmacology of ca v . channel blockers and -ht receptor antagonists has been suggested [ ] . in fact, both classes of compounds are capable of antagonizing -htinduced and high k + -induced contractions in rat aorta rings. sarpogrelate, a -ht receptor antagonist registered in japan, china, and south korea, is used to improve vascular function in patients with peripheral artery disease and symptoms related to raynaud's syndrome, which is characterized by reduced blood flow to the fingers and toes caused by vessel tightening or spasms in the cold [ ] [ ] [ ] . as ca v . channel blockers, such as nifedipine, have recently been confirmed as useful agents to reduce the frequency, duration, severity of attacks, pain, and disability associated with raynaud's syndrome [ ] , the aim of the present study was to analyze the effect of ritanserin on the vascular ca v . channel. although the functional effects of ritanserin on blood vessels have been extensively investigated, its possible interaction with ca v . channels, which are fundamental regulators of vascular muscle tone and function [ ] , has never been investigated. therefore, patch-clamp, functional, and moleculardocking analyses of ritanserin effects were performed on rat tail arteries. all animal care and experimental protocols conformed to the european union guidelines for the care and use of laboratory animals (european union directive / /eu) and were approved by the italian department of health ( / -pr). male wistar rats ( - g, charles river italia, calco, italy) were anaesthetized (i.p.) with a mixture of zoletil ® ( . mg·kg − tiletamine and . mg·kg − zolazepam; virbac srl, milan, italy) and rompun® ( mg·kg − xylazine; bayer, milan, italy), decapitated and exsanguinated. the tail was isolated immediately, cleaned of skin and placed in physiological solution (namely an external solution or modified krebs-henseleit solution; see sections "cell isolation procedure" and "functional experiments", respectively). the tail main artery was dissected free of its connective tissue and cells or rings prepared as detailed in sections "cell isolation procedure" and "functional experiments", respectively. cell isolation procedure smooth muscle cells were freshly isolated from the tail main artery under the following conditions. a -mm long piece of artery was incubated at °c for - min in ml of . mm ca + external solution (in mm: nacl, . kcl, hepes, glucose, . mgcl , and na-pyruvate; ph . ) containing mm taurine, which replaced an equimolar amount of nacl, . mg·ml − collagenase (type xi), mg·ml − soybean trypsin inhibitor, and mg·ml − bovine serum albumin. this solution was gently bubbled with a % o - % co gas mixture to stir the enzyme solution, as previously described [ ] . cells stored in . mm ca + external solution containing mm taurine and . mg·ml − bovine serum albumin at °c under a normal atmosphere were used for experiments within days after isolation [ ] . whole-cell patch clamp recordings cells were continuously superfused with external solution containing . mm ca + and mm tetraethylammonium (tea) using a peristaltic pump (lkb , bromma, sweden) at a flow rate of µl·min − . the conventional whole-cell patch-clamp method was employed to voltage clamp smooth muscle cells. recording electrodes were pulled from borosilicate glass capillaries (wpi, berlin, germany) and fire-polished to obtain a pipette resistance of - mΩ when filled with internal solution. the internal solution (pca . ) consisted of (in mm): cscl, hepes, egta, mgcl , cacl , na-pyruvate, succinic acid, oxaloacetic acid, na atp, and phosphocreatine; the ph was adjusted to . with csoh. an axopatch b patch-clamp amplifier (molecular devices corporation, sunnyvale, ca, usa) was used to generate and apply voltage pulses to the clamped cells and record the corresponding membrane currents. at the beginning of each experiment, the junction potential between the pipette and bath solution was electronically adjusted to zero. current signals, after compensation for whole-cell capacitance and series resistance (between % and %), were low-pass filtered at khz and digitized at khz prior to being stored on the computer hard disk. electrophysiological responses were tested at room temperature ( - °c). the i ca . was recorded in an external solution containing mm tea and mm ca + . the current was elicited with ms clamp pulses ( . hz) to mv from a v h of either − or − mv. data were collected once the current amplitude had been stabilized (usually - min after the whole-cell configuration had been obtained). then the various experimental protocols were performed as detailed below. under these conditions, the current, which did not run down during the following min [ ] , was carried almost entirely by ca v . channels [ , ] . steady-state activation curves were derived from the current-voltage relationships. conductance (g) was calculated from the equation g = i ca . /(e m −e rev ), where i ca . is the peak current elicited by depolarizing test pulses between − and mv from a v h of − mv; e m is the membrane potential; and e rev is the reversal potential ( mv, as estimated by the nernst equation). g max is the maximal ca + conductance (calculated at potentials ≤ mv). the g/g max ratio was plotted against the membrane potential and fitted to the boltzmann equation [ ] . steady-state inactivation curves were obtained using a doublepulse protocol. once various levels of the conditioning potential had been applied for s, followed by a short ( ms) return to the v h of − mv, a test pulse ( ms) to mv was delivered to evoke the current. the delay between the conditioning potential and the test pulse allowed the full or near-complete deactivation of the channels, simultaneously avoiding partial recovery from inactivation. k + currents were blocked with mm tea in the external solution and cs + in the internal solution. current values were corrected for leakage and residual outward currents using µm nifedipine, which completely blocked i ca . . the osmolarity of the mm tea-containing and mm ca + -containing external solution ( mosmol) and that of the internal solution ( mosmol) were measured with an osmometer (osmostat om , menarini diagnostics, florence, italy). the hepes buffer system present in the cell storage medium (i.e., external solution) may alter the response of vascular smooth muscle to vasoconstricting agents such as adrenaline and angiotensin ii [ ] . therefore, in the functional experiments, the rings and cells were continuously superfused at °c with a modified krebs-henseleit, hepes-free solution containing (in mm): nacl, . kcl, . cacl , . mgso , . kh po , nahco , and . glucose bubbled with a % o - % co gas mixture to create a ph of . . cells were randomly selected among those, phase-dense, presenting an elongated shape (i.e., relaxed). single-cell shortening was quantified using imagej by means of the analyze/measure plugin with the straight-line tool (ver . r, nih, http://imagej.nih.gov/ij/download.html). the length was measured in the same cell before and after drug challenge. ritanserin blocks the vascular ca v . channel f fusi et al. two millimiter long rings, endothelium-denuded, were obtained from the tail main artery and mounted in a homemade plexiglass support for tension recording as previously described [ ] with slight modifications. the endothelium was removed by gently rubbing the lumen of the rings with a very thin roughsurfaced tungsten wire. rings were immersed in a double chambered organ bath at °c filled with a modified krebs-henseleit solution. contractile tension was recorded using an isometric force transducer (ugo basile, comerio, italy) connected to a digital powerlab data acquisition system (power-lab / ; adinstruments, castle hill, australia) and analyzed by labchart pro version . . for windows software (adinstruments). at the beginning of the experiment, a preload of g ( g/mm) was applied to each ring. after an equilibration period of min, the rings were contracted with either mm kcl or µm phenylephrine until reproducible responses to each stimulus were obtained. the absence of a functional endothelium was confirmed by the lack of carbachol-induced relaxation of rings precontracted with phenylephrine. ritanserin vasoactivity was assessed on rings either depolarized with mm kcl or stimulated with nm bay k in the presence of mm kcl. a concentration-relaxation curve for ritanserin was subsequently constructed. muscle tension was evaluated as a percent of the initial response to mm kcl or bay k , which was taken as %. a high kcl concentration was achieved by directly adding kcl from a . m stock solution, to the organ bath solution, as neither efficacy nor potency of vasodilators is significantly affected by the resultant increase in the osmolarity compared to preparations in which the osmolarity is preserved [ ] . the chemicals used included collagenase (type xi), trypsin inhibitor, bovine serum albumin, tea chloride, hepes, taurine, (s)-(-)-methyl- , -dihydro- , -dimethyl- -nitro- -( -trifluoromethylphenyl)pyridine- -carboxylate (bay k ), phenylephrine, ritanserin, and nifedipine (sigma chimica, milan, italy); ketanserin [janssen, ceva logistics italia srl, stradella (pv), italy]. bay k and nifedipine, dissolved directly in ethanol, and ritanserin, dissolved directly in dmso, were diluted at least times prior to use. control experiments confirmed that no response was induced in vascular preparations when dmso or ethanol at the final concentration used in the above dilutions ( . %, v/v) was added alone (data not shown). final drug concentrations are stated in the text. statistical analysis acquisition and analysis of the data were accomplished using pclamp . . . software (molecular devices corporation, sunnyvale, ca, usa) and graphpad prism version . (graphpad software inc., san diego, ca, usa). data are reported as the mean ± sem; n is the number of cells/ rings analyzed isolated from at least three animals. statistical analyses and significance, as measured by student's t-test for either paired or unpaired samples (two-tailed) and repeated measures anova followed by dunnett's or bonferroni's posttest, were obtained using graphpad prism version . . posttests were performed only when anova found a significant value of f and no variance in homogeneity. in all comparisons, p < . was considered significant. the pharmacological response to ritanserin, described in terms of pic (the −log of the ic value, i.e., the drug concentration reducing the response by %), was obtained by nonlinear regression analysis. the homology d model of the ca v . channel pore domain was utilized as previously described [ ] . docking of the ligands ritanserin, ketanserin, and bay k was simulated by a flexible side chain protocol with autodock vinaxb [ ] . the ritanserin, ketanserin, and bay k structures were downloaded from the pubchem database in sdf format (pubchem cid , , and , respectively) [ ] , while the pdbqt file was created by using autodock vina v. . . tools [ ] . multiple ligand-protein interaction maps were generated using the protein-ligand interaction profiler (plip) [ ] . pymol was used as the molecular graphics system (the pymol molecular graphics system, version . schrödinger, llc.). effect of ritanserin and ketanserin on i ca . this series of experiments were carried out to evaluate the effect of ritanserin and ketanserin (fig. a) on i ca . . figure b shows recordings of the inward current elicited with a clamp pulse to mv from a v h of − mv under control conditions and after the addition of cumulative concentrations of ritanserin, which inhibited peak i ca . in a concentration-dependent manner with a pic (m) value of . ± . (n = ; fig. c ). in contrast, ketanserin-induced inhibition of current amplitude was b average traces (recorded from five cells) of i ca . , elicited with ms clamp pulses to mv from a v h of − mv, measured in the absence (control) or presence of cumulative concentrations of ritanserin. c concentration-dependent effect of ritanserin measured in the absence (control) and presence of nm bay k . the concentration-response curve of ketanserin is also shown. on the ordinate scale, the current amplitude is reported as a percentage of the value recorded just before the addition of the first concentration of ritanserin or ketanserin. the curves show the best fit of the points. data points are the mean ± sem (n = - ). *p < . vs. ritanserin, studentʼs t test for unpaired samples significantly lower than that of its analog ritanserin and, at the maximal concentration assessed ( µm), ketanserin inhibited the current amplitude by only~ %. to investigate whether the dihydropyridine-binding site on the channel protein was involved in the ca + antagonistic activity of ritanserin, the potential functional interaction of ritanserin and the ca v . channel stimulator bay k was assessed. in myocytes challenged with nm bay k , i ca . increased to % ± % of the control (n = ). as shown in fig. c , pretreatment with bay k caused a significant rightward shift of the ritanserin concentration-response curve (pic (m) value of . ± . , n = ; p = . vs. control). characterization of the effects of ritanserin on i ca . a biophysical and pharmacological analysis was carried out to clarify the mechanism underlying the ritanserin-induced inhibition of i ca . and its activity was described at the channel protein. figure a illustrates the time course of the effects of µm ritanserin or vehicle ( . % dmso) on the current recorded at . hz from a v h of − mv to a test potential of mv. after i ca . reached steady values, the addition of ritanserin to the bath solution produced a gradual decrease in the current amplitude that reached a plateau in~ min. in contrast, dmso had no effect on the current amplitude. ritanserin-induced inhibition of i ca . was partially reversed upon drug washout (fig. a) and was not affected by the membrane potential. in fact, when v h was shifted to − mv, the residual current amplitude ( . % ± . % of control, n = ) was similar to that recorded at a v h of − mv ( . % ± . % of control, n = ; p = . ). i ca . evoked at mv from a v h of either − or − mv was activated and then declined with a time course that could be fitted by a monoexponential function. ritanserin ( µm) significantly accelerated only the τ of inactivation recorded at a v h of − mv (fig. b) . the current-voltage relationship (fig. a) shows that µm ritanserin significantly decreased the peak inward current in the range of membrane potential values from − to mv, shifting the apparent maximum by mv in the hyperpolarizing direction without varying the threshold at approximately − mv. the voltage dependence of ritanserin inhibition was further investigated by analyzing the steady-state inactivation and activation curves for i ca . . the steady-state activation curves (fig. b) , calculated from the current-voltage relationships shown in fig. a , were fitted with the boltzmann equation. ritanserin neither shifted the % activation potential (− . ± . mv for the control, and − . ± . mv for µm ritanserin, n = ; p = . , student's t test for paired samples) nor affected the slope factor ( . ± . and . ± . mv, respectively; p = . ). the apparent dissociation constant of ritanserin for inactivated channels (k i ) was determined by the shift of ca v . channel steady-state availability as a function of ritanserin concentration at a v h of − mv [ ] . ritanserin significantly shifted the steadystate inactivation curve to more hyperpolarizing potentials in a concentration-dependent manner ( fig. b ; p = . , repeated measures anova). the % inactivation potential changed from − . ± . mv (n = , control) to − . ± . mv ( µm ritanserin, p > . ), − . ± . mv ( µm ritanserin, p < . ), and − . ± . mv ( µm ritanserin, p < . , dunnett's posttest). the slope factor, however, was not affected by ritanserin (− . ± . , − . ± . , − . ± . , and − . ± . mv, respectively; p > . ). the k i was estimated by plotting the % inactivation potentials as a function of ritanserin concentration. this relationship was fitted to the equation where v control and k control are the values of the % inactivation potential and the slope measured under control conditions, respectively. the value of k i thus determined was . ± . µm (n = ), which was not significantly different from the k r reported above ( . ± . µm, n = ; p = . , student's t test for unpaired samples). the shift of the inactivation curve caused by µm ritanserin led to a marked reduction in the ca + window current that peaked at approximately − mv (with a relative amplitude of . ) compared with the peak at approximately − mv (relative amplitude . ) observed under control conditions. to assess whether ritanserin inhibition of i ca . was frequencydependent, the current was recorded during depolarizing pulses ms in length to mv from a v h of − mv applied at a stimulation frequency of hz. ritanserin, at a concentration of µm, produced a frequency-dependent block of i ca . (fig. ) . this frequency dependence, calculated by normalizing the current amplitude evoked by the th applied stimulus against that induced by the first step pulse, was significantly greater than that observed under control conditions. in silico docking was carried out to define the interaction of ritanserin with the rat ca v . channel α c subunit. the lowest energy poses of ritanserin, ketanserin, and bay k showed gibbs free-energy values (Δg) of − . , − . , and − . kcal·mol − , respectively. the computational analysis established that the three compounds were placed in the same binding region, though in different binding pockets (fig. ). in particular, ritanserin and ketanserin bound to the same site with good superposition; however, differences in their structure gave rise to different residue interaction networks. plip analysis indicated that ritanserin formed hydrophobic interactions with leu- , val- , leu- , and phe- , a hydrogen bond with tyr- , a π-stacking interaction with phe- , and halogen bonds with thr- and ile- (fig. a) , whereas ketanserin formed hydrophobic interactions with leu- , met- , tyr- , ile- , phe- , a π-stacking interaction with phe- , and a halogen bond with thr- . conversely, bay k gave rise to hydrophobic interactions with thr- , phe- , phe- , and ile- , and formed a strong hydrogen bond with tyr- (fig. b) . the ligand-protein interaction networks showed that the orientation of tyr- present in the binding pockets accommodating either bay k or ritanserin plays a key role in the binding of the latter to the ca v . channel α c subunit. only the tyr- orientation shown in fig. c was able to form a stable . Å-long hydrogen bond with ritanserin; in contrast, the tyr- orientation shown in fig. d , which gave rise to the formation of a stronger . Å long hydrogen bond with bay k , was favored by the presence of bay k in the pocket. effect of ritanserin on freshly isolated rat caudal artery myocytes the contractile effect of ritanserin was evaluated by recording the morphological changes of freshly isolated cells (shortening with formation of membranous evaginations; see ref. [ ] ). as shown in fig. a , under the experimental conditions used for patch-clamp recordings, i.e., in the presence of mm ca + and mm tea, the addition of ritanserin in the range concentration of - μm fig. effect of ritanserin on the frequency dependence of i ca . in single vascular myocytes. in the absence of ritanserin, depolarizing ms clamp pulses to mv from v h of − mv were applied at hz. ritanserin ( µm) was added just after the delivery of the first train of pulses; min later, the same protocol was repeated. the peak amplitude elicited by the first train pulse (in either the absence or presence of ritanserin) was taken as % to better appreciate the frequency-dependent block of i ca . . data points are the mean ± sem (n = ). *p < . vs. the time-matched control, student's t test for paired samples fig. surface-cartoon representation of the homology model of the ca v . structure. domains i-iv are colored yellow, gray, pink, and green, respectively. ritanserin (blue sticks), ketanserin (orange sticks), and bay k (red sticks) are posed inside their binding pockets caused cell contraction. tea did not evoke any evident contraction per se, which is in line with what was previously observed with mm k + [ ] . conversely, the addition of ritanserin did not elicit contractions in cells dialyzed under the whole-cell configuration. similar results were obtained under the experimental conditions used for functional experiments (i.e., . mm ca + and no tea in the bath; fig. b ). superfusion with the sole vehicle, i.e., . % dmso, did not affect the length of the myocytes ( . ± . µm, control; . ± . µm, . % dmso min; . ± . µm, . % dmso min; p > . ); however, it shortened significantly upon the addition of . mm atp ( . ± . µm, n = ; p < . ). in rat caudal artery rings, the addition of cumulative concentrations of ritanserin did not evoke an increase in basal tone (data not shown). in preparations precontracted with mm kcl ( ± mg, n = ), ritanserin induced a concentration-dependent relaxation with a pic (m) value of . ± . (n = ) and a maximal inhibitory effect of . % ± . % (n = ) (fig. a, b) . ketanserin was less effective than ritanserin on mm kclcontracted rings ( ± mg, n = ), causing a relaxation of . % ± . % (n = ; p = . ). drug washout for~ - min caused a partial recovery of the µm phenylephrine-induced contraction, which amounted to . % ± . % of the control (ritanserin; n = ) and . % ± . % of the control (ketanserin; n = ; p = . ). noticeably, mm kcl-induced contraction recovered to only . % ± . % of the control (n = ) following a - min washout of ritanserin (p < . vs. phenylephrine). ritanserin concentration-dependently relaxed rings precontracted by nm bay k ( ± mg, n = ); however, the pic (m) value was lower than that recorded in the depolarized preparations ( . ± . , n = ; p < . ). the present investigation provides the first direct electrophysiological evidence that ritanserin is a vascular ca v . channel blocker, as demonstrated by the inhibitory effect produced by the drug on i ca . . the major findings supporting this conclusion are as follows: ( ) in single vascular myocytes, ritanserin inhibited i ca . in a concentration-dependent manner; ( ) this inhibition was antagonized by the ca v . stimulator bay k and was likely due to the interaction of ritanserin with the channel protein; ( ) ritanserin stabilized the ca v . channel in its inactivated state; and ( ) since ritanserin relaxed vascular smooth muscle contraction resulting from the opening of ca v . channels, the i ca . blockade is supposed to have functional relevance and supports previous data obtained in vascular and nonvascular tissues [ ] [ ] [ ] , where such mechanism of action was hypothesized to account for the relaxant effects of the drug. the potency of ritanserin was diminished when ca v . channels were stimulated by bay k , in analogy with what was observed with nifedipine and bay k , which are both dihydropyridines and share the same binding site on ca v . channel α c subunit [ ] . this observation provides compelling evidence for a mutual interaction of ritanserin and bay k at the channel protein. the rightward shift of the ritanserin inhibition curve by bay k might be because bay k and ritanserin bind to channel receptor sites that are in close proximity to each other or are allosterically linked [ ] . support for this hypothesis is aroused from the in silico analysis (see below). ritanserin-induced inhibition of i ca . observed at . hz-a frequency that allows full recovery between pulses from ca v . channel inactivation in rat tail artery myocytes [ ] -was tonic in nature and developed independently of channel activation [ ] . this is interpreted as a consequence of the selective inhibition of the resting channel state. furthermore, ritanserin, like verapamil [ ] , inhibited i ca . in a frequency-dependent fashion (usedependent block), and similarly to nicardipine [ ] shifted the channel availability toward more hyperpolarizing potentials. these effects are conventionally interpreted as a consequence of a high affinity drug binding and stabilization of inactivated channels [ ] . this phenomenon, however, was not voltage-dependent. in fact, the ritanserin apparent dissociation constant for the inactivated channel (k i ) was not significantly different from that for the closed channel (k r ). this hypothesis is further supported by the similar current inhibition recorded at v h values of − and − mv. stabilization of the inactivated state, however, seemed to follow a slow kinetics, since the i ca . inactivation, observed during the ms long depolarizing step, was slightly affected by ritanserin fig. docking of ritanserin and bay k into the ca v . channel α c subunit. view of a ritanserin and b bay k (blue and red sticks, respectively) interacting with the binding pocket residues (orange sticks). the hydrogen bonds, hydrophobic interactions, and π-stacking are displayed as purple, gray, and green dotted lines, respectively. c, d enlarged view showing the role played by the tyr- residue (orange sticks) in the binding of c ritanserin and d bay k . the hydrogen bond is displayed as a purple dotted line only at a v h of − mv. finally, it is conceivable that ritanserin could cause ca v . channel inactivation by increasing basal cytoplasmic ca + levels. however, this hypothesis can be ruled out, as the drug did not increase the resting smooth muscle tone of artery rings when added to the organ bath in the absence of other stimulating agents. taken together, these findings indicate that within the frame of the "state-dependent pharmacology" of the channel, three different mechanisms, operated simultaneously by ritanserin, are responsible for ca v . channel block: stateindependent (tonic), weak state-dependent open channel inhibition (the faster ca v . channel inactivation kinetics observed in the presence of the drug, reported also for other ca v . channel blockers such as dihydropyridines and phenylalkylamines) [ ] , and state-dependent inactivated channel stabilization. the leftward shift of the steady-state inactivation curve operated by ritanserin, caused a marked reduction of the window current. this current is physiologically relevant because is thought to be largely responsible for both generation and regulation of vascular smooth muscle tone [ ] . therefore, when preincubated in vitro with the isolated preparations, ritanserin may cause relaxation per se via a reduction of the window current. on the other hand, although ritanserin shifted the voltage at which the maximum of the current-voltage relationship occurred by mv, neither the slope nor the % activation potential were affected by the drug, thus indicating that the sensitivity of the channel activation mechanism to the membrane voltage was only modestly altered. docking results demonstrated that, similar to dihydropyridine ca v . channel blockers [ ] , ritanserin bound to the outer, lipidfacing surface of the pore in the intersubunit crevice formed by neighboring tilted s -s helices and the p-loop of domains iii and iv, a region involved in ca v . inactivation [ , ] . noticeably, ritanserin and bay k bound to the same region, though to different, very closely situated pockets, analogous to what was previously observed with the pka inhibitor h- [ ] . the tyr- residue, located in the overlapping area, was crucial for the interaction of the two molecules with the channel protein. the conformation of the residue shown in fig. c allowed ritanserin binding. conversely, when the residue assumed the conformation shown in fig. d only bay k was able to dock. additionally, the tyr- residue conformation is important in shaping the channel pore state [ , ] indicating that the conformation favored by ritanserin stabilizes the closed state, while that favored by bay k stabilizes the open state of the channel. finally, the two different docking poses and hydrogen bond lengths likely explain the decreased ca + antagonist potency of ritanserin recorded in the presence of bay k . the lower inhibitory effect of ketanserin compared to ritanserin could be due to its different interaction network with binding pocket residues and, in particular, to the lack of interaction with the key tyr- residue. ritanserin inhibited i ca . in the same cells where it induced a contractile effect. noticeably, dialysis of the cytoplasm (as in the case of the conventional whole-cell method) prevented cell contraction but not inhibition of i ca . . this observation suggests that contraction was mediated by a rise in cytoplasmic ca + concentration (which, in the whole-cell configuration, is buffered by the high level of egta present in the pipette solution) and/or by diffusible intracellular factors. on the other hand, ritanserin inhibition of i ca . seems to be related to a direct interaction of the drug with the channel protein, although the possible involvement of intracellular signaling pathways surviving dialysis cannot be ruled out. taken together, these results indicate that the drug is able to activate both vasoconstricting and vasorelaxant mechanisms, the former prevailing in single cells and the latter in intact tissue either in resting conditions or under the presence of stimulating agents such as high concentrations of k + . this apparent discrepancy, previously observed with the rat toxicant norbormide [ ] and described with high k + [ ] likely follows the disruption of the extracellular matrix and ensuing isolation of single smooth muscle cells, leading to the loss of focal contact tension that in turn considerably alters cell signaling systems [ ] . moreover, this hypothesis argues that the relaxation effect elicited by ritanserin in the intact tissue may be underestimated, i.e., inhibition of the ca v . channel by itself would induce a more pronounced vasorelaxation. ca v . channel inhibition was also observed in intact tissue under conditions of full membrane depolarization, i.e., in vascular rings depolarized with high k + concentrations. under experimental conditions similar to those represented by voltage-clamp pulses of depolarization applied to evoke i ca . ritanserin caused relaxation and its potency and efficacy were consistent with those calculated in the patch-clamp experiments. furthermore, this vasorelaxant effect was significantly antagonized by the ca v . channel agonist bay k , which is in perfect agreement with what was observed in single myocytes, thus supporting the hypothesis of a mutual interaction of ritanserin and bay k at the channel protein. finally, in rat caudal artery rings, the myorelaxant effect of the drug was only partially reverted by washout, similar to what was observed in the patch-clamp experiments. the partial reversibility of ritanserin i ca . antagonism observed in the present study suggested a relatively strong interaction of the drug with the channel protein. in fact, recovery of high k + -induced contraction (essentially dependent on the opening of ca v . channels) was significantly lower than that correlated to phenylephrine (only partly dependent on the opening of ca v . channels). furthermore, this effect was similar to that previously reported in isolated dog cardiac tissue [ ] , where the effects of ritanserin could not be fully reversed even after h of repeated washout with physiological solution. interestingly, ketanserin, when assessed on both smooth muscle active tone and i ca . amplitude, showed a much lower activity compared to its analog ritanserin. in silico docking and postdocking analyses, displaying a higher number and stronger type of interactions toward the binding pocket for ritanserin compared to ketanserin, well supported these experimental observations. taken together, these data strengthen the hypothesis of the existence of a rather specific ritanserin structure-ca v . channel blocking activity relationship. in conclusion, the present electrophysiological, functional, and in silico data point to ritanserin as a vasorelaxant and ca v . channel blocking agent. this observation may open a new avenue for the treatment of raynaud's syndrome. in this disorder, either ca v . channel blockers, such as nifedipine [ ] , or -ht receptor antagonists, such as sarpogrelate [ ] [ ] [ ] , are effective therapeutic agents. therefore, the ca v . channel and -ht receptor antagonist ritanserin may represent an interesting scaffold to develop bifunctional defense drugs for the treatment of raynaud's phenomenon, as opposed to ketanserin, which was previously defined as not clinically beneficial for the treatment of this disorder in progressive systemic sclerosis [ ] . the discovery of a contractile pathway activated by the drug suggests that ritanserin derivatives, possibly deprived of the contractile property, would give rise to relaxing agents that are more potent than the parent compound itself. finally, in search of effective drugs for the treatment of reynaud's syndrome, forthcoming analysis of some -ht receptor antagonists will clarify whether other agents besides ritanserin emerge as scaffolds worthy of further pharmaceutical development. receptor-binding properties in vitro and in vivo of ritanserin: a very potent and long acting serotonin-s antagonist efficacy and acceptability of acute treatments for persistent depressive disorder: a network meta-analysis efficacy of antidepressants in treating the negative symptoms of chronic schizophrenia: meta-analysis a randomized, double-blind, placebo-controlled study of ritanserin pharmacotherapy for cocaine dependence effects of ritanserin on transmembrane action potentials in canine purkinje fibres dual activities of ritanserin and r as dgka inhibitors and serotonin receptor antagonists diacylglycerol kinase α inactivation is an integral component of the costimulatory pathway that amplifies tcr signals targeting the mesenchymal subtype in glioblastoma and other cancers via inhibition of diacylglycerol kinase alpha the ligand binding landscape of diacylglycerol kinases chemoproteomic discovery of a ritanserin-targeted kinase network mediating apoptotic cell death of lung tumor cells comparative pharmacological profile of ritanserin and ketanserin allosteric properties of the -ht receptor system of the rat tail artery. ritanserin and methysergide are not competitive -ht receptor antagonists but allosteric modulators in vivo and in vitro activity of selective -hydroxytryptamine receptor antagonists the interaction between ca + , verapamil and ketanserin in rat tail artery aorta effects of -ht receptor antagonists on responses to potassium depolarization, in rat isolated aorta overlap in the pharmacology of l-type ca + -channel blockers and -ht receptor antagonists in rat aorta sarpogrelate hydrochloride for raynaud's phenomenon of patients with collagen diseases effects of sarpogrelate hydrochloride on skin ulcers and quality of life in patients with systemic sclerosis sarpogrelate hydrochloride therapy for raynaud's phenomenon in patients with systemic sclerosis calcium channel blockers for primary and secondary raynaud's phenomenon. cochrane database syst rev the physiology, pathology, and pharmacology of voltage-gated calcium channels and their future therapeutic potential cardamonin is a bifunctional vasodilator that inhibits ca v . current and stimulates k ca . current in rat tail artery myocytes l-type ca + channel current characteristics are preserved in rat tail artery myocytes after oneday storage mechanism of myricetin stimulation of vascular ltype ca + current characterization of voltage-gated calcium currents in freshly isolated smooth muscle cells from rat tail main artery measurement of cellular excitability by whole cell patch clamp technique adverse effects of artificial buffers on contractile responses of arterial and venous smooth muscle vasorelaxant properties of norbormide, a selective vasoconstrictor agent for the rat microvasculature influence of depolarization on vasorelaxant potency and efficacy of ca + entry blockers, k + channel openers, nitrate derivatives, salbutamol and papaverine in rat aortic rings the vasodilator papaverine stimulates l-type ca + current in rat tail artery myocytes via a pka-dependent mechanism autodock vinaxb: implementation of xbsf, new empirical halogen bond scoring function, into autodock vina pubchem substance and compound databases autodock and autodocktools : automated docking with selective receptor flexibility plip: fully automated protein-ligand interaction profiler lidocaine block of cardiac sodium channels preparation of functional smooth muscle cells from the rabbit aorta functional, electrophysiological and molecular docking analysis of the modulation of ca v . channels in rat vascular myocytes by murrayafoline a molecular determinants of drug binding and action on l-type calcium channels quercetin as a novel activator of l-type ca + channels in rat tail artery smooth muscle cells pharmacological and physiological significance of ion channels and factors that modulate them in vascular tissues regulation and modulation of calcium channels in cardiac, skeletal, and smooth muscle cells nitrendipine block of cardiac calcium channels: high-affinity binding to the inactivated state contribution of transient and sustained calcium influx, and sensitization to depolarization-induced contractions of the intact mouse aorta structural basis for inhibition of a voltage-gated ca + channel by ca + antagonist drugs structural model for dihydropyridine binding to l-type calcium channels identification of "on-off residues" in rat ca v . α c subunit channel using in silico analysis and docking simulation ca(v) . channel current block by the pka inhibitor h- in rat tail artery myocytes via a pka-independent mechanism: electrophysiological, functional, and molecular docking studies ca + entry blocking and contractility promoting actions of norbormide in single rat caudal artery myocytes regulation of smooth muscle calcium sensitivity: kcl as a calcium sensitizing stimulus ketanserin for raynaud's phenomenon in progressive systemic sclerosis we wish to thank dr. a. ahmed for the assistance in some preliminary experiments. ff and sb designed the research; ff, at, and sb performed the research; ff and at analyzed the data; ff, os, ss, and sb wrote the paper; and gs critically revised the paper. competing interests the authors declare no competing interests. key: cord- -fuwp qt authors: lu, chen-chen; hu, ze-bo; wang, ru; hong, ze-hui; lu, jian; chen, pei-pei; zhang, jia-xiu; li, xue-qi; yuan, ben-yin; huang, si-jia; ruan, xiong-zhong; liu, bi-cheng; ma, kun-ling title: gut microbiota dysbiosis-induced activation of the intrarenal renin–angiotensin system is involved in kidney injuries in rat diabetic nephropathy date: - - journal: acta pharmacol sin doi: . /s - - - sha: doc_id: cord_uid: fuwp qt some studies have shown that gut microbiota along with its metabolites is closely associated with diabetic mellitus (dm). in this study we explored the relationship between gut microbiota and kidney injuries of early diabetic nephropathy (dn) and its underlying mechanisms. male sd rats were intraperitoneally injected with streptozotocin to induce dm. dm rats were orally administered compound broad-spectrum antibiotics for weeks. after the rats were sacrificed, their blood, urine, feces, and renal tissues were harvested for analyses. we found that compared with the control rats, dm rats had abnormal intestinal microflora, increased plasma acetate levels, increased proteinuria, thickened glomerular basement membrane, and podocyte foot process effacement in the kidneys. furthermore, the protein levels of angiotensin ii, angiotensin-converting enzyme, and angiotensin ii type receptor in the kidneys of dm rats were significantly increased. administration of broad-spectrum antibiotics in dm rats not only completely killed most intestinal microflora, but also significantly lowered the plasma acetate levels, inhibited intrarenal ras activation, and attenuated kidney damage. finally, we showed that plasma acetate levels were positively correlated with intrarenal angiotensin ii protein expression (r = . , p < . ). in conclusion, excessive acetate produced by disturbed gut microbiota might be involved in the kidney injuries of early dn through activating intrarenal ras. as a chronic microvascular complication of diabetes, diabetic nephropathy (dn) has become one of the major causes leading to the death of diabetic patients. according to the th idf diabetes atlas, million adults were diagnosed with diabetes worldwide in , and this population has been expected to grow by % by [ ] . the rapid growth of diabetes has led to a dramatically increasing prevalence of dn. in china, due to the rapid development of the social economy and lifestyle changes, the prevalence of diabetes is also on the rise. according to the latest survey data in china, the prevalence of diabetes in adults is . %, and the total number of diabetic patients is as high as million, nearly half of which also have dn [ ] . however, due to the limited diagnosis of early dn, most of the newly diagnosed patients have already progressed to stage iii or iv, which is basically irreversible. therefore, it is of great importance to clarify the pathophysiological changes in the early stages of dn and formulate intervention strategies for clinical diagnosis and treatment. the activation of the intrarenal renin-angiotensin system (ras) has long been considered one of the initiators of dn. it has been reported that under diabetic conditions, the circulating ras is normal or decreased, while local ras is highly activated in the kidney. in addition, renal tissue is sensitive to angiotensin ii (ang ii), leading to renal vasoconstriction, higher resistance of the glomerular efferent artery, and increased sodium and water reabsorption, all resulting in increased blood pressure and glomerular hypertension [ ] . in addition, ang ii can also promote the phenotypic transformation of glomerular endothelial cells and podocytes, the deposition of extracellular matrix, and the secretion of inflammatory and profibrotic chemokines and factors, accelerating the progression of dn [ ] [ ] [ ] [ ] . in recent years, the effect of gut microbiota on diabetes and its complications has aroused great interest. the gut microbiota of humans weighs~ . - . kg, including~ trillion bacteria, the distribution density of which increases gradually from the proximal end to the distal intestine. the composition of gut microbiota in the host is associated with several factors, such as genetic and environmental influence and long-term dietary patterns, and this microbial community usually manifests as a state of equilibrium between different groups in the gut. while the host provides nutrients to the gut microbiota, the latter helps digest complex carbohydrates, producing immune molecules, short-chain fatty acids (scfas) and other metabolites that exert immune and metabolic functions [ , ] . studies have revealed notable differences in the gut microbiota between diabetic and healthy people [ ] . in the intestines of healthy people, there are rich butyrate-producing bacteria, such as escherichia coli, clostridium, etc. in patients with type diabetes (t dm), butyrateproducing bacteria are significantly reduced compared with increasing opportunistic pathogens [ ] . karlsson et al. [ ] have shown that postmenopausal women with t dm in europe have significant insulin resistance, partly because of a significant decrease in the abundance of faecalibacterium prausnitzii and roseburia, which are known as dwellers and butyrate producers of the human gut [ ] and have been linked to improved insulin sensitivity and diabetes [ , ] . larsen et al. [ ] found that the abundance of intestinal firmicutes in adult male patients with t dm decreased significantly, while the abundance of bacteroides and proteobacteria increased, and the ratio of bacteroides/ firmicutes was correlated with patients' glucose tolerance and blood-glucose (bg) levels. compared with healthy controls, the abundance of firmicutes increased in t dm patients, while the abundance of bacteroidetes decreased [ , ] . in animal studies, treatment with a prebiotic (oligofructose) in high-fat-fed diabetic mice not only increased the bifidobacterial content of their guts but also improved their glucose tolerance and insulin resistance as well [ ] . therefore, the gut microbiota might be closely related to the occurrence and development of diabetes. recent studies have found that under the stimulation of injurious factors, the gut microbiota could produce excessive scfas such as acetate, mediate immune disorders and chronic inflammatory reactions of the host, and promote the occurrence of diseases such as diabetes, obesity, and inflammatory bowel disease [ ] [ ] [ ] . the gut microbiota of high-fat-fed rats has been reported to promote insulin secretion and aggravate insulin resistance by synthesizing large amounts of acetate [ ] . studies have shown that scfas are involved in physiological pathways by binding to their receptors, g protein-coupled receptors (gpcrs) and olfactory receptors (olfr). reportedly, functional receptors of scfas include gpr , gpr , gpr , olfr [ , ] and so on. brown et al. [ ] found that the smooth muscle cells of renal arteries express scfa receptors, and among them, gpr and olfr are relatively abundant. intestinal-derived propionic acid can bind to the renal arteriolar olfr and activate intrarenal ras, further increasing the secretion of renin and angiotensin and thus regulating circulating and glomerular pressure. therefore, we speculated that in the development of diabetes, the gut microbiota was likely to produce excessive scfas, especially acetate, which could bind to renal-related signal receptors, thus activating intrarenal ras and mediating the early pathophysiological processes of dn. experimental animals and measurement of general parameters eight-week-old healthy male sprague-dawley rats were kept under the following conditions: a constant -h photoperiod, temperature of - °c, and free access to food and water. after weeks of adaptive feeding, these sd rats were randomly divided into three groups: the control group, diabetic mellitus (dm) group, and diabetic rats treated with antibiotics (dm + ab) (n = ). the latter two groups were intraperitoneally injected with streptozotocin at a dose of . mg/kg (sigma, usa), and bg levels were measured days after the injection to confirm the establishment of diabetic models. all rats of the three groups were normally fed, while the dm + ab group were orally given compound antibiotic solution, consisting of g/l ampicillin, g/l neomycin, . g/l vancomycin, and . g/l amphotericin b. all rats were sacrificed after weeks, with blood, urine, feces, and renal tissues harvested (kidney weights were measured at the time of sacrifice). the concentrations of bg were determined by a bg meter (shanghai johnson & johnson medical devices company). serum creatinine and blood urea nitrogen (bun) were measured by an automatic analyzer (hitachi tokyo, japan). the rats were kept alone in metabolic cages to collect their -h urine samples, of which the level of -h urinary protein was quantitatively analyzed by the lowry assay. the measurement of blood pressure in the three groups was performed at the animal core facility of nanjing medical university. the procedures for the animal experiments were approved by the ethical committee of southeast university and followed the latest version of the declaration of helsinki. after the kidneys were removed, the tissues were decapsulated and fixed in % paraformaldehyde and glutaraldehyde. after h, fixed tissues were embedded in paraffin for observation of the pathological changes under light microscopy (olympus, japan) or embedded in % lanthanum nitrate for ultramicrostructural observation of the podocytes and glomerular basement membrane (gbm) under electron microscopy (jem- , japan). the tissues were cut into -μm-thick slices and then stained with periodic acid-schiff (pas) solution and wheat germ agglutinin (wga) after removing the paraffin. immunofluorescent staining of the tissue slices was performed using primary antibodies against wilms' tumor (wt- ) and nephrin (santa cruz, usa) and examined by laser confocal microscopy (leica, germany). the levels of plasma acetate were measured by gas chromatography. briefly, mmol/l -methylvaleric acid (macklin, china) was used as an internal standard stock solution, while mm acetic acid (sigma-aldrich, usa) was used as the acetate standard stock solution. the diluent and extracting solvent were ethyl acetate. different concentrations of acetate standards were prepared with mmol/l internal standard. a total of μl of plasma was spiked with μl of mm stock solution of internal standard and acidified with μl of hydrochloric acid. after shaking for s, the mixture was sonicated for min and placed at °c until layering. after centrifugation for min at a speed of r/min , the organic phase was filtrated using a . -μm filter. a -μl injection of standards and filtrated samples was used for gas chromatography analysis with a db- column ( m × . mm, agilent, usa) at a speed of ml/min. the split ratio was : . the carrier gas was nitrogen. the initial temperature was °c, which increased to °c ( °c/min). it was then heated to °c ( °c/min), gradually increasing to °c and waiting for min. the standard curve was made according to the concentration and peak area of acetate standards to that of the internal standard. the plasma acetate concentration was then quantified. s ribosomal dna (rdna) sequencing analysis the gene sequencing of gut microbiota was performed using s rdna sequencing technology. fresh fecal specimens were collected using sterile tweezers and tubes and stored at − °c. the total dna of the fecal bacteria was extracted according to the manual of the qiaamp dna stool mini kit (qiagen, hilden, germany). briefly, feces were homogenized and lysed in asl buffer. the mixture was centrifuged at r/min for min. an inhibitex tablet was dissolved in the supernatant, followed by centrifugation. proteinase k and ethanol were added to the supernatant. lysates were then loaded onto the qiaamp spin column with a qiaamp membrane. the column was efficiently washed in two steps with repeated centrifugation. lastly, purified dna was eluted in low-salt buffer. pcr amplification of the variable region - of bacterial s rdna was conducted through double eight cycles to yield detectable products. the amplicons were mixed and purified to construct the gene library after quantification by real-time pcr. sequencing was performed on the illumina miseq × bp platform. operational taxonomic unit picking ( % nucleotide sequence identity) was assigned using the ribosomal database project classifier. chimeric sequences were removed using uchime. the alpha diversity analysis was performed with mothur (version . . ), while the beta diversity analysis was performed with r program (version . . ). the levels of renin, ang І, and ang ii in the plasma of the three groups were measured by a radioimmunoassay kit (beijing north institute of biological technology, china) in accordance with the instructions. the method for determining the level of plasma renin activity (pra) is as previously described [ ] : the optimal dose of trypsin (invitrogen, usa) added was confirmed by constructing a dose-response curve, which was used for subsequent determination. plasma samples were treated with trypsin and kept at °c, ph . , for min for activation, after which trypsin inhibitor was added for min at room temperature to terminate the activation. the levels of renin activation before and after the addition of trypsin were measured, and the pra level of each sample was the total renin level by trypsin activation minus the renin level before trypsin addition. western blot analysis proteins were extracted from kidney tissues, and the protein concentration of each sample was measured. by adding lysate, loading buffer, and ddh o, the protein concentration and volume of each sample were equal, and all samples were boiled at °c for min for storage. gel electrophoresis was performed in a sodium dodecyl sulfate-polyacrylamide system, and gel transfer was performed using polyvinylidene fluoride membranes. the membranes were then immersed in blocking buffer for h at room temperature and incubated overnight at °c in primary antibodies against angiotensin-converting enzyme (ace), angiotensinogen (agt), ang ii, and ang ii type receptor (at ) (santa cruz, usa). after washing with . % tris buffered saline and tween, the membranes were incubated in horseradish peroxidase-conjugated secondary antibodies for h at room temperature. protein expression was observed using the ecl system (bio-rad, usa). statistical analysis all data were processed using spss . (ibm, usa). measurement data were expressed as the mean ± standard deviation (sd), t-test was used for statistical comparisons among the data of three groups, and the spearman correlation test was used for correlation analysis. p values < . were considered statistically significant. general parameters of the three groups compared with the controls, the bg level of the dm group was significantly elevated, confirming that the stz-induced diabetic model was established. compared with the dm group, treatment with antibiotics caused an obviously reduced bg level in the dm + ab group (fig. a) , suggesting that gut microbiota might be closely related to the body's high bg levels under diabetic conditions. the plasma insulin level in the three groups showed an opposite trend compared with that of bg (fig. b) . changes in gut microbiota in the three groups the results of s rdna gene sequencing showed that there were significant differences in the bacterial composition and abundance of gut microbiota between the control and dm groups. after the application of broad-spectrum antibiotics, most of the gut microbiota in the dm + ab group was killed (fig. a) . the subgroup sequencing analysis results of gut microbiota in each group have shown that the abundance of blautia, roseburia, and paraprevotella in the colon of the dm group was significantly increased compared with that of the controls (fig. b-d) , while the abundance of bacteroides was relatively decreased, suggesting that the composition and abundance of gut microbiota have both changed under diabetic status. the results of the dm + ab group further confirmed the bactericidal effect of the treatment with broad-spectrum antibiotics (fig. e) . treatment with antibiotics significantly reduced the level of plasma acetate the results of gas chromatography analysis demonstrated significantly increasing plasma levels of acetate in the dm group, which might be due to the abnormalities of gut microbiota under diabetic conditions. antibiotic intervention significantly lowered the plasma acetate level in the dm + ab group (fig. ) . the effect of gut microbiota on renal injury of incipient dn compared with the controls, the ratio of kidney weight-to-body weight in both the dm group and the dm + ab group was significantly higher (fig. a) , indicative of renal hypertrophy under the state of diabetes. in terms of proteinuria, we observed a significantly increasing level of -h urine protein in the dm group compared with that of the control group. after the antibiotic intervention, the dm + ab group had a significant reduction in -h urine protein compared with that of the dm group (p < . ) (fig. b) . in addition, compared with the control group, the level of bun increased in both the dm group and the dm + ab group (p < . ), but there was no significant difference in blood creatinine among the three groups (fig. c, d) , suggesting that this abnormality of gut microbiota has not yet developed to a sufficient degree to exert obvious effects on renal functions. we next observed renal pathological changes to further evaluate the degree of renal injury in the three groups. the results of pas staining showed mild mesangial expansion among the kidneys in the dm group compared with that in the dm + ab group (fig. a, b) . under electron microscopy, ultramicrostructural changes in the glomerular filtration membrane in each group could be observed. the gbm in the dm group was thickened, and there was fusion of fenestrated endothelium, along with partly merged and missing podocyte foot processes, which corresponds to the degree of renal injury in early dn. after the antibiotic intervention, the basement membrane thickening, endothelial fusion, and podocyte injury in the dm + ab group recovered to some extent (fig. c) . we performed wga immunofluorescence staining to observe the changes in glomerular endothelium glycocalyx. under laser confocal microscopy, we found that compared with the control group, the thickness of the glomerular endothelium was significantly reduced in the dm group. in the dm + ab group, the reduced glycocalyx was repaired after the antibiotic intervention (fig. d) . the immunofluorescence staining results also showed significantly decreased expression of glomerular podocyte-specific protein wt- and nephrin in the dm group compared with that in the control group, which was relatively recovered in the dm + ab group (fig. e-g) , suggesting that unbalanced gut microbiota might be a key factor resulting in injuries to the glomerular filtration membrane in early dn. intrarenal ras is activated in early dn the measurement of circulating ras in the three groups showed no significant differences in pra and the level of ang І (fig. a, b) . however, compared with the dm group, the concentration of ang ii in the circulation was significantly reduced (fig. c) . the western blot results to evaluate the degree of intrarenal ras activation showed that compared with the control group, the protein expression of ace, ang ii, and at r in the kidney of the dm group was significantly increased, and antibiotic treatment showed a suppressing effect on these three ras-activating indicators (fig. d, e) . the expression of agt showed an opposite trend, which might be a result of negative-feedback adjustment considering its role as a rate-limiting enzyme. this result suggests that the dysbiosis of the gut microbiota may be involved in the intrarenal ras activation of early dn. correlation analysis between plasma acetate levels and intrarenal ang ii expression to investigate the causal relationship between the dysbiosis of gut microbiota and intrarenal ras activation, we further analyzed the correlation between the plasma acetate concentration and intrarenal ang ii protein expression as determined by western blot (fig. f) . a positive correlation was observed (r = . , p < . ). most kidney diseases are characterized by an initial injury, followed by compensatory growth and associated functional alterations, usually manifested as renal hypertrophy [ ] . stages І and ii of dn are mainly characterized by glomerular hypertension, hyperfiltration, and renal hypertrophy [ ] . renal hypertrophy is an important and significant marker of structural and functional changes in the kidney in early dn, mainly characterized by glomerular cell hypertrophy, thickening of the basement membrane, and an increase in the mesangial matrix. in this study, we confirmed that the experimental animals had developed corresponding renal injury under diabetic conditions by pathological observation. the ras can be divided into the circulatory and local ras depending on its location of synthesis and action. in circulating plasma acetate concentration (µ µmol/l) ras, ang ii is produced under the serial effects of renin secreted by the kidney, angiotensin from the liver, and ace located in vascular endothelial cells. in certain organs or cells, the synthesis of ras components is independent. ang ii can be produced in the intercellular space [ ] under the effect of enzymes other than renin and ace (such as chymase) and exerts many pathophysiological effects by activating at r [ ] . the activation of ras has always been considered an important factor in the development of dn, and local ras seems to be more involved compared with the circulating ras [ ] . the kidney itself contains all the ras components [ ] . high glucose has been reported to promote the production of ang ii, which in turn leads to glomerular hyperfiltration and high permeability, as well as extracellular matrix deposition [ ] . clinical application of ras inhibitors could significantly retard the development of proteinuria in dn patients and lower the incidence of esrd. as a current first-line treatment for dn, the efficacy of ras inhibitors in preventing dn is still very limited. the rising prevalence of dn has suggested that a deeper understanding of the molecular mechanisms underlying dns is required to seek better treatment. the relationship between gut microbiota and many metabolic diseases has been a hotpot for research in recent years. under normal circumstances, firmicutes and bacteroidetes take up a large proportion of the whole microbiota in the gut, while the composition of other phyla varies individually due to several factors, such as genetics, diet, and antibiotic use. however, under pathological conditions, the species and abundance of the host gut microbiota would change significantly, mainly characterized by decreasing normally dominant bacteria and increasing pathogenic bacteria. it has also been shown in this study that compared with the control group, there was microecological dysbiosis of gut microbiota in the intestinal tract of diabetic rats. the application of antibiotic intervention is a common method used in studies of gut microbiota and its role in the development of diseases. in this study, we observed that the intervention of antibiotics affected many aspects of the dn animal model, from basic functions and pathological changes in the kidneys to the amount of products released from gut microbiota. the use of mixed antibiotics has ensured the killing effect of the bacteria in the intestinal tract of experimental animals, and in the meantime, it could also attenuate the renal injury of early dn, including reducing the amount of -h urine protein and mitigating the injury to the glomerular filtration membrane and the pathological changes in the tubules. scfas are a major product of the fermentation of carbohydrates by gut microbiota, and there are three main types: acetate, d the changes in glomerular endothelium glycocalyx were assessed by wga staining (original magnification, × ). e wt- and nephrin protein expression was evaluated by immunofluorescent staining (original magnification, × ). the arrows indicate glomerular endothelium glycocalyx. f quantitation of immunofluorescence staining for wt- . *p < . ; compared with the control; ## p < . compared with dm. g quantitation of immunofluorescence staining for nephrin. ***p < . ; compared with the control; # p < . compared with dm. propionate, and butyrate. the butyrate-producing microbiota is essential in maintaining the balance of the intestinal environment in humans. however, the number of butyrate-producing microbiota in diabetic patients is significantly reduced, while the number of other opportunistic pathogens shows an increasing trend. there is evidence that a butyrate-producing clostridium genus could exert antidiabetic effects by increasing the production of butyrate and upregulating the expression of scfa receptors in the gut [ ] . as a main scfa product of gut microbiota, acetate has demonstrated an intricate effect on the internal environment. it has been reported that acetate is almost undetectable in the blood of germ-free mice [ ] , while the content of acetate in animals with a high-fat diet is significantly increased, indicating that the level of acetate might be a potential indicator of the activity of gut microbiota. this study has shown that antibiotic intervention significantly reduced the relatively higher plasma level of acetate in diabetic rats, considering the alleviated renal lesions after antibiotic intervention. it can be speculated that overproduction of acetate might be adverse to the development of early dn. however, the causal relationship between gut microbiota dysbiosis and the development of dn remains to be elucidated, and more explorations may provide a new perspective and therapeutic target for the future diagnosis and treatment of dn. ras activation has been considered one of the important initiating factors in the early development of dn, yet the exact associations between gut microbiota and ras activation remain to be elucidated. pluznick et al. [ ] found that signals from the gut microbiota, i.e., scfas, could be received by corresponding receptors expressed at renal small arterioles, further regulating the secretion of renin, which was involved in maintaining glomerular pressure. this process could be blocked by antibiotics or knockout of the scfa receptor. considering the characteristics of glomerular hypertension and hyperfiltration in the early stage of dn, the disordered gut microbiota is likely to generate excessive scfas, which bind to corresponding receptors in the kidney and regulate ras, thus promoting the pathological changes in early dn. in this study, we have shown that in the early stage of dn, the expression of ras in the kidney was significantly elevated, indicating ras activation at this stage. after antibiotic intervention, the level of circulating ang ii was lowered, and the expression of ras within the kidney was also significantly weakened, suggesting that there might be a causal relationship between the dysbiosis of gut microbiota and intrarenal ras activation in early dn. therapeutic intervention could be applied to change the composition of gut microbiota, focusing on the preservation of beneficial phyla, to create renoprotective prospects [ ] . intrarenal ras is activated in early dn. plasma renin activity level (a), ang i level (b), and ang ii level (c). the protein expression levels of ras were measured by western blotting (d). the histograms represent the mean ± sd of the densitometric scans of the protein bands normalized to β-actin (e). correlation analysis of plasma acetate levels and intrarenal ang ii expression levels (f) (r = . , p < . ). *p < . compared with the control group, # p < . compared with the dm group. in summary, we have established a dn model to observe the changes in gut microbiota and its metabolite acetate in early dn and to further investigate the association between these changes and ras activation in the kidney to elucidate the underlying mechanism of early renal injury in dn. further exploration of the causal relationship and intricate mechanism of gut microbiota and ras activation in the early development of dn are required to develop new prevention strategies for clinical early dn. idf diabetes atlas: global estimates of diabetes prevalence for and projections for prevalence and control of diabetes in chinese adults upregulation of renal renin-angiotensin system in mouse diabetic nephropathy diabetic nephropathy: the role of inflammation in fibroblast activation and kidney fibrosis immunity and inflammation in diabetic kidney disease: translating mechanisms to biomarkers and treatment targets oxidative stress in diabetic nephropathy with early chronic kidney disease role of the intrarenal renin-angiotensin system in the progression of renal disease signals from the gut microbiota to distant organs in physiology and disease unraveling the environmental and genetic interactions in atherosclerosis: central role of the gut microbiota microbiota and diabetes: an evolving relationship a metagenome-wide association study of gut microbiota in type diabetes gut metagenome in european women with normal, impaired and diabetic glucose control diversity of human colonic butyrateproducing bacteria revealed by analysis of the butyryl-coa:acetate coatransferase gene differential adaptation of human gut microbiota to bariatric surgery-induced weight loss: links with metabolic and low-grade inflammation markers transfer of intestinal microbiota from lean donors increases insulin sensitivity in individuals with metabolic syndrome gut microbiota in human adults with type diabetes differs from nondiabetic adults gut microbiota in children with type diabetes differs from that in healthy children: a case-control study toward defining the autoimmune microbiome for type diabetes selective increases of bifidobacteria in gut microflora improve high-fat-diet-induced diabetes in mice through a mechanism associated with endotoxaemia the neuropharmacology of butyrate: the bread and butter of the microbiota-gutbrain axis? regulation of inflammation by short chain fatty acids short-chain fatty acids activate gpr and gpr on intestinal epithelial cells to promote inflammatory responses in mice acetate mediates a microbiome-brain-beta-cell axis to promote metabolic syndrome olfactory receptor responding to gut microbiota-derived signals plays a role in renin secretion and blood pressure regulation immunocytochemical localization of na+ channels in rat kidney medulla plasma and renal renin concentrations in adult sheep after prenatal betamethasone exposure progression of renal disease and renal hypertrophy the stages in diabetic renal disease. with emphasis on the stage of incipient diabetic nephropathy the intracrine renin-angiotensin system pleiotropic at receptor signaling pathways mediating physiological and pathogenic actions of angiotensin ii angiotensinconverting enzyme amplification limited to the circulation does not protect mice from development of diabetic nephropathy paracrine regulation of the renal microcirculation the intrarenal renin-angiotensin system and diabetic nephropathy anti-diabetic effects of clostridium butyricum cgmcc . through promoting the growth of gut butyrate-producing bacteria in type diabetic mice gut microbiota in cardiovascular health and disease key: cord- -spiuqngp authors: huang, yuan; yang, chan; xu, xin-feng; xu, wei; liu, shu-wen title: structural and functional properties of sars-cov- spike protein: potential antivirus drug development for covid- date: - - journal: acta pharmacol sin doi: . /s - - - sha: doc_id: cord_uid: spiuqngp coronavirus disease is a newly emerging infectious disease currently spreading across the world. it is caused by a novel coronavirus, severe acute respiratory syndrome coronavirus (sars-cov- ). the spike (s) protein of sars-cov- , which plays a key role in the receptor recognition and cell membrane fusion process, is composed of two subunits, s and s . the s subunit contains a receptor-binding domain that recognizes and binds to the host receptor angiotensin-converting enzyme , while the s subunit mediates viral cell membrane fusion by forming a six-helical bundle via the two-heptad repeat domain. in this review, we highlight recent research advance in the structure, function and development of antivirus drugs targeting the s protein. the epidemic of novel coronavirus disease (covid- ) was caused by a new coronavirus occurred in december , and now has spread worldwide and turned into a global pandemic [ ] . the covid- was quickly discovered to be caused by a coronavirus later named severe acute respiratory syndrome coronavirus (sars-cov- ) [ ] , which belongs to the β coronavirus family. it is the seventh known coronavirus to infect humans; four of these coronaviruses ( e, nl , oc , and hku ) only cause slight symptoms of the common cold. conversely, the other three, sars-cov, mers-cov, and sars-cov- , are able to cause severe symptoms and even death, with fatality rates of %, %, and %, respectively. although a large number of studies and clinical trials are being launched on covid- around the world [ , ] , no evidence from randomized clinical trials has shown that any potential therapy improves outcomes in patients [ ] . as the epidemic spreads, it is critical to find a specific therapeutic for covid- , and vaccines targeting various sars-cov- proteins are under development. sars-cov- is a single-stranded rna-enveloped virus [ ] . an rna-based metagenomic next-generation sequencing approach has been applied to characterize its entire genome, which is , bp in length (genbank no. mn ), encoding amino acids [ ] . gene fragments express structural and nonstructural proteins. the s, e, m, and n genes encode structural proteins, whereas nonstructural proteins, such as -chymotrypsinlike protease, papain-like protease, and rna-dependent rna polymerase, are encoded by the orf region [ ] . a large number of glycosylated s proteins cover the surface of sars-cov- and bind to the host cell receptor angiotensinconverting enzyme (ace ), mediating viral cell entry [ ] . when the s protein binds to the receptor, tm protease serine (tmprss ), a type tm serine protease located on the host cell membrane, promotes virus entry into the cell by activating the s protein. once the virus enters the cell, the viral rna is released, polyproteins are translated from the rna genome, and replication and transcription of the viral rna genome occur via protein cleavage and assembly of the replicase-transcriptase complex. viral rna is replicated, and structural proteins are synthesized, assembled, and packaged in the host cell, after which viral particles are released (fig. d) [ ] . these proteins are critical to the viral life cycle and provide potential targets for drug therapies. for example, ace -based peptide, clpro inhibitor ( clpro- ), and a novel vinylsulfone protease inhibitor have been experimentally demonstrated to be effective against sars-cov- [ ] . the sars-cov- s protein is highly conserved among all human coronaviruses (hcovs) and is involved in receptor recognition, viral attachment, and entry into host cells. due to its indispensable functions, it represents one of the most important targets for covid- vaccine and therapeutic research. in this review, we summarize advances in research of the sars-cov- s protein and its therapeutic targeting. with a size of - kda, the s protein consists of an extracellular n-terminus, a transmembrane (tm) domain anchored in the viral membrane, and a short intracellular c-terminal segment [ ] . s normally exists in a metastable, prefusion conformation; once the virus interacts with the host cell, extensive structural rearrangement of the s protein occurs, allowing the virus to fuse with the host cell membrane. the spikes are coated with polysaccharide molecules to camouflage them, evading surveillance of the host immune system during entry [ ] . the total length of sars-cov- s is aa and consists of a signal peptide (amino acids - ) located at the n-terminus, the s subunit ( - residues), and the s subunit ( - residues); the last two regions are responsible for receptor binding and membrane fusion, respectively. in the s subunit, there is an n-terminal domain ( - residues) and a receptor-binding domain (rbd, - residues); the fusion peptide (fp) ( - residues), heptapeptide repeat sequence (hr ) ( - residues), hr ( - residues), tm domain ( - residues), and cytoplasm domain ( - residues) comprise the s subunit ( fig. a) [ ] . s protein trimers visually form a characteristic bulbous, crown-like halo surrounding the viral particle (fig. a) . based on the structure of coronavirus s protein monomers, the s and s subunits form the bulbous head and stalk region [ ] . the structure of the sars-cov- trimeric s protein has been determined by cryo-electron microscopy at the atomic level, revealing different conformations of the s rbd domain in opened and closed states and its corresponding functions (fig. b , c) [ , ] . in the native state, the cov s protein exists as an inactive precursor. during viral infection, target cell proteases activate the s protein by cleaving it into s and s subunits [ ] , which is necessary for activating the membrane fusion domain after viral entry into target cells [ ] . similar to other coronaviruses, the s protein of sars-cov- is cleaved into s and s subunits by cellular proteases, and the serine protease tmprss is used as a protein primer. although the cleavage site of sars-cov is known, that of sars-cov- s has not yet been reported [ , ] . structure of the s subunit the binding of virus particles to cell receptors on the surface of the host cell is the initiation of virus infection; therefore, receptor recognition is an important determinant of viral entry and a drug design target. rbd situated in the s subunit binds to the cell receptor ace in the region of aminopeptidase n. the s region contains the ntd and ctd, and atomic details at the binding interface demonstrate key residue substitutions in sars-cov- -ctd. in addition, the sars-cov- s ctd binding interface has more residues that directly interact with the receptor ace than does sars-rbd ( versus ), and a larger surface area is buried with sars-cov- s ctd in complex with ace than with sars s rbd. mutations of key residues play an important role in enhancing the interaction with ace . f in sars-cov- , instead of i in sars rbd, forms strong aromatic-aromatic interactions with ace y , and e in sars-cov- -ctd, instead of p in sars rbd, forms ionic interactions with k , which leads to higher affinity for receptor binding than rbd of sars-cov ( fig. d) [ , , , ] . the rbd region is a critical target for neutralizing antibodies (nabs), and sars-cov- and sars-cov rbd are~ %- % similar in sequence. nine ace -contacting residues in cov rbd are fully conserved, and four are partially conserved. analysis of the rbm (receptor-binding motif, a portion of rbd making direct contacts with ace ) of sars-cov and sars-cov- revealed that most residues essential for ace binding in the sars-cov s protein are conserved in the sars-cov- s protein. however, some studies showed that murine monoclonal antibodies (mabs) and polyclonal antibodies against sars-rbd are unable to interact with the sars-cov- s protein, revealing differences in antigenicity between sars-cov and sars-cov- [ ] . similarly, a sars-cov rbd-specific antibody failed to block infection mediated by the s protein of sl-cov-shc [ ] , which suggests that the s rbd may not be an ideal drug target due to the highly mutable characteristic of broad-spectrum anti-cov drugs. structure of the s subunit the s subunit, composed successively of a fp, hr , hr , tm domain, and cytoplasmic domain fusion (ct), is responsible for viral fusion and entry. fp is a short segment of - conserved amino acids of the viral family, composed mainly of hydrophobic residues, such as glycine (g) or alanine (a), which anchor to the target membrane when the s protein adopts the prehairpin conformation. previous research has shown that fp plays an essential role in mediating b-c the s protein rbd closed and opened status. d the s protein binds to ace with opened rbd in the s subunit. e the six-helix structure formed by hr and hr of the s subunit. membrane fusion by disrupting and connecting lipid bilayers of the host cell membrane [ ] . hr and hr are composed of a repetitive heptapeptide: hpphcpc, where h is a hydrophobic or traditionally bulky residue, p is a polar or hydrophilic residue, and c is another charged residue [ ] . hr and hr form the six-helical bundle ( -hb) (fig. e) , which is essential for the viral fusion and entry function of the s subunit [ ] . hr is located at the c-terminus of a hydrophobic fp, and hr is located at the n-terminus of the tm domain [ ] . the downstream tm domain anchors the s protein to the viral membrane, and the s subunit ends in a ct tail [ ] . rbd binds to ace , and s changes conformation by inserting fp into the target cell membrane, exposing the prehairpin coiledcoil of the hr domain and triggering interaction between the hr domain and hr trimer to form -hb, thus bringing the viral envelope and cell membrane into proximity for viral fusion and entry [ ] . hr forms a homotrimeric assembly in which three highly conserved hydrophobic grooves on the surface that bind to hr are exposed. the hr domain forms both a rigid helix and a flexible loop to interact with the hr domain. in the postfusion hairpin conformation of covs, there are many strong interactions between the hr and hr domains inside the helical region, which is designated the "fusion core region" (hr core and hr core regions, respectively). targeting the heptad repeat (hr) has attracted the greatest interest in therapeutic drug discovery. the s protein is an important target protein for the development of specific drugs, while the s rbd domain is part of a highly mutable region and is not an ideal target site for broad-spectrum antiviral inhibitor development [ ] . in contrast, the hr region of the s subunit plays an essential role in hcov infections and is conserved among hcovs, as is the mode of interaction between hr and hr [ ] . a synthetic peptide derived from the stem region of the zikv envelope protein was demonstrated in to potently inhibit infection by zikv and other flaviviruses in vitro [ ] , implying antiviral efficiency of peptides derived from conserved regions of viral proteins. peptides derived from the hr region of class i viral fusion proteins of enveloped viruses competitively bind to viral hr and effectively inhibit viral infection [ ] . therefore, hr is a promising target for the development of fusion inhibitors against sars-cov- infection. the s protein on the surface of the virus is a key factor involved in infection. it is a trimeric class i tm glycoprotein responsible for viral entry, and it is present in all kinds of hcovs, as well as in other viruses such as hiv (hiv glycoprotein , env), influenza virus (influenza hemagglutinin, ha), paramyxovirus (paramyxovirus f), and ebola (ebola virus glycoprotein) [ ] . similar to other coronaviruses, the s protein of sars-cov- mediates receptor recognition, cell attachment, and fusion during viral infection [ , , , [ ] [ ] [ ] . the trimer of the s protein located on the surface of the viral envelope is the basic unit by which the s protein binds to the receptor [ , ] . the s domain contains the rbd, which is mainly responsible for binding of the virus to the receptor, while the s domain mainly contains the hr domain, including hr and hr , which is closely related to virus fusion [ ] . receptor binding as mentioned above, the sars-cov- s protein binds to the host cell by recognizing the receptor ace [ ] . ace is a homolog of ace, which converts angiotensin i to angiotensin - [ ] . ace is distributed mainly in the lung, intestine, heart, and kidney, and alveolar epithelial type ii cells are the major expressing cells [ ] . ace is also a known receptor for sars-cov. the s subunit of the sars-cov s protein binds with ace to promote the formation of endosomes, which triggers viral fusion activity under low ph (fig. a, b) [ ] . interaction between the s protein and ace can be used to identify intermediate hosts of sars-cov- , as ace from different species, such as amphibians, birds, and mammals, has a conserved primary structure [ ] . luan et al. compared the binding affinities between ace and sars-cov- s from mammals, birds, snakes, and turtles and found that the ace of bovidae and cricetidae interacted well with sars-cov- s rbd but that ace from snakes and turtles could not. the s protein binds to ace through the rbd region of the s subunit, mediating viral attachment to host cells in the form of a trimer [ ] . sars-cov- s binds to human ace with a dissociation constant (k d ) of . nm, though that of sars-cov s is . nm [ ] , indicating that sars-cov- s is more sensitive to ace than is sars-cov s. through the identification of sars-cov- proteins, researchers found~ % difference in s between sars-cov- and sars-cov, whereas that of rbd is~ % [ ] . viral fusion viral fusion refers to fusion of the viral membrane and host cell membrane, resulting in the release of the viral genome into the host cell. cleavage of the sars-cov- s and s subunits is the basis of fusion. the s protein is cleaved into two parts, the s subunit and s subunit, by host proteases, and the subunits exist in a noncovalent form until viral fusion occurs [ ] . researchers have found that the specific furin cleavage site is located in the cleavage site of sars-cov- but not in other sarslike covs [ , ] . mutation of the cleavage site in sars-cov- or sars-like covs has revealed that the s protein of sars-cov- exists in an uncleaved state but that the others are mainly in a cleaved state. sars-cov- s has multiple furin cleavage sites, which increases the probability of being cleaved by furin-like proteases and thereby enhances its infectivity [ , ] . the furin-like cleavage domain is also present in highly pathogenic influenza virus and is related to its pathogenicity, as observed in the avian influenza outbreak in hong kong in [ , ] . in addition, host cell proteases such as tmprss are essential for s protein priming, and they have been shown to be activated in the entry of sars-cov and influenza a virus [ , , ] . another host cell protease that has been proven to cleave viral s protein is trypsin [ ] . in summary, the s protein of sars-cov- is similar to that of sars-cov, and host cell proteases are essential for promoting s protein cleavage of both sars-cov- and sars-cov. the presence of a specific furin cleavage site on sars-cov- s might be one reason that sars-cov- is more contagious than sars-cov. the formation of -hb is essential for viral fusion. the fp in the n-terminus of sars-cov- and the two hr domains on s is essential for viral fusion [ ] . after cleavage of the s protein, the fp of sars-cov- is exposed and triggers viral fusion. under the action of some special ligands, the fusion protein undergoes a conformational change and then inserts into the host cell membrane (fig. c) [ ] . for example, the ligand for influenza a virus is h + , while the ligand for hiv is a coreceptor such as ccr or cxcr [ ] . the distance between the viral membrane and host cell membrane is shortened, and the hr domain of the s protein is in close proximity to the host cell membrane, whereas the hr domain is closer to the viral membrane side. then, hr folds back to hr , the two hr domains form a six-helix structure in an antiparallel format of the fusion core, the viral membrane is pulled toward the host cell membrane and tightly binds to it, and the two membranes fuse [ ] . the fundamental role of the s protein in viral infection indicates that it is a potential target for vaccine development, drug development targeting sars-cov- s y huang et al. antibody-blocking therapy, and small molecule inhibitors. considering the similarity with sars-cov and mers-cov, potential nabs and inhibitors targeting sars-cov- s are summarized below (fig. ) . antibodies based on the sars-cov- s protein the s protein is the main antigen component in all structural proteins of sars-cov- . unlike other functional proteins of sas-cov- , it is responsible for inducing the host immune response, and nabs targeting the s protein can induce protective immunity against viral infection. similar to sars-cov and mers-cov, research on nabs of sars-cov- mainly includes mabs, antigenbinding fragments, single-chain variable region fragments, and single-domain antibodies (nbs), which target s rbd, s -ntd, or s regions to prevent s -mediated fusion [ , ] . on the other hand, multiple sars-cov- vaccine types are under development, including rna/dna-based formulations, recombinant viral epitopes, adenovirus-based vectors, and purified inactivated virus [ ] . the sequence and striking structural similarity between the sars-cov- and sars-cov s proteins emphasize the close relationship between these two viruses, which provides the possibility to treat covid- with antibodies targeting the sars-cov s protein [ ] . compared with sars-cov- rbd, sars-cov- interacts with hace via the c-terminal domain (sars-cov- -ctd), showing higher affinity for receptor binding. rbd can induce highly potent nab responses and has the potential to be developed as an effective and safe subunit vaccine against sars-cov- . sars-cov s polyclonal antibodies obtained from immunized mice completely inhibited the invasion of sars-cov s-mlv (murine leukemia virus), whereas the invasion rate of sars-cov- s-mlv was reduced to~ % [ ] . the polyclonal anti-sars s antibody t inhibits the entry of sars-cov s but not that of sars-cov- s pseudovirus particles [ ] . consistently, recent studies have reported similar results, showing that three sars rbd-directed mabs, s , m , and r, were unable to bind to sars-cov- rbd [ , , ] . on the other hand, several mabs have shown promising results in neutralizing sars-cov- . cr , a sars-cov-specific human mab, binds potently with sars-cov- (k d of . nm, measured by bli in octetred ), suggesting that cr has the potential to be developed as candidate therapeutic, alone or in combination with other nabs, for the prevention and treatment of sars-cov- infection [ ] . a mab targeting s prepared from immunized transgenic mice expressing human ig variable heavy and light chains has recently been shown to neutralize both sars-cov- and sars-cov infections via an unknown mechanism that is independent of the blockade of rbd-hace interaction [ ] . recently, many human blocking mabs ( mab- b , mab- d , d , n , n , s , p c- f , p b- f , b , h ) have been successfully cloned from single memory b cells from recovered covid- patients [ ] [ ] [ ] [ ] [ ] [ ] . these mabs specifically bind to sars-cov- s to effectively neutralize infection. in addition, sera from sars patients during rehabilitation or animals specifically immunized with sars-cov s may cross-neutralize sars-cov- and reduce s protein-mediated sars-cov- entry (fig. ) [ ] . the stability of the sars-cov- s protein is lower than that of sars-cov s [ ] . the mapping of multiple s sequences of the subgenus sarbecovirus underscores that the s fusion region is more conserved than the s subunit and that the s subunit is more exposed at the viral surface [ ] . the sars-cov s subunit plays a key role in mediating virus-cell fusion and its integration into host cells, where hr and hr interact to form -hb, thus enabling the virus to bind to and fuse with the cell membrane [ ] . sequence alignment shows that sars-cov- hr has the same sequence as sars-cov hr . therefore, sars-cov- hr p ( - residues) was designed to inhibit sars-cov- fusion and entry into a target cell. surprisingly, hr p showed inhibitory activity against sars-cov- s-mediated fusion and sars-cov- pseudovirus, with ic values of . and . μm, respectively [ ] . notably, ek is a pancoronavirus fusion inhibitor targeting the hr domain of hcov s [ ] . the x-ray crystal structure of the -hb core of the sars-cov- s subunit hr and hr domains has been solved, indicating that several mutant residues in the hr region may be related to enhanced interaction in the hr region [ ] . subsequently, ek c , a lipopeptide derived from ek , was generated and verified to inhibit sars-cov- s-mediated cell-cell fusion. as expected, the entry of sars-cov- s pseudovirus was also inhibited by ek c , with an ic of . nm,~ -fold more potent than the original ek peptide. another sequence-based lipopeptide fusion inhibitor, ipb , potently inhibits sars-cov- s protein-mediated cell-cell fusion and pseudovirus infection [ ] . in addition to peptide fusion inhibitors, nelfinavir mesylate (viracept), a currently prescribed anti-hiv protease inhibitor, suppresses both sars-cov- s and sars-cov s-mediated cell-cell fusion. viracept is the first reported small molecule fusion inhibitor in addition to peptide fusion inhibitors. moreover, nelfinavir may inhibit the function of tmprss involved in activation of the s protein [ ] . this discovery makes possible clinical applications of anti-sars-cov- therapeutics, especially in the early stage of infection. protease inhibitors targeting sars-cov- s cleavage sites sars-cov- entry requires cleavage of the s protein at the s /s and s sites. proteolysis by tmprss and cathepsin b and l plays an important role in priming sars-cov- s for entry. camostat mesilate is a potent serine protease inhibitor of tmprss . utilizing research on the sars-cov and sars-cov- cell entry mechanism, it has been demonstrated that sars-cov- cellular entry can be blocked by camostat mesilate [ , ] . there are currently five clinical trials registered to evaluate the efficacy of camostat mesilate (clinicaltrials.gov identifier: nct , nct , nct , nct , nct ). in addition, cathepsins in lysosomes are crucial for sars-cov entry via endocytosis. e- d, an inhibitor of cathepsin l, blocks infection with sars-cov and sars-cov- psv [ ] [ ] [ ] . future trials with covid- patients may help to confirm the efficacy of e- d therapy. phosphatidylinositol -phosphate -kinase (pikfyve) is the main enzyme synthesizing pi ( , ) p in early endosomes [ ] . apilimod, a potent inhibitor of pikfyve , can significantly reduce the entry of sars-cov s pseudovirus into /hace cells via early endosomes in a dose-dependent manner [ ] . treating / hace cells with another pikfyve inhibitor, ym [ ] , also had a similar effect. moreover, a major downstream effector of pi ( , )p , two-pore channel subtype (tpc ) [ ] , is important for sars-cov- entry, and tetrandrine (an inhibitor of tpc ) inhibits the activity of sars-cov- s pseudovirus. furin (proprotein convertase (pc) subtilisin kexin , pcsk ), as a member of the pc family, catalyzes the hydrolysis of peptide and protein substrates at paired basic residues [ ] . strikingly, sars-cov- s harbors a furin cleavage site ( - residues) at the s / s boundary, which may increase the efficiency of sars-cov- transmission [ ] . the furin-like cleavage site in the s protein of sars-cov- may have implications for the viral life cycle and pathogenicity. therefore, furin inhibitors can be used as a drug therapy for sars-cov- [ ] . patent literature since describes the use of furin or its inhibitors in the treatment of diseases, and some furin inhibitors that have been reported, including α- -pdx (α -antitrypsin portland) [ ] , hexa-d-arginine(d r) [ ] , serpin proteinase inhibitor (pi ) [ ] , and a peptidomimetic furin inhibitor [ ] . the sars-cov- s protein binds to the host cell receptor and induces virus-cell membrane fusion, which plays a vital role in the process of virus invasion. moreover, the high affinity between the s protein and ace increases the infectivity of sars-cov- . mammals including pangolins, pets (dogs and cats), and members of cricetidae may be important for determining key residues for association with s from sars-cov and sars-cov- [ ] . further drug development targeting sars-cov- s y huang et al. understanding of the structure and function of sars-cov- s will allow for additional information regarding invasion and pathogenesis of the virus, which will support the discovery of antiviral therapeutics and precision vaccine design. structural information will also assist in evaluating mutations of the sars-cov- s protein and will help in determining whether these residues have surface exposure and map to known antibody epitopes of s proteins from other coronaviruses. in addition, structural knowledge ensures that the proteins produced by constructs are homogeneous and participate in the prefusion conformation, which should maintain the most neutralizationsensitive epitopes when used as a candidate vaccine or b-cell probe for isolating neutralizing human mabs. furthermore, atomic-level details will enable the design and screening of small molecules that inhibit fusion. since sars-cov- and sars-cov rbd domains share % amino acid sequence identity, future work will be necessary to evaluate whether any of these abs neutralize newly emerged coronavirus. overall, interaction between the s protein of sars-cov- and ace should be further studied to contribute elucidation of the mechanism of sars-cov- infection. similarly, focusing on high expression of the s protein or its receptor binding region is also of great significance for the development of vaccines. the s subunit of sars-cov- shows % sequence homology with the sars-cov s domain and is structurally conserved. therefore, the development of antibodies targeting this functional motif may cross-bind and neutralize these two viruses and related covs. antiviral peptides prevent sars-cov- membrane fusion and can potentially be used for the prevention and treatment of infection. it is worth mentioning that ek c , which targets the highly conserved hr domain of the s subunit, is expected to have therapeutic potential against sars-cov- . more importantly, ek c can be used as a nasal drop, which increases its medicinal properties, it possesses a high genetic barrier to resistance, and does not easily induce drug-resistant mutations. on the other hand, peptide fusion inhibitors may not be widely used clinically and have low bioavailability. therefore, the development of oral small molecule fusion inhibitors is a major direction. in the course of virus epidemics, the ability to adapt to external pressure is an important factor affecting the spread of the virus. regarding the envelope s protein, recombination or mutation in the gene of its rbd can occur to promote transmission between different hosts and lead to a higher fatality rate [ ] . mutation of the aspartate (d) at position to glycine (g ) results in a more pathogenic strain of sars-cov- [ ] , which makes it more difficult to develop antibodies or vaccines that target nonconservative regions. to effectively prevent disease, combinations of different mabs that identify different epitopes on the sars-cov- s surface can be assessed to neutralize a wide range of isolates, including escape mutants [ ] . currently, no specific therapeutic or prophylactic has been used clinically to treat or prevent sars-cov- infection. nonspecific antiviral drugs, such as ifn-α (recombinant human ifn-α b, ifn-α a), remdesivir, chloroquine, favipiravir, and lopinavir-ritonavir (aluvia), have been clinically used to treat covid- in china [ ] . nevertheless, niaid-vrc scientists are developing a candidate vaccine expressing sars-cov- s protein in mrna vaccine platform technology. clinical trials of the vaccine are expected in the coming months. continued strengthening of the monitoring of the sars-cov- s protein is of great significance for subsequent new drug development and protection against covid- . a novel coronavirus from patients with pneumonia in china analysis of therapeutic targets for sars-cov- and discovery of potential drugs by computational methods research and development on therapeutic agents and vaccines for covid- and related human coronavirus diseases pharmacologic treatments for coronavirus disease (covid- ): a review genomic characterisation and epidemiology of novel coronavirus: implications for virus origins and receptor binding rna based mngs approach identifies a novel human coronavirus from two individual pneumonia cases in wuhan outbreak genomic characterization of the novel human-pathogenic coronavirus isolated from a patient with atypical pneumonia after visiting wuhan functional assessment of cell entry and receptor usage for sars-cov- and other lineage b betacoronaviruses coronaviruses: an overview of their replication and pathogenesis learning from the past: possible urgent prevention and treatment options for severe acute respiratory infections caused by -ncov the coronavirus spike protein is a class i virus fusion protein: structural and functional characterization of the fusion core complex site-specific glycan analysis of the sars-cov- spike fusion mechanism of -ncov and fusion inhibitors targeting hr domain in spike protein coronavirus membrane fusion mechanism offers a potential target for antiviral development cryo-em structure of the -ncov spike in the prefusion conformation structure, function, and antigenicity of the sars-cov- spike glycoprotein tmprss activates the human coronavirus e for cathepsin-independent host cell entry and is expressed in viral target cells in the respiratory epithelium sars-cov- cell entry depends on ace and tmprss and is blocked by a clinically proven protease inhibitor cleavage of spike protein of sars coronavirus by protease factor xa is associated with viral infectivity structural and functional basis of sars-cov- entry by using human ace structure of the sars-cov- spike receptor-binding domain bound to the ace receptor a pan-coronavirus fusion inhibitor targeting the hr domain of human coronavirus spike physiological and molecular triggers for sars-cov membrane fusion and entry into host cells heptad repeat sequences are located adjacent to hydrophobic regions in several types of virus fusion glycoproteins preliminary bioinformatics studies on the design of a synthetic vaccine and a preventative peptidomimetic antagonist against the sars-cov- ( -ncov, covid- ) coronavirus peptide-based membrane fusion inhibitors targeting hcov- e spike protein hr and hr domains bat-to-human: spike features determining 'host jump' of coronaviruses sars-cov, mers-cov, and beyond interaction between heptad repeat and regions in spike protein of sars-associated coronavirus: implications for virus fusogenic mechanism and identification of fusion inhibitors a peptide-based viral inactivator inhibits zika virus infection in pregnant mice and fetuses structural basis for membrane fusion by enveloped viruses cryo-electron microscopy structures of the sars-cov spike glycoprotein reveal a prerequisite conformational state for receptor binding coronavirus spike protein and tropism changes structural basis for the recognition of sars-cov- by full-length human ace origin and evolution of pathogenic coronaviruses a novel angiotensin-converting enzyme-related carboxypeptidase (ace ) converts angiotensin i to angiotensin - angiotensin-converting enzyme (ace ) as a sars-cov- receptor: molecular mechanisms and potential therapeutic target cell entry mechanisms of sars-cov- structure analysis of the receptor binding of -ncov receptor recognition by the novel coronavirus from wuhan: an analysis based on decade-long structural studies of sars coronavirus structural basis for human coronavirus attachment to sialic acid receptors the spike glycoprotein of the new coronavirus -ncov contains a furin-like cleavage site absent in cov of the same clade sars-cov- , sars-cov, and mers-cov: a comparative overview a review on the cleavage priming of the spike protein on coronavirus by angiotensin-converting enzyme- and furin host cell proteases: critical determinants of coronavirus tropism and pathogenesis human influenza a h n virus related to a highly pathogenic avian influenza virus role of host cellular proteases in the pathogenesis of influenza and influenza-induced multiple organ failure tmprss and adam cleave ace differentially and only proteolysis by tmprss augments entry driven by the severe acute respiratory syndrome coronavirus spike protein tmprss is the major activating protease of influenza a virus in primary human airway cells and influenza b virus in human type ii pneumocytes characterization of spike glycoprotein of sars-cov- on virus entry and its immune cross-reactivity with sars-cov biochemical analysis of coronavirus spike glycoprotein conformational intermediates during membrane fusion viral membrane fusion mechanisms of viral membrane fusion and its inhibition covid- , an emerging coronavirus infection: advances and prospects in designing and developing vaccines, immunotherapeutics, and therapeutics novel antibody epitopes dominate the antigenicity of spike glycoprotein in sars-cov- compared to sars-cov rapid development of an inactivated vaccine candidate for sars-cov- purified coronavirus spike protein nanoparticles induce coronavirus neutralizing antibodies in mice potent binding of novel coronavirus spike protein by a sars coronavirus-specific human monoclonal antibody a human monoclonal antibody blocking sars-cov- infection human monoclonal antibodies block the binding of sars-cov- spike protein to angiotensin converting enzyme receptor identification of fully human single-domain antibodies against sars-cov- structural and functional analysis of a potent sarbecovirus neutralizing antibody potent human neutralizing antibodies elicited by sars-cov- infection a potent neutralizing human antibody reveals the n-terminal domain of the spike protein of sars-cov- as a site of vulnerability inhibition of sars-cov- (previously -ncov) infection by a highly potent pan-coronavirus fusion inhibitor targeting its spike protein that harbors a high capacity to mediate membrane fusion design of potent membrane fusion inhibitors against sars-cov- , an emerging coronavirus with high fusogenic activity the anti-hiv drug nelfinavir mesylate (viracept) is a potent inhibitor of cell fusion caused by the sars-cov- spike (s) glycoprotein warranting further evaluation as an antiviral against covid- infections camostat mesilate therapy for covid- alisporivir inhibits mers-and sars-coronavirus replication in cell culture, but not sars-coronavirus infection in a mouse model but not hcov-nl , utilizes cathepsins to infect cells-viral entry. nidoviruses: toward control of sars and other nidovirus glycopeptide antibiotics potently inhibit cathepsin l in the late endosome/lysosome and block the entry of ebola virus, middle east respiratory syndrome coronavirus (mers-cov), and severe acute respiratory syndrome coronavirus (sars-cov) the phosphatidylinositol- -phosphate -kinase inhibitor apilimod blocks filoviral entry and infection inhibition of pikfyve using ym suppresses the growth of liver cancer via the induction of autophagy two-pore channels control ebola virus host cell entry and are drug targets for disease treatment proprotein convertases in health and disease drug development targeting sars-cov- clinical features of patients infected with novel coronavirus in wuhan furin inhibition reduces vascular remodeling and atherosclerotic lesion progression in mice furin inhibitor d r suppresses epithelial-mesenchymal transition in sw and patu cells via the hippo-yap signaling pathway the serpin proteinase inhibitor : an endogenous furin inhibitor released from human platelets peptidomimetic furin inhibitor mi- in combination with oseltamivir and ribavirin efficiently blocks propagation of highly pathogenic avian influenza viruses and delays high level oseltamivir resistance in mdck cells alteration of brain network topology in hiv-associated neurocognitive disorder: a novel functional connectivity perspective the establishment of reference sequence for sars-cov- and variation analysis sars-cov- viral spike g mutation exhibits higher case fatality rate perspectives on therapeutic neutralizing antibodies against the novel coronavirus sars-cov- remdesivir and chloroquine effectively inhibit the recently emerged novel coronavirus ( -ncov) in vitro this project was supported by grants from guangzhou science and technology program (# to wx), the fund of natural science foundation of guangdong province (# a to wx), and grants from major scientific and technological projects of guangdong province (# b to swl). competing interests: the authors declare no competing interests. key: cord- -wfdt vs authors: zhang, sai-long; li, zhi-yong; wang, dong-sheng; xu, tian-ying; fan, mao-bing; cheng, ming-he; miao, chao-yu title: aggravated ulcerative colitis caused by intestinal metrnl deficiency is associated with reduced autophagy in epithelial cells date: - - journal: acta pharmacol sin doi: . /s - - - sha: doc_id: cord_uid: wfdt vs metrnl is a newly identified secreted protein highly expressed in the intestinal epithelium. this study aimed to explore the role and mechanism of intestinal epithelial metrnl in ulcerative colitis. metrnl(−/−) (intestinal epithelial cell-specific metrnl knockout) mice did not display any phenotypes of colitis under basal conditions. however, under administration of % dextran sodium sulfate (dss) drinking water, colitis was more severe in metrnl(−/−) mice than in wt mice, as indicated by comparisons of body weight loss, the presence of occult or gross blood per rectum, stool consistency, shrinkage in the colon, intestinal damage, and serum levels of inflammatory factors. dss-induced colitis activated autophagy in the colon. this activation was partially inhibited by intestinal epithelial metrnl deficiency, as indicated by a decrease in beclin- and lc -ii/i and an increase in p in dss-treated metrnl(−/−) mice compared with wt mice. these phenomena were further confirmed by observation of autophagosomes and immunofluorescence staining for lc in epithelial cells. the autophagy-related ampk-mtor-p s k pathway was also activated in dss-induced colitis, and this pathway was partially blocked by intestinal epithelial metrnl deficiency, as indicated by a decrease in ampk phosphorylation and an increase in mtor and p s k phosphorylation in dss-treated metrnl(−/−) mice compared with wt mice. therefore, metrnl deficiency deteriorated ulcerative colitis at least partially through inhibition of autophagy via the ampk-mtor-p s k pathway, suggesting that metrnl is a therapeutic target for ulcerative colitis. ulcerative colitis is a type of inflammatory bowel disease. in recent years, research on its etiology and pathogenesis has received extensive attention [ ] [ ] [ ] . for the treatment of ulcerative colitis, surgery, anti-infection agents, glucocorticoids, and immunosuppressive agents are often used. however, these are symptomatic treatments, and long-term side effects are likely to cause disease recurrence [ , ] . autophagy is an important self-protection mechanism and is a cellular metabolic pathway that relies on lysosomes. previous studies have shown that autophagy plays a protective role in a variety of diseases, including cancer and inflammatory and immune diseases [ ] [ ] [ ] . in recent years, it has been reported that there is a close relationship between autophagy and inflammation, especially with certain anti-inflammatory effects [ ] [ ] [ ] . in addition, it has been reported that activation of autophagy can ameliorate intestinal inflammation; thus, the induction of autophagy is expected to become a new strategy for the treatment of ulcerative colitis [ ] . metrnl, which is also known as cometin or subfatin, is a newly discovered secreted protein containing amino acids with a nh -terminal signal peptide of amino acids [ ] . our previous study has shown that metrnl is a new adipokine antagonizing insulin resistance through the pparγ signaling pathway [ , ] . metrnl in adipose tissue can promote adipocyte differentiation, improve metabolism, inhibit inflammation, regulate fat function, and alleviate insulin resistance caused by obesity [ , , ] . through the examination of metrnl expression in various tissues, we have found that metrnl is highly expressed in both human and mouse gastrointestinal tissues, especially in intestinal epithelial cells [ ] . a recent study has demonstrated that the metrnl adipokine ameliorates spontaneous colitis in il- knockout mice by attenuating mesenteric adipose tissue lesions [ ] . due to the high expression of metrnl in intestinal epithelial cells, we speculate that intestinal epithelial metrnl plays a role in the regulation of ulcerative colitis development. in the present study, using a conditional knockout mouse model with a specific knockout of metrnl in intestinal epithelial cells (metrnl −/− ), we explored the role of intestinal epithelial metrnl in the development of dextran sodium sulfate (dss)induced colitis, an accepted experimental model of ulcerative colitis. we also examined the possible involvement of autophagy in the role of metrnl. generation of intestinal epithelial cell-specific metrnl knockout mice all animal experiments were performed in accordance with the national institutes of health guide for the care and use of laboratory animals and were approved by the animal ethical committee of the second military medical university. metrnl loxp/loxp mice were generated as we described elsewhere [ ] . villin-cre mice [b . cg-tg(vil -cre) gum/j; jax stock ] were used to generate intestinal epithelial cell-specific metrnl knockout (metrnl −/− ) mice. the breeding strategy for metrnl −/− mice was described elsewhere [ ] . the littermate metrnl loxp/loxp mice were used as the corresponding wild-type (wt) controls. cell culture and treatment caco- cells were provided by the cell bank of chinese academy of sciences (shanghai, china) and cultured in dulbecco's modified eagle's medium with % fetal bovine serum. for certain groups, lipopolysaccharide (lps) ( ng/ml, sigma-aldrich, st. louis, mo, usa) and/or chloroquine (cq) ( μm, sigma-aldrich) were administered for h for stimulation. lentivirus-mediated knockdown of metrnl in cells lentiviruses encoding small hairpin rna (shrna) directed against human metrnl were constructed by shanghai bio-link company (shanghai, china). briefly, the following three shrna target sequences were designed: ′-gggcgctcattgttaacct- ′, ′-gc ttccagtatgagctgatga- ′, and ′-cacgctttagtgactttcaa a- ′. both the recombinant lentivirus encoding shrna targeting metrnl and the lentivirus encoding scrambled shrna were prepared and titered to transfection units (tu)/ml. the knockdown efficiencies of the three target sequences were assessed in caco- cells with real-time pcr. the last target sequence displayed the highest knockdown efficiency and was used in the present study [ ] . quantification of mrna by real-time pcr total rna was extracted from various tissues using trizol (invitrogen, carlsbad, ca, usa), and real-time pcr was performed using an abi real-time pcr system (applied biosystems) as previously described [ ] . a final μl reaction mixture included μl sybr green, μl cdna template, and μl primers. the average threshold cycle (ct) was determined from duplicate reactions, the target gene expression was normalized to gapdh, and the quantitative measurements were obtained using the ΔΔct method. all primers are listed with their sequences in table . histological staining hematoxylin and eosin (h&e) staining was performed as previously described [ ] . briefly, the intestine samples used for histological analysis were fixed with % phosphate-buffered paraformaldehyde for h and embedded in paraffin. sections with a thickness of μm were prepared and stained with h&e. the colon was immersed in % paraformaldehyde at °c for h, cryoprotected in %- % (wt/vol) paraformaldehyde, and cut into frozen coronal slices ( -μm thickness) in cryostat (cm s; leica microsystems, bannockburn, il, usa). colon sections were washed with × pbs three times for min each. then, . % (wt/vol) triton x- and blocking serum were added successively and incubated for min and h, respectively. the specific primary antibodies were incubated at °c overnight ( table ). after being washed three times with × pbs, the sections were incubated with the corresponding secondary antibody (alexa -conjugated and cy conjugated) (jackson immuno research inc., west grove, pa, usa) for h at room temperature and protected from light. nuclei were stained with dapi (vector laboratories, burlingame, ca, usa) for min. fluorescence of colon sections was examined under a fluoview fv confocal laser scanning microscope (olympus, japan). immunoblot analysis protein extraction and immunoblotting were performed as described previously [ ] . briefly, proteins were extracted from the tissue using a standard extraction reagent supplemented with a protease inhibitor (kangchen; shanghai, china). protein concentrations were determined using a bca protein assay kit (beyotime institute of biotechnology; haimen, china). proteins were separated using sds-page, electrotransferred to nitrocellulose membranes and incubated with a primary antibody for - h at °c ( table ). the samples were then incubated with an irdye cw-conjugated secondary antibody (rockland; gilbertsville, pa, usa) for h at °c. images were acquired with the odyssey infrared imaging system (li-cor bioscience; lincoln, ne, usa). all immunoblotting experiments were repeated at least three times. enzyme-linked immunosorbent assay (elisa) serum samples were obtained as previously described [ ] . the levels of il- β, il- , and tnf-α in serum samples were quantified using commercial elisa kits (r&d system, new york, ny, usa). transmission electron microscopy colonic tissues were separated and fixed overnight at °c in . % glutaraldehyde in . m pbs and then post fixed in % buffered osmium tetroxide for h. specimens were processed using routine procedures and examined under a transmission electron microscope (h- ; hitachi, tokyo, japan). an inflammatory bowel disease model was induced in wt and metrnl −/− mice with % or % dss (molecular weight from , to , kda, mp biomedicals llc, santa ana, ca, usa) dissolved in drinking water given ad libitum ( - days) as previously described [ ] . disease activity index and histological analysis in mice body weight, the presence of occult or gross blood per rectum, stool consistency, and colon length were determined by two investigators blinded to the treatment groups. the disease activity index was determined by combining the scores of (i) body weight loss, (ii) stool inconsistency, and (iii) blood in the stool and dividing by [ ] . body weight changes are shown as a loss of baseline body weight. postmortem, the colon was removed, and pieces of colonic tissue were used for ex vivo analysis. for histology, rings of certain parts of the colon were fixed in % buffered formalin and embedded in paraffin. sections were stained with h&e according to standard protocols. histological scoring was performed by a pathologist in a blinded way. a focally increased number of inflammatory cells in the lamina propria was scored as , a confluence of inflammatory cells extending into the submucosa as and a transmural extension of the infiltrate as . for tissue damage, discrete lymphoepithelial lesions were scored as , mucosal erosions as , and extensive mucosal damage and/or extension through deeper structures of the bowel wall as . two equally weighted subscores (cell infiltration and tissue damage) were added, and the combined histological colitis severity score ranged from to . statistical analysis all data are presented as the mean ± sem and were analyzed using prism . software (graphpad software). statistical significance was determined by a two-tailed student's t test. p < . was considered statistically significant. (fig. a) . h&e staining of colon sections showed no significant morphological differences between metrnl −/− and wt mice (fig. b) . to further verify whether intestinal inflammation is induced after the conditional knockout of metrnl, we performed an assay for inflammatory factors in the colon. the results showed no differences in il- , il- , and tnf-α between metrnl −/− and wt mice (fig. c, d) . these results indicate that colitis is not present after intestinal epithelial deficiency of metrnl alone. three percent dss drinking water induces colitis on the th day before establishing the dss-induced colitis model, to choose the optimal observation time and dss administration concentration, we gave % dss and % dss drinking water to wt mice and observed the survival time of these mice at these two dss concentrations. fig. a shows that one mouse died on day in the % dss group and that all mice died after days of dosing. in the % dss group, no mice died during the experimental period. there were no significant changes in body weight loss of mice in the % dss group, while the body weight of mice in the % dss group was significantly reduced on the th day (fig. b ). in addition, the % dss group showed no significant changes in gross parameters such as disease activity index and colon length, whereas the % dss group had increased disease activity index and shortened colon length when compared with those in the control group (fig. c, d) . histological morphology exhibited the phenotypes of colitis with obvious tissue erosion in the % dss group (fig. e) . moreover, the expression of metrnl was comparable between the % dss and control groups. therefore, we selected % dss and days of dosing as the experimental conditions for subsequent experiments. after drinking % dss water for days, both wt and metrnl −/− mice developed severe illness characterized by the presence of sustained weight loss, bloody diarrhea, and severe colon inflammation, and these characteristics were associated with hyperemia, ulceration, and bowel wall thickening leading to further reduction in colon length. during this process, metrnl −/− fig. consuming % dss drinking water for days induces ulcerative colitis in wild-type mice. a survival curve in mice treated with % or % dss drinking water for days. n = . b-e body weight loss, disease activity index, colon length, and histological morphology after consuming % or % dss drinking water for days. n = . *p < . . f metrnl mrna expression in mice treated with % or drinking water. mice showed significantly worse symptoms of weight loss, bloody diarrhea, and colon inflammation, with aggravated inflammatory infiltration in mucosa and submucosa (fig. a-e) . to rule out that the above differences were caused by the intake of different amounts of % dss by the mice, we also measured the drinking water of the two groups of mice. the results showed no significant differences between the two groups (fig. c) . to further verify the proinflammatory effects of metrnl deficiency after dss treatment, we tested inflammatory factors in mouse serum. serum tnf-α, il- , and il- β levels remained comparable between wt and metrnl −/− mice without dss administration and were elevated in both groups after dss administration. moreover, tnf-α, il- , and il- β levels were significantly higher in dsstreated metrnl −/− mice than in wt mice (fig. a-c) . furthermore, we investigated the levels of these inflammatory factors in the colon of metrnl −/− and wt mice treated with dss. tnf-α, il- , and il- β mrna levels were significantly higher in dss-treated metrnl −/− mice than in wt mice (fig. d-f ). we examined autophagy-related proteins and found that the level of autophagy in the metrnl −/− mice induced by % dss was significantly lower than that in the wt mice. beclin- and lc -ii/i expression levels decreased and p expression levels increased in metrnl −/− mice induced by % dss (fig. a) . to further verify that lc -ii was increased by enhancing the formation of autophagosomes, we measured autophagic flux by using the lysosomal inhibitor cq. consistent with the results from animal models, the in vitro results showed that beclin- and lc -ii/i expression levels decreased and p expression levels increased in the metrnl shrna group compared with the control group under lps treatment. notably, cq treatment abrogated the lps-induced differences in autophagy levels between the metrnl shrna and control groups (fig. b) . further observations were obtained by transmission electron microscopy. the number of autophagosomes remained consistent between wt and metrnl −/− mice without dss administration and was increased in both groups after dss administration. moreover, the number of autophagosomes was lower in dss-treated metrnl −/− mice than in wt mice (fig. a) . to further verify the above results, we performed immunofluorescence on four groups of colon sections. the results showed that in the intestinal epithelial cell-specific marker cd -labeled cells, the expression of lc in the % dss-treated metrnl −/− mice was significantly lower than that in the % dss-treated wt mice (fig. b) . metrnl deficiency affects the autophagy-related ampk-mtor-p s k pathway in dss-induced colitis we further examined the effects of metrnl on autophagy-related pathways. the results showed that dss induced an increase in p-ampk and a decrease in mtor and p s k in wt mice. these effects were significantly reduced in metrnl −/− mice (fig. ) , suggesting that the dss-induced activation of the ampk-mtor-p s k pathway was partially blocked by metrnl deficiency in the intestinal epithelium. the present study demonstrates, for the first time, a protective role for metrnl in murine dss-induced colitis. this protection seems to be due to the regulation of metrnl on the autophagy of intestinal epithelial cells in dss-induced colitis. the conditional knockout of metrnl in intestinal epithelial cells can downregulate autophagy levels in dss-induced colitis through inhibition of the ampk-mtor-p s k pathway, thereby aggravating intestinal inflammation (fig. ) . our lab screened for new adipokines in a global gene expression profiling of different adipose depots with bioinformatic methods in . metrnl was identified as a novel adipokine by our group [ ] . jorgensen et al. described metrnl as a neurotrophic factor similar to meteorin (metrn) in [ ] . thus far, there are only a few studies on the function of metrnl. our previous study demonstrated that adipocyte metrnl improves insulin sensitivity through the pparγ pathway in an autocrine/paracrine manner. jorgensen et al. [ ] reported the neurotrophic activity of metrnl in neurite outgrowth and neuroblast migration in vitro and in the survival of spiral ganglion neurons in vivo. watanabe et al. [ ] reported that metrnl is a latent process gene for cell differentiation and neurite extension length. rao et al. [ ] showed that metrnl could promote browning of the subcutaneous and epididymal white adipose tissue. there is a recent report on metrnl ameliorating crohn's disease, and it focused on the crosstalk of mesenteric adipose tissue and intestine. the study found that metrnl administration attenuated mesenteric fat tissue lesions by promoting adipocyte function and differentiation partly through activating the stat /ppar-γ signaling pathway [ ] . although their research is different from ours, it indicated that metrnl could truly affect inflammatory bowel disease; more directly, the administration of metrnl has a protective effect on inflammatory bowel disease. with the deepening of research on inflammatory bowel disease in recent years, there have been an increasing number of reports of autophagy affecting intestinal inflammation. in particular, inflammatory bowel disease susceptibility gene studies have revealed that multiple autophagy-related genes and their single nucleotide polymorphism sites, such as atg l , nod , and irgm, are associated with the pathogenesis of inflammatory bowel disease [ ] [ ] [ ] [ ] . moreover, currently, there are no reports about metrnl and autophagy; therefore, we tested the role of metrnl in autophagy-related proteins in dss-induced colitis mice. to further clarify whether lc -ii was increased by enhancing the formation of autophagosomes or by decreasing the degradation of autolysosomes, autophagic flux was measured after the addition of cq, a lysosomal blocker, to inhibit the formation of autophagosomes. unsurprisingly, cq increased autophagy in both the metrnl shrna and control groups. in addition, cq abrogated the lps-induced differences in autophagy levels between the metrnl shrna and control groups, suggesting that lc -ii increased due to the enhanced formation of autophagosomes instead of the decreased degradation of autolysosomes. for the related mechanism of autophagy, current studies indicate that the ampk-mtor-p s k pathway is an important activating signaling pathway [ , ] . ampk is an amp-activated serine/threonine kinase that plays an important regulatory role in the metabolic process. amp and adp inhibit ampk dephosphorylation and promote ampk activation, thereby activating ampk-dependent signaling pathways [ ] . it has been reported that the activation of the ampk-mtor-p s k signaling pathway can significantly induce or increase autophagy levels in a variety of cells and diseases [ , ] . consistent with those reports, in dss-induced colitis, autophagy is induced by dss in wt mice at least partially through this signaling pathway [ , ] . there are shortcomings of this study. first, the protective function of autophagy on colitis is still under debate, and whether autophagy mediates the beneficial effects of metrnl in colitis is not proven. second, the signaling pathways activating autophagy are multiple and complex. in addition to the ampk-mtor-p s k signaling pathway, there are also other pathways, such as the pi k/akt/mtor pathway, that can trigger autophagy but are not examined in the present study [ , ] . these deficiencies need to be clarified in future research. in summary, the present study shows that metrnl deficiency aggravates dss-induced colitis, which is associated with the regulation of autophagy by metrnl through the ampk-mtor-p s k pathway. these findings reveal that metrnl is a potential therapeutic target for ulcerative colitis. fig. the autophagy-related ampk-mtor-p s k pathway is impaired in dss-treated metrnl −/− mice. the p-ampk/ampk, p-mtor/ mtor, and p-p s k/p s k ratios were determined by western blots. n = . *p < . . topical therapy in ulcerative colitis: always a bridesmaid but never a bride? ulcerative colitis artemisinin analogue sm ameliorates dss-induced mouse ulcerative colitis via suppressing neutrophils and macrophages ulcerative colitis guidelines for the management of inflammatory bowel disease in adults endophilin a regulates calciumactivated chloride channel activity via selective autophagy-mediated tmem a degradation wx , a novel iap antagonist, induces tumor cell autophagy via activating ros-foxo pathway eukaryotic elongation factor- kinase regulates the cross-talk between autophagy and pyroptosis in doxorubicin-treated human melanoma cells in vitro autophagy proteins regulate innate immune responses by inhibiting the release of mitochondrial dna mediated by the nalp inflammasome modulation of inflammation by autophagy: consequences for human disease the roles of macrophage autophagy in atherosclerosis stimulation of autophagy prevents intestinal mucosal inflammation and ameliorates murine colitis metrnl: a secreted protein with new emerging functions adipocyte metrnl antagonizes insulin resistance through ppargamma signaling subfatin is a novel adipokine and unlike meteorin in adipose and brain expression meteorin-like is a hormone that regulates immune-adipose interactions to increase beige fat thermogenesis intestinal metrnl released into the gut lumen acts as a local regulator for gut antimicrobial peptides the adipokine metrnl ameliorates chronic colitis in il- −/− mice by attenuating mesenteric adipose tissue lesions during spontaneous colitis rhein lysinate increases the median survival time of samp mice: protective role in the kidney arrb /beta-arrestin- mediates neuroprotection through coordination of becn -dependent autophagy in cerebral ischemia evaluation of two commercial enzyme-linked immunosorbent assay kits for the detection of human circulating metrnl involvement of nitric oxide with activation of tolllike receptor signaling in mice with dextran sodium sulfate-induced colitis pdgf and tgf-beta promote tenascin-c expression in subepithelial myofibroblasts and contribute to intestinal mucosal protection in mice cometin is a novel neurotrophic factor that promotes neurite outgrowth and neuroblast migration in vitro and supports survival of spiral ganglion neurons in vivo latent process genes for cell differentiation are common decoders of neurite extension length loss of the autophagy protein atg l enhances endotoxin-induced il- beta production a key role for autophagy and the autophagy gene atg l in mouse and human intestinal paneth cells nod and nod direct autophagy by recruiting atg l to the plasma membrane at the site of bacterial entry deletion polymorphism upstream of irgm associated with altered irgm expression and crohn's disease osteoprotegerin inhibit osteoclast differentiation and bone resorption by enhancing autophagy via ampk/mtor/ p s k signaling pathway in vitro radiosensitization of metformin in pancreatic cancer cells via abrogating the g checkpoint and inhibiting dna damage repair guidelines for the use and interpretation of assays for monitoring autophagy activating cannabinoid receptor protects against diabetic cardiomyopathy through autophagy induction -nonylphenol induces autophagy and attenuates mtor-p s k/ ebp signaling by modulating ampk activation in sertoli cells induction of autophagy contributes to the neuroprotection of nicotinamide phosphoribosyltransferase in cerebral ischemia autophagy in ischemic stroke apelin inhibits the proliferation and migration of rat pasmcs via the activation of pi k/akt/mtor signal and the inhibition of autophagy under hypoxia this study was supported by grants from the national natural science foundation of china (no. and no. ). slz and zyl performed most of the experiments and data analyses and wrote the paper. dsw, tyx, mbf, and mhc performed some experiments and data analyses. cym designed the study, performed data analysis, and wrote and revised the paper. competing interests: the authors declare no competing interests. key: cord- -wvf swyj authors: sun, shu-zhen; cao, hong; yao, na; zhao, ling-ling; zhu, xiao-fang; ni, er-an; zhu, qi; zhu, wei-zhong title: β-arrestin mediates arginine vasopressin-induced il- induction via the erk( / )-nf-κb signal pathway in murine hearts date: - - journal: acta pharmacol sin doi: . /s - - -y sha: doc_id: cord_uid: wvf swyj evidence to date suggests that β-arrestins act beyond their role as adapter proteins. arginine vasopressin (avp) may be a factor in inflammation and fibrosis in the pathogenesis of heart failure. in the present study we investigated the effect of avp on inflammatory cytokine il- production in murine hearts and the impact of β-arrestin -dependent signaling on avp-induced il- production. we found that administration of avp ( . u/kg, iv) markedly increased the levels of il- mrna in rat hearts with the maximum level occurred at h. in β-arrestin ko mouse hearts, deletion of β-arrestin decreased avp-induced il- mrna expression. we then performed in vitro experiments in adult rat cardiac fibroblasts (arcfs). we found that avp ( (− )– (− ) m) dose-dependently increased the expression of il- mrna and protein, activation of nf-κb signaling and erk( / ) phosphorylation, whereas knockdown of β-arrestin blocked avp-induced il- increase, nf-κb activation and erk( / ) phosphorylation. pharmacological blockade of erk( / ) using pd diminished avp-induced nf-κb activation and il- production. the selective v( a) receptor antagonist sr effectively blocked avp-induced nf-κb phosphorylation and activation as well as il- expression in arcfs. in avp-treated mice, pre-injection of sr ( mg/kg, iv) abolished avp-induced nf-κb activation and il- production in hearts. the above results suggest that avp induces il- induction in murine hearts via the v( a) receptor-mediated β-arrestin /erk( / )/nf-κb pathway, thus reveal a novel mechanism of myocardial inflammation in heart failure involving the v( a)/β-arrestin /erk( / )/nf-κb signaling pathway. gpcrs transduce various environmental signals and coordinate cellular responses to environmental stimuli. β-arrestin and are adapter proteins that primarily terminate gpcr signaling via receptor desensitization and internalization [ , ] . recent evidence has demonstrated that these proteins also act as scaffold proteins for multiple signaling molecules. for example, these proteins link gpcr signaling to erk / , akt, and src kinases [ ] and interact with the transcriptional factor nf-κb [ , ] . therefore, β-arrestins have emerged as key regulators of cellular functions [ ] , including controlling gpcrdependent chemotactic signals in immune cells during inflammatory reactions. the il- family of cytokines, including il- , has been implicated in cardiac pressure overload and postischemic remodeling [ ] [ ] [ ] . recent studies have demonstrated that il- plasma levels are elevated during congestive heart failure [ ] , which suggests that il- can be used as a marker of inflammation in acute and chronic myocardial injury [ ] . the increased production of cytokines, particularly inflammatory cytokines, such as tumor necrosis factor alpha (tnfα), interleukin- (il- ) and il- , is at least partially responsible for heart dysfunction in patients with heart failure and cardiac remodeling [ , ] . arginine vasopressin (avp) is secreted in response to hypovolemic or cardiac stress. accordingly, avp receptors are also found in some immune cells, such as rat b lymphocytes and thymic epithelial cells [ ] . therefore, avp may stimulate the production of cytokines and antibodies in an autocrine manner during inflammation. the physiological effects of avp are mediated via binding to specific membrane receptors on target cells. three vasopressin receptor subtypes (v a r, v r, and v b r [also termed v r]) have been identified in humans [ ] [ ] [ ] . all of these receptors belong to the gpcr superfamily [ , ] . however, only v a r is found in cardiac myocytes [ ] and cardiac fibroblasts [ , ] . gα q -coupled v a r typically induces protein kinase c, which is an activator of gene programming for cardiac hypertrophy [ ] , but it also interacts with gpcr kinase (grk) isoforms [ ] , primarily grk [ , ] . grks activate g proteinindependent signaling pathways in addition to having a clearly defined role in receptor desensitization [ , ] . these pathways are related to the regulation of cardiac hypertrophy and apoptosis [ ] and the promotion of cardioprotective extracellular signal-regulated kinase / (erk / ) signal transduction via β-arrestin [ ] . however, little is known about the inflammatory regulation of β-arrestin in the heart. the present study investigated the effect of avp on il- production in murine hearts and the impact of β-arrestin -dependent signaling on avp-mediated il- production. the animal care and use committee of nantong university approved all of the procedures. adult sprague-dawley rats were obtained from the animal center of nantong university (nantong, china). β-arrestin ko mice were gifted by the laboratory of dr. lefkowitz (duke university, durham, nc, usa). dulbecco's modified eagle's medium (dmem), penicillin and streptomycin were purchased from invitrogen (gaithersburg, md, usa). arginine vasopressin (avp, v ) was purchased from sigma-aldrich (st. louis, mo, usa). the v a r selective antagonist sr was purchased from tocris bioscience (minneapolis, mn, usa). a rat il- elisa kit (#bms ) was purchased from thermofisher scientific (waltham, ma, usa). the adenovirus containing gfp was a gift from dr. yibin wang (university of california, los angeles, ca, usa). the adenovirus containing human β-arrestin was created by fubio (shanghai, china). the lentivirus containing shrna targeting β-arrestin was purchased from genechem (shanghai, china). nf-κb luciferase (e ) and renilla luciferase (e ) were purchased from promega (madison, mi, usa). anti-p-nf-κb (# ) and anti-β-arrestin ( s) antibodies were purchased from cell signaling technology (danvers, ma, usa). antibodies against nf-κb (sc- ) and gapdh (sc- ) were obtained from santa cruz biotechnology (dallas, tx, usa). male rats and mice ( - weeks old) were administered . u/kg avp via the tail vein. murine heart tissues were harvested for rna and protein isolation at designated timepoints after avp administration. the animal care and use committee of nantong university approved all of the procedures. cell culture and adenoviral or lentiviral infection arcfs were prepared from the hearts of adult ( -to -weekold) sprague-dawley rats as previously described [ ] . the cells were cultured in dmem containing % fbs and -u/ml penicillin-streptomycin for - days before being passaged. when the cells reached % confluence, they were passaged at a ratio of : with . % trypsin. experiments were consistently performed on cells from passages - . the serum was replaced with serum-free medium, and the arcfs were transfected with an adenovirus containing β-arrestin or a lentivirus containing shrna targeting β-arrestin at an moi of . medium with or without treatment agent was added after h according to the experimental design. transient transfection and luciferase gene reporter assay arcfs ( × cells/well) were plated in -well plates. the reporter plasmid was transfected when the confluency of the growing cells reached %- % using lipofectamine . cotransfection was performed with . μg of an nf-κb luciferase plasmid and . μg of a renilla luciferase plasmid. the cells transfected with the nf-κb-luciferase plasmid ( . μg) and the renilla control vector ( . μg) but not incubated with avp were used to measure basal activity. the transfected cells were further cultured for h, serum-starved overnight and cultured in the presence of avp for h. a luminometer (thermofisher scientific, waltham, ma, usa) was used to measure luciferase activation based on the manufacturer's instructions. quantitative pcr (qpcr) for analyzing il- mrna levels and rt-pcr for analyzing the levels of vasopressin receptor subtypes rt-pcr fresh tissue samples were collected from -week-old male sprague-dawley rats. the messenger rna (mrna) levels of v a r, v b r, and v r in arcfs and the neonatal rat brain were assessed using rt-pcr, as described previously [ ] . an rna extraction kit was used to extract total rna from fresh tissues and cultured arcfs. a reverse transcription synthesis kit (takara, dalian, china) was used to prepare complementary dna (cdna). all procedures were performed based on the manufacturer's instructions. the reverse-transcribed cdna was amplified using pcr under standard conditions with specific primers. cdna amplification was performed with an initial step of min at °c followed by cycles of s at °c, s at - °c, min at °c, and a final extension of min at °c. the pcr products were analyzed using agarose gel electrophoresis and scanning densitometry. primers for the receptor subtypes and β-actin are shown in table . qpcr arcfs ( × cells/well) were plated in -cm culture dishes. after the cells reached quiescence at % confluency, total rna was extracted from the cultured nrcfs using trizol reagent (promega, madison, wi, usa) after stimulation with avp and pd , according to the experimental design. cdna was amplified using a step-one real time pcr system (applied biosystems, foster city, ca, usa). the double-stranded dnaspecific dye sybrgreen i was incorporated into the pcr buffer provided in the quantitech sybr pcr kit (qiagen, valencia, ca, usa) to allow for the quantitative detection of the pcr product in a -μl reaction volume. the primer sequences used to detect murine il- , gapdh, and β-actin are shown in table . the following temperature profile was used for the reaction: °c for min, cycles of denaturation at °c for s, annealing at °c for min, and extension at °c for s. gapdh and β-actin were used as rna loading controls for rat and mouse il- , respectively. the data were analyzed using the Δct method ( -ΔΔct ) [ ] . western blotting cells were treated with avp for - min, washed twice with icecold pbs and lysed with μl of ice-cold lysis buffer ( mm tris-hcl, mm nacl, mm egta, % triton x- , mg·ml − leupeptin, mm phenylmethylsulfonyl fluoride, mg/ml aprotinin, mm naf, and mm na vo ). after centrifugation at × g for min at °c, equal amounts of total cell lysates ( μg of protein) were loaded onto %- % sds-page gels, and immunoblotting for phosphorylated nf-κb, nf-κb, β-arrestin , phosphorylated erk / , and gapdh was performed. elisa for il- heart homogenates or cell culture supernatant samples ( μl) were used to measure il- using an elisa kit, as described in the kit manual. the analytical sensitivity of the kit was . pg/ml and . - pg/ml, respectively. the data and statistical analyses complied with the recommendations on experimental design and analysis in pharmacology [ ] . an experimenter who was blinded to the experimental protocol randomized the animals into groups in the animal experiments on avp-induced il- production. the number of rats in each group in the in vivo study was less than needed to reduce the number of euthanized animals because the difference between the control and treatment groups was significant in a preliminary study. randomization was not applicable in any of the other experiments. comparisons were performed using one-or two-way anova followed by bonferroni's test. all values are presented as the means ± sem. p < . was considered statistically significant. to determine whether avp evokes il- induction in the murine myocardium, avp ( . u·kg − ) was administered via the tail veins of the rats. the level of troponin i in the serum was not increased h after avp administration (data not shown). avp-evoked il- expression in the heart, and the maximum expression of il- mrna and protein occurred h ( fig. a , b) after avp administration. notably, the deletion of β-arrestin reduced avp-induced il- mrna expression (fig. c) in β-arrestin ko mouse hearts (supporting information data s ). to further investigate the mechanistic role of β-arrestin in avpmediated il- induction, we performed in vitro experiments using cultured adult rat cardiac fibroblasts (arcfs). avp-induced il- expression in a dose-and time-dependent manner in cultured arcfs (fig. ) . the silencing of β-arrestin with shrna ( fig. a and supporting information data ) or the overexpression of β-arrestin with a lentiviral vector ( fig. b and supporting information data ) fig. administration of avp promotes the expression of il- mrna and protein in murine hearts. animals were administered . u/kg avp or . % nacl as vehicle via the tail vein. total rna and protein lysates were isolated from the myocardium of the left ventricle to measure il- mrna at h after the administration of avp. a, b avp induced the elevation of il- mrna and protein levels in the rat myocardium. elisa for il- protein and qpcr for il- mrna were performed in triplicate to ensure the reliability of the individual values. c, deletion of β-arrestin reduced the avp-induced il- elevation in the mouse myocardium. the upper panels in (a, c) are representative images of the qpcr products, and the lower panels show the data expressed as the means ± sem of four (rats) or five (mice) separate experiments. *p < . , **p < . , ***p < . vs. vehicle, ## p < . were used to manipulate the levels of β-arrestin . the silencing of β-arrestin suppressed the avp-induced mrna and protein levels of il- in arcfs (fig. c, d) . notably, the overexpression of β-arrestin did not alter the induction of il- expression. these results suggest that β-arrestin is necessary, but not sufficient, for the avp-induced induction of il- in murine hearts. β-arrestin is required for avp-induced nf-κb activation our previous study demonstrated that avp induces il- production via nf-κb signaling in neonatal rat cardiac fibroblasts [ ] and cultured arcfs (fig. ) . to determine whether β-arrestin is involved in the avp-mediated induction of nf-κb activation, we measured the levels of phosphorylated nf-κb and the luciferase activity of nf-κb after the cells were transfected with lentiviral or adenoviral for h, the starved cells were stimulated with − m avp for h or h, respectively and harvested for total rna and supernatant. il- mrna was measured by qpcr as described in the methods. the supernatant was collected to measure il- using elisa as described in the methods. notably, the overexpression of β-arrestin did not alter the induction of il- expression. *p < . , **p < . vs. control, n = . ## p < . . ns: no significance under different conditions. we found that the silencing of β-arrestin by shrna efficiently suppressed avp-induced nf-κb phosphorylation (fig. a, b) and nf-κb luciferase activity (fig. c ). in contrast, the overexpression of β-arrestin did not alter the activation or phosphorylation levels of nf-κb (fig. b, c) . notably, avp-evoked nf-κb activity in a pkc-independent manner, as a pkc inhibitor did not block avp-induced nf-κb activity in arcfs (supporting information data ). these results suggest that β-arrestin is necessary, but not sufficient, for avp-evoked nf-κb signaling, which is consistent with its effect on avp-induced il- production. β-arrestin -dependent erk / phosphorylation is essential for avp-induced nf-κb and il- production β-arrestin is a critical component in the mediation of the β adrenergic stimulation of erk / signaling [ ] . we next tested our hypothesis that β-arrestin -dependent erk / signaling is responsible for avp activity in the heart. avp induced the phosphorylation of erk / in a pd -sensitive manner in cultured arcfs and intact rat hearts (fig. ) . the deletion of βarrestin in β-arrestin knockout mouse hearts or using β-arrestin shrna or β-arrestin adenovirus reduced the avp-evoked phosphorylation of erk / , and the overexpression of β-arrestin did not further enhance avp-induced erk / activation. these results suggest that β-arrestin is required for the avp-induced phosphorylation of erk / . accordingly, β-arrestin deficiency and erk / inhibition with pd abolished the avp-induced activation of nf-κb (fig. a) and induction of il- expression (fig. b) in mouse hearts. a pd -sensitive pathway mediated avp-induced il- expression and nf-κb phosphorylation in mouse hearts, which was evidenced by the inhibition of nf-κb phosphorylation and the induction of il- by pd (fig. ) . taken together, these data support the hypothesis that β-arrestin -mediated erk / signaling contributes to the avp-induced nf-κb/il- inflammatory response in the heart. we used rt-pcr and demonstrated that the v a receptor is the only vasopressin receptor subtype in arcfs (fig. a) . notably, the v a receptor-selective inhibitor sr efficiently blocked avp-induced nf-κb phosphorylation with an ic of . ± . nm (fig. b) , nf-κb activation with an ic of . ± . nm (fig. c) , and il- expression with an ic of . ± . nm in arcfs (fig. d . notably, μm of sr had no effect on basal il- expression or nf-κb activation in arcfs. similarly, pretreating mice with mg/kg sr prior to avp administration ( . u/kg) diminished nf-κb activation (fig. f) and il- production (fig. e, g) . the cytokine il- is important for immune system regulation, inflammatory responses and cardiovascular remodeling. il- has a low baseline mrna expression level in cardiac fibroblasts and is absent in cardiomyocytes, but il- levels are stimulated by β ar [ ] , hypoxia [ ] or coculture with macrophages [ ] . the following results were found in the present study: ( ) avp increased the mrna and protein levels of il- in murine hearts; ( ) the silencing or deletion of β-arrestin reduced avp-induced il- production, nf-κb activation and erk / phosphorylation; ( ) the pharmacological inhibition of erk / signaling diminished avp-induced nf-κb activation and il- production; and ( ) the blockade of the v a receptor by the selective antagonist sr abolished avp-evoked nf-κb phosphorylation and il- induction in intact hearts and arcfs. avp is an antidiuretic hormone that is secreted by the hypothalamus-pituitary-adrenal axis. avp significantly affects vasoconstriction, immune regulation and body temperature regulation. ventricular remodeling occurs when end-stage heart failure develops, and inflammation is an important mechanism for the development of ventricular remodeling. when heart failure occurs, the concentration of catecholamines increases, which induces cardiac fibroblasts to secrete the pro-inflammatory factor il- via β adrenergic receptors [ ] . il- is involved in the remodeling of the extracellular matrix of cardiomyocytes, which induces or aggravates the development of ventricular remodeling, which promotes chronic heart failure [ ] . the level of avp may be used as an early warning sign of the disease, and it may provide new ideas for the treatment of cardiovascular diseases in the future. gpcr-or stress-stimulated il- secretion from cardiac fibroblasts may lead to cardiac inflammation, fibroblast proliferation and cardiac remodeling. data from our and other studies have demonstrated that avp promotes the proliferation of cardiac fibroblasts [ , [ ] [ ] [ ] [ ] . we report for the first time that avp induces il- production in the murine myocardium. avp-induced transforming growth factor-beta (tgf-beta ) secretion is fig. v a r mediates avp-induced nf-κb activation and il- induction in arcfs and rat hearts. a the v a receptor subtype, but not the v b or the v subtype, exists in arcfs, as measured using rt-pcr. total rna was isolated from arcfs and rat brains. rt-pcr was performed with primers designed to target each subtype of the avp receptor. similar results were reproduced at least three times. b-d the inhibition of the v a subtype by sr abolished avp-evoked nf-κb phosphorylation (b) and activation (c) and il- mrna production (d) in arcfs. starved cells were pretreated with . nm- . μm sr (a v a blocker) for h and further stimulated with . μm avp for h (b), h (c) and h (d). the upper panel in b is a representative blot, and the lower panel shows the data expressed as the means ± sem of three separate experiments. **p < . vs. control, ## p < . vs. avp alone. e-g sr blocked the avp-induced expression of il- and nf-κb phosphorylation in rat hearts. after being pretreated with sr ( . mg·kg − , ip) for h, the animals were administered . u· kg − avp via the tail vein for h. the left ventricle was collected to measure nf-κb phosphorylation at h after the administration of avp. the data are expressed as the means ± sem of four separate experiments. *p < . vs. vehicle, # p < . , ## p < . vs. avp alone. sr: sr responsible for cardiac fibroblast-myofibroblast transformation [ ] , but whether the induction of il- plays a role in cardiac fibroblast proliferation requires further study. a large number of studies have confirmed that β-arrestin is primarily involved in negative feedback regulation, such as the desensitization and internalization of gpcrs and seven transmembrane receptors ( tmrs), and recent studies have demonstrated that β-arrestin also participates in g protein-independent signaling pathways, which also mediate the physiological and pathological functions of cells. for example, β-arrestin and β-arrestin mediate protective β -adrenergic signaling in cardiac myocytes [ ] . due to their various biochemical functions in the regulation of critical physiological and pathophysiological processes, including inflammation, β-arrestins have become a drug target in human diseases, such as heart failure. heart failure is marked by increased avp levels in the systemic circulation and local cardiac tissues [ ] . therefore, the present findings highlight il- as the effector cytokine downstream of β-arrestin that may link elevated avp levels to inflammatory cardiac damage and, ultimately, cardiac remodeling. β-arrestin plays a regulatory role as a scaffold protein in a variety of cell models and pathological conditions. the cardioprotective effect of β-arrestin may occur via gpcr-dependent [ ] and gpcrindependent [ , ] signaling pathways. it was recently reported that β-arrestin regultes the immune response in immune cells via the nf-κb signaling pathway. β-arrestin directly interacts with iκbα (an nf-κb inhibitor and the key molecule in innate and adaptive immunity) [ ] and prevents the phosphorylation and degradation of iκbα [ , ] . consequently, β-arrestin effectively modulates nf-κb activation and the expression of nf-κb target genes [ , ] . moreover, the stimulation of β -adrenergic receptors significantly enhances the interaction between β-arrestin and iκbα and greatly promotes the stabilization of iκbα by β-arrestin , which indicates that β-arrestin mediates crosstalk between β -adrenergic receptors and nf-κb signaling pathways [ ] . this interaction suggests a novel mechanism for the regulation of the immune system by the sympathetic nervous system. in contrast, witherow et al. found that the overexpression of β-arrestin or β-arrestin leads to a marked inhibition of nf-κb activity. conversely, the suppression of the expression of β-arrestin , but not of β-arrestin , using rna interference leads to a threefold increase in tnf-stimulated nf-κb activity [ ] . the direct interaction between β-arrestin and nf-κb regulates nf-κb signaling, and the present study demonstrates that the phosphorylation of nf-κb is mediated by β-arrestin signaling via erk / activation in cardiac cells. it is well established that nf-κb regulates the expression of inflammatory factors (including il- ) and the initiation and progression of myocardial fibrosis [ , ] . the present study demonstrated that avp, as an endogenous pro-inflammatory factor, activates nf-κb in fibroblasts, as evidenced by nf-κb phosphorylation, nuclear translocation and luciferase activity. pdtc is an inhibitor of nf-κb that inhibits the inflammatory effect of avp to a certain extent via the inhibition of the activation of nf-κb p and alleviates avp-induced inflammation and organ damage. therefore, the proinflammatory effect of avp depends on the regulation of nf-κb to some extent. by targeting the mechanisms of nf-κb regulation, we hope to develop new ideas for the treatment of inflammation and myocardial fibrosis in cardiovascular diseases. there are three vasopressin receptor subtypes, namely, v a, v b , and v . v b is widely distributed in the central system, and v is concentrated in the renal collecting duct. early studies by our laboratory revealed that only v a receptors are present in neonatal rat cardiac fibroblasts. the distribution and function of the three receptors are different. the primary roles of the v a receptor are vasoconstriction, platelet aggregation, blood pressure regulation, and hepatic glycogen metabolism [ ] . the v a receptor mediated the avp-induced inflammatory response in this experiment, and its selective antagonist sr significantly inhibited this response. our data further demonstrate that a selective v a receptor blocker efficiently abolishes avp-induced nf-κb signaling and il- production in intact hearts, which suggests that the v a subtype mediates avp-evoked inflammation in the murine myocardium. β-arrestin /erk / /nf-κb signaling may not be specific to the v a receptor because cardiomyocytes also contain other gq-coupled receptors, which could induce the activation of such a pathway and participate in the inflammatory response in the heart. notably, the gq-pkc pathway was not involved in the v a /avp-mediated activation of nf-κb in our study (as shown in supplementary information data ) or in cellular protection via a pkc-independent pathway in our previous study [ ] . however, the avp-mediated activation of erk / has been previously demonstrated in h c cells [ , ] and cardiomyocytes [ ] . therefore, further study on whether different signaling mechanisms between gq-pkc and β-arrestin mediates the diverse biological functions evoked by avp is valuable because avp is increased during the development of heart failure. in summary, our data reveal an important role of β-arrestin in the avp-mediated inflammatory response, which highlights a novel inflammatory mechanism whereby avp induces il- production in intact murine hearts via a β-arrestin /erk / /nf-κb pathway. these findings shed new light on our molecular understanding of inflammation in heart failure and possibly other cardiac stress conditions. molecular mechanisms of g protein-coupled receptor signaling: role of g protein-coupled receptor kinases and arrestins in receptor desensitization and resensitization the role of beta-arrestins in the termination and transduction of g-protein-coupled receptor signals beta-arrestins and cell signaling identification of beta-arrestin as a g protein-coupled receptor-stimulated regulator of nf-kappab pathways beta-arrestin inhibits nf-kappab activity by means of its interaction with the nf-kappab inhibitor ikappabalpha g protein-coupled receptors. iii. new roles for receptor kinases and beta-arrestins in receptor signaling and desensitization identification of distinct cterminal domains of the bombyx adipokinetic hormone receptor that are essential for receptor export, phosphorylation and internalization arrestin mediates vasopressin-induced il- induction sz sun et al g-protein receptor kinase regulates the cannabinoid receptor -induced up-regulation of serotonin a receptors arginine vasopressin enhances cell survival via a g protein-coupled receptor kinase /beta-arrestin / extracellular-regulated kinase / -dependent pathway in h c cells inflammatory markers and risk of heart failure in elderly subjects without prior myocardial infarction: the framingham heart study proinflammatory cytokines: predictors of a failing heart? negative inotropic effects of cytokines on the heart mediated by nitric oxide continuous activation ofgp , a signaltransducing receptor component for interleukin -related cytokines, causes myocardial hypertrophy in mice corticotropin-releasing hormone immunoreactivity in human t and b cells and macrophages: colocalization with arginine vasopressin cloning and characterization of the human v pituitary vasopressin receptor cloning and characterization of a vasopressin v receptor and possible link to nephrogenic diabetes insipidus molecular cloning and expression of a rat v a arginine vasopressin receptor molecular pharmacology and modeling of vasopressin receptors molecular biology of vasopressin receptors vasopressin promotes cardiomyocyte hypertrophy via the vasopressin v a receptor in neonatal mice grk /beta-arrestin mediates arginine vasopressin-induced cardiac fibroblast proliferation arginine vasopressin receptor signaling and functional outcomes in heart failure controlled and cardiac-restricted overexpression of the arginine vasopressin v a receptor causes reversible left ventricular dysfunction through galphaq-mediated cell signaling beta-adrenergic receptormediated cardiac contractility is inhibited via vasopressin type a-receptordependent signaling vasopressin v a receptor mediates cell proliferation through grk -egfr-erk / pathway in a r cells regulation of receptor trafficking by grks and arrestins g protein-coupled receptor kinases in normal and failing myocardium grk mythology: g-protein receptor kinases in cardiovascular disease cell-specific roles of grk in onset and severity of hypoxic-ischemic brain damage in neonatal mice , , ', -tetrahydroxystilbene- -o-beta-d-glucoside inhibits angiotensin ii-induced cardiac fibroblast proliferation via suppression of the reactive oxygen species-extracellular signal-regulated kinase / pathway analyzing real-time pcr data by the comparative c(t) method experimental design and analysis and their reporting: new guidance for publication in bjp grk mediates arginine vasopressininduced interleukin- production via nuclear factor-kappab signaling neonatal rat cardiac fibroblast betaarrestin-dependent, g protein-independent erk / activation by the beta adrenergic receptor paroxetine is a direct inhibitor of g protein-coupled receptor kinase and increases myocardial contractility hypoxia-stimulated cardiac fibroblast production of il- promotes myocardial fibrosis via the tgf-beta signaling pathway macrophage-stimulated cardiac fibroblast production of il- is essential for tgf beta/smad activation and cardiac fibrosis induced by angiotensin ii increased systemic inflammation and oxidative stress in patients with worsening congestive heart failure: improvement after short-term inotropic support recombinant human interleukin- receptor antagonist promotes m microglia biased cytokines and chemokines following human traumatic brain injury arginine vasopressin stimulates proliferation of adult rat cardiac fibroblasts via protein kinase c-extracellular signal-regulated kinase / pathway effects of arginine vasopressin on growth of rat cardiac fibroblasts: role of v receptor mitogenic effect of arginine vasopressin on adult rat cardiac fibroblast: involvement of pkc-erk / pathway the secretion patterns and roles of cardiac and circulating arginine vasopressin during the development of heart failure effects of arginine vasopressin on differentiation of cardiac fibroblasts into myofibroblasts ang-( - ) is an endogenous beta-arrestin-biased agonist of the at receptor with protective action in cardiac hypertrophy beta-arrestin improves post-myocardial infarction heart failure via sarco(endo)plasmic reticulum ca + -atpase-dependent positive inotropy in cardiomyocytes galphai is required for carvedilol-induced beta adrenergic receptor beta-arrestin biased signaling hypertension cmrgo. role of aldosterone in angiotensin ii-induced cardiac and aortic inflammation, fibrosis, and hypertrophy blockade of nf-kappab improves cardiac function and survival after myocardial infarction delayed nootropic effects of arginine vasopressin after early postnatal chronic administration to albino rat pups signal transduction of arginine vasopressin-induced arachidonic acid release in h c cardiac myoblasts: role of ca + and the protein kinase c-dependent activation of p mitogen-activated protein kinase stimulation of / kda mitogen-activated protein kinases by arginine vasopressin in rat cardiomyocytes the authors thank dr. yibin wang (university of california, los angeles) for kindly providing the β-gfp adenovirus. this study was supported by the national natural science foundation of china (no. , no. , no. ) and the nantong university co-innovation program (ntu - ). szs and wzz contributed to the conception of the work, the interpretation of the data and the critical revision of the paper. szs, ny, xfz, qz, llz, ean and hc contributed to data acquisition. szs and wzz drafted and finalized the paper. all authors contributed to the final approval of the manuscript. the online version of this article (https://doi.org/ . /s - - -y) contains supplementary material, which is available to authorized users. the authors declare that they have no conflict of interest. key: cord- -h gv g authors: wei, xiao-min; wumaier, gulinuer; zhu, ning; dong, liang; li, cheng-wei; xia, jing-wen; zhang, you-zhi; zhang, peng; zhang, xiu-juan; zhang, yuan-yuan; li, sheng-qing title: protein tyrosine phosphatase l represses endothelial-mesenchymal transition by inhibiting il- β/nf-κb/snail signaling date: - - journal: acta pharmacol sin doi: . /s - - -x sha: doc_id: cord_uid: h gv g endothelial–mesenchymal transition (enmt) plays a pivotal role in various diseases, including pulmonary hypertension (ph), and transcription factors like snail are key regulators of enmt. in this study we investigated how these factors were regulated by ph risk factors (e.g. inflammation and hypoxia) in human umbilical vein endothelial cells (huvecs). we showed that treatment with interleukin β (il‐ β) induced enmt of huvecs via activation of nf-κb/snail pathway, which was further exacerbated by knockdown of protein tyrosine phosphatase l (ptpl ). we demonstrated that ptpl inhibited nf-κb/snail through dephosphorylating and stabilizing iκbα. il‐ β or hypoxia could downregulate ptpl expression in huvecs. the deregulation of ptpl /nf-κb signaling was validated in a monocrotaline-induced rat ph (mct-ph) model and clinical ph specimens. our findings provide novel insights into the regulatory mechanisms of enmt, and have implications for identifying new therapeutic targets for clinical ph. endothelial-mesenchymal transition (enmt) is a cellular process characterized by the loss of endothelial features and the acquisition of mesenchymal, fibroblast, or stem-cell-like characteristics [ ] . it is defined by the loss of cellular adhesion and the cytoskeletal reorganization of actin and intermediate filaments that convert apical-basal polarity to front end-back end polarity to form spindle-shaped cells. during this transformation, there is a marked decrease in endothelial biomarkers such as cd and cd , as well as increased expression of mesenchymal biomarkers such as α-sma and sm- α [ ] . enmt plays a pivotal role in various pathologic conditions, such as atherosclerosis [ ] , tumors [ , ] , vein graft failure [ ] , and fibrosis in key organs [ ] [ ] [ ] . a recent study has shown that abnormal differentiation of enmt-derived sm-like cells results in pulmonary hypertension (ph) [ ] [ ] [ ] . however, the underlying mechanism remains to be fully elucidated. inflammation and hypoxia contribute to the pathogenesis of various types of ph, especially group iii ph. expression of nuclear factor-κb (nf-κb) and the pro-inflammatory cytokine interleukin- β (il- β) is elevated in the lung tissue of a rat model of monocrotaline-induced ph (mct-ph), and nf-κb activation may contribute to the development of this condition [ ] . il- β could induce enmt by activating the nf-κb pathway [ ] . the nf-κbregulated gene snail is a key regulator of enmt, cell adhesion, and proliferation [ , ] . these findings highlight the critical role of nf-κb/snail signaling in enmt. protein tyrosine phosphatase l (ptpl ) is a high-molecularweight ( kda) nonreceptor-type phosphatase. it contains multiple domains, providing numerous potential interfaces [ ] . previous studies have shown that ptpl plays an important role in the development of tumors [ ] [ ] [ ] and idiopathic pulmonary fibrosis [ ] . recent studies have also shown that ptpl inhibits emt in hepatocellular carcinoma [ ] . however, the regulatory role and underlying molecular mechanisms of ptpl in ph, a disorder attributed necessarily to enmt of vascular endothelial cells, are not clearly elucidated. here, we investigated the role of ptpl in il- βinduced enmt using human umbilical vein endothelial cells (huvecs). our findings suggest that ptpl represses enmt by stabilizing iκbα, and therefore inhibiting the il- β/nf-κb/snail pathway. it may help identify new potential drug targets for ph. animals were randomly divided into two groups (n = /group). the control group rats received phosphate-buffered saline (pbs). the mct group rats received mct ( mg/kg; sigma, st. louis, mo, usa). all animals were raised under a h: h light-dark cycle and were freely supplied with food and water. the room temperature was maintained at °c, and the bedding was changed once per week. hemodynamic analysis and tissue preparation in the rat model of mct-ph, rat pulmonary vascular and hemodynamic alterations and rv remodeling occurred days after mct injection. at that point, the right jugular vein was carefully isolated. a specially shaped catheter linked to the powerlab system (ad instruments, bella vista, nsw, australia) was inserted into the right ventricle (rv) via this vein, and right ventricular systolic pressure (rvsp) was recorded. then, sternotomy was performed, and rats were perfused with paraformaldehyde. both lungs and the heart were harvested. the rv and the left ventricle plus septum (lv + s) were weighed, and the rv/lv + s weight ratio was determined as an indication of right ventricular hypertrophy. next, the lower lobe of the right lung was sectioned into -mm-thick slices and soaked in a % formalin solution (ph . ). the other parts were kept at − °c for further experiments. hematoxylin and eosin staining the fixed lungs were sliced in the mid-sagittal plane, embedded in paraffin, and cut into~ -μm-thick sections with a microtome. then, the sections were placed on glass slides, stained with hematoxylin and eosin (he) for morphological analysis, and visualized under an olympus bx microscope (tokyo, japan). cell culture and induction of enmt huvecs (passages - ; sciencell research laboratories, california, usa) were cultured in endothelial cell medium (ecm). to induce enmt, recombinant il- β ( ng/ml; peprotech) in . % fetal bovine serum medium was used to treat the cells for h. human pulmonary artery endothelial cells (hpaecs) (passages - ; sciencell research laboratories, california, usa) were cultured in ecm. cells were maintained in a humidified atmosphere at °c in % co . immunofluorescence staining cells were fixed with % paraformaldehyde for min at room temperature. the cells were then incubated with primary antibodies against α-sma and cd (abcam, cambridge, ma, usa) overnight at °c and subsequently with alexa fluorconjugated secondary antibodies (invitrogen, carlsbad, ca) for h at °c. after nucleus staining with ʹ, -diamidino- -phenylindole (dapi) dye, cells were examined under a fluorescence microscope. a minimum of three replicates were performed for each examination. the lentiviral shuttle vector for the ptpl -targeted short hairpin rna, sh-ptpl , and the packaging vectors, including pmd . g and pspax, were purchased from addgene (cambridge, ma, usa). they were transfected into hek t cells for packaging and amplification of lentivirus. for transient transduction, cells at approximately % confluence were transduced with the lentiviral vectors at a multiplicity of infection of for h. afterward, the media were changed to fresh ecm, and the transfected cells were cultured for an additional h. quantitative rt-pcr total rna was extracted from cultured cells using trizol reagent (invitrogen, life technologies, carlsbad, ca, usa). reverse transcription was performed using a first-strand cdna reverse transcription kit (takara, otsu, japan), and real-time pcr was performed using a sybr green/rox qpcr master mix kit according to the manufacturer's instructions (takara, otsu, japan). the relative quantification was determined using the ΔΔct method with β-actin as a reference gene. all primer sequences are shown in table . proteins from cultured cells were extracted with radioimmunoprecipitation assay lysis buffer and quantified with the bicinchoninic acid protein assay kit. then, the proteins were separated with sodium dodecyl sulfate-polyacrylamide gel electrophoresis (sds-page) and transferred to a polyvinylidene fluoride transfer membrane. the membranes were blocked in % nonfat dry milk for h, followed by incubation overnight at °c with primary antibodies. the membranes were then washed and incubated with secondary antibodies, washed with tris-buffered saline and tween ( . %), and treated with electrochemiluminescence reagents for blot detection. blots were quantified by densitometry using imagej (nih image, bethesda, md). the primary antibodies used are as follows: cd ( : ), α-sma ( : ), cd ( : ), sm- α ( : ), iκbα ( : ), phospho-iκbα (y , : ), and βactin ( : ) were purchased from abcam (cambridge, ma, usa); ptpl ( : ) was purchased from novus biologicals; and nf-κb ( : ), phospho-nf-κb (p , : ), and snail ( : ) were purchased from cell signaling technology (danvers, ma, usa). secondary horseradish-peroxidase-conjugated antibodies were purchased from jackson immunoresearch laboratories ( : , dilution; immunoresearch laboratories, inc., west grove, pa, usa). immunohistochemical assay all the specimens were fixed with % paraformaldehyde and embedded with paraffin. four-micrometer-thick sections were cut and transferred to glass slides coated with g/l polylysine. the immunohistochemical (ihc) staining was performed as described tube formation assays huvecs were harvested and suspended in ecm. forty-eight-well plates were coated with matrigel (bd biosciences), which was allowed to polymerize at °c in a % co chamber for min. huvecs ( × cells/ml) were added to each chamber, treated as indicated, and incubated for h at °c in % co , followed by observation of capillary-like tube formation using phase-contrast microscopy. we analyzed the change in the mrna profiles between hpaecs that were treated with and without hypoxia for h. total rna was isolated using trizol reagent (invitrogen, carlsbad, ca, usa) according to the manufacturer's instructions. cdna library preparation was performed using the rna-seq sample prep kit (illumina, neb, ipswich, ma, usa) according to the manufacturer's instructions. for the qc step, an agilent bioanalyzer and an abi steponeplus real-time pcr system were used to qualify and quantify the sample library. each cdna library was amplified once before sequencing. sequencing was performed on an illumina hiseq x ten at biotecan co., ltd sequencer (shanghai, china). the rna-seq data were analyzed as previously described [ , ] . differentially expressed genes (with a fold change over . and p < . ) were selected. statistical analysis data are presented as the means ± sems. all experiments were repeated at least three times. statistical analysis was performed with graphpad prism . software. comparisons among groups were made using one-way analysis of variance or student's t-test. p values < . were considered significant. il- β induces enmt in huvecs by activating the nf-κb/snail pathway exposure of huvecs to il- β ( ng/ml) for h caused an obvious alteration in cellular morphology from a polygonal, cobblestone-like shape to a more spindle and fibroblast-like shape (fig. a) . quantitative rt-pcr (qrt-pcr) showed that il- β treatment reduced the mrna levels of endothelial markers (cd and cd were greatly reduced) and increased those of the mesenchymal markers α-sma and sm- α (fig. b) . to further confirm that enmt was induced by il- β, the protein levels of the endothelial or mesenchymal markers were examined by western blot assay and immunofluorescent staining. as a result, cd and cd were significantly downregulated, and α-sma and sm- α were remarkably upregulated by il- β treatment (fig. c, d) . in addition, huvec tube formation was significantly inhibited by il- β (fig. e) . consistent with the transition to a mesenchymal phenotype, huvecs treated with il- β exhibited enhanced migration capabilities when compared with untreated cells (fig. f) . exposure of huvecs to il- β activated nf-κb signaling and increased the level of snail (fig. g) . these results suggest that il- β likely elicits enmt of huvecs via nf-κb/snail signaling. we next examined the regulatory role of ptpl in the il- βinduced enmt of huvecs. ptpl was knocked down in huvecs via lentiviral vector-based sh-rnas (fig. a-c) . consequently, ptpl knockdown increased the mrna and protein expression of α-sma and sm- α and decreased cd and cd expression (fig. d, e) . the huvec tube formation capacity was significantly inhibited after ptpl silencing (fig. f ). in accordance with the transition to a mesenchymal phenotype, ptpl -knockdown huvecs displayed enhanced migration abilities compared with the control cells (fig. g) . control and ptpl -knockdown huvecs were further subjected to treatment with il- β ( ng/ml) for h. we found that knockdown of ptpl synergized with il- β to upregulate the mesenchymal proteins α-sma and sm- α and impair the expression of the endothelial biomarkers cd and cd (fig. h) . similarly, ptpl -knockdown huvecs showed much higher nf-κb activity and snail levels than control cells in response to treatment with il- β (fig. i) . these data indicate that ptpl represses the il- β-induced enmt of huvecs by counteracting nf-κb/snail signaling. ptpl inhibits the nf-κb signaling pathway by stabilizing iκbα ptpl has been predicted to dephosphorylate various proteins, including iκbα [ ] . we thus investigated whether ptpl inhibits nf-κb activity and impedes the enmt of huvecs by targeting iκbα. indeed, ptpl was coimmunoprecipitated with iκbα from huvec lysates (fig. a) . while il- β induced the phosphorylation of iκbα at tyrosine and triggered its degradation in huvecs, ptpl knockdown further promoted the phosphorylation and downregulation of the iκbα protein (fig. b) . therefore, ptpl may attenuate nf-κb signaling by dephosphorylating iκbα and increasing its half-life in vascular endothelial cells. ptpl is downregulated by il- β and hypoxia in human vascular endothelial cells we next asked how the expression of ptpl was regulated in vascular endothelial cells. we found that treatment of huvecs with il- β significantly reduced the levels of ptpl (fig. a, b) . tissue hypoxia is a risk factor for diverse clinical disorders, e.g., group ph, that pathologically involves emt [ , ] or enmt [ ] [ ] [ ] . we thus probed whether hypoxia plays a regulatory role in ptpl expression in vascular endothelial cells. our rna-seq results showed that hypoxia treatment of hpaecs induced robust changes in the expression profile of genes, including ptpl (fig. c) . the downregulation of ptpl by hypoxia in huvecs was further verified by western blot (fig. d) . these results show that ptpl downregulation by inflammatory factors or hypoxia contributes to the enmt of vascular endothelial cells. we evaluated the involvement of ptpl and the nf-κb pathway in the pathogenesis of ph. a rat model of mct-induced ph (mct-ph) was established via exposure to mct for weeks, as verified by significantly increased rvsp (fig. a) and mean pulmonary arterial pressure (mpap) (fig. b) and a significantly increased ratio of right ventricle to left ventricle plus septum (rv/lv + s) weight (fig. c) . to validate nf-κb activation in vivo, the relative mrna levels of the endothelial markers cd and cd and the mesenchymal markers α-sma and sm- -α in control and il- βtreated huvecs were assessed by qrt-pcr (each bar represents the mean ± sem. **p < . , ***p < . vs. the control group). c the protein levels of cd , cd , α-sma, and sm- -α in control and il- β-treated huvecs were determined by western blot analysis. (each bar represents the mean ± sem. *p < . , **p < . , ***p < . vs. the control group). d immunofluorescent staining of cd (green) and α-sma (red) expression in control and il- β-treated huvecs. e tube formation assays for unstimulated and il- β-exposed huvecs (each bar represents the mean ± sem. *p < . vs. the control group). f transwell assay to assess the migration of unstimulated and il- β-exposed huvecs (wach bar represents the mean ± sem. ***p < . vs. the control group). g the protein levels of p-nf-κb (p ), nf-κb, and snail were determined by western blot analysis using lysates from unstimulated and il- β-exposed huvecs (each bar represents the mean ± sem. *p < . , ***p < . vs. the control group) ptpl suppresses enmt by inhibiting il- β/nf-κb/snail xm wei et al. we generated a rat model of mct-ph characterized by abnormal media thickening (fig. d) . quantitative rt-pcr showed that the expression of pro-inflammatory cytokines, such as tumor necrosis factor-α (tnf-α), interleukin (il)- and interleukin (il)- β, was increased, whereas the level of ptpl was reduced in the lung tissue of mct-ph rats compared to control rats (fig. e, f) . we also found that the level of phosphorylated nf-κb protein was significantly elevated in the lung tissue of mct-ph rats compared to control rats (fig. g ). in addition, we collected lung tissue samples from five patients with ph and examined the expression of ptpl in the lung tissues of different patients by immunohistochemical staining. the results showed that ptpl levels were lower in the pulmonary arterial endothelial cells of ph patients than in control lung cancer patients (fig. h) . therefore, ptpl might suppress the development of ph via inhibition of nf-κb activity. ) and western blot (c, e) (each bar represents the mean ± sem. *p < . , **p < . , ***p < . , ****p < . vs. the sh-mock group). f tube formation assay for cells in a (each bar represents the mean ± sem. *p < . vs. the sh-mock group). g cell migration was assessed by transwell assays (each bar represents the mean ± sem. ****p < . vs. the sh-mock group). h, i the cells in a were treated with il- β ( ng/ml) and were subjected to western blot analysis (each bar represents the mean ± sem. *p < . , **p < . , ***p < . vs. the sh-mock group; # p < . , ## p < . vs. the sh-ptpl group) huvecs were treated with il- β ( ng/ml) for h and subjected to qrt-pcr (a) and western blot analysis (b) (each bar represents the mean ± sem. *p < . , **p < . vs. the control group). c heatmap of rna-seq data in hpaecs after hypoxia treatment (t: hypoxia; c: normoxia). d western blot analysis of huvecs exposed to hypoxia ( % o , % n , and % co ) for h (each bar represents the mean ± sem. *p < . vs. the normoxia group) in this study, we demonstrated that ptpl suppresses the il- βinduced enmt process in huvecs by impairing nf-κb/snail signaling. it has been established that enmt plays critical roles in ph [ , , ] . il- β is one of the most important proinflammatory cytokines that elicit enmt [ ] [ ] [ ] . we found that huvecs exposed to il- β could undergo enmt, as verified by the transition of cells to a spindle-like shape, varied expression of endothelial and fibroblast markers, increased migration ability, and reduced tube formation capacity. consistent with studies reporting that the nf-κb/snail pathway plays an important role in emt [ , ] , we found that il- β induced enmt in huvecs through activation of nf-κb and upregulation of snail. the activity of nf-κb is regulated by divergent stimuli dependent upon the intracellular signaling context [ , ] , which prompted us to explore the regulatory mechanisms of nf-κb activation in the inflammatory factor-induced enmt of vascular endothelial cells. ptpl is a nonreceptor-type protein tyrosine phosphatase involved in various cellular signal pathways. previous studies have found that ptpl acts as both a tumor promoter [ ] and a suppressor [ ] . a previous study showed that ptpl overexpression significantly inhibited the progression of hepatocellular carcinoma cells by inhibiting emt [ ] . in this study, we found that ptpl could inhibit il- β-induced enmt in huvecs, and knockdown of ptpl could activate nf-κb signaling and increase the level of snail. unlike previous studies showing that ptpl controls nf-κb activation via p ntr [ ] , we found that ptpl attenuates nf-κb activation by dephosphorylating iκbα at tyrosine and consequently increasing the half-life of iκbα. these findings are in agreement with a recent report that ptpl could target iκbα in ovarian cancer cells [ ] . nonetheless, given the wide crosstalk between intracellular signaling pathways and the relatively low number of protein tyrosine phosphatases compared with protein tyrosine kinases, we cannot rule out that ptpl also regulates enmt by targeting other pathways in vascular endothelial cells. both inflammation [ ] [ ] [ ] and hypoxia [ , ] contribute to the pathogenesis of various forms of ph. our study showed that hypoxia or il- β could downregulate ptpl in vascular endothelial cells, consistent with a previous finding that ptpl expression was impeded upon exposure of prostate cancer cells to hypoxia [ ] . we also found that the expression of inflammatory cytokines such as tnf-α, il- , and il- β was increased in the lung tissue of a rat model of mct-ph, adding weight to the probability that the inflammatory microenvironment and endothelial ptpl in the , and rv/(lv + s) (c) data were obtained from each group and plotted (each bar represents the mean ± sem. ****p < . vs. the control group). d he staining of paraffin-fixed lung sections for morphological analysis of pulmonary arteries from control or mct-ph rats. e-g pulmonary artery samples from control or mct-ph rats were used for qrt-pcr (e, f) and western blot (g) analyses. h immunohistochemical staining of lung tissues from ph patients and control lung cancer patients. bar, μm (each bar represents the mean ± sem. **p < . , ****p < . . vs. the control group) lung form a feedback circuit to control enmt and the pathogenesis of ph. together, the present findings demonstrated a suppressive role of ptpl in enmt and the deregulation of ptpl /nf-κb signaling in the pathogenesis of ph. epithelial-to-mesenchymal and endothelial-to-mesenchymal transition: from cardiovascular development to disease endothelial-mesenchymal transition and its contribution to the emergence of stem cell phenotype endothelial-tomesenchymal transition drives atherosclerosis progression endothelial deficiency of l reduces tumor angiogenesis and promotes vessel normalization the role of endothelial-to-mesenchymal transition in cancer progression tgf-beta signaling mediates endothelial-to-mesenchymal transition (endmt) during vein graft remodeling relaxin inhibits cardiac fibrosis and endothelial-mesenchymal transition via the notch pathway fibroblasts in kidney fibrosis emerge via endothelial-to-mesenchymal transition endothelial-mesenchymal transition in bleomycin-induced pulmonary fibrosis endothelial-to-mesenchymal transition: an evolving paradigm and a promising therapeutic target in pah targeted gene delivery of bmpr attenuates pulmonary hypertension endothelial-like cells in chronic thromboembolic pulmonary hypertension: crosstalk with myofibroblast-like cells betaine attenuates monocrotalineinduced pulmonary arterial hypertension in rats via inhibiting inflammatory response il- β and tgfβ synergistically induce endothelial to mesenchymal transition in an nfκb-dependent manner antimetastatic effects of celastrus orbiculatus on human gastric adenocarcinoma by inhibiting epithelialmesenchymal transition and nf-kappab/snail signaling pathway curcumin inhibits lps-induced emt through downregulation of nf-kappab-snail signaling in breast cancer cells genetic characterization of fas-associated phosphatase- as a putative tumor suppressor gene on chromosome q . in hepatocellular carcinoma expression of the putative tumor suppressor gene ptpn /ptpl is an independent prognostic marker for overall survival in breast cancer protein tyrosine phosphatase ptpn negatively regulates her /erbb malignant signaling high ptpn expression in high grade serous ovarian carcinoma is associated with a better patient outcome protein tyrosine phosphatase-n promotes myofibroblast resistance to apoptosis in idiopathic pulmonary fibrosis tumour-suppressive role of ptpn in hepatocellular carcinoma and its clinical significance in pulmonary arterial hypertension, reduced bmpr promotes endothelial-tomesenchymal transition via hmga and its target slug upregulation of hydroxysteroid sulfotransferase b b promotes hepatic oval cell proliferation by modulating oxysterol-induced lxr activation in a mouse model of liver injury transcriptomics and proteomics analyses of anti-cancer mechanisms of tr -an active fraction from xinjiang bactrian camel milk in esophageal carcinoma cell association of protein-tyrosine phosphatase ptp-bas with the transcription-factor-inhibitory protein ikappabalpha through interaction between the pdz domain and ankyrin repeats hypoxia-induced epithelial-mesenchymal transition and fibrosis for the development of breast capsular contracture metformin inhibits epithelial-tomesenchymal transition of keloid fibroblasts via the hif- alpha/pkm signaling pathway rhoj promotes hypoxia induced endothelial-tomesenchymal transition by activating wdr expression autophagy mediates -methoxyestradiolinhibited scleroderma collagen synthesis and endothelial-to-mesenchymal transition induced by hypoxia a hypoxia-induced vascular endothelial-to-mesenchymal transition in development of radiationinduced pulmonary fibrosis endothelial-to-mesenchymal transition in pulmonary hypertension endothelial to mesenchymal transition contributes to endothelial dysfunction in pulmonary arterial hypertension interleukin- induces major phenotypic changes in human skin microvascular endothelial cells endothelial mesenchymal transformation mediated by il- beta-induced fgf- in corneal endothelial cells inflammatory cytokines induce the transformation of human dermal microvascular endothelial cells into myofibroblasts: a potential role in skin fibrogenesis enterolactone modulates the erk/nf-kappab/snail signaling pathway in triple-negative breast cancer cell line mda-mb- to revert the tgf-beta-induced epithelial-mesenchymal transition apigenin inhibits epithelialmesenchymal transition of human colon cancer cells through nf-kappab/snail signaling pathway activators and target genes of rel/nf-kappab transcription factors good cop, bad cop: the different faces of nf-kappab ptpl is a direct transcriptional target of ews-fli and modulates ewing's sarcoma tumorigenesis the putative tumor suppressor gene ptpn /ptpl induces apoptosis through insulin receptor substrate- dephosphorylation functional interaction of fas-associated phosphatase- (fap- ) with p (ntr) and their effect on nf-kappab activation protein tyrosine phosphatase l inhibits high-grade serous ovarian carcinoma progression by targeting ikappabalpha increased interleukin- and interleukin- serum concentrations in severe primary pulmonary hypertension elevated levels of inflammatory cytokines predict survival in idiopathic and familial pulmonary arterial hypertension proinflammatory cytokine levels are linked to death in pulmonary arterial hypertension hypoxia inhibits expression and function of mitochondrial thioredoxin to promote pulmonary hypertension severe emphysema in the su /hypoxia rat model of pulmonary hypertension hypoxia leads to significant changes in alternative splicing and elevated expression of clk splice factor kinases in pc prostate cancer cells this work was supported by the national natural science foundation of china (nos. , , , and ) and the chinese postdoctoral science foundation (no. m ). we are grateful for the financial support from the national natural science foundation (no. , , , ). sql designed the experiments and prepared the initial manuscript. xmw and gw conducted the experiments. ld and nz cultivated the cells. cwl and jwx were responsible for the experimental calculations. yzz, xjz, yyz, and pz were involved in the experimental analysis. competing interests: the authors declare no competing interests. key: cord- -c jr ga authors: zhang, le-le; guo, jing; jiang, xiao-ming; chen, xiu-ping; wang, yi-tao; li, ao; lin, li-gen; li, hua; lu, jin-jian title: identification of nagilactone e as a protein synthesis inhibitor with anticancer activity date: - - journal: acta pharmacol sin doi: . /s - - - sha: doc_id: cord_uid: c jr ga norditerpenoids and dinorditerpenoids represent diterpenoids widely distributed in the genus podocarpus with notable chemical structures and biological activities. we previously reported that nagilactone e (nle), a dinorditerpenoid isolated from podocarpus nagi, possessed anticancer effects against lung cancer cells in vitro. in this study we investigated the in vivo effect of nle against lung cancer as well as the underlying mechanisms. we administered nle ( mg·kg(− )·d(− ), ip) to cb- /scid mice bearing human lung cancer cell line a xenograft for weeks. we found that nle administration significantly suppressed the tumor growth without obvious adverse effects. thereafter, rna sequencing (rna-seq) analysis was performed to study the mechanisms of nle. the effects of nle on a cells have been illustrated by go and pathway enrichment analyses. cmap dataset analysis supported nle to be a potential protein synthesis inhibitor. the inhibitory effect of nle on synthesis of total de novo protein was confirmed in click-it assay. using the pcdna -rluc-polires-fluc luciferase assay we further demonstrated that nle inhibited both cap-dependent and cap-independent translation. finally, molecular docking revealed the low-energy binding conformations of nle and its potential target riok . in conclusion, nle is a protein synthesis inhibitor with anticancer activity. norditerpenoids and dinorditerpenoids are diterpenoids widely distributed in the genus podocarpus with notable chemical structures [ , ] . to date, a number of norditerpenoids and dinorditerpenoids have been identified and isolated from the genus podocarpus, and some have been documented to possess various biological activities, such as antifungal, anti-inflammatory, and antiatherosclerotic activities [ ] [ ] [ ] . previously, we reported that nagilactone e (nle), a dinorditerpenoid isolated from the seeds of podocarpus nagi, exhibits anticancer activity in lung cancer in vitro. nle treatment inhibited the proliferation of lung cancer cells via g phase cell cycle arrest mediated by the downregulation of cyclin b [ ] . in addition, nle inhibited tgf-β induced epithelial-mesenchymal transition (emt), migration and invasion in a cells via inhibition of the tgf-β-smad / signaling pathway [ ] . however, the anticancer effects of nle have not been studied in vivo, and the precise mechanisms of nle remain to be further clarified. investigations of the mechanisms of natural products are generally difficult due to their diverse biological activities and multiple targets. rapidly developed modern systematic methods, including network pharmacology and sequencing technologies, light the way for mechanistic studies and the further development of natural products [ , ] . in recent years, a series of massively parallel "next generation" sequencing technologies have been well developed and dramatically changed the landscape of the genetic, biological, and medical sciences [ , ] . rna sequencing (rna-seq) analysis is a mature and powerful sequencing-based approach for the characterization and quantification of transcriptomes [ ] . it enables researchers to obtain precise and comprehensive information on absolute transcript expression levels within a single experiment. rna-seq together with systematic bioinformatic analysis have been increasingly used for various applications, including investigations of the mechanisms of drug action and gene function [ , ] . in this study, the in vivo anticancer effects of nle were studied in a mouse xenograft model. human lung cancer a cells treated with nle were subjected to rna-seq and bioinformatic analyses. nle was shown to inhibit protein synthesis, and the inhibitory effect of nle on de novo protein synthesis in a cells was further confirmed using the click-it assay. reagents nle with a purity of approximately . % was isolated from the seeds of p. nagi as documented in a previous study [ ] . cycloheximide; anti-rabbit igg; anti-mouse igg; and primary antibodies against nrf , p , stat , atf and gapdh were obtained from cell signaling technology (beverly, ma, usa). primary antibody against β-actin was purchased from santa cruz biotechnology (dallas, ca, usa). ripa lysis buffer was procured from beyotime biotechnology (shanghai, china). cisplatin was purchased from selleck chemicals (houston, tx, usa). the reagents used for the click-it assay, click-it™ aha (l-azidohomoalanine), biotin alkyne (peg carboxamide-propargyl biotin), a click-it™ protein reaction buffer kit, and anti-streptavidin-hrp, were purchased from life technologies (eugene, or, usa). the bicistronic reporter plasmid pcdna -rluc-polires-fluc constructed by nahum sonenberg was obtained from addgene (plasmid # ) [ ] . cell culture human a lung cancer cells were acquired from the american type culture collection (atcc, rockville, md, usa). a cells were cultured in rpmi- medium containing % fetal bovine serum and % penicillin-streptomycin (gibco, thermo fisher scientific, carlsbad, ca, usa). the in vivo assays were performed at tongji medical college, huazhong university of science and technology. all of the animal experiments were approved by the laboratory animal center of huazhong university of science and technology and the ethics committee. cb- /scid mice (male, weight - g) were provided by beijing vital river laboratory animal technology co., ltd (beijing, china). human lung cancer a cells ( × cells) were used to subcutaneously inoculate the right armpit of each mouse. seven days later, the mice were randomly divided into different groups and intraperitoneally administered one of the following treatments daily for weeks: ( ) vehicle control group (n = ), ( ) mg/kg/d nle treatment group (n = ). the nle dose was selected according to the results of preliminary experiments. nle was dissolved in a solution of polyoxyethylene castor oil:ethanol ( : , v/v) at a concentration of mg/ml and sonicated for min to promote dissolution of the compound, which was diluted with physiological saline immediately before use. cisplatin was administered at . mg/kg every other day as a positive control. tumor sizes were measured with vernier calipers, and tumor volumes were calculated with the following formula: a × b × π/ , where a is the length and b is the width. the weights of the mice were recorded every other day. after the mice were sacrificed, tumor weight was measured. hematoxylin-eosin (h&e) staining was applied to analyze pathological changes in the liver, spleen and kidney. rna-seq analysis exponentially growing a cells were trypsinized, resuspended, seeded into six-well plates and cultured for h. the cells were treated with or μm nle for another h. then, total rna was extracted from the cells by using trizol reagent (life technologies, shanghai, china). quality control (qc) of the rna samples was carried out using agarose gel electrophoresis and an agilent bioanalyzer. cdna libraries were constructed from the rna samples using the illumina truseq mrna library kit, and × paired-end rna-seq was performed by wuxi apptec (shanghai, china) using an illumina hiseq x sequencing system. gene ontology (go) and pathway enrichment analyses differentially expressed genes (degs) were subjected to go and pathway enrichment analyses. gene set enrichment analysis of go terms and kyoto encyclopedia of genes and genomes (kegg) pathway analysis were performed using the online david . bioinformatics resources. quantitative real-time pcr assay total rna was extracted from whole cells with trizol reagent, and cdna was then synthesized by using a transcriptor first strand cdna synthesis kit (roche, germany). qpcr was carried out using a sybr green kit (roche, germany), and results were detected by using the mx p quantitative pcr system (agilent technologies, usa). the primers used are listed in table . western blotting analysis western blotting analysis was performed to determine the expression levels of the proteins as documented in a previous study [ ] . click-it assay exponentially growing a cells were trypsinized, resuspended, seeded into six-well plates and grown to approximately % confluence. then, the cells were pretreated with nle, chx, or cis for min. after treatment, the medium was removed, and the cells were washed three times with pbs. then, methionine-free rpmi- medium (with nle/chx/cis) was added and incubated for another min. then, the labeling compound aha was added to the medium at a final concentration of μm and incubated for h. after washing three times with pbs, cell lysates were harvested by the addition of lysis buffer ( % sodium dodecyl sulfate in mm tris-hcl, ph . ). after centrifugation, the protein contents of the cell lysates were detected using a bca tm protein assay kit. click reactions were carried out by using μg of cell lysate, biotin alkyne, and a click-it™ protein reaction buffer kit according to the manufacturer's instructions. thereafter, the biotinylated proteins were purified via methanol/chloroform extraction and subjected to western blotting. membranes were probed with anti-streptavidin-hrp antibody. the unprocessed lysates were also subjected to western blotting analysis and probed for β-actin. protein expression was detected using ecl select western blot detection reagent (ge healthcare, buckinghamshire, uk). luciferase assay exponentially growing a cells were transfected with pcdna -rluc-polires-fluc plasmid using turbofect transfection reagent (thermo scientific, lithuania). after transfection for h, the cells were trypsinized, seeded in -well plates, incubated overnight, and further treated with nle or chx for h. luciferase activity was measured using a dual-luciferase reporter assay system (promega, madison, wi, usa) in accordance with the manufacturer's instructions. [ ] . the ligand-binding pocket was selected with graphical tools in the icm software to create the boundaries for the docking search. the chemical structure of the compound was input with mol files for docking. in the docking calculation, default parameters were applied to calculate the potential energy maps of the receptor. the compound was imported into the icm and filed as an index project. conformational sampling was based on the monte carlo procedure, and finally, the ligand with the lowest energy and most favorable orientation was selected. statistical analysis data are displayed as the mean ± standard deviation. statistical analyses were conducted by using one-way analysis of variance with graphpad prism (graphpad software, inc., ca, usa). p < . and p < . indicated statistically significant differences. an a cell lung cancer xenograft mouse model was constructed using cb- /scid mice to evaluate the anticancer efficacy of nle in vivo. as shown in fig. a , the intraperitoneal injection of mg/kg/d nle markedly suppressed the growth of the tumors. after weeks of treatment, all of the mice in the nle treatment group (n = ) as well as the control group (n = ) were alive. however, in the cisplatin treatment group, mouse body weight had decreased significantly, and cisplatin treatment was terminated since two mice in the cisplatin treatment group died after days of treatment. mice in the control and nle treatment groups were then sacrificed, their tumors were photographed, and the average tumor weights were measured (fig. b) . the tumor weight of the control group was . ± . g, whereas that of the nle-treated group was . ± . g, representing a significant reduction in weight by . %. compared with the body weights of mice in the control group, nle treatment did not have an obvious effect on mouse body weight (fig. c) . moreover, h&e staining indicated obvious pathological damage in the liver tissue sections of the model group, indicating lesion metastasis of a orthotopic tumor to the liver tissue (fig. d) . nle treatment reversed tumor metastasis and ameliorated hepatic pathology. overall, these results indicated that nle could suppress tumor growth in vivo. rna-seq analysis of differentially expressed genes to identify degs in a cells after nle treatment, rna-seq analysis was performed. gene abundance estimation was carried out using rsem software (v . . ) [ ] . tens of thousands of expressed genes were detected, and the r package edger (v . . ) was used to compare the expression levels between the sample pairs (nle vs control groups) [ ] . the criteria for deg detection in this study were a fold change > and false discovery rate < . . a heatmap of the gene expression levels of all the samples is shown in fig. a. a total of genes were specifically upregulated, and genes were specifically downregulated in a cells treated with nle compared with control cells. to better understand the potential biological processes associated with the effects of nle in lung cancer a cells, go analysis was performed using the online david . bioinformatics resource. among "biological process" (bp) terms, the upregulated genes were mainly enriched in the regulation of transcription, rrna processing and ribosome biogenesis terms, while the downregulated genes were mainly enriched in the oxidationreduction, cholesterol biosynthetic processes, and lipid metabolic process terms. with respect to "molecular function" (mf) terms, the upregulated genes were mainly enriched in terms related to binding (rna, nucleic acid, protein, dna, etc.), while the downregulated genes were mainly enriched in the calcium ion binding and oxidoreductase activity terms. the top most significantly enriched bp and mf terms are listed in fig. b , c, respectively. thereafter, kegg pathway enrichment analysis of the degs was carried out. the top most significantly enriched kegg pathways in a cells after nle treatment are listed in fig. d . among these significantly enriched pathways, ribosome biogenesis in eukaryotes appeared to be the most enriched pathway in the upregulated genes, while metabolic pathways appeared to be the most enriched pathways in the downregulated genes. in addition, other pathways (rna transport, lysosome, circadian rhythm, biosynthesis of antibiotics and steroids) were enriched in the degs, indicating the multiple functions of nle in lung cancer a cells. analysis of the rna-seq data using the cmap dataset the connectivity map (cmap) dataset (https://clue.io/cmap) is a powerful and convenient database that contains data on transcriptional responses to chemical, genetic and disease perturbation in multiple cell lines [ ] [ ] [ ] . the cmap dataset can help characterize new chemical entities as well as understand their potential molecular mechanisms by using gene expression signatures derived from treatment with a small molecule. to clarify the mechanisms underlying the anticancer effect of nle in a lung cancer cells, we further analyzed the degs using the cmap dataset. briefly, the top most upregulated genes (p < . ) and top most downregulated genes (p < . ) were subjected to cmap database analysis to discover compounds with similar effects on gene expression. among the results obtained from cmap analysis, the "median score" referred to the connectivity score, which was used to rank the similarity between instances in the database with nle. those at the top of the ranking with higher scores were more similar to nle (the redder the color, the higher the degree of similarity) based on their gene expression signatures. surprisingly, the highest connectivity score among perturbational classes was assigned to protein synthesis inhibitors ( . ) (fig. a) . specifically, the highest connectivity score was assigned to emetine ( . ) (a protein synthesis inhibitor) (fig. b ). in addition, the following three protein synthesis inhibitors were given relatively high scores of . , . , and . : cephaeline, homoharringtonine, and cycloheximide, respectively (fig. b) . in total, the nle gene expression signatures were most similar to those of protein synthesis inhibitors, indicating that nle is a potential protein synthesis inhibitor. since short-lived proteins exhibit a short half-life, their expression levels might decrease rapidly upon the inhibition of de novo protein synthesis. rna-seq analysis revealed a large number of genes that were upregulated after nle treatment. herein, the short-lived proteins nrf , p , stat and atf were selected for further verification to confirm their upregulated expression, as shown by rna-seq. briefly, a cells were treated with nle ( , , and μm) for or h, and then the mrna and protein levels of nrf , p , stat , and atf were determined by qpcr and western blot analyses, respectively. as shown in fig. a , the mrna levels of nrf , p , stat , and atf were upregulated after nle treatment, which was consistent with the results obtained by rna-seq analysis. in contrast, the protein levels of nrf , p , stat , and atf were obviously decreased after nle treatment (fig. b) , indicating that nle might inhibit de novo synthesis of these proteins. the click-it assay confirmed nle as a protein synthesis inhibitor to confirm the results obtained by cmap dataset analysis as well as the inhibitory effect of nle on the expression of short-lived proteins, the inhibitory effect of nle on de novo protein synthesis in lung cancer a cells was further assessed by using the click-it assay. for the click-it assay, methionine-free medium was utilized for cell pretreatment and drug treatment; thus, newly synthesized proteins would be tagged with the labeling compound aha, an amino acid analog of methionine containing an azido moiety, since methionine is the amino acid universally used to initiate new protein synthesis [ , ] . thereafter, biotinylation was carried out by using a copper-catalyzed "click" alkyne reaction, and biotin was subsequently detected with hrp-coupled streptavidin. herein, chx and cis were used as positive and negative controls, respectively. as shown in fig. a , compared with the control group, treatment with the methionine analog aha for h followed by biotinylation allowed the remarkable visualization of newly synthesized proteins with ultra streptavidin-hrp antibody. cis pretreatment had no effect on the visualization of newly synthesized proteins, while pretreatment with the well-known protein synthesis inhibitor chx ( μm) completely abolished de novo protein synthesis. in addition, pretreatment with μm nle partially inhibited de novo protein synthesis, while μm nle pretreatment completely abolished de novo protein synthesis in a cells. the dual pcdna -rluc-polires-fluc reporter luciferase assay was performed to evaluate the effects of nle on cap-dependent and cap-independent translation. in the pcdna -rluc-polires-fluc plasmid, the rluc cistron undergoes cap-dependent translation, while the fluc cistron undergoes cap-independent translation as it is directed by the poliovirus ires [ ] . as shown in fig. b, nle ( and μm) significantly inhibited the fluorescence intensity of both firefly and renilla luciferase as well as positive control cells treated with chx ( μm), indicating that nle inhibited both cap-dependent and cap-independent translation in a cells. to discover the targets of nle, molecular docking was carried out. several proteins have been identified as potential targets of nle, among which riok exhibited the highest binding affinity with nle; therefore, the inhibitory effect of nle on riok was elucidated. molecular docking was performed with icm-pro . . modeling software (molsoft llc, san diego, ca) [ ] with the crystal structure of the riok kinase domain as the template (pdb code: hk ) [ ] . nle at the lowest-energy binding conformation is shown in fig. a . as seen from the generated docking model, nle fit well into a relatively shallow pocket formed between the winged helix-turn-helix (whth) domain and n-terminal lobe (nlobe) of riok . the binding of nle in this allosteric site was suggested to lock the kinase in an inactive conformation, thus inhibiting its activity. this binding pocket was relatively hydrophobic, and hydrophobic interactions were predicted between phe and ring a, tyr and ring c, and ser and ring b. in addition, several hydrogen-binding interactions between arg with the carbonyl group of ring d and met and lys with the carbonyl group of ring c were predicted (fig. b) . we previously reported the inhibitory effects of nle on the proliferation, tgf-β -induced emt, migration and invasion of lung cancer cells in vitro [ , ] . the in vivo study herein further confirmed that the intraperitoneal injection of mg/kg/d nle suppressed tumor growth in a xenograft model without obvious toxicity. moreover, nle treatment also inhibited tumor metastasis, as evidenced by h&e staining. thus, nle has been demonstrated to be an effective anticancer candidate both in vitro and in vivo. however, during this study, we also found that nle showed relatively poor solubility in solvent, which might somehow have affected the efficacy of nle in this in vivo study. thus, in future work, structural modification based on analysis of the structureactivity relationship or pharmaceutical approaches, such as nanoparticle technology and micelle preparation, should be taken into consideration to further improve the solubility and drug-like properties of nle [ ] . to further clarify the mechanisms of nle, rna-seq and bioinformatic analyses were carried out. in particular, as evidenced by cmap dataset analysis, a number of substances and proteins were determined to produce gene expression signatures markedly similar to that following nle treatment. emetine is an fdaapproved compound for amebiasis that has been documented for decades as a protein synthesis inhibitor via its binding to the s fig. cmap dataset analysis of rna-seq data suggested that nle is a protein synthesis inhibitor. the top most upregulated genes (p < . ) and top most downregulated genes (p < . ) were recruited and analyzed using the cmap database to compare them with differentially expressed genes classified by perturbational class (a) and compound (b). subunit of the ribosome [ , ] . meanwhile, very high connectivity scores (scores > . ) were also awarded to a class of well-known protein synthesis inhibitors, such as cephaeline, the structural desmethyl analog of cephaeline [ ] ; homoharringtonine; and the most commonly used protein synthesis inhibitor cycloheximide [ ] . thereafter, the inhibitory effect of nle on de novo protein synthesis in a cells was further confirmed using the click-it assay. chx is a both cap-dependent and capindependent protein synthesis inhibitor [ ] . we further performed a luciferase assay with the reporter plasmid pcdna -rluc-polires-fluc and found that nle and chx inhibited both cap-dependent and cap-independent translation. the results of our previous study indicated that nle might inhibit the translation of cyclin b since it decreased cyclin b protein levels without inhibiting the transcription or promoting the degradation of cyclin b [ ] . moreover, in our study on the inhibitory effects of nle on tgf-β -induced emt in nsclc cells, we found that nle treatment inhibited the expression of tβri and a series of mesenchymal and invasiveness markers, and we also deduced that de novo protein synthesis inhibition might partially contribute to the inhibitory effects of nle on some regulators responsible for the tgf-β induced upregulation of tβri [ ] . the results obtained here are also consistent with our previous hypothesis. previously, by comparing cytotoxicity profiles, nagilactone c, an analog of nle, was reported to be a translation inhibitor [ ] . nagilactone c inhibited the elongation of protein synthesis without preventing ribosome binding to mrna templates. nagilactone c treatment inhibited the eef- α-dependent binding of aminoacyl-trna to the ribosomal a site, as well as peptidyl transferase activity [ ] . nle was also demonstrated to be a protein synthesis inhibitor in our study. moreover, the precise effects of nle on absolute transcript expression levels were clarified by using rna-seq. systematic bioinformatic analysis through go (bp and mf terms) and kegg pathway analysis has provided comprehensive gene expression signatures following treatment with nle and can help us better understand the fig. nle inhibited the expression of short-lived proteins. a cells were treated with nle for or h. a the mrna levels of nrf , p , stat , and atf were determined by qpcr assay. b the protein levels of nrf , p , stat , and atf were detected by western blot analysis. *p < . , **p < . , compared with the control group. nle inhibited both cap-dependent and cap-independent de novo protein synthesis. a a cells were pretreated with nle, chx, or cis at the indicated concentration for min, then washed three times with pbs and further cultured in methionine-free rpmi medium (with nle/chx/cis) for another min. then, the labeling compound aha was added to the medium at a final concentration of μm for h of treatment. aha was withheld from a control well to assess the background signal. after washing three times with pbs, cell lysates were harvested, and click reactions were performed using μg of cell lysate, biotin alkyne, and a click-it™ protein reaction buffer kit. the biotinylated proteins were purified and subjected to western blotting. b a cells were transfected with the pcdna -rluc-polires-fluc plasmid. after transfection for h, the cells were trypsinized, seeded in -well plates, incubated overnight, and further treated with nle or chx for h. luciferase activity was measured using the dual-luciferase reporter assay system. biological activities and mechanisms of nle. according to the results, more than one thousand genes were specifically upregulated after nle treatment. in our opinion, some of these genes might be upregulated via a feedback mechanism. moreover, nle might promote the expression of a number of genes, as it is a natural compound with multiple targets. molecular docking was carried out for target prediction, and nle exhibited an outstanding binding affinity with riok . riok belongs to an evolutionarily conserved family of atypical kinases that are related to the ribosome maturation process [ ] . a recent study indicated that the expression level of riok can predict clinical outcome in nsclc and might serve as a target for nsclc therapy [ ] . moreover, we then performed kaplan-meier analysis of riok expression and overall survival in some other cancer types. kaplan-meier survival curves indicated that high riok expression was correlated with poorer overall survival compared with low riok expression in patients with liver hepatocellular carcinoma, kidney renal papillary cell carcinoma, head and neck squamous cell carcinoma, and bladder carcinoma (fig. s ). based on these results, we deduce that riok is a potential target of nle, which should be further confirmed by surface plasmon resonance, isothermal titration calorimetry, or other techniques in future work. in summary, nle was demonstrated to suppress tumor growth in a xenograft model without obvious toxicity. rna-seq analysis was carried out to further investigate the underlying mechanisms of nle. the effects of nle on a cells were illustrated by go and pathway enrichment analyses. cmap dataset analysis supported nle as a protein synthesis inhibitor, which was further confirmed by the click-it assay. molecular docking predicted riok as a target of nle. taken together, the results of our study demonstrated that nle is a protein synthesis inhibitor. norditerpenoids and dinorditerpenoids from the seeds of podocarpus nagi as cytotoxic agents and autophagy inducers antiproliferative constituents of the roots of ethiopian podocarpus falcatus and structure revision of α-hydroxynagilactone f and nagilactone i effect of nagilactone e on cell morphology and glucan biosynthesis in budding yeast saccharomyces cerevisiae new podolactones from the seeds of podocarpus nagi and their anti-inflammatory effect a novel small molecule liver x receptor transcriptional regulator, nagilactone b, suppresses atherosclerosis in apoe-deficient mice downregulation of cyclin b mediates nagilactone e-induced g phase cell cycle arrest in non-small cell lung cancer cells nagilactone e suppresses tgf-β -induced epithelial-mesenchymal transition, migration and invasion in non-small cell lung cancer cells systems pharmacology strategies for anticancer drug discovery based on natural products target identification and mechanism of action in chemical biology and drug discovery ten years of next-generation sequencing technology next generation sequencing technology: advances and applications. bba-mol basis dis uncovering the complexity of transcriptomes with rna-seq using transcriptome sequencing to identify mechanisms of drug action and resistance gfpt -expressing cancer-associated fibroblasts mediate metabolic reprogramming in human lung adenocarcinoma e-bp , a new member of the eukaryotic initiation factor e-binding protein family effects of alisol b -acetate on ovarian cancer cells: g phase cell cycle arrest, apoptosis, migration and invasion inhibition icm-a new method for protein modeling and design: applications to docking and structure prediction from the distorted native conformation edger: a bioconductor package for differential expression analysis of digital gene expression data differential expression analysis for sequence count data the connectivity map: using gene-expression signatures to connect small molecules, genes, and disease the connectivity map the connectivity map: a new tool for biomedical research application of mass spectrometry profiling to establish brusatol as an inhibitor of global protein synthesis a metabolic alkene reporter for spatiotemporally controlled imaging of newly synthesized proteins in mammalian cells crystal structure of human riok bound to a specific inhibitor anticancer drug discovery from chinese medicinal herbs enzymic and nonenzymic translocation by yeast polysomes the molecular basis of emetine resistance in chinese hamster ovary cells: alteration in the s ribosomal subunit emetine inhibits zika and ebola virus infections through two molecular mechanisms: inhibiting viral replication and decreasing viral entry cycloheximide: aspects of inhibition of protein synthesis in mammalian cells brusatol overcomes chemoresistance through inhibition of protein translation eukaryotic protein synthesis inhibitors identified by comparison of cytotoxicity profiles high expression of riok and nob predict human non-small cell lung cancer outcomes llz and jjl designed this study. lgl provided the compound nle. llz and jg performed the experiments. llz, hl and jjl drafted the paper. llz, xmj, hl and jjl analyzed the data. all the authors participated in the revision and improvement of the paper. the online version of this article (https://doi.org/ . /s - - - ) contains supplementary material, which is available to authorized users.competing interests: the authors declare no competing interests. key: cord- -dyk mr q authors: zheng, yong; deng, yan; gao, jian-mei; lv, chun; lang, ling-hu; shi, jing-shan; yu, chang-yin; gong, qi-hai title: icariside ii inhibits lipopolysaccharide-induced inflammation and amyloid production in rat astrocytes by regulating ikk/iκb/nf-κb/bace signaling pathway date: - - journal: acta pharmacol sin doi: . /s - - - sha: doc_id: cord_uid: dyk mr q β-amyloid (aβ) is one of the inducing factors of astrocytes activation and neuroinflammation, and it is also a crucial factor for the development of alzheimer’s disease (ad). icariside ii (ics ii) is an active component isolated from a traditional chinese herb epimedium, which has shown to attnuate lipopolysaccharide (lps)-induced neuroinflammation through regulation of nf-κb signaling pathway. in this study we investigated the effects of ics ii on lps-induced astrocytes activation and aβ accumulation. primary rat astrocytes were pretreated with ics ii ( , , and μm) or dexamethasone (dxms, μm) for h, thereafter, treated with lps for another h. we found that ics ii pretreatment dose dependently mitigated the levels of tumor necrosis factor-alpha (tnf-α), interleukin- beta (il- β), inducible nitric oxide synthase (inos), cyclooxygenase- (cox- ) in the astrocytes. moreover, ics ii not only exerted the inhibitory effect on lps-induced iκb-α degradation and nf-κb activation, but also decreased the levels of aβ( – ), aβ( – ), amyloid precursor protein (app) and beta secretase (bace ) in the astrocytes. interestingly, molecular docking revealed that ics ii might directly bind to bace . it is concluded that ics ii has potential value as a new therapeutic agent to treat neuroinflammation-related diseases, such as ad. alzheimer's disease (ad) is a progressive neurodegenerative disease characterized by beta-amyloid (aβ) peptide fibrils, which are extracellular depositions of a particular protein and are accompanied by extensive neuroinflammation [ ] [ ] [ ] . a number of studies have reported that inflammation, which may precede amyloid deposition, exerts vital effects in the pathogenesis of ad [ , ] . moreover, inflammatory mediators increase the expression of amyloid precursor protein (app) and the formation of aβ and upregulate beta-secretase activity [ ] . therefore, antiinflammatory drugs may prevent or treat ad by inhibiting neuroinflammation, thereby reducing the production or deposition of aβ [ ] [ ] [ ] [ ] . however, there are still no ideal antiinflammatory drugs to prevent or treat ad. astrocytes and microglia are important components of homeostasis in the brain [ ] . when the brain is exposed to undesirable environmental conditions, both astrocytes and microglia, which are crucial perpetrators of inflammation and potential neuronal dysfunction [ , ] , acquire special "response" or "activation" phenotypes [ , ] . in ad, the interaction between microglia and astrocytes may be of great significance for the development of neurodegenerative disease. in particular, astrocytes represent the most plentiful cell type in the brain and play an imperative role in maintaining the homeostasis of the central nervous system [ ] [ ] [ ] . under physiological conditions, astrocytes play a role in supporting and separating nerve cells [ ] [ ] [ ] [ ] . nevertheless, under neuroinflammatory conditions, the excessive activation of astrocytes is involved in the inflammatory response through its ability to release multiple molecules and further lead to neurodegenerative diseases [ ] [ ] [ ] . epimedium is a traditional chinese herb used for the treatment of cardiovascular diseases, osteoporosis, and sexual and neurological disorders [ ] . icariside ii (ics ii) is known as one of the major active pharmaceutical ingredients of epimedium, and it has been indicated to have an extensive range of pharmacological effects, including antiinflammatory [ , ] , anticancer [ , ] , antioxidative [ , ] , and antiaging activities [ ] . our previous study found that ics ii attenuates streptozotocin-or aβ - -induced cognitive deficits in rats by increasing the number of surviving neurons in the hippocampus [ ] or inhibiting neuronal apoptosis and reducing pde protein expression [ ] . in our previous in vivo studies, we proved that ics ii exerts beneficial effects on lipopolysaccharide (lps)-induced neuroinflammation by regulating the tlr /myd /nf-κb signaling pathway in rats and inhibiting lps-induced astrocyte overactivation [ ] . however, whether ics ii can suppress neuroinflammatory responses in vitro remains unclear. thus, in the current study, we investigated the effects of ics ii on lipopolysaccharide (lps)-induced neuroinflammation in primary astrocytes and the underlying molecular mechanism. ics ii (purity ≥ %) was obtained from nanjing zelang medical technology corporation ltd (nanjing, china). dmem/f was obtained from hyclone (logan, ut, usa), and fetal bovine serum (fbs) was purchased from gibco (thermo fisher scientific, ma, usa). dexamethasone (dxms) and lps (l ) (escherichia coli o :b ) were purchased from sigma-aldrich (st louis, mo, usa). dxms and lps were dissolved in normal saline at concentrations of mm and mg/ml, respectively, and ics ii was dissolved in dimethyl sulfoxide (dmso) at a concentration of mm. anticyclooxygenase- (cox- ) (#ab ), anti-nitric oxide synthase (inos) (#ab ), antinuclear factor-κb (nf-κb) (p ) (#ab ), anti-p-nf-κb (p ) (#ab ), anti-iκb-α (#ab ), anti-ikk-α (#ab ), anti-p-ikk-α (#ab ), anti-ikk-β (#ab ), anti-p-ikk-β (#ab ), and anti-β-site app cleaving enzyme (bace ) (#ab ) antibodies were purchased from abcam (cambridge, uk). anti-app (#ab b) was purchased from bbi life sciences corporation (shanghai, china). tumor necrosis factor-alpha (tnf-α) enzyme-linked immunosorbent assay (elisa) kits (an interleukin- beta (il- β) elisa kit, an aβ - elisa assay kit (jl ), and an aβ - elisa kit (jl )) were purchased from shanghai jianglai biotechnology (shanghai, china). a nitric oxide (no) detection kit (s ) was purchased from beyotime biotechnology (shanghai, china). astrocyte culture and drug treatment sprague-dawley rats ( ± g) were housed under a -h-light/ dark cycle at a humidity of % ± % and a temperature of °c. all animals were given free access to water and food. animal experiments were performed in compliance with the state committee of science and technology of the people's republic of china order no. of november , , and the protocol was approved by the experimental animal ethics committee of the zunyi medical university. primary astrocytes were extracted from the safe concentration range of ics ii within h (n = ). c the safe concentration range of ics ii within h (n = ). d the safe concentration range of ics ii within h (n = ). * p < . , ** p < . versus control group neonatal rat brains as previously described [ ] . primary astrocytes were isolated from the cerebral cortex of -h-old neonatal rats. briefly, suckling rats were repeatedly disinfected with % alcohol three times, and then the brains were removed, and the brain tissues were separated. then, the cerebral cortex was collected under low temperatures, and the meninges and blood vessels were slightly removed. the cerebral cortex was fully dissociated by the addition of ml of trypsin, the flask was treated with polylysine, and the suspension was spread in a culture flask at a cell density of × cells/ml. the differential adherence method was used to remove other types of cells in the tissue, and then astrocytes were cultured in dmem/f containing % fbs. the dmem/f was changed every days until the th day. the primary cells were shaken at °c at a constant temperature to remove other glial cells, such as microglia and oligodendrocytes. thereafter, the astrocytes were fixed with % paraformaldehyde, incubated with an anti-gfap antibody ( : , abcam), and visualized using an alexa fluor-conjugated secondary antibody ( : , abcam). more than % of the cultured astrocytes were identified by gfap staining, and the astrocytes were pretreated with different concentrations of ics ii or dxms ( μm) for h. thereafter, they were treated with lps for another h. since dxms is an effective and classical antiinflammatory drug, it was selected as a positive control drug to evaluate the antiinflammatory effect of ics ii, and μm was chosen as the desired concentration of dxms according to a previous study [ ] . cell viability was assessed using the -[ , -dimethylthiazol- -yl]- , -diphenyl tetrazolium bromide (mtt) assay as described in our previous study [ ] . in brief, astrocytes were seeded in a -well plate at × cells/well and treated as described above. then, μl of mtt ( mg/ml) was added to fbs-free medium and cultured for another h. the mtt was removed, and the cells were lysed with μl of dmso in each well. the optical density was measured at nm using a microplate reader. the results of the treatments were expressed as a percentage of the control. in brief, astrocytes were inoculated into -well plates at a density of × cells/well and pretreated with ics ii ( , , μm) for h. lps ( mg/ml) and ics ii were added to the plates for h. thereafter, the culture medium was collected and centrifuged for min at × g. the supernatants were then collected and used to measure tnf-α, il- β, aβ - , and aβ - levels using elisa kits. all data were obtained from at least three independent experiments. measurement of nitric oxide (no) astrocytes were inoculated in -well plates at a density of × cells/well and pretreated with ics ii ( , , μm) for h. lps ( mg/ml) and ics ii were added to the plates for h. the accumulation of nitrite in the supernatant was evaluated using the griess reaction. each μl of supernatant was reacted with an equal volume of griess reagent and cultured for min at room temperature. the absorbance was detected in a microplate absorbance reader at a wavelength of nm, and a series of known concentrations of sodium nitrite was utilized as a standard. western blot analysis western blot analysis was performed as described in our previous study [ ] . briefly, astrocytes were treated as mentioned above, homogenized with protein extraction solution, and lysed for min on ice. the lysate was centrifuged for min at × g. ) . then, the blots were subjected to the corresponding horseradish peroxidase-conjugated anti-rabbit or mouse immunoglobulin g. thereafter, immunoreactive proteins were measured using an enhanced chemiluminescence western blotting detection system. statistical analysis all data were analyzed by spss . statistics software and are expressed as the mean ± sd. the data were analyzed using oneway anova followed by the post hoc least significant difference. all data were obtained from at least three independent experiments. p < . was considered significant. we extracted the cultured cells and identified them by staining for gfap, which is a specific marker of astrocytes [ ] . the results showed that astrocytes made up % of the cultured cells (fig. a) . furthermore, the results showed that ics ii ( . , . , . , . , , or μm) for , , or h had no effect on the astrocytes; however, ics ii ( , , or μm) for , , or h was cytotoxic to the astrocytes. since the concentration of dmso used to dissolve ics ii in the experiment did not exceed . %, it was assumed that the concentration of dmso itself was not toxic to the cells and that the toxicity to the cells therefore resulted from the high concentration of ics ii. thus, a concentration of ics below μm was considered to be the safe concentration range (fig. b-d) . thereafter, , , and μm ics ii were used in subsequent experiments, and dxms ( μm) was used as a positive control agent. the effect of ics ii on the lps-induced production of tnf-α and il- β in astrocytes was determined using an elisa assay. the results showed that ics ii ( , , or μm) slightly mitigated the production of tnf-α (fig. a) and il- β (fig. b) . following lps stimulation, the production of tnf-α and il- β was substantially elevated compared with that in the control group, while ics ii markedly alleviated lps-induced tnf-α and il- β overproduction in a concentration-dependent manner. the effect of ics ii on lps-induced no production and inos and cox- expression in astrocytes was determined using the griess reaction and western blot analysis, respectively. the results showed that ics ii decreased lps-induced no production in astrocytes (fig. c) . moreover, inos and cox- expression levels were dramatically increased after lps insult. however, ics ii ( , , and μm) induced a concentration-dependent decrease in the expression of inos and cox- in astrocytes compared with that in the lps group ( fig. d-f ). the results suggested that ics ii not only suppressed the lpsinduced phosphorylation of p (fig. a, b) but also prevented the nuclear translocation of p (fig. c-f ). furthermore, ics ii also suppressed the lps-induced degradation of iκb-α (fig. a, b) , ikk-α, and ikk-β (fig. c, d) . these results indicate that ics ii mitigates the lps-induced activation of nf-κb via inhibiting iκb-α phosphorylation and the translocation of p to the nucleus. the effect of inflammation on amyloid formation in vitro was also investigated because neuroinflammation can cause amyloid production, whereas the aberrant activation of astrocytes is a major source of neuroinflammation. astrocytes provide both mechanical and metabolic support to neurons, regulating the environment in which they function. to determine the relationship between neuroinflammation and amyloidogenesis, we investigated whether the antiinflammatory effect of ics ii can result in antiamyloidogenesis. as shown in fig. a , b, when the cells were unstimulated, they expressed low protein levels of app and bace , whereas the protein expression of app and bace increased in response to lps after h. in addition, ics ii also decreased lps-induced aβ - and aβ - secretion into the culture media of astrocytes (fig. c, d) . in astrocytes, we also found that ics ii inhibited the lps-induced expression of app and bace , as well as aβ - and aβ - levels in a concentration-dependent manner. these results further indicate that the amyloidogenic pathway can be promoted by neuroinflammatory stimulation and that the antiinflammatory effect of ics ii can result in antiamyloidogenesis. interestingly, mock molecular docking was used to verify whether ics ii binds to bace protein, and the results showed that the binding energy of ics ii and bace was − . kcal/mol, which confirmed that ics ii can bind to bace , as the standard threshold for a molecule to bind to a protein is thought to be greater than or equal to − kcal/mol (fig. a, b) . we further investigated the supposed binding modes and interactions within the amino acid pocket, including gly , gly , gly , thr , tyr , gly , lys , asp , and phe (fig. c, d) . taken together, these findings show that ics ii may directly affect bace and thus exert neuroprotective effects. the present study revealed that ( ) ics ii protects against lpsinduced inflammation in primary-cultured astrocytes; ( ) the inhibitory effect of ics ii is due to regulation of the ikk/iκb/nf-κb signaling pathway; and ( ) ics ii decreases aβ - and aβ - levels by downregulating app and bace expression (fig. ) . lps is an effective component of gram-negative bacilli endotoxin, which can cause a series of inflammatory reactions in the body and is widely applied to establish animal or cellular inflammatory models. moreover, astrocytes represent the most abundant cell type in the central nervous system, and they exert a variety of physiological functions through close association with neurons and other brain structures. similar to microglia, the immune and inflammatory properties of astrocytes, which can promote the secretion of various neuroinflammatory factors, such as tnf-α and il- β, are activated by lps. accumulating evidence has demonstrated that the production of multiple neuroinflammatory factors, including tnf-α, il- β, and inos, can activate the nf-κb pathway, resulting in neuroinflammation [ ] . moreover, the excessive production of neuroinflammatory factors leads to neuronal damage through the activation of glial cells. notably, astrocytes play a key role in central nervous system inflammation by producing cytokines, such as tnf-α, il- β, and no, leading to neuronal injury [ ] . therefore, the suppression of astrocyte activation may be an effective treatment for neuroinflammation-related diseases [ ] . our findings showed that lps induces an increase in proinflammatory factors, which is consistent with the results of previous studies [ , ] . ics ii significantly inhibited the lps-induced accumulation of tnf-α, il- β, no, inos, and cox- in primary culture astrocytes. in addition, these effects were not due to the cytotoxic effect of ics ii, as evidenced by the observation that there was no effect on astrocytes at concentrations up to μm. moreover, ics ii also inhibited inos and cox- protein notably, nf-κb not only regulates a variety of inflammatory factors, such as il- β and tnf-α but also plays a key role in mediating cox- and inos expression [ ] . nf-κb (p /p ) heterodimers exist in the cytoplasm of resting cells. however, under stimulation by lps, the phosphorylation of ikk was induced, which subsequently phosphorylates the iκb protein, resulting in the release of nf-κb (p /p ) heterodimers. then, nf-κb-p translocates from the cytoplasm to the nucleus, thereby promoting the release of proinflammatory factors, including tnf-α and il- [ , ] . consistent with these studies, the present study showed that lps induces the phosphorylation of nf-κb-p , iκb, and ikk. however, ics ii abolished these changes, suggesting that ics ii exerts potent antiinflammatory effects, at least partly through modulating the ikk/iκb/nf-κb pathway. app is a membrane-intrinsic protein expressed in a variety of tissues and is closely related to ad. app can be cleaved by α, β, and γ proteases, and the continuous action of β-protease and γprotease can cause app to be cleaved to produce aβ [ ] . however, aβ can cause the formation of senile plaques in the brain and the apoptosis of neuronal cells, thereby causing ad. most importantly, mounting evidence has shown that neuroinflammation is related to the accumulation of aβ in the brains of ad patients [ , ] . in addition, app is first cleaved by β-secretase at its β-cleavage site and is then proteolyzed by the second enzyme, γ-secretase, to produce aβ - and aβ - . in particular, the aβ - and aβ - peptides are involved in the amyloidogenic pathway; aβ - is the most plentiful species, and aβ - shows the strongest neurotoxicity [ , ] . of note, bace is an important rate-limiting enzyme in the app-aβ pathway and plays an important role in the production of aβ. bace was overexpressed in the brains of animal models of ad [ ] . in contrast, in the brains of bace knockout mice, the levels of aβ and sappβ are decreased [ ] . in addition, activated astrocytes that release mediators can induce app and bace expression, thereby stimulating aβ production [ ] [ ] [ ] [ ] . notably, nf-κb mediates the accumulation of app and the expression of bace to increase aβ production [ , ] . in this study, lps not only augmented the production of aβ - and aβ - but also upregulated app and bace , and these findings are consistent with those of previous studies [ , ] . however, ics ii reversed these changes, revealing that ics ii exerts inhibitory effects on neuroinflammation via suppressing the nf-κb/bace signaling pathway, and this was also evidenced by molecular docking. in conclusion, the current study revealed that ics ii exerts inhibitory effects on lps-induced inflammation in astrocytes through the ikk/iκb/nf-κb/bace signaling pathway, and thus ics ii may be a promising therapeutic agent for neuroinflammatory diseases, including ad. elevated cns inflammation in patients with preclinical alzheimer's disease is 'friendly fire' in the brain provoking alzheimer's disease? modulation of inflammation in transgenic models of alzheimer's disease review: systemic inflammation and alzheimer's disease rodent models of neuroinflammation for alzheimer's disease inflammation combined with ischemia produces myelin injury and plaque-like aggregates of myelin, amyloid-beta and abetapp in adult rat brain inflammation, antiinflammatory agents, and alzheimer's disease: the last years effects of non-steroidal anti-inflammatory drug treatments on cognitive decline vary by phase of pre-clinical alzheimer disease: findings from the randomized controlled alzheimer's disease anti-inflammatory prevention trial nonsteroidal anti-inflammatory drugs and peroxisome proliferator-activated receptor-gamma agonists modulate immunostimulated processing of amyloid precursor protein through regulation of beta-secretase nsaid exposure and risk of alzheimer's disease: an updated meta-analysis from cohort studies brain inflammatory cascade controlled by gut-derived molecules synergistic actions of microglia and astrocytes in the progression of alzheimer's disease immune memory in the brain transient acidification and subsequent proinflammatory cytokine stimulation of astrocytes induce distinct activation phenotypes autotaxin downregulates lps-induced microglia activation and pro-inflammatory cytokines production emerging roles of astrocytes in neural circuit development astrocytes mediate synapse elimination through megf and mertk pathways snapshot: astrocytes in health and disease astrogliopathology in neurological, neurodevelopmental and psychiatric disorders astrocytic activation generates de novo neuronal potentiation and memory enhancement stimulating astrocytes to remember does rapid and physiological astrocyte-neuron signalling amplify epileptic activity? elusive roles for reactive astrocytes in neurodegenerative diseases astrocyte physiopathology: at the crossroads of intercellular networking, inflammation and cell death the contribution of activated astrocytes to abeta production: implications for alzheimer's disease pathogenesis chemical and pharmacological investigations of epimedium species: a survey icariside ii, a broad-spectrum anti-cancer agent, reverses beta-amyloid-induced cognitive impairment through reducing inflammation and apoptosis in rats icariside ii, a novel phosphodiesterase- inhibitor, attenuates streptozotocin-induced cognitive deficits in rats synergistic anti-cancer effects of icariin and temozolomide in glioblastoma icariin regulates the proliferation and apoptosis of human ovarian cancer cells through microrna- by targeting pten, reck and bcl- icariside ii, a novel phosphodiesterase inhibitor, protects against h o -induced pc cells death by inhibiting mitochondria-mediated autophagy icariside ii alleviates oxygen-glucose deprivation and reoxygenation-induced pc cell oxidative injury by activating nrf / sirt signaling pathway icariin, a natural flavonol glycoside, extends healthspan in mice icariside ii, a phosphodiesterase- inhibitor, attenuates beta-amyloid-induced cognitive deficits via bdnf/trkb/creb signaling icariside ii attenuates lipopolysaccharide-induced neuroinflammation through inhibiting tlr /myd / nf-kappab pathway in rats inhibitory effect of ent-sauchinone on amyloidogenesis via inhibition of stat -mediated nf-kappab activation in cultured astrocytes and microglial bv- cells dexamethasone inhibits inducible nitric-oxide synthase expression and nitric oxide production by destabilizing mrna in lipopolysaccharide-treated macrophages trilobatin protects against oxidative injury in neuronal pc cells through regulating mitochondrial ros homeostasis mediated by ampk/nrf /sirt signaling pathway role of gfap in cns injuries a review: inflammatory process in alzheimer's disease, role of cytokines traf upregulation in spinal astrocytes maintains neuropathic pain by integrating tnf-alpha and il- beta signaling neuroinflammation in the pathogenesis of alzheimer's disease. a rational framework for the search of novel therapeutic approaches anti-neuroinflammatory effect of mc , a novel coumarin compound from condiment murraya, through inhibiting lipopolysaccharide-induced traf -tak -nf-kappab, p /erk mapks and jak -stat /stat pathways trilobatin attenuates the lpsmediated inflammatory response by suppressing the nf-kappab signaling pathway tangeretin exerts antineuroinflammatory effects via nf-kappab modulation in lipopolysaccharidestimulated microglial cells modifications and trafficking of app in the pathogenesis of alzheimer's disease accelerated amyloid deposition in the brains of transgenic mice coexpressing mutant presenilin and amyloid precursor proteins amyloid-beta protein dimers isolated directly from alzheimer's brains impair synaptic plasticity and memory beta-amyloid neurotoxicity requires fibril formation and is inhibited by congo red amyloid-beta peptides are generated in mitochondria-associated endoplasmic reticulum membranes morin reverses neuropathological and cognitive impairments in appswe/ps de mice by targeting multiple pathogenic mechanisms in vivo beta-secretase inhibition leads to brain abeta lowering and increased alpha-secretase processing of amyloid precursor protein without effect on neuregulin- inflammation in alzheimer disease: driving force, bystander or beneficial response large-scale production of soluble recombinant amyloid-beta peptide - using cold-inducible expression system regional and sub-regional differences in hippocampal gabaergic neuronal vulnerability in the tgcrnd mouse model of alzheimer's disease chitosan oligosaccharides inhibit/ disaggregate fibrils and attenuate amyloid beta-mediated neurotoxicity gene structure and organization of the human beta-secretase (bace) promoter increased nf-kappab signalling up-regulates bace expression and its therapeutic potential in alzheimer's disease neuro-inflammation induced by lipopolysaccharide causes cognitive impairment through enhancement of beta-amyloid generation qhg and jss designed the experimental protocols. yz carried out all the studies except the western blot analysis, which was performed by cl, lhl, and yd. yz wrote the manuscript with help from qhg, cyy, and jmg. competing interests: the authors declare no competing interests. key: cord- -cu qcen authors: qi, fei-long; wang, mei-fang; li, bo-zhao; lu, ze-fang; nie, guang-jun; li, su-ping title: reversal of the immunosuppressive tumor microenvironment by nanoparticle-based activation of immune-associated cells date: - - journal: acta pharmacol sin doi: . /s - - - sha: doc_id: cord_uid: cu qcen immunotherapy that activates the host immune system to reverse immunosuppression has emerged as a new generation of cancer treatment in both preclinical studies and clinical trials. although immunotherapy has shown significant achievements in the treatment of various cancers, it faces challenges that limit its further evolution such as poor permeation and modest responsiveness. the development of nanoparticle drug delivery system has provided an opportunity to overcome these drawbacks and to achieve optimized immunotherapy. based on the research of our group, we here introduce the new strategies being employed using nanoscale intelligent drug delivery systems to enhance the effects of cancer immunotherapy. we also provide a perspective on the further possible application of nanoparticles in more effective antitumor immunotherapy. effective treatment of cancer is an ongoing challenge for both basic researchers and clinicians worldwide. traditional chemotherapy and radiotherapy have serious disadvantages, such as systemic toxicity and multidrug resistance [ ] [ ] [ ] [ ] . in more recent years, cancer immunotherapy, which effectively kills cancer cells by enhancing immune system function in patients, has emerged as a new therapeutic approach [ , ] . it is well known that the tumor microenvironment maintains an immunosuppressive state [ , ] . there are many reasons for this, including: ( ) tumor cells hyperactivate immune checkpoints, escaping immune surveillance [ ] ; ( ) tumor antigens that are heterogeneous and have high mutation rates make surrounding immune cells unable to recognize and kill tumor cells [ ] ; and ( ) tumor cells have low immunogenicity and are not easily recognized by antigenpresenting cells (apcs) [ ] . therefore, the intrinsic immune system cannot clear tumor cells in the same manner that it eliminates ordinary foreign substances. many drawbacks of traditional therapy have been overcome by this new emerging therapy, which exhibits a rapid response, powerful effects, and long-lasting efficacy [ , ] . however, one drawback of immunotherapy is its unsatisfactory effects on deep solid tumors [ ] . consequently, to improve cancer immunotherapy, there is an urgent demand for novel strategies to reverse and remodel immunosuppressive microenvironments [ ] . the development of nanomedicine has provided a novel approach to enhance immunotherapeutic efficacy and minimize adverse toxicities [ , ] . in contrast to free drugs, nanodrugs ( - nm) have a prolonged plasma half-life and high accumulation in tumor sites due to passive or/and active targeting effects [ ] . moreover, the easy functionalization of nanodrug surfaces makes it possible to release drugs solely in response to the tumor microenvironment, ensuring high biosafety and improved therapeutic efficiency [ ] . considering these outstanding advantages, nanomedicine presents a promising strategy for advancing tumor immunotherapy. in recent years, our group has been committed to the investigation of intelligent nanomedicine to regulate the tumor immune microenvironment [ , ] . this review introduces three aspects of cancer immunotherapy based on the work of our group and provides a perspective on the combination of nanomedicine and immunotherapy. system improves the therapeutic efficiency of immune checkpoint inhibitors in the tumor microenvironment under physiological conditions, immune checkpoint pathways maintain immune balance and protect against autoimmune diseases [ , ] . however, tumor cells exploit this function, causing immune cells to recognize them as normal cells. immune checkpoint inhibitors interfere with the camouflage of the surface of tumor cells and disrupt the receptor-ligand interaction so that t cells can be activated to eliminate tumor cells. ctla- was the first immune checkpoint protein discovered to antagonize a t cell costimulatory signal and turn off t cells as a negative regulator [ ] . the continuous success of pd- /pd-l inhibitors has attracted widespread attention in recent years [ , ] . many commercially available pd- /pd-l inhibitors have been used clinically with inspiring curative effects [ ] . however, due to the dynamic and complicated nature of the tumor environment, only a small number of tumor-infiltrating lymphocytes are recruited to tumor sites [ ] . in addition, the rapid proliferation of tumor cells and abnormality of tumor blood vessels lead to hypoxia in tumor tissues and overexpression of hypoxia-inducible factor (hif)- α, consequently resulting in chemoresistance and poor immunotherapeutic outcomes [ , ] . accordingly, remodeling the tumor microenvironment will be important to enhance the therapeutic efficacy of immune checkpoint inhibitors [ , ] . many nanodrug delivery systems have achieved encouraging therapeutic efficacy in tumor inhibition through the codelivery of immune checkpoint inhibitors with other drugs or compounds expected to regulate the tumor microenvironment [ , ] . to enhance the efficacy of anti-pd- antibodies, many strategies involving hypoxia alleviation and combination with other immunomodulatory drugs have been proposed. zou et al. prepared a multifunctional biomimetic nanoplatform containing zeolitic imidazolate framework (zif)- coated with a tumor cell membrane as the guidance part and antigen stimulation to codeliver catalase and doxorubicin [ ] . oxygen generation by catalysis of catalase could alleviate hypoxic conditions and enhance the antitumor effects of doxorubicin. consistent with previous research, this nanoplatform was shown to decrease the expression of pd-l on the tumor cell membrane by inhibiting hif- α [ ] . encouraged by this outcome, zou et al. immediately combined this nanoplatform with an anti-pd- antibody for immunotherapy. the effects of the combination therapy were significant compared with those of other treatments, indicating that hypoxia alleviation using nanomedicine in the tumor microenvironment could enhance the effects of immunotherapy. in addition, zhang et al. generated engineered magnetosomes to achieve synergistic antitumor effects by integrating a checkpoint antibody and a tgf-β inhibitor into fe o magnetic nanoclusters. in this system, the anti-pd- antibody for activating exhausted t cells and the tgf-β inhibitor for modulating macrophage polarization from the m phenotype to the m phenotype had a synergistic effect that created an immunogenic microenvironment. this treatment could increase the amount of h o in polarized m macrophages, while fe o magnetic nanoclusters induced the fenton reaction to produce reactive oxygen species (ros). the generated ros subsequently resulted in lethal ferroptosis in tumor cells. the anticancer effects were evaluated in seven different tumor models, and all of the results were extraordinarily satisfactory [ ] . in addition to overexpression of pd- /pd-l and ctla , overexpression of indoleamine , -dioxygenase (ido) in tumors has also been shown to be associated with immune tolerance [ ] . ido is a unique rate-limiting enzyme that regulates metabolism of tryptophan into kynurenine and is widely expressed in most organs except the liver [ ] . tryptophan is an essential amino acid that influences the proliferation and activation of t cells through two distinct molecular stress-response pathways (the gcn /eif- α and glk /mtorc pathways) [ , ] . therefore, a reduction in the tryptophan level in the tumor microenvironment is not conducive to effector t cell activity [ , ] . in addition, kynurenine products produced by ido- are able to bind to aryl hydrocarbon receptors (ahr) as an endogenous ligand. activation of ahr induces the differentiation of regulatory t cells (tregs), which suppress antitumor immunity and help tumors escape immune surveillance [ ] . therefore, several ido inhibitors have been developed to prevent both the consumption of tryptophan and the accumulation of kynurenine, including the first-line ido inhibitor nlg [ ] . in our prior studies, we designed an amphiphilic peptide consisting of deap, plglag (a short peptide substrate of mmp- ), and d ppa- (a bioactive d-peptide antagonist of pd-l ) [ ] . this peptide self-assembled into nanoparticles to encapsulate nlg in the hydrophobic domain under physiological ph conditions. intravenously administered nanoparticles swelled and exposed specific peptide substrates to abundant mmp- after protonation of deap in the acidic tumor microenvironment. following structural disintegration of the nanoparticles, nlg and d ppa- were consequently released to inhibit the metabolism of tryptophan and block pd-l , respectively (fig. ) . transmission electron microscopy showed that the peptide-assembling nanoparticles could be hydrolyzed rapidly in the tumor microenvironment. in vivo treatment indicated that the combination immunotherapeutic group showed the greatest antitumor efficacy and promoted t cell replication and activation to the greatest extent. natural killer (nk) cells, which account for %- % of the circulating lymphocytes in the innate immune system, can spontaneously lyse tumor cells without requiring prior sensitization [ ] ; therefore, nk cells have unique roles in cancer immune surveillance and clearance [ , ] . following activation counterbalanced by various receptors, nk cells express activating receptors, including natural cytotoxicity receptors (e.g., nkp and nkp ) and fc-gamma receptors (e.g., cd ), that recognize tumor cells or virus-infected cells [ ] . in addition, nk cells are also equipped with some inhibitory receptors, such as killer immunoglobulin-like receptors, to protect normal cells [ ] . finally, nk cells also express some cytokine (e.g., il- and tgf-β) receptors, which regulate their function [ , ] . nk cells recognize foreign antigens through cell stress-induced recognition and missing-self recognition, which blocks the transmission of inhibitory signals or upregulates interactions between activating receptors and stress ligands on tumor cells [ , ] . after recognition, perforin and granzyme b are released by activated nk cells, resulting in rapid tumor cell lysis [ ] . however, many clinical and preclinical studies have reported dysfunction of nk cells, especially in various solid tumors [ ] , which is primarily due to the immunosuppressive microenvironment [ ] . nk cells directly lyse tumor cells via antibody-dependent cellmediated cytotoxicity (adcc), wherein antibodies play key roles in the activation and adcc of nk cells [ , ] . fcγriiia (cd ), which is abundantly expressed on the nk cell surface, has been reported to trigger adcc by specifically binding to an antibody-coated antigen [ , ] . however, the heterogeneity of tumor antigens severely limits this recognition process, resulting in a reduction in the adcc effect [ ] . therefore, our group combined fc fragments or therapeutic antibodies with ph (low) insertion peptides (phlips) [ ] . a weakly acidic ph has been validated as the most significant feature of many solid tumors and has been widely applied in nanoparticle construction [ ] . under low-ph conditions, phlips can transform coil conformations into α-helices and anchor themselves into tumor cell membranes so that the fc fragments or therapeutic antibodies are exposed [ ] . the subsequent recognition and binding of recruited nk cells can then trigger adcc more efficiently. in vitro, phlip-fc-mediated adcc at ph . is superior to that at ph . . in vivo, both primary tumor and metastatic disease models showed enhanced therapeutic effects, and the number of activated nk cells was increased more than three times with phlip-fc therapy than with control treatment and led to distinctly outstanding therapeutic effects (fig. ) . given the excellent insertion capacity of phlips, our group is also investigating whether phlips can be applied to car-t cell therapy, increasing the possible applications of this therapy in solid tumors. zheng et al. also designed immunomodulatory core-shell assembled nanoparticles to activate nk cells in situ. the phresponsive shell pmpc-b-papm/glu can disassemble following exposure to the acidic tumor microenvironment. the naked bifunctional core nbsa-pba-igg immediately binds sialic acid expressed on the tumor cell membrane to allow igg to activate nk cells. these safe and effective immunomodulatory nanoparticles can avoid immune-related adverse events in nontarget tissues and exert immunotherapeutic effects exclusively on tumors [ ] . nanoparticle-engineered nk cells have also been extensively investigated, including nk cells with expression of an egfrtargeted car on the cell membrane realized through gene delivery and recruitment and infiltration of nk cells into tumors elevated by magnetic traction [ , ] . all of these approaches indicate that nanoparticles have broad applications in nk cellmediated cancer immunotherapy. dendritic cells (dcs) are the primary apcs in the human body [ ] . they have two responsibilities in the immune system; first, they catch and process tumor antigens, and second, they present processed antigens and stimulate t cells or other lymphocytes [ , ] . dcs are classified as immature and mature [ ] . only mature dcs possess the ability to induce antitumor immunity [ ] . as a consequence, activated dcs may be the key to reversing the immunosuppressive tumor microenvironment [ ] [ ] [ ] . it is generally accepted that tumor vaccines enable targeted delivery of tumor-associated antigens (taas) or adjuvants to dcs and induce long-lasting antitumor immune responses [ ] [ ] [ ] [ ] . nevertheless, it is difficult for dcs to absorb soluble antigens; phagocytosis of antigen-loaded nanoparticles is preferred [ ] [ ] [ ] . nanoparticles with sizes similar to those of pathogens can simulate endocytosis of taas [ ] [ ] [ ] . on account of this advantage, the application of nanoparticles will be important in the development of tumor vaccines delivering taas to dcs [ ] [ ] [ ] . in recent years, biologically derived nanomaterials, such as exosomes, have received increasing attention [ ] . exosomes, which are vesicles secreted by parent cells, are widely used in disease diagnosis and treatment [ ] [ ] [ ] [ ] . in our previous study, we prepared two types of dc-derived membrane vesicles (dc-mvs) that bore antigens from b or llc tumor cells [ ] . a dc-mv vaccine could lead to the activation of specific cytotoxic t lymphocytes and exhibited powerful antitumor effects when administered to tumor-bearing mice. consequently, dc-mvs as antitumor vaccines exhibit an excellent antigen presentation ability. in tumor rejection studies, dc-mvs containing dual tumor antigens have been shown to be superior to those containing a single antigen (fig. a) . recently, biomimetic nanoparticles coated with cancer cell membranes have emerged as a rising star in nanomedicine [ , ] . because of the abundance of tumor antigens on membranes, these nanoparticles are more suitable for application as tumor vaccines [ , ] . kroll et al. constructed a tumor vaccine that used the b -f mouse melanoma cell membrane as the shell exposing an external tumor antigen to encapsulate the core, which was a plga nanoparticle that encapsulated cpg, a common adjuvant [ ] . the mature apcs and activated t cells triggered by this vaccine eliminated tumor cells efficiently in female c bl/ nhsd mice. while exploring whether the immune activation caused by this vaccine enhances immunotherapeutic efficiency, researchers found that combination of this vaccine with both anti-ctla and anti-pd antibodies significantly reinforced the curative effects compared with checkpoint blockade cocktail alone (fig. b) . therefore, tumor vaccines can be considered an effective tool to relieve immunosuppression and increase immunotherapeutic efficiency, especially with the development of personalized tumor vaccines in the future [ ] [ ] [ ] . in addition to use of the tumor membrane, the application of bacteria in cancer immunotherapy has also received widespread attention [ ] . we currently have unpublished data showing that a hybrid membrane-based tumor vaccine that consists of both a tumor membrane and a bacterial cytoplasmic membrane exhibits great potential as an adjuvant. nanoparticle drug delivery systems have been widely used as well-known drug carriers in tumor immunotherapy. in addition to the three areas described above, our group continues to conceive nanomedicine strategies to achieve the following: promote the polarization of macrophages from the m phenotype to the m phenotype, eliminate the physical barrier around tumors prior to immune activation, and inhibit the differentiation of myeloidderived suppressor cells. the combination of immunotherapy with other strategies is believed to produce optimal results. nanodelivery systems can subtly integrate different strategies to target the tumor microenvironment and release drugs or effectors. however, some nanoparticles are too complex to be applied for clinical diagnosis and therapy. to solve this problem, in our view, nanoparticles should be designed with a multifunctional component serving as both the bioactive and building elements. for instance, it is better to construct peptide-based self-assembled nanoparticles with an amphipathic peptide against pd-l than to conjugate an anti-pd-l antibody to presynthesized nanoparticles through additional building elements for blocking pd-l [ , ] . therefore, the construction of nanomedicine materials should never be regarded as only the integration of many components. as the components and steps increase, the probability of us achieving large-scale stable production and fast translation into clinical practice decreases. as mentioned by researchers in the medical field, we should always remember that our original intention is to invent a real drug to save lives, not to create sophisticated artwork. achieving the most powerful effects with the simplest system should be the most important consideration in future nanomedicine development. an approach to chemotherapy-associated toxicity molecular chess? hallmarks of anti-cancer drug resistance protection against radiotherapy-induced toxicity functions and mechanisms of circular rnas in cancer radiotherapy and chemotherapy resistance cancer immunotherapy: the beginning of the end of cancer cancer immunotherapy comes of age immunosuppressive strategies that are mediated by tumor cells immune crosstalk in cancer progression and metastatic spread: a complex conversation role of the tumor microenvironment in pd-l /pd- -mediated tumor immune escape elements of cancer immunity and the cancer-immune set point potential applications of nanoparticles for tumor microenvironment remodeling to ameliorate cancer immunotherapy cancer immunotherapy: opportunities and challenges in the rapidly evolving clinical landscape the journey from discoveries in fundamental immunology to cancer immunotherapy car-t cells: the long and winding road to solid tumors the influence of microenvironment on tumor immunotherapy combining nanomedicine and immunotherapy improving cancer immunotherapy through nanotechnology targeting cancer cells with nanotherapeutics and nanodiagnostics: current status and future perspectives nanotechnology platforms for cancer immunotherapy smart nanotherapeutic targeting of tumor vasculature tumor microenvironment targeting and responsive peptide-based nanoformulations for improved tumor therapy the application of nanotechnology in immune checkpoint blockade for cancer treatment immune checkpoint blockade: a common denominator approach to cancer therapy a new member of the immunoglobulin superfamily-ctla- immune checkpoint blockade therapy for cancer: an overview of fda-approved immune checkpoint inhibitors the blockade of immune checkpoints in cancer immunotherapy current perspectives in cancer immunotherapy ways to enhance lymphocyte trafficking into tumors and fitness of tumor infiltrating lymphocytes hypoxia-modified cancer cell metabolism hypoxic stress: obstacles and opportunities for innovative immunotherapy of cancer the next hurdle in cancer immunotherapy: overcoming the non-tcell-inflamed tumor microenvironment immunogenic cell death in cancer and infectious disease regulatory t celltargeted hybrid nanoparticles combined with immuno-checkpoint blockage for cancer immunotherapy dual targeting nanoparticle stimulates the immune system to inhibit tumor growth a multifunctional biomimetic nanoplatform for relieving hypoxia to enhance chemotherapy and inhibit the pd- /pd-l axis mir- inhibited aerobic glycolysis in gastric cancer via hif- alpha regulation engineering magnetosomes for ferroptosis/immunomodulation synergism in cancer targeting indoleamine- , -dioxygenase in cancer: scientific rationale and clinical evidence the role of indoleamine , -dioxygenase in immune suppression and autoimmunity gcn kinase in t cells mediates proliferative arrest and anergy induction in response to indoleamine , -dioxygenase ido inhibits a tryptophan sufficiency signal that stimulates mtor: a novel ido effector pathway targeted by d- -methyl-tryptophan ido as a drug target for cancer immunotherapy: recent developments in ido inhibitors discovery ido in the tumor microenvironment: inflammation, counter-regulation, and tolerance discovery of ido inhibitors:from bench to bedside sequentially responsive therapeutic peptide assembling nanoparticles for dual-targeted cancer immunotherapy natural killer cells and tumor metastasis targeting natural killer cells in cancer immunotherapy nk cells: an attractive candidate for cancer therapy controlling natural killer cell responses: integration of signals for activation and inhibition activating and inhibitory receptors of natural killer cells natural killer cells: development, maturation, and clinical utilization natural killer cells and other innate lymphoid cells in cancer kir downregulation by il- / / unleashes human nk cells from kir/hla-i inhibition and enhances killing of tumor cells mechanisms of natural killer cell-mediated cellular cytotoxicity natural killer cell dysfunction in hepatocellular carcinoma and nk cell-based immunotherapy natural killer cell-based immunotherapy for cancer: advances and prospects nk cell-fc receptors advance tumor immunotherapy nkmediated antibody-dependent cell-mediated cytotoxicity in solid tumors: biological evidence and clinical perspectives targeting nk-cell checkpoints for cancer immunotherapy antibody therapy of cancer enhanced natural killer cell immunotherapy by rationally assembling fc fragments of antibodies onto tumor membranes tumor microenvironmentactivatable prodrug vesicles for nanoenabled cancer chemoimmunotherapy combining immunogenic cell death induction and cd blockade applications of phlip technology for cancer imaging and therapy in situ modification of the tumor cell surface with immunomodulating nanoparticles for effective suppression of tumor growth in mice multifunctional nanoparticles for genetic engineering and bioimaging of natural killer (nk) cell therapeutics magnetic delivery of fe o @-polydopamine nanoparticle-loaded natural killer cells suggest a promising anticancer treatment dysfunction of antigen processing and presentation by dendritic cells in cancer dendritic cell-based immunotherapy dendritic cell therapy in cancer treatment; the stateof-the-art properties of immature and mature dendritic cells: phenotype, morphology, phagocytosis, and migration taking dendritic cells into medicine dendritic cells and cancer immunotherapy empowering dendritic cell cancer vaccination: the role of combinatorial strategies tissue-resident memory cd + t cells amplify anti-tumor immunity by triggering antigen spreading through dendritic cells vaccination of patients with b-cell lymphoma using autologous antigen-pulsed dendritic cells dendritic cell-based immunotherapy: state of the art and beyond re-engineered cd receptor enables potent pharmacological activation of dendritic-cell cancer vaccines in vivo cancer immunotherapy: dendritic-cell vaccines on the move engineering nanoparticles for targeted remodeling of the tumor microenvironment to improve cancer immunotherapy nanoparticle approaches against bacterial infections nanoparticle-based immunotherapy for cancer vaccine delivery: a matter of size, geometry, kinetics and molecular patterns nanoparticle-based vaccine delivery for cancer immunotherapy virus-sized vaccine delivery systems nanoparticles in vaccine delivery targeting human dendritic cells in situ to improve vaccines intratumoral dendritic cells in the anti-tumor immune response exosome biochemistry and advanced nanotechnology for next-generation theranostic platforms a microfluidic exosearch chip for multiplexed exosome detection towards blood-based ovarian cancer diagnosis a sensitive detection assay based on signal amplification technology for alzheimer's disease's early biomarker in exosome tumor exosome-based nanoparticles are efficient drug carriers for chemotherapy dendritic cells loaded with tumor derived exosomes for cancer immunotherapy a membrane vesicle-based dual vaccine against melanoma and lewis lung carcinoma biomimetic metal-organic framework nanoparticles for cooperative combination of antiangiogenesis and photodynamic therapy for enhanced efficacy biomimetic amplification of nanoparticle homing to tumors biomimetic nanoparticle vaccines for cancer therapy biohybrid vaccines for improved treatment of aggressive melanoma with checkpoint inhibitor nanoparticulate delivery of cancer cell membrane elicits multiantigenic antitumor immunity cancer immunotherapy and breaking immune tolerance: new approaches to an old challenge personalized tumor vaccines keep cancer in check vaccines combined with immune checkpoint antibodies promote cytotoxic t-cell activity and tumor eradication adjuvant effect of bacterial outer membrane vesicles with penta-acylated lipopolysaccharide on antigen-specific t cell priming this work was supported by grants from the national key r&d program of china ( yfe and yfa ) and the national natural science foundation of china ( and ). competing interests: the authors declare no competing interests. key: cord- -p fdc authors: liu, can-zhao; li, fei-ya; lv, xiao-fei; ma, ming-ming; li, xiang-yu; lin, cai-xia; wang, guan-lei; guan, yong-yuan title: endophilin a regulates calcium-activated chloride channel activity via selective autophagy-mediated tmem a degradation date: - - journal: acta pharmacol sin doi: . /s - - - sha: doc_id: cord_uid: p fdc tmem a ca( +)-activated chloride channel (cacc) plays an essential role in vascular homeostasis. in this study we investigated the molecular mechanisms underlying downregulation of tmem a cacc activity during hypertension. in cultured basilar artery smooth muscle cells (basmcs) isolated from k c renohypertesive rats, treatment with angiotensin ii ( . − μm) dose-dependently increased endophilin a levels and decreased tmem a expression. similar phenomenon was observed in basilar artery isolated from k c rats. we then used whole-cell recording to examine whether endophilin a could regulate tmem a cacc activity in basmcs and found that knockdown of endophilin a significantly enhanced cacc activity, whereas overexpression of endophilin a produced the opposite effect. overexpression of endophilin a did not affect the tmem a mrna level, but markedly decreased tmem a protein level in basmcs by inducing ubiquitination and autophagy of tmem a. ubiquitin-binding receptor p (sqstm ) could bind to ubiquitinated tmem a and resulted in a process of tmem a proteolysis in autophagosome/lysosome. these data provide new insights into the regulation of tmem a cacc activity by endophilin a in basmcs, which partly explains the mechanism of angiotensin-ii-induced tmem a inhibition during hypertension-induced vascular remodeling. tmem a/ano is the molecular identity of the ca + -activated chloride channel (cacc) in eukaryotic cells [ ] [ ] [ ] [ ] . the tmem amediated cacc has been reported to regulate a variety of physiological functions, including neuronal excitability, nociception, transepithelial ion transport, and gastrointestinal peristalsis [ ] [ ] [ ] [ ] [ ] . our recent study found that cacc activity in the cardiovascular system, as well as tmem a expression level, was decreased in basilar artery smooth muscle cells (basmcs) isolated from twokidney two-clip ( k c) renohypertensive rats, possibly caused by a high concentration of angiotensin ii during hypertension [ ] . although diverse physiological and pathological functions have been described, regulatory mechanisms of the tmem a channel have rarely been reported. calmodulin, one of the many modulators, has been identified as a factor contributing to the ca + dependence of tmem a channel activity [ ] [ ] [ ] . recently, another intriguing study reported that secreted calcium-activated chloride channel regulator could modulate tmem a-dependent calcium-activated chloride currents by stabilizing tmem a on the cell surface in hek t cells [ ] , which gives rise to new insight into seeking proper adaptor proteins required for tmem a cacc modulation. a recent proteomics study identified several endogenous proteins implicated in protein trafficking, folding, and stability that interact with tmem a, including snare complexes such as syntaxins and syntaxin-binding proteins, as well as ezrin-radixin-moesin scaffolding proteins [ ] . these studies reveal the importance of the regulation of tmem a distribution and stability on cacc activity. identification of appropriate modulators in different tissues is crucial to precisely understand tmem a functions. endophilin a is a member of the endophilin protein family that is widely expressed in eukaryotic cells and profoundly exists in smooth muscle cells compared with the other family members [ ] . endophilins are composed of two conserved functional domains, including a sizeable n-terminal bar (bin-amphiphysin-rvs) domain, which can induce and stabilize membrane curvature upon dimerization, and a c-terminal sh (src homology ) domain, which can recognize and bind to proline-rich domains (prds). there are other undiscovered domains that mediate essential functions as well [ ] [ ] [ ] . evidence has shown that endophilin a plays a vital role in regulating clathrin-independent endocytosis and clathrindependent endocytosis due to its unique protein structure [ ] . in addition, amino acid residues - mediate the direct interaction of the endophilin-ca + channel complex, whereas e , which is flanked with a prd, is required to form a binding site for ca + and can affect the conformation of the endophilin a protein in a ca + -dependent manner, eventually modulating the formation of the endophilin a -ca + channel complex [ ] . based on multiple related functions of endophilin a , we tested the hypothesis that endophilin a could regulate tmem a cacc activity. in this study, we identified endophilin a as a vital regulator involved in tmem a cacc activity in smooth muscle cells. for primary basmc isolation, -week-old sprague-dawley rats were used. endophilin a transgenic mice were generated as described previously [ ] . all experiments were approved by and conducted in accordance with the ethical guidelines of the sun yat-sen university animal experimentation committee and were in complete compliance with the national institutes of health guide for the care and use of laboratory animals. efforts were made to minimize any pain or discomfort, and the minimum number of animals was used. cell culture rat basmcs were isolated and cultured from rat basilar arteries using the explant method as previously described [ , ] . briefly, -week-old male sprague-dawley rats ( ~ g) were anesthetized with pentobarbital sodium ( mg/kg, intraperitoneally). the basilar arteries were collected immediately and immersed in krebs solution containing u/l penicillin and mg/l streptomycin. after the connective tissue was cleaned, the basilar arteries were cut into small pieces~ . mm long and the vessel segments were placed on the surface of the culture dish. the dish was then incubated in dulbecco's modified essential medium/f- supplemented with % fetal calf serum at °c and % co . passages - of the cultured basmcs were used for experiments. ad-endophilin a was designed and produced by sunbio medical biotechnology (shanghai, china). briefly, cultured basmcs at % confluence were infected with adenovirus encoding endophilin a for h and then the cells were washed with phosphatebuffered saline (pbs) and incubated in fresh culture medium before experimentation. table s ) against rat endophilin a and p were synthesized (invitrogen, life technologies). briefly, sirna was diluted in μl culture medium and mixed with μl hiperfect reagent (qiagen, valencia, ca, usa) for min at room temperature. the mixture was then added to the cells and incubated for h in serum-free medium (the final sirna concentration was nm). finally, the cells were washed once with pbs and incubated in standard culture medium for another h. the sirna efficiency was detected by western blotting (supplementary fig. s ). reverse-transcription pcr briefly, total rna was isolated using trizol reagent (invitrogen tm , life technologies) according to the manufacturer's instructions. one picogram of total rna was reverse-transcribed in a total volume of μl on a thermal cycler (thermo scientific, waltham, ma, usa) according to the instructions of the one-step rt-pcr kit (qiagen, valencia, ca, usa). reactants underwent reverse transcription ( °c, min), an initial denaturation ( °c, min), amplification cycles ( °c, s; °c, s; °c, s), and a final extension ( °c, min). the specific primers are listed in supplementary table s . western blotting analysis cells or tissues were lysed with ripa lysis buffer containing a protease inhibitor cocktail (merck, germany). protein was separated with % sds-polyacrylamide gel electrophoresis (page) and transformed to a polyvinylidene difluoride (pvdf) membrane. after blocking with %- % milk at room temperature for h, the membrane was incubated with primary antibody (supplementary table s ) at °c overnight and then incubated for h with secondary antibody at room temperature. final detection was carried out with a chemiluminescent horseradish peroxidase substrate (millipore) by using a bio-rad molecular imager, chemidoc xrs+. the densities of the target bands were determined by image lab . . co-immunoprecipitation cell lysates were incubated with precoupled antibodies bound to protein g beads overnight at °c. the beads were centrifuged and washed, and then boiled in protein sds sample buffer. samples were resolved on % sds-page gels and transferred to pvdf membranes. the bound proteins were determined by immunoblotting with the indicated antibodies. the ca + -activated cl − channel currents were recorded with an axopatch b amplifier (axon instruments, foster city, ca, usa) using the whole-cell recording technique at room temperature. patch pipettes were made from borosilicate capillary tubes with a sutter p- horizontal puller (sutter instrument, novato, ca, usa). the resistance of the pipettes was - mΩ after filling with pipette solution. the currents were elicited with voltage steps from − mv to + mv in a + mv increment for ms with an interval of s from a holding potential of − mv. currents were filtered at khz and sampled at khz using pclamp . software (axon instruments). the extracellular solution contained (mm): nmdg-cl , kcl , cacl . , mgso , hepes , glucose and ph was adjusted to . with nmdg. the pipette solution contained (mm): cscl , mg·atp , mgcl . , hepes , egta , cacl . and ph was adjusted to . with csoh. the intracellular ca + concentration was nm. basmcs were seeded onto poly-lysine-treated dishes and transfected with pegfp-lc and red fluorescent protein (rfp) endophilin a . after h of incubation, the cells were fixed with % paraformaldehyde. after permeabilization with blocking buffer (pbs containing % normal donkey serum, % bovine serum albumin), the cells were incubated overnight in blocking buffer at °c with the indicated antibodies (supplementary table s ). subsequently, the dishes were rinsed in washing buffer (pbs with . % tx- ) and incubated for h at room temperature with fluorescently conjugated secondary antibodies and hoechst diluted in blocking buffer. pictures were acquired on a confocal microscope (fluoview fv , olympus). statistical analysis all statistical analyses were performed using graphpad prism (graphpad software, la jolla, ca, usa). all data are expressed as the mean ± sd. statistical differences were assessed with the unpaired two-tailed student's t-test for two experimental groups and one-way and two-way analysis of variance for more than two groups. p < . was considered statistically significant. endophilin a and tmem a ca + -activated chloride channel cz liu et al. endophilin a is upregulated during hypertension we previously identified tmem a as the molecular identity of the cacc in smooth muscle cells and found that cacc activity was associated with a high concentration of angiotensin ii in the basilar artery of the k c renohypertensive rat model. in this study, by employing basmcs isolated from a k c renohypertensive rat model, we found that angiotensin ii could increase endophilin a levels and decrease tmem a expression in a dose-dependent manner (fig. a) . similar results were also found in basilar arteries isolated from the k c model. as shown in fig. b , a significant increase in endophilin a was observed in -week-old k c renohypertensive rat basilar arteries compared with sham control arteries. there have been reports on the association between endophilin a and calcium regulation, as well as the association between endophilin a and tmem a expression during hypertension, and we therefore wanted to test the effects of endophilin a on tmem a cacc activity. endophilin a modulates the activity of caccs to determine whether endophilin a could regulate tmem a cacc activity, we carried out experiments to record i cacc . basmcs were transfected with endophilin a fluorescent-labeled sirna or nonspecific, scrambled rna. i cacc of the fluorescent-labeled cells was recorded by whole-cell patch clamp electrophysiology. in the presence of nm intracellular ca + and a physiological concentration of extracellular cl − , a robust outward rectifying current was recorded in the endophilin a sirna group compared with the nonspecific, scrambled rna or untransfected group (fig. a) . by contrast, overexpression of endophilin a significantly decreased cacc activity according to the recording of i cacc of green fluorescent protein (gfp)-expressing cells infected with endophilin a -gfp adenovirus or gfp control (fig. b) . similar results were found in basmcs freshly isolated from endophilin a transgenic mice [ ] and their littermate controls (fig. c) . these data suggest that endophilin a can modulate cacc activity in smooth muscle cells. endophilin a regulates the expression of tmem a protein next, to evaluate how endophilin a modulates tmem a cacc activity in smooth muscle cells, we determined the mrna and protein levels of tmem a with overexpression or knockdown of endophilin a . the quantitative reverse-transcription pcr (qrt-pcr) results showed that there was no significant effect on tmem a mrna level by adenovirus-mediated endophilin a overexpression or endophilin a knockdown (fig. a) . however, the western blotting results revealed that the tmem a protein level was significantly decreased following adenovirus-mediated overexpression of endophilin a , whereas knockdown of endophilin a increased the protein level of tmem a (fig. b) . the expression of tmem a was also reduced in the basilar arteries isolated from endophilin a transgenic mice (fig. d) . furthermore, the calcium-activated cl − current was decreased in basmcs freshly isolated from endophilin a transgenic mice compared with wt mice (fig. c) . together, these data implicate that endophilin a plays a causal role in modulating tmem a cacc activity and modulates tmem a protein expression by regulating tmem a posttranslational modification rather than transcriptional regulation. upregulation of tmem a ubiquitination by endophilin a as a prerequisite for selective autophagy-mediated degradation in our study, we showed that angiotensin ii induced a decrease in tmem a protein in basmcs. to elucidate how angiotensin ii regulates tmem a protein expression, we detected the effects of tmem a ubiquitination following angiotensin ii stimulation. the results showed that tmem a ubiquitination was significantly increased following stimulation of angiotensin ii, whereas the expression level of tmem a was decreased (fig. a) . similar results were found in the basilar arteries isolated from the hypertension model in vivo (fig. b) . considering the significant upregulation of endophilin a in the basilar arteries isolated from the hypertension model, we hypothesized that overexpression of endophilin a could reduce tmem a protein expression by modulating tmem a ubiquitination, which would further lead to downregulation of cacc activation. by immunoprecipitating tmem a with ubiquitin, we found that overexpression of endophilin a resulted in robust polyubiquitination of tmem a (fig. c) . to identify the exact degradation mechanism by which endophilin a regulated tmem a protein homeostasis, we used concanamycin a, a specific inhibitor of v-type atpases that can inhibit lysosome activity, to evaluate its effect on tmem a protein levels. as shown in fig. a , the suppression of tmem a by endophilin a was significantly reversed by concanamycin a. similar results were found by recruiting bafilomycin a , another lysosome inhibitor that is involved in the maturation of autophagic vacuoles, as demonstrated by inhibiting fusion between autophagosomes and lysosomes (fig. b) . however, no significant difference could be detected when the cells were pretreated with or without the proteasome inhibitor mg (supplementary fig. s ). these data suggest that the modification of tmem a ubiquitination tends to be delivered to the lysosome and eventually degrades in the autophagosome/lysosome. to confirm whether endophilin a was a regulator of autophagy in basmcs and whether autophagosomes/lysosomes represent a major method by which tmem a is degraded, we detected lc i/ii conversion and gfp-lc puncta, which are both hallmarks of autophagosome activation. significantly increased expression levels of lc and evoked conversion of lc i to lc ii were observed with adenovirus-mediated endophilin a overexpression (fig. c) . obvious gfp-lc puncta were found in smc cells that were cotransfected with an rfp-endophilin a plasmid and a gfp-lc plasmid (fig. d) . we also detected autophagy flux levels to confirm this observation by using bafilomycin a and the results revealed that overexpressing endophilin a dramatically increased autophagy flux compared with the lacz control ( supplementary fig. s ). these data suggest the critical role of endophilin a in regulating autophagosome/lysosome activity, which is associated with tmem a degradation. p (sqstm ) is required as a bridge coupling ubiquitinated tmem a and autophagosomes/lysosomes to further understand how ubiquitinated tmem a is delivered to autophagosomes/lysosomes, we tested the interaction between tmem a and p (sqstm ), a ubiquitin-binding receptor that can bind ub and autophagosome-associated ub-like (ubl) proteins such as lc , which mediates wharfing of ubiquitinated protein to the autophagosome. co-immunoprecipitation experiments showed that p (sqstm ) could bind to both endophilin a and tmem a, implying the potential essential roles of p (sqstm ) in crosslinking ubiquitinated tmem a and autophagosome-associated ubl proteins (fig. a, b) . to shed light on the significance of p (sqstm ) in the degradation of ubiquitinated tmem a, we took advantage of p (sqstm ) sirna to knock down endogenous p . similar to concanamycin a and bafilomycin a , the disruption of p (sqstm ) caused a significant reversal of the degradation of tmem a mediated by endophilin a (fig. c ). in addition, the immunofluorescence results showed that puncta positive for lc , as well as p with tmem a, were increased in adenovirus-mediated endophilin a overexpressing cells (supplementary fig. s ). altogether, these data suggest that ubiquitinated tmem a modulated by endophilin a is recognized by p (sqstm ) and is finally delivered to endophilin a and tmem a ca + -activated chloride channel cz liu et al. fig. expression of endophilin a and tmem a in hypertensive rat basilar artery and basmcs in response to angiotensin ii stimuli. a basmcs were incubated with angiotensin ii at concentrations indicated above for h; tmem a and endophilin a expression were detected by western blotting. n = ; *p < . vs. control (con). b two-kidney two-clip ( k c) renovascular stroke-prone hypertensive rats were operated weeks later; tmem a and endophilin a expression in sham operation (sham and hypertension (hyper) basilar artery were detected by western blotting). n = ; *p < . vs. sham our previous study demonstrated that tmem a played a significant role in vascular remodeling by inhibiting angiotensin ii-induced proliferation in rat basilar smooth muscle cells. the activation of cacc and expression of tmem a protein were negatively correlated with a medial cross-sectional area in the basilar artery during hypertension [ ] . however, how tmem a channel activity is negatively regulated during hypertensioninduced remodeling is still unclear. in this study, we found that endophilin a was significantly increased in basilar arteries isolated from a k c renohypertensive rat model and in basmcs following angiotensin ii stimulus, and this result was accompanied by a downregulation of tmem a protein expression. furthermore, by using the whole-cell patch clamp method, we verified that endophilin a could modulate cacc activity, which implies that endophilin a is an important regulator of cacc during hypertension. we also found that overexpression of endophilin a largely decreased the expression of tmem a protein, whereas its mrna level did not change. these data suggest that endophilin a regulates the posttranslational modification of tmem a protein. endophilin a is a protein with a proline-rich peptide ligand for sh domains [ ] . proteins with this functional domain are commonly involved in protein ubiquitination as previously reported [ ] [ ] [ ] . therefore, we speculated that endophilin a might play a vital role in tmem a ubiquitination during hypertension. the data showed that endophilin a undeniably enhanced tmem a ubiquitination following angiotensin ii stimulus. although ubiquitination, the process of transferring the target to the proteasome where most cytosolic and nuclear proteins are degraded, is the hallmark of protein degradation, the ability of the proteasome is restrictive due to its inability to degrade most membrane and extracellular proteins, especially oligomeric and aggregated proteins taken up by endocytosis [ , ] . tmem a is also known to be an oligomeric membrane protein complex similar to many other ion channels, which raises the view that the formation of high-weight tmem a oligomeric complexes in the membrane might not result in sufficient degradation in the proteasome [ ] [ ] [ ] . therefore, we hypothesized that the modification of tmem a ubiquitination could be degraded through ubiquitin-mediated selective autophagy. by using concanamycin a and bafilomycin a , our results revealed that autophagosome inhibitors reversed the downregulation effect of tmem a mediated by endophilin a . moreover, fig. c cells cotransfected with rfp-endophilin a and gfp-lc plasmids revealed more significant lc puncta in comparison with rfp vector and gfp-lc group. statistically significant differences with p-value < . (*). d overexpression of endophilin a increased the expression of lc and enhanced conversion of lc i to lc ii. n = , *p < . vs. con endophilin a increased the formation of autophagosomes and autophagy in basmcs. interestingly, it has been reported that endophilin b (bif- ), another member of the endophilin family, is involved in the regulation of autophagy and is required for induced autophagy in parkinson's disease [ ] [ ] [ ] [ ] . in our study, we found that endophilin a participated in the modulation of autophagy in vascular smooth muscle cells (vsmcs). however, how endophilin a regulates cell autophagy and the following functions in vsmcs requires further study. furthermore, we demonstrated ubiquitin-binding receptor p (sqstm ) as a critical link that bridged ubiquitinated tmem a to autophagosomes [ ] [ ] [ ] [ ] and that knockdown of p (sqstm ) could reverse the downregulation of tmem a modulated by endophilin a . taken together, our studies disclose the associations between endophilin a and tmem a during hypertension. we found that angiotensin ii upregulated endophilin a expression and facilitated the inhibition of tmem a by ubiquitin-mediated selective degradation, ultimately modulating cacc activity. our data provide new insights into the mechanisms by which endophilin a regulates cacc activity and angiotensin-ii-mediated inhibition of tmem a during hypertension-induced vascular remodeling. future studies are warranted to determine the process of autophagy regulated by endophilin a in hypertension by using genetic models. p (sqstm ) could interact with endophilin a and tmem a. a co-immunoprecipitation analysis of endophilin a and p (sqstm ) protein interaction in basmcs. b co-immunoprecipitation analysis of tmem a and p (sqstm ) protein interaction in basmcs. the normal igg was used as a control. c effects of p knockdown on tmem a expression modulated by endophilin a . n = , *p < . vs. con. ## p < . vs. ad-endo endophilin a and tmem a ca + -activated chloride channel cz liu et al. tmem a, a membrane protein associated with calcium-dependent chloride channel activity expression cloning of tmem a as a calcium-activated chloride channel subunit tmem a confers receptoractivated calcium-dependent chloride conductance structure and function of tmem proteins (anoctamins) the acute nociceptive signals induced by bradykinin in rat sensory neurons are mediated by inhibition of mtype k + channels and activation of ca + -activated cl − channels the calcium-activated chloride channel anoctamin acts as a heat sensor in nociceptive neurons expression of anoctamin /tmem a by interstitial cells of cajal is fundamental for slow wave activity in gastrointestinal muscles activation of the cl − channel ano by localized calcium signals in nociceptive sensory neurons requires coupling with the ip receptor calcium-activated chloride channel tmem a modulates mucin secretion and airway smooth muscle contraction downregulation of tmem a calcium-activated chloride channel contributes to cerebrovascular remodeling during hypertension by promoting basilar smooth muscle cell proliferation a comprehensive search for calcium binding sites critical for tmem a calcium-activated chloride channel activity preassociated apocalmodulin mediates ca + -dependent sensitization of activation and inactivation of tmem a/ b ca + -gated cl -channels dynamic modulation of ano / tmem a hco − permeability by ca + /calmodulin secreted clca modulates tmem a to activate ca + -dependent chloride currents in human cells anoctamin (tmem a) ca + -activated chloride channel stoichiometrically interacts with an ezrin-radixin-moesin network structure of the sh domain of rat endophilin a single point mutation in bin/ amphiphysin/rvs (bar) sequence of endophilin impairs dimerization, membrane shaping, and src homology domain-mediated partnership endophilin-a functions in membrane scission in clathrin-independent endocytosis endophilin functions as a membranebending molecule and is delivered to endocytic zones by exocytosis recruitment of endophilin to clathrin-coated pit necks is required for efficient vesicle uncoating after fission formation of an endophilin-ca + channel complex is critical for clathrin-mediated synaptic vesicle endocytosis endophilin a influences volumeregulated chloride current by mediating clc- trafficking in vascular smooth muscle cells simvastatin ameliorates rat cerebrovascular remodeling during hypertension via inhibition of volumeregulated chloride channel threonine phosphorylation in clc- is required for angiotensin ii-induced cl current and migration in cultured vascular smooth muscle cells sh domains from a subset of bar proteins define a ubl-binding domain and implicate parkin in synaptic ubiquitination the rsp ubiquitin ligase binds to and ubiquitinates members of the yeast cin -endophilin complex, sla -rvs role of autophagy, sqstm , sh glb , and trim in the turnover of nicotinic acetylcholine receptors a role for nbr in autophagosomal degradation of ubiquitinated substrates selective autophagy: ubiquitin-mediated recognition and beyond identification of a dimerization domain in the tmem a calcium-activated chloride channel (cacc) characterization of the oligomeric structure of the ca + -activated cl -channel ano / tmem a the multiple expression of ca + -activated cl -channels via homo-and hetero-dimer formation of tmem a splicing variants in murine portal vein bif- interacts with beclin through uvrag and regulates autophagy and tumorigenesis bargaining membranes for autophagosome formation: regulation of autophagy and tumorigenesis by bif- /endophilin b bif- /endophilin b : a candidate for crescent driving force in autophagy cdk -mediated phosphorylation of endophilin b is required for induced autophagy in models of parkinson's disease signal integration and diversification through the p scaffold protein homeostatic levels of p control cytoplasmic inclusion body formation in autophagy-deficient mice role of ubiquitin-and ubl-binding proteins in cell signaling p /sqstm binds directly to atg /lc to facilitate degradation of ubiquitinated protein aggregates by autophagy czl and fyl conceived and designed the experiments. czl, fyl, xfl, mmm, xyl, and cxl performed the experiments. czl and fyl analyzed the data. czl, fyl, glw, and yyg wrote the paper. the online version of this article (https://doi.org/ . /s - - - ) contains supplementary material, which is available to authorized users.competing interests: the authors declare no competing interests. key: cord- -xm ftrw authors: zhao, wu-li; xing, yan; ye, cheng; qiu, yu-han; li, yi; liu, xiu-jun; wang, meng-yan; bi, chong-wen; song, dan-qing; shao, rong-guang title: the novel quinolizidine derivate imb-hdc inhibits stat a phosphorylation at and and promotes dna breakage and cell apoptosis via blocking stat a nuclear translocation date: - - journal: acta pharmacol sin doi: . /s - - - sha: doc_id: cord_uid: xm ftrw sophoridine is a quinolizidine natural product and the exploration of its derivatives has been carried out, and the potent anticancer compound imb-hdc was acquired. although previous studies have revealed that some sophoridine derivatives could induce dna breakage, the underlying mechanisms of inhibition of dna damage repair (atr inactivation) and the apoptosis independent of p , have not been elucidated. our research reveals a novel dna response mechanism different from general dna-damaging agents, and that sophoridine derivate inhibits the phosphorylation of tyr and ser of stat a to induce the lessened shuttle from the cytoplasm to the nucleus, and leads to the decreased nuclear stat a and subsequently inhibits the expression of stat a target gene rad that contributes to the checkpoint activation, thus inhibiting atr activation. meanwhile, imb-hdc that induced the diminished expression of stat a target gene contributes to proliferation and leads to apoptosis. more importantly, we give the first evidence that promoting the effect of tyr phosphorylation on nuclear location and subsequent stat a target gene transcription depends on ser increased or unchanged phosphorylation and was not correlated with ser phosphorylation. introduction sophoridine (fig. a , left) was a quinolizidine natural product and was abstracted from sophora alopecuroides. it plays an important role in anti-inflammatory [ , ] , antivirus, and improvement of the cardiac action [ ] . its antitumor activity was ascertained in , and in sophoridine acquired the approval of clinical study [ , ] . in , sophoridine injection was approved by chinese fda and used to treat malignant trophoblastic tumors [ ] . although sophoridine as the anticancer agent was approved, its anticancer effect was only observed in malignant trophoblastic tumors. to improve the anticancer effect of sophoridine, a series of structure modifications based on sophoridine were performed, and the anticancer activities of the corresponding derivatives were examined. among these derivatives, sophoridinic acid with a three-ring structure scaffold displayed the potent antiproliferative activities compared with their parent and other derivatives. not only could three-ring structure compounds exert potent inhibitory effect on malignant trophoblastic tumor, but could also inhibit the growth of other tumor cell lines effectively, such as hepatic carcinoma, colon cancer, pancreatic cancer, etc. [ , ] . based on sophoridinic acid with three-ring structure, we made a series of modifications and acquired the associated derivatives. subsequent anticancer activity screening showed that the novel structure compound imb-hdc (fig. a ) possessed the stronger anticancer activity compared with its parent or other structure derivatives with three-ring structure. in addition, compared with other derivatives, imb-hdc also shows good water solubility, lower toxicity, and synthesizes easily, suggesting that it is worthy to be further explored as the anticancer compound. although many studies on the anticancer mechanisms of sophoridine or its derivatives have been performed, they could induce dna breakage and cell apoptosis and these changes were independent of p , and found that dna damage response was inhibited (such as atr inactivation); although p and atr activation were vital in dna damage repair (ddr) [ ] , the underlying molecular mechanisms of these aberrant events different from conventional dna-damaging compounds had not been elucidated clearly. stat a is a key transcription factor and signaling protein that mediates diverse cellular responses to cytokines and growth factors [ ] . in response to cytokines and growth factors, stat a is phosphorylated by the receptor-associated kinases, and then forms homo-or heterodimers that translocate to the cell nucleus where they act as the transcription activator [ , ] . stat a could trigger the transcription of some genes such as bcl- , clycind , pim, c-fos, and β-catenin to regulate proliferation and apoptosis. in addition to promoting proliferation activity, stat a also revealed that it participated in the ddr, and it could upregulate the expression of ddr genes, such as rad , a vital component in homologous recombination of dna during double-strand break repair, and its deletion could induce checkpoint inactivation to subsequently suppress atr activation [ ] . generally, as a target molecule of p in response to ddr, stat a expression increased as a result of p stability and then plays the promoting repair of dna damage in ddr. given that the biological effect of stat a was dependent on its target gene expression, stat a nuclear location was the key to the transcriptional initiation of its target gene and biological function preformation. previous reports showed that tyrosine phosphorylation (y ) of stat a could allow its dimerization, and phosphorylation (y ) was a prerequisite for nucleus location. some reports revealed that serine residues of stat a (s and s , corresponding to murine s and s ) are phosphorylated in human myeloid malignancies including acute myeloid leukemia, and s phosphorylation, and the combined phosphorylation of s and s was necessary for the development of chronic myeloid leukemia, and sole s phosphorylation has no effect on disease development [ ] . the combined effect of the more vital phosphorylation site y with other phosphorylation sites such as s or s on cell proliferation, especially on dna damage response, had not been reported in solid tumor. in this report, we demonstrated the combined phosphorylation effect of y with s on stat a nuclear location and target gene expression, and verified that imb-hdc could intensively give rise to dna breakage and cell apoptosis, and decreased stat a nuclear location, and give the first evidence to demonstrate that the combined decrease in y and s phosphorylation was a prerequisite for the lessened stat a nuclear location treated by imb-hdc in solid tumors, and that is the key for imb-hdc anticancer effect exertion. anti-γ-h ax, anti-cleaved caspase , anti-cleaved caspase , anti-parp, anti-p , anti-rad , anti-phospho-stat a (tyr ), anti-stat a, and anti-c-myc antibodies were purchased from cell signaling technology (danvers, ma, usa). anti-caspase , anti-caspase , and anti-caspase were purchased from abcam (cambridge, uk). anti-phospho-stat a (ser ) and antiphospho-stat a (ser ) were purchased from origene technologies (rockville, md, usa). the anti-β-actin antibody was obtained from sigma-aldrich (saint louis, mo, usa), and peroxidase-conjugated goat anti-mouse or goat anti-rabbit secondary antibodies were purchased from zsgq-bio company (beijing, china). anti-γ-h ax (ser ), conjugated with fluorescein isothiocyanate (fitc), was purchased from bd company (franklin lakes, nj, usa). -hydroxystaurosporine (ucn- ) and colcemid were purchased from sigma-aldrich (saint louis, mo, usa). human non-small-cell lung cancer cell a , human pancreatic cancer cells aspc- and mia-paca- , human hepatoma cell hepg , human breast cancer cell mcf- , human colon carcinoma cells hct- and hct , human breast cancer cell mda-mb- , human ovarian cancer cell skov , human choriocarcinoma cell jeg , human colon carcinoma cells hct p ko, and human leukemia cell k were obtained either from cell center of the institute of basic medical sciences or from atcc. a , aspc- , mia-paca- , hepg , mcf- , hct , mda-mb- , hct p ko, and jeg cells were all cultured in the dmem with % fbs at % co at °c. k cells was cultured in rpmi- medium with % fbs. anchorage-dependent cells in the exponential growth phase were harvested with a . % trypsin- . % edta solution and resuspended in the specified medium. suspension cells were subcultured regularly. only single cells with viability over % (trypan blue exclusion) were used. cell growth inhibition assay cell growth inhibition was determined by using a srb assay [ ] . cells were seeded in -well plates at × - × /well and treated with the increasing concentrations of compound imb-hdc and incubated for h or h, then cells were fixed with % trichloroacetic acid (tca) (sigma-aldrich, saint louis, mo, usa), and then . % (w/v) srb in acetic acid ( %) was added. srb-bound cells were solubilized with mm trizma base. the absorbance was read at nm. the growth inhibition (%) was calculated at each concentration, and the ic was calculated by sigmaplot. assays were repeated three times, and the results were shown as mean and sd. colony-formation assay colony-formation assay was performed as described in ref. [ ] . briefly, hct- or mia-paca- cells were seeded in a six-well plate at - /well, after h escalating doses of imb-hdc were added and continued to be incubated with cancer cells for days, then cells were fixed with methanol for min, and stained with . % crystal violet for min at room temperature. the colony is defined to consist of at least cells. visible colonies are counted. colony-formation rate = (number of colonies/number of seeded cells) × %. comet assay dna breakage was measured with a neutral comet assay (trevigen, gaithersburg, md, usa) as described in the manufacturer's procedures and the literature. the treated cells were embedded in agarose on a slide and subjected to lysis followed by electrophoresis under neutral conditions. during electrophoresis, the damaged and fragmented negatively charged dna migrated away from the nucleus toward the anode. the amount of migrated dna was a measure of the extent of dna damage. to detect dna, the slides were stained with sybr gold (life technology, inchinnan business park, paisley, uk) staining solution. the slides were examined by fluorescence microscopy, and the results were analyzed with the comet analysis software casp to quantify dna damage [ ] . for each drug concentration, three independent assays were conducted, in which comet tails were analyzed in a minimum of randomly selected cells in each assay, and the parameter reflecting the dna damage was represented as tl (tail length) [ ] . western blot whole-cell lysates were used for immunoblotting as described previously [ ] . the cells were lysed using a lysis buffer ( mmol/l tris-hcl, ph . , mmol/l sodium chloride, . % triton x- , . % sodium deoxycholate, . % sds, and protease inhibitor). twenty micrograms of protein lysate was resolved by sds-page and analyzed by immunoblotting with the specified antibodies (the dilution ratio of all primary antibodies was : , and the secondary antibody was : ; usually μl of the above-mentioned antibodies were used in the assay). the immunoreactive signals were revealed using the enhanced chemiluminescence (millipore, billerica, ma, usa) method and visualized with a chemiimager imaging system (alpha innotech, san leandro, ca, usa). immunofluorescence γ-h ax was detected by immunofluorescent microscopy as described in ref. [ ] . cells were treated with different concentrations of imb-hdc and fixed in % paraformaldehyde, and then permeabilized with . % triton x- (sigma-aldrich, saint louis, mo, usa) in pbs containing % bsa. cells were incubated with γ-h ax (phospho-ser ) antibody fitc-conjugated at -fold dilution in % bsa in pbs overnight at °c and mounted with mounting medium vectashield (vector laboratories, orton southgate peterborough, uk) and sealed with nail polish. γ-h ax and nuclear staining were viewed with a olympus fv confocal laser scanning microscope. analysis of cell cycle distribution flow cytometric analysis was performed as described in our previous report [ ] . cells were treated with imb-hdc and collected at various time points; then they were harvested and fixed with % ethanol at − °c overnight. cells were stained with propidium iodide ( mg/ml) and rnase a ( mg/ml) at °c for min. the dna content was analyzed with a facscan flow cytometer (coulterepics xl, fullerton, ca, usa). cell apoptosis assay quality analysis of apoptotic cells was accomplished with annexin v-fitc apoptosis detection kit (beyotime, haimen, jiangsu, china) as described previously [ , ] . briefly, cells were treated with increasing concentrations of imb-hdc, and after h cells were collected for flow cytometry analysis by facscan flow cytometry. cytogenetic analysis cytogenetic analysis was performed as described previously [ ] . briefly, colcemid ( ng/ml) was added to cell cultures h prior to harvest. cells were collected and fixed in fresh methanol/acetic acid ( : , v/v) at room temperature. after three washes with ice-cold methanol/acetic acid ( : , v/v), cells were spread onto slides and airdried overnight to stain with dapi, and at least metaphase spreads were analyzed on three separate slides for each sample. semiquantitative real-time pcr rna was extrated from untreated and imb-hdc-treated cells expressing wild-type or mutant stat variants using trizol reagent (invitrogen, waltham, ma, usa). cdna synthesis was performed with oligo (dt) or random primers by using a quantscript rt kit (tiangen). real-time pcr was performed with ssoadvanced sybr greensupermix (bio-rad) and primers for bcl- [ ] and c-myc (forward ′-aacaggaactatgacctcg- ′, and reverse ′-agcagctcgaatttcttc- ′), cyclind (forward ′-acaa acagatcatccgcaaacac- ′ and reverse ′-tgttggggctcct caggttc- ′), pim- (forward ′-tcttctggcaggtgctg- ′ and reverse ′-ggtagcgaatccactctg- ′), osm (forward ′-agatacc tgagcccacacagacag- ′ and reverse ′-atcgtcccattccctga agacc- ′), and c-fos (forward ′-cgaagggaacggaataagatgg- ′ and reverse ′-agacctccagtcaaatccaggg- ′). primers for target gene expression of stat were normalized to gapdh. transfection and establishment of cell lines stably expressing stat a and its mutant cells were collected and seeded in a six-well plate with × cells, and transfected with -μg constructs according to the manufacturer's instructions with lipo (invitrogen, waltham, ma, usa). post transfection of pcmv -stat a-flag or its mutants, cells were exposed to µg/ml g (invitrogen, waltham, ma, usa) and screened for stable stat a and its mutant-expressing cells. (yfp)-tagged stat a and mutant's protein were examined by immunofluorescent laser-scanning microscopy (olympus ix , -fold magnification). in vivo antitumor activity the in vivo efficacy of imb-hdc was evaluated with hepg xenografts in nude mouse. nude mice were purchased from laboratory of experimental animals, chinese academy of medical sciences & peking union medical college, and all animal studies were approved by the ethical board of cancer hospital chinese academy of medical sciences. in total, × hepg cells or cells stably expressing stat a or its mutants were suspended in μl of pbs and inoculated s.c. in the right armpits of nude mice. after weeks, the tumors were removed from the nude mice and dissected aseptically in sterile saline. pieces of tumor tissue ( mm in size) were then transplanted into the right armpits of nude mice with a trocar. tumor-bearing mice were randomly divided into four groups (n = ), when the tumor size was~ mm . imb-hdc ( , , or mg/kg) was administered by intraperitoneal injection every second day until the mice were killed. tumor size was measured every days, and tumor volume was determined as length × width / . mice were killed when the tumor volumes of the control group reached mm ; the tumor loads and tissues were isolated and used in further assays. h&e staining and detection of apoptosis of tissue sections tumor and various tissues from imb-hdc-administered mice were excised and preserved in liquid nitrogen until frozen sections were produced. h&e staining was performed as described in ref. [ ] . tumor tissues were prepared as homogenates to detect the corresponding proteins by western blot. quantification and statistical analysis results were expressed as mean±sd and analyzed using either two-tailed student's t test or -way analysis of variance (anova) in graphpad prism . software to assess statistical significance. p values < . were considered to be statistically significant. to evaluate the effect of imb-hdc on tumor cells, μm imb-hdc was added into the human colon carcinoma cell hct- , and cell morphology was observed at , , and h. the cells were shrunk at h and the spread of some cells was restricted; up to h almost all cells could not spread. when the cells were treated for up to h, some broken cells and cell debris were observed (fig. b) . after h of treatment, most hct- cells disappeared, and large amounts of cell debris were visible (data not shown), indicating a potent anticancer activity of imb-hdc. to evaluate the dose-dependent effect of imb-hdc, μm, . μm, . μm, . μm, and . μm imb-hdc were added into the human colon carcinoma cell hct- , human leukemia cell k , and human hepatoma carcinoma cell hepg , and cell vitality was detected after incubation for h. the results showed that imb-hdc significantly inhibited cell growth in a dose-dependent manner (fig. c) . at the concentration of . μm, cell survival rate was~ % of control and decreased to % at . μm after imb-hdc treatment for h in k cells (fig. c, left) ; when the concentration was up to μm, the survival rate was only % of control, indicating an obvious dose-dependent manner. correspondingly, we also observed similar dose-dependent effects of imb-hdc in hct- cells and hepg cells (fig. c, medium and right). meanwhile, we observed the time-dependent effect with μm imb-hdc at h, h, and h (fig. d, left) , and at h the vital cell counts in hct- cells were %, and up to h and h the viable cells decreased to % and %. in mia-paca- cells treated at μm, at h, h, and h the viable cells were, respectively, %, %, and % of the control (fig. d, medium) , and hepg also got the corresponding time-dependent effect (fig. d, right) . given that colony-formation analysis is the gold standard of cytotoxic agent effect, we utilized colony-formation assay to further assess the inhibitory effect of imb-hdc. hct- cells or mia-paca- cells were seeded in a six-well plate at - cells/ well and incubated with escalating concentrations of imb-hdc ( , . , . , . , and μm) for days prior to colony count. the results turned out that imb-hdc could inhibit colony formation of tumor cells in a dose-dependent manner. at . μm, colonyformation rate was~ % of control and decreased to % at . μm, and at . μm the colony rate was only % of control and % at μm, suggesting an obvious dose-dependent inhibitory effect on hct- cells (fig. e) . correspondingly, we also observed similar effects in mia-paca- cells following exposure to imb-hdc (fig. e) . in addition, we also detected the inhibitory effect of imb-hdc on other cancer cells, and the results showed that imb-hdc could exhibit a spectral anticancer effect on various cells, including lung cancer, ovarian cancer, chorionic carcinoma of uterine, hepatoma, and breast cancer and leukemia (fig. f) , indicating a widely anticancer activity. correspondingly, we also detected the effect of sophoridinol (the parent of imb-hdc) on these tumor cells, and found that there was almost no inhibitory effect at μm on seven tumor cell lines (fig. g) , but at the same concentration three-ring structure scafflold derivate imb-hdc exhibited over % inhibitory effect in all tumor cells. these results showed that imb-hdc possessed a stronger anticancer effect compared with its parent sophoridinol. imb-hdc induces cell apoptosis via the mitochondria-mediated apoptotic pathway cell apoptosis was assessed by flow cytometric analysis, and the results showed that imb-hdc could extensively induce hct- cells apoptosis in a dose-dependent manner, and at lower concentrations of μm and μm, the total apoptotic cells were . % and % of the untreated cells, respectively, and at the higher concentration of μm, the ratio was up to % (fig. a) , indicating that imb-hdc plays its anticancer role in apoptosis inducement. then we examined the corresponding apoptosis-signaling pathways, and the results turned out that imb-hdc could lead to the final apoptotic signal protein caspase and mitochondriamediated apoptotic signal protein caspase activation, but the signal proteins of non-mitochondrial-mediated apoptosis, such as caspase , caspase , and caspase , were not activated in hct- and hepg cells (fig. b) . as the release of cytochrome c from the mitochondria to cytoplasm is essential in mitochondria-mediated apoptosis, we extracted the mitochondria and cytoplasm separately and detected the content of cytoplasmic cytochrome c, and the results suggested that cytochrome c was released to the cytoplasm, indicating that imb-hdc-induced cancer cells apoptosis was mitochondrial-mediated (fig. c) . imb-hdc induces tumor cells dna breakage, γ-h ax foci formation, but does not activate p that is indispensable in ddr previous reports showed that imb-hdc analog could induce dna breakage [ , ] , and then we evaluated imb-hdc effect on dna breakage. neutral single-cell gel electrophoresis was used to evaluate its effect on dna breakage. the results showed that comet cells with comet tail were all visualized (fig. a) , indicating that imb-hdc could induce cell dna double-strand breakage. the quantitative data revealed that imb-hdc caused the tail length increase in a dose-dependent manner in dna breakage. at lower dose of imb-hdc, the tail length was about -fold of control, and at the highest concentration of μm, the tail length was up to fold of control, indicating an extensive double dna strand breakage. given that the histone variant h ax is phosphorylated rapidly on ser (γ-h ax) in response to dna double-strand breakage in cells, and it is a maker of dna breakage [ ] , we examined the γ-h ax level and the results showed that the γ-h ax expression significantly increased in the addition of imb-hdc and its elevation was in a dose-dependent manner (fig. b) . then we utilized immunofluorescent analysis to further examine whether γ-h ax foci were formed (-h ax foci represent a dna damage site in the nucleus) [ ] in the presence of imb-hdc, and the results showed that imb-hdc could cause the abundant formation of γ-h ax foci in the nuclei of hct- cells after h of treatment (fig. c) . meanwhile, we observed that imb-hdc could induce g /m-phase arrest (fig. d ). all the above results indicated that imb-hdc could significantly lead to dna breakage in a short time and these damages had been persistent in response to imb-hdc. in dna damage response, acting as the pivotal linker to accept signals from upstream ddr sensors (such as atr) to the downstream effector to exert dna repair, generally p plays the indispensable role in sensors transmission. when p was activated by its upstream signal, p stability was enhanced and subsequently triggered its target gene expression to induce dna repair, apoptosis, and cycle arrest [ , , , , ] . in our study, although we observed dna breakage and apoptosis following the addition of imb-hdc, we did not observe p expression increase, and atr activation was blocked that is different from the general dna-damaging agents (fig. b) , suggesting that a novel ddr mechanism implicated in imb-hdc induced ddr and apoptosis, and the induced apoptosis was independent of p . based on the above-mentioned results, the dna breakage was the dominant factor underlying anticancer activity of imb-hdc. to observe dna damage more intuitively, in line with the previous report [ ] , we performed cytogenetic analysis of hct- cells treated with colcemid to force cells to stay in the metaphase. this could allow to observe chromosomal damage, as only in a metaphase chromosomes are relaxed and the damage could be easily observed [ , , ] . ucn- , a chk kinase inhibitor, was used to abrogate imb-hdc-induced g arrest [ ] . ucn- could allow g -arrested cells to proceed through mitosis and into the metaphase to observe the chromosome damage, and our results showed that nm ucn- treatment alone had no discernible effects on cell cycle distribution (fig. a) . by scoring wellresolved metaphases, we detected an average of four chromatidtype aberrations, including gaps and breaks, per cell (fig. b) . in contrast, fewer aberrations were visualized in untreated cells and ucn- -treated cells alone (data not shown). our previous chip results of sophoridine derivate (data not shown) whose structure was similar to imb-hdc showed that the expression of stat a target genes decreased; meanwhile recent data showed that in ddr stat a is an essential molecule in contribution to ddr, and its silence contributed to anticancer effect of dna-damaging therapy [ ] . thus, we speculated that stat a also might be involved in imb-hdc-induced dna damage and apoptosis. in ddr, p expression increased and then initiated stat a expression in response to dna damage. subsequently, stat a translocates into the nucleus to arouse rad expression, it is involved in the search for homology and strand-pairing stages in homologous recombination of dna breakage [ , ] to activate atr to promote broken dna repair. in our study, we observed that imb-hdc only could induce minor expression change in stat a or p (figs. b, a), but stat a target gene rad obviously decreased (fig. a) ; correspondingly we also detected the translational level of other stat a target genes associated with apoptosis and proliferation, such as cyclind , c-myc, bcl- , c-fos, and pim- , and the results revealed that their mrna levels all decreased (fig. b) . these data suggested that although stat a expression did not obviously change, its target gene expression associated with apoptosis and proliferation decreased significantly, hinting that there might be an unconventional mechanism involved in imb-hdc anticancer mechanism. acting as a transcriptional factor, stat a activity exertion needs nuclear location. considering that the total stat a slightly decreased and expression of its target gene decreased significantly, thus we hypothesized that there might be the less nuclear stat a to induce the expression decrease of its target gene, then we examined nuclear stat a content, and found that the content of nuclear stat a significantly decreased by % of control, and the nuclear-cytoplasmic ratio of stat a was lowered down by %, indicating an obvious movement from the cytoplasm to the nucleus, and imb-hdc could suppress stat a nuclear translocation (fig. c) . then we used the construct egfp-stat a to transfect hct- cells, and detected imb-hdc-mediated nuclear location effect with the newly expressed stat a. the results demonstrated that imb-hdc really potentiated newly synthesized stat a to remain in the cytoplasm (fig. d) . furthermore, we utilized confocal assay to assess the stat a location with stat a antibody, and found that nuclear stat a was almost not observed after imb-hdc treatment (fig. e) . these results revealed that imb-hdc could lessen stat a nuclear translocation. since the total stat a level did not change and nuclear stat a decreased obviously, we speculated that there might be a movement from the cytoplasm to nucleus. tyrosine n (y ) of stat a is known to be a vital factor to promote stat a nuclear translocation by inducement of stat a dimer formation, and most attention has been paid to y [ ] . but recent reports showed that stat a serine phosphorylation (s and s ) has been implicated in stat a nuclear location to promote leukemogenesis, and s and s phosphorylation is a prerequisite during bm transplantation. then we detected the phosphorylation of y , s , and in the addition of imb-hdc in hct- cells. the results showed that imb-hdc could significantly depress the phosphorylation of y and s in a time-dependent manner, and up to h their phosphorylation was not nearly visualized (fig. a) , but the level of phosphorylation did not change up to h (fig. a) . imb-hdc depresses stat a nuclear translocation, transcriptional activity, and triggers dna breakage and apoptosis via blocking and phosphorylation then we wanted to verify whether lessened phosphorylation of and of stat a plays the role in imb-hdc-induced nuclear fig. imb-hdc leads to cell dna breakage, γ-h ax foci formation, and g /m arrest, but inhibits p activation. a hepg cells were treated by increasing concentrations of imb-hdc for h, and alkaline comet electrophoresis assays were performed. the quantitative analysis was performed with the comet analysis software casp, and the parameter tl (tail length) was employed to evaluate dna damage. assays were repeated three times, and the mean value of tl was normalized to the control ( μm). b hepg cells were treated with elevating doses of imb-hdc for h or elevating treated time with μm imb-hdc (down); indicated proteins were detected by western blot analysis. c hepg cells were treated with imb-hdc for h, γ-h ax foci were tested with γ-h ax antibody conjugated with fitc, the nucleaus was stained with dapi, and cells were observed with a confocal laser scanning microscope. d hepg cells were treated with imb-hdc, and at the indicated time points cells were collected for cycle analysis. mean±sd. *p < . ; ** p < . . scale bar represents μm. translocation, subsequent transcriptional activity, dna breakage, and apoptosis, and thus constructed the consecutive activation mutant y e, in which y was mutated to glutamic acid (e) and was consecutively phosphated to lead to stat a location in the nucleus [ ] . meanwhile according to the above-mentioned results, we constructed the mutant s d in which site s was mutated to aspartic acid (d) and was consecutively phosphorylated, and the mutants y e + s d in which y and s were both consecutively phosphated, and y e + s a in which y was consecutively phosphated but s was not phosphated. the verification of these mutations is shown in fig. a (down) . the results showed that sole y e and y e + s d all could rescue imb-hdc-induced growth inhibition, indicating that sole or both of y and s might be the targets of imb-hdc. to further validate whether s is also the target of imb-hdc, we further compared the effect of between y e + s a (y was consecutively phosphated, but s was not phosphated) and y e + s d in addition of imb-hdc; the results showed that y e + s a did not reverse imb-hdcinduced growth inhibition effect, indicating that s is essential for imb-hdc effect and s is also the target of imb-hdc. correspondingly, we also examined the effect of decreased phosphorylation of s and y on nuclear translocation of stat a in addition of imb-hdc, and subsequent transcription level of stat a target gene by transfecting egfp-stat a and its mutants. similar to the above-mentioned assays, we observed that consecutive phosphorylation alone could rescue imb-hdc-induced lessened nuclear stat a (fig. b) . in y e + s a-transfected cells, we also observed that y e ( consecutive phosphorylation) that induced the enhancement of stat a nuclear location was rescued by inactivation in addition of imb-hdc, indicating that the combined decrease in and phosphorylation was vital to imb-hdc-induced shortened nuclear location of stat a. these results mentioned above further demonstrated that decreased phosphorylation of is also required for the function exertion of our compound in addition to decreased phosphorylation of . imb-hdc suppresses the phosphorylation of y and s in stat a and blocks nuclear translocation of stat a. a hepg cells were treated with μm imb-hdc at various time points, and indicated protein was detected with western blot. b hepg cells were treated with increasing doses of imb-hdc, rna was extracted, and the mrna level of indicated gene was examined with real-time pcr. c imb-hdc was added into hepg cells, and the cytoplasm and nuclear protein were extracted separately for western blot and indicated proteins were detected. d stat a-egfp was transfected into hct- cells prior to the addition of imb-hdc, and cells were observed with immunofluorescence microscopy. e hepg cells were treated with imb-hdc for h, stat a antibody was added into cells prior to the addition of a secondary antibody conjugated with fitc, nucleus was stained with dapi, and cells were observed with a confocal laser scanning microscope. mean±sd. *p < . , **p< . vs control. scale bar represents μm. fig. imb-hdc depresses stat a nuclear translocation, transcriptional activity, and triggers dna breakage and apoptosis via blocking and phosphorylation. hct- cells were transfected with stat a-egfp or its mutants prior to the addition of imb-hdc, and cells vitality was examined with srb assay, and indicated proteins were detected with western blotting (a). stat a location was observed with immunofluorescence microscopy (b), mrna levels of stat a target gene were detected with real-time pcr (c), and apoptosis-associated or indicated proteins were detected with western blot (d). comet assay to examine dna breakage was assayed (e), and dna damage proteins were detected (f). mean±sd. *p< . vs y e. then, we assayed the transcriptional level of target gene of stat a associated with proliferation, and the results showed that its transcriptional levels were consistent with stat a nuclear location in fig. b (fig. c) , indicating that decreased phosphorylation of and was necessary for imb-hdc anticancer effect. although sole also could play a role in imb-hdcinduced stat a nuclear location and growth inhibition, these effects depend on decreased phosphorylation, and sole did not affect imb-hdc activity. the apoptosis data and rad changes also supported the above-mentioned results (fig. d) . then we utilized comet assays to detect the dna breakage and got the similar results that consecutive activation could rescue imb-hdc-induced dna breakage, but this rescue could be reversed by inactive mutation, indicating that combined decrease of and phosphorylation was essential to imb-hdc-mediated dna breakage (fig. e) . γ-h ax, the represent protein marker of dna breakage, was examined and the corresponding results were observed, and the changes also support the comet data of the mutants (fig. f ). all the above-mentioned results demonstrated that imb-hdc depressed stat a nuclear translocation, transcriptional activity, and triggers dna breakage and apoptosis via blocking and phosphorylation imb-hdc-induced proliferation inhibition depends on the decreased phosphorylation of and in vivo next, in a tumor xenograft nude mouse model, we examined imb-hdc anticancer efficacy. the results indicated that imb-hdc could suppress tumor xenograft proliferation, and at mg/kg the inhibitory rate was~ %, while at . mg/kg the inhibitory rate was~ % (fig. a) . the body weight curves indicated that the animals tolerated well the imb-hdc dosages administered (fig. b) . when the tumor sizes reached mm , the mice were killed, and tumors and various tissues were excised to be used for further analysis. tissues were prepared as sections for h&e staining, and tumor tissues were used for wb analysis. the results showed that imb-hdc could significantly induce cleaved parp and reduced phosphorylation of and , indicating that imb-hdc could induce apoptosis and inhibit and phosphorylation in vivo nude mice (n = ) bearing hepg xenografts were administered imb-hdc i.p. × every second day after tumor implantation. the results were recorded every days after tumor implantation with all mice alive. mice were killed when the tumor volume of the control group reached mm ; the tumor loads and other tissues were isolated and prepared sections. a tumor volume was measured by calipers twice per week in the indicated days. mean±sd. **p< . . b body weights of miceharboring tumors were monitored twice per week in the indicated days. c western blot for analysis of p-stat a and apoptotic-associated proteins from various treatment groups. d h&e staining of tissue sections from various treatment groups. e stable y e-stat a and y e + s a-stat a-expressing cells were inoculated in the armpits of nude mice, and when the tumor volume reached mm , imb-hdc was administered for days once per day, and tumor volume was measured by calipers twice per week in the indicated days, and the relative volume to stat a was calibrated and the volume-treated day curve was depicted. scale bar represents μm. (fig. c) . h&e staining of various tissues showed that there were no obvious pathologic changes observed between control and administrative groups (fig. d) . then we inoculated stable y e or y e + s a-expressing cells in the right armpits of nude mice, and when the tumor volume reached mm , imb-hdc was administered every second day, and tumor volume was measured. the in vivo results were in line with cell results, that is, when s was mutated into alanine and not be phosphorylated, the promoting proliferation activity of y e ( consecutive phosphorylation) was eliminated, and phosphorylation of both and was required for imb-hdc anticancer activity induction (fig. e) . the chinese traditional medicine fufang kushen injection was approved as an anticancer agent in , and the main active ingredient of kushen extract is sophoridine and it exhibits the anticancer effect. based on the original sophoridine structure, our lab made a series of optimization, modification, and synthetized the corresponding sophoridine derivatives, and found that imb-hdc possessed the potent anticancer activity in various tumor cell lines in vitro and in vivo. h&e staining of various tissues disclosed that there was no obvious toxicity, indicating the safety of imb-hdc (fig. ) . although previous studies revealed that sophoridine derivatives could induce dna breakage, apoptosis, and inhibit dna repair, further studies about how to trigger apoptosis and inhibit dna repair did not elucidate clearly. our studies found that p , the pivotal linker to accept signals from upstream ddr sensor and transmit these signals to the downstream effector to exert dna repair or apoptosis, could not be activated and its expression did not change, indicating that there might be a novel mechanism involved in imb-hdc-induced dna damage response. our previous chip assay analysis showed that the level of several stat a target genes decreased; thus, we speculated that stat a might be implicated in imb-hdc-induced apoptosis and dna breakage in tumor cells. stat a expression has been studied closely to tumor, such as prostate cancer, colorectal cancer, and head and neck cancer [ ] [ ] [ ] . aberrant continual activity of stat has been causally linked to human leukemias and solid tumor formation [ ] . unphosphorylated or inactive stat a may suppress tumor growth in colorectal cancer, and active stat a expression in premalignant and tumor lesions has shown potential as a prognostic marker in oral squamous cell carcinoma [ , ] . inhibition of stat a/b has resulted in large-scale apoptotic death [ ] . stat a classically can sense cytokine and growth factor signals in the cytoplasm and deliver those signals to responsive genes in the nucleus to regulate dna damage, proliferation, differentiation, growth, and apoptosis [ ] . movement between cytoplasmic and nuclear compartments is thereby central to their biological function. although in our study we did not observe that stat a expression decreased and that is consistent with p unchanged expression as the target gene of p , we found that after imb-hdc treatment nuclear stat a content was obviously lowered and cytoplasm stat a increased, indicating that imb-hdc might promote stat a nuclear translocation via regulating its movement between the cytoplasm and nucleus. correspondingly, we also observed the expression of stat a target genes associated with apoptosis and genes beneficial to dna repair gene rad eliminated. rad is an enhancing dna repair protein and its elimination will attribute to dna breakage, and that might a contributor to imb-hdc-induced dna damage. other target genes of stat a such as bcl- , c-myc, and cyclind decrease would contribute to growth inhibition and apoptosis, and these changes might be involved in imb-hdc-induced anticancer effect on tumor cells. although tyrosine phosphorylation (y ) of stat a is known to be beneficial for the dimer formation that is prerequisite to stat a shuttle from the cytoplasm to nucleus, recent reports showed that phosphorylation of s and s was also implicated in the stat a shuttle. but the combined effect of y and other two serines on stat a nuclear location was not further reported in solid tumors. in our report, we visualized that imb-hdc inhibits significantly the phosphorylation of y and s , and had a little effect on the phosphorylation of s , indicating that the decreased phosphorylation of y and s might be implicated in lessened stat a nuclear location. our further study revealed that consecutive phosphorylation at y alone could reverse imb-hdc-mediated lessened stat a nuclear location, dna breakage, and growth inhibition, but this rescue could be inhibited by s consecutive unphosphorylation, indicating that although y alone could play its role, its function still depends on s phosphorylation. simultaneous phosphorylation decrease of y and s is indispensable to imb-hdc anticancer effects on tumor cells, indicating a novel mechanism of phosphorylation of both y and s on stat a nuclear location. although for decades the anticancer study of sophoridine and its derivate has been going on, the molecular mechanism of how to induce apoptosis and dna breakage has not been clarified. our results for the first time showed that imb-hdc could inhibit the phosphorylation of stat a y and s , and subsequently block the shuttle of stat a from the cytoplasm to nucleus, and then decreased the expression of stat a target gene associated with apoptosis and proliferation to exert anticancer activity. meanwhile stat a target gene rad expression also decreased and subsequently inhibited atr activation. furthermore, we first verified that although phosphorylation of stat a y is indispensable to stat a nuclear shuttle, this effect also depends on s phosphorylated state (unchanged or elevated phosphorylation), and s also plays a key role in stat a nuclear shuttle. this mechanistic exploration is extremely vital to the combination of drug therapy, such as the inhibitory effect of imb-hdc on stat a and phosphorylation, hinting that imb-hdc could be used in combination with other kinase inhibitors, and the inhibitory effect of imb-hdc on ddr provides the probability that imb-hdc could be used in combination with dna-damaging agents. regarding the further mechanism of imb-hdc to block the phosphorylation of stat a, we speculated that it might be a result that imb-hdc hampers stat a phosphorylation, either imb-hdc inhibits stat a kinase enzyme activity or hinders the binding of stat a with its kinase, and in the future we will continue to investigate them. imb-hdc possesses good safety profiles, good druggable characteristics, high solubility, the special chemical scaffold, and good pk profile in vivo [ , ] , suggesting that it is a promising agent and worthy to be explored further [ ] . the anti-hbv study of sophoridine in vitro the effect of sopho ra alopecuroides on cv b in vitro effects of sophoridine on cardiac function and myocardial ultrastructure of rats with myocardial infarction the effect of sophoridine on tumor cells sophoridine is a new antitumor medicine with new molecular structure synthesis and biological evaluation of -n-p-chlorobenzyl sophoridinol derivatives as a novel family of anticancer agents sophoridinol derivative d induces tumor cells apoptosis by topoisomerase -mediated dna breakage stats: transcriptional control and biological impact stat a/b blockade sensitizes prostate cancer to radiation through inhibition of rad and dna repair pak-dependent stat serine phosphorylation is required for bcr-abl-induced leukemogenesis myofibrillogenesis regulator- promotes cell adhesion and migration in human hepatoma cells mr- blocks the megakaryocytic differentiation and transition of cml from chronic phase to blast crisis through mek dephosphorylation the dual topoisomerase inhibitor a preferentially and specially targets topoisomerase alpha by enhancing prestrand and post-strand cleavage and inhibiting dna religation single cell gel electrophoresis assay: methodology and applications cyclizing-berberine a induces g /m arrest and apoptosis by activating yap phosphorylation (ser ) regulation of the g /m transition by p p in the dna-damage-repair process hematoxylin and eosin staining of tissue and cell sections inhibition of topoisomerase iialpha and g cell cycle arrest by nk , a novel benzo[c]phenanthridine currently in clinical trials the chk protein kinase and the cdc c regulatory pathways are targets of the anticancer agent ucn- role of the p /p system in the response of human colon carcinoma cells to doxorubicin pharmacodynamics of cytarabine alone and in combination with -hydroxystaurosporine (ucn- ) in aml blasts in vitro and during a clinical trial regulation of p in response to dna damage arf and atm/atr cooperate in p -mediated apoptosis upon oncogenic stress phase i trial of ucn- in combination with topotecan in patients with advanced solid cancers: a princess margaret hospital phase ii consortium study unraveling the mechanism of brca in homologous recombination rad inactivation is synthetically lethal with brca deficiency src inhibits the hippo tumor suppressor pathway through tyrosine phosphorylation of lats synthesis and biological evaluation of sophoridinol derivatives as a novel family of potential anticancer agents wlz, dqs, and rgs contributed to the experimental design; wlz, yx, myw, rgs, and other authors contributed to the acquisition and analysis of the data; rgs reviewed the paper; wlz and rgs obtained the funding; wlz wrote the paper. wlz, rgs, and dqs contributed to the experimental design; wlz, yx, yhq, cy, yl, xjl, myw, and cwb contributed to the acquisition and analysis of the data; rgs reviewed the paper; wlz and rgs obtained the funding; wlz wrote the paper. competing interests: the authors declare no competing interests.consent for publication: all mentioned authors were involved in the writing of this paper and gave consent for its publication. key: cord- -yp g r authors: zhang, na; zhao, shuang-shuang; zhang, yi-xuan; wang, yu-cheng; shao, rong-guang; wang, ju-xian; he, hong-wei title: a novel biphenyl compound imb-s ameliorates hepatic fibrosis in bdl rats by suppressing sp -mediated integrin αv expression date: - - journal: acta pharmacol sin doi: . /s - - - sha: doc_id: cord_uid: yp g r chronic tissue injury with fibrosis results in the disruption of tissue architecture, organ dysfunction, and eventual organ failure. therefore, the development of effective antifibrotic drugs is urgently required. imb-s is novel biphenyl compound derived from bifendate (biphenyldicarboxylate) that is used for the treatment of chronic hepatitis in china. in the current study we investigated the potential of imb-s as an antihepatic fibrosis agent. in bile duct ligation (bdl) rat model, oral administration of imb-s ( mg· kg(− )· d(− ), for days) significantly ameliorated bdl-induced liver necrosis, bile duct proliferation, and collagen accumulation. we then showed that imb-s treatment markedly suppressed the tgf-β/smad pathway in human hepatic stellate cell line lx and mouse primary hscs, as well as in liver samples of bdl rats, thus inhibiting the transcription of most fibrogenesis-associated genes, including tgf-β , col a , and acta . furthermore, imb-s treatment significantly suppressed the expression of integrin αv at the mrna and protein levels in tgf-β-treated lx cells and liver samples of bdl rats. using integrin αv overexpression and silencing, we demonstrated that integrin αv activity correlated positively with the activation of tgf-β/smad pathway. based on dual luciferase assay and dna affinity precipitation assay, we revealed that imb-s inactivated integrin αv through competitively inhibiting the binding of sp , a transcription factor, to the integrin αv (itgav) promoter (− /− bp). these results suggest that imb-s inhibits hscs activation and liver fibrosis through sp -integrin αv signaling, and imb-s may be a promising candidate to combat hepatic fibrosis in the future. liver fibrosis, which is characterized by excessive extracellular matrix (ecm) deposition resulting from chronic liver injury of different etiologies, represents a major health problem worldwide. the development of fibrosis is the first step toward the progression to cirrhosis and its complications, including portal hypertension, organ failure, and hepatocellular carcinoma [ ] . nonalcoholic steatohepatitis, viral infection, and alcohol are the most common causes of hepatic fibrosis, and asians have a high prevalence of this condition due to frequent infection of viral hepatitis b or c [ ] . hepatic stellate cells (hscs) reside in the space of disse in the liver and are the predominant fibrogenic cell type that produces ecm in response to various injuries. the inhibition of hsc activation is proposed as a therapeutic strategy for hepatic fibrosis treatment. the hsc activation process is induced by various stimulatory factors. among them, tgf-β plays a central role. in this regard, abolishing tgf-β synthesis or tgf-β mediated signaling pathways has been shown to decrease fibrosis that has been implicated in experimental models and patients [ , ] . integrins, a family of transmembrane cell adhesion molecules, are composed of noncovalently linked heterodimers of αsubunits and β-subunits that form at least combinations in mammals [ ] . the main roles of integrins are promoting the attachment of cells to ecm proteins and transporting signals from the ecm to the cells and vice versa [ ] . thus, integrins provide a major node of communication between the ecm, inflammatory cells, fibroblasts, and parenchymal cells. as such, integrins are intimately involved in the initiation, maintenance, and resolution of tissue fibrosis. modulation of members of the αv integrin family has exhibited profound effects on fibrosis in multiple organs and disease states. as reported, modulation of the subunit of αv, which forms heterodimers with the β , β , β , β , and β subunits, has exhibited profound effects on fibrosis in multiple organs and disease states [ ] [ ] [ ] [ ] [ ] . moreover, specifically depleting integrin αv in hscs could inhibit the progression of hepatic fibrosis, which strongly verified the vital function of integrin αv in fibrosis [ ] . at the same time, integrin αv has been demonstrated to play a key role in the activation of latent tgf-β through interacting with a linear arginine-glycine-aspartic acid (rgd) motif present in the lap [ ] . therefore, the therapeutic targeting of specific integrin αv represents a promising avenue for treating a broad range of fibrotic diseases. in our previous studies, we identified a novel biphenyl compound, imb-s , through our collagen type i α (col Α ) promoter screening model [ ] . imb-s (s ) is derived from bifendate (biphenyldicarboxylate), which is used for the treatment of chronic hepatitis in china [ , ] . in this article, we aimed to explore the antifibrotic potential of imb-s and its related mechanisms. reagents and antibodies imb-s was synthesized autonomously in our laboratory, and its purity was more than % (hplc). the pcdna . -klf plasmid was purchased from youbio (changsha, china), and pcmv -myc-itgav was purchased from sino biological inc. (beijing, china); pcdna . -sp was kindly provided by dr li wang (institute of medicinal biotechnology, china). real-time pcr master mix was purchased from roche (indianapolis, usa). integrin αv antibody was purchased from bd biosciences (becton, usa). antibodies against sp , phosphor-smad / , smad , and gapdh were purchased from cell signaling technology (danvers, usa). anti-α-sma and anti-col a antibodies were obtained from abnova (taipei, chinese taiwan). anti-tgf-β antibody and recombinant tgf-β protein were acquired from r&d systems (minneapolis, usa). cell culture and western blot human hepatic stellate lx cells were cultured in dmem/ glutamax i medium (invitrogen, usa) with % fbs and % penicillin/streptomycin and incubated in a humidified atmosphere with % co at °c. no mycoplasma contamination was found in lx cells using dapi staining. for western blot analysis, cells were washed twice with ice-cold pbs and lysed in ripa buffer supplemented with protease inhibitor cocktail (roche). proteins were separated by sds-page and transferred to pvdf membranes. the membranes were probed with the appropriate primary antibodies and an hrp-conjugated secondary antibody. finally, blots were visualized using the tanon system (tanon, shanghai, china). gapdh was used as the internal control. small interfering rna (sirna) knockdown and transfection lx cells were transfected with commercially available sirna targeting integrin αv and sp (santa cruz) following the manufacturer's instructions. one day before transfection, the medium of subconfluent lx cells was replaced with fresh dmem/glutamax i medium without penicillin/streptomycin. the cells were incubated with a complex formed by sirna ( nm), transfection reagent (lipofectamine rnaimax, invitrogen), and transfection medium (opti-mem i, invitrogen) for h at °c. a scrambled sirna sequence (santa cruz proprietary target sequence) was used as a control. the transfection efficiency was confirmed by western blot, as described above. real-time pcr total rna from lx cells or liver tissue samples was extracted using trizol reagent. complementary dna (cdna) was generated using a transcriptor first strand cdna synthesis kit (roche). the relative expression levels of specific genes were determined with an abi fast real-time pcr system (thermo fisher, usa). data were calculated by the cycle threshold ( ΔΔ ct) method and normalized to gapdh. the primers for the target genes were obtained from applied biosystems (foster city, usa). male adult sprague-dawley rats weighing - g were purchased from the laboratory animal center (academy of military medical sciences, china). the rats were divided randomly into three groups: the sham group (n = ), bile duct ligation (bdl) group (n = ), and imb-s group (n = ). in the sham group, a - cm midline incision in the abdomen was made and then closed. in the bdl and imb-s groups, the bile duct was doubly ligated using - silk sutures after midline laparotomy and transected at . - . cm distal to the last bifurcation. one day after surgery, the rats in the imb-s group were given mg/kg imb-s by oral administration for days. the rats in the bdl group were given saline as a control. on the last day, the rats were sacrificed under anesthesia by co after an overnight fast. serum, urine, liver samples, and bile of the bdl and imb-s groups were collected for further analyses. all animal experiments were approved by the institutional animal care and use committee of the institute of medicinal biotechnology & chinese academy of medical sciences. serum, urine, and bile biochemistry serum alanine aminotransferase (alt), aspartate aminotransferase (ast), total bilirubin (tbil), cholesterol (cho), low-density lipoprotein cholesterol (ldl), high-density lipoprotein cholesterol (hdl), and total bile acid (tba); urine tbil and tba; and bile tbil and tba values were analyzed using a hitachi chemistry analyzer with detection kits from zhongsheng beikong biotechnology (beijing, china) according to the manufacturer's instructions. liver histology and tissue biochemistry formalin-fixed liver tissue was embedded in paraffin and stained with hematoxylin and eosin (h&e) or sirius red. liver bile duct proliferation and necrosis were quantified on a to scale in a blinded manner using a leica dm microscope. histological sections from each animal were observed at low magnification (× objective lens, olympus, ix ) in a blinded manner to analyze the percentage of the fibrotic area. the contents of hydroxyproline liver tissues were detected utilizing kits from nanjing jiancheng bioengineering institute (nanjing, china) according to the manufacturer's instructions. mouse primary hsc precipitation mouse primary hscs were isolated according to the protocol of ingmar mederacke [ ] . the process includes three sequential stages: (i) in situ perfusion of mouse liver through the hepatic portal vein with egta, pronase, and collagenase iv solutions; (ii) subsequent in vitro digestion with pronase/collagenase iv/dnase i for~ min; and (iii) density gradient-based separation of hscs from other hepatic cell populations in the presence of % nycodenz stock solution. cell purity was observed by fluorescence microscopy and determined by immunofluorescence staining for desmin. primary hscs were cultured in dmem with % fbs and % penicillin/streptomycin. the reporter construct contained the itgav promoter spanning the region of − bp to + bp. the region was amplified using pcr from the genomic dna of lx cells and cloned into the pgl . basic vector at the kpni and bglii sites (promega, usa), and the constructed reporter was named pgl . - . in addition to pgl . - , four short luciferase reporter variants, including pgl . - (− to + ), pgl . - (− to + ), pgl . - (− to + ), and pgl . - (− to + ), were constructed as above. in the construction, the following primers were used: integrin αv/kpni − : ′-ggccggtacctccaggtagactggt tgtcatgt- ′; integrin αv/kpni − : ′-ggccggtaccgtccaca caatgcacttaaa- ′; imb-s ameliorates hepatic fibrosis by sp -integrin αv n zhang et al. integrin αv/kpni − : ′-tttggtaccgcaagaggctatgct- ′; integrin αv/kpni − : ′-tttggtacccctccttccaggtctcct cc- ′; integrin αv/kpni − : ′-aaccagatctagctcctgagcctg- ′; integrin αv/bglii + : ′-ggccagatctactgtccacgtctagg ttgaagg- ′. each construct together with prl-tk was transfected into lx cells. the promoter activity was detected using the dual luciferase assay system (promega) according to the manufacturer's instructions. dna affinity precipitation assay (dapa) the ′ biotin end-labeled sense and antisense oligonucleotides of tgcgctgctgtccccgccccgcgcgctctg, which contain the binding sequence tccccgccccg (− /− bp) of the itgav promoter, were custom made by life technologies. the ′ biotin end-labeled sense and antisense oligonucleotides tgcgctgc tgcgcgctctg, which do not have the binding sequence, were used as a negative control. nuclear extract from lx cells was preincubated with dapa buffer ( mm kcl; mm hepes, ph . ; mm mgcl ; . % glycerol; . mm edta; mm dtt; and . % triton x- ) for min on ice. one micromolar of biotin-labeled ds oligonucleotides was added for preincubation and further incubated for min on ice. then, the dna/protein complexes were incubated with neutravidin-coated agarose beads (pierce chemical co., il) that were preequilibrated in dapa buffer for h at °c. complexed proteins were examined by western blot analysis with anti-sp antibody [ ] . all experiments were repeated at least three times, and the data are presented as the means ± sd. differences between groups were assessed for significance with two-tailed student's t tests. p < . indicated significant differences between groups. after imb-s (fig. a) administration for days, several biochemical hepatic fibrosis indices were analyzed and are shown in table . the serum levels of alt and ast in the bdl group were fig. imb-s ameliorates bdl-induced liver injury in rats. a chemical structure of imb-s . b liver samples collected from three groups (sham, bdl, and imb-s ) were examined by h&e staining, and representative pathological images are shown (× , n = per group). c, d quantitative assessment of liver necrosis and bile duct proliferation. *p < . , **p < . vs the sham group; # p < . vs the bdl group. e, f liver samples collected from three groups (sham, bdl, and imb-s ) were examined by sirius red staining, representative images of sirius red staining (× , n = per group) and positive staining scores. g hydroxyproline content in liver samples was determined by a hydroxyproline assay kit. data are expressed as the mean ± sd of three independent experiments, *p < . vs the sham group; # p < . vs the bdl group. imb-s ameliorates hepatic fibrosis by sp -integrin αv n zhang et al. significantly elevated compared with the sham group, indicating the generation of a successful rat model. a statistical reduction in alt and ast was observed after treatment with imb-s ( mg/ kg). no significant differences in ldl, tbil, and tba were found between the bdl and imb-s groups, suggesting that imb-s might not influence bile acid synthesis or transportation. h&e staining demonstrated that the histological structures of the bdl group were severely damaged, accompanied by extensive parenchyma necrosis and newly formed bile ducts (fig. b) . after imb-s treatment, the pathological changes were substantially reduced (fig. b) . blinded assessment showed that the imb-s group had significantly lower scores for parenchymal necrosis and bile duct proliferation than the bdl group (fig. c, d) . next, the antifibrotic effect of imb-s was further assessed by sirius red staining. in bdl rats, sirius red-positive collagen fibrils extended not only to the portal areas but also to the hepatic parenchyma. however, collagen accumulation was strongly attenuated after imb-s treatment (fig. e, f) . in addition, we observed that imb-s dramatically reduced the hydroxyproline expression in the liver tissues caused by the bdl operation (fig. g) . taken together, these data indicated that imb-s can alleviate bdl-induced liver injury in rats. imb-s significantly inhibits hsc activation in cell models and rat fibrotic livers to further verify the liver protective activity of imb-s , we first detected its effect on the tgf-β/smad pathway, whose activation promotes the transcription of most fibrogenesis-associated genes. srb analysis revealed that imb-s had little cytotoxicity in lx cells even at a relatively high concentration of . mm (fig. a) . therefore, the nontoxic concentrations of . and . mm were chosen in the following studies. tgf-β ( ng/ml) was used to preactivate lx cells for h, followed by imb-s ( . and . mm) treatment for another h. the relative mrna levels and protein expression of several biomarkers of activated hscs, including tgf-β , col a , and α-sma (encoded by acta ), were largely repressed by imb-s (fig. b, c) . in addition, the phosphorylation of downstream smad / was also reduced by imb-s (fig. c) . consistent results were observed in rat liver tissues (fig. d, e) , primary mouse hscs (fig. f, g) , and the rat hepatic stellate cell line hsc-t (data not shown). altogether, these findings indicated that imb-s could inhibit hsc activation in both cell models and animal models, which confirmed its inhibition of hepatic fibrosis progression. as a subunit of the integrin family, integrin αv is essential for hsc activation. we first examined integrin αv activity after imb-s treatment. in normal or tgf-β -activated lx cells, the mrna and protein levels of integrin αv were both reduced after imb-s treatment (fig. a, b) . furthermore, imb-s administration also reversed the integrin αv upregulation in bdl rats (fig. c, d) . these data suggested that integrin αv expression was negatively regulated by imb-s . in order to confirm the role of integrin αv in imb-s regulation, we used sirna-itgav to knockdown its expression in lx cells. similar to imb-s administration, the activation of tgf-β , col a , α-sma, and p-smad / was suppressed by sirna-itgav (fig. e ). in addition, we further overexpressed integrin αv in lx cells using pcmv -myc-itgav. as expected, the inhibitory effect of imb-s was reversed by integrin αv overexpression (fig. f) . as evidenced by knockdown and overexpression, integrin αv may be a target of imb-s in hsc activation, and the effect of imb-s on repressing integrin αv may contribute to the suppression of hscs and consequently hepatic fibrosis. imb-s regulates the expression of integrin αv at the − /− bp promoter region to study the underlying mechanism of integrin αv expression in response to imb-s treatment, we first examined changes in the transcriptional activity of itgav. we constructed the itgav promoter (full length of bp) with the luciferase reporter gene using the pgl . vector and named it pgl . - . itgav promoter activity was reflected by the fluorescence intensity. we transfected lx cells with pgl . - and the control vector pgl . , and the fluorescence results showed that itgav promoter activity was suppressed by imb-s (fig. a) . to find the accurate promoter region on which imb-s acts, we transfected lx cells with pgl . - and four truncation mutants (pgl . - , pgl . - , pgl . - , and pgl . . based on the luciferase intensity, we observed that imb-s induced a significant decrease in the pgl . - , pgl . - , pgl . - , and pgl . - groups. however, when lx cells were transfected with pgl . - in which the region between − bp and − bp was missing, imb-s -mediated downregulation of the itgav promoter was completely abolished (fig. b, pgl . - vs pgl . . these results suggested that imb-s inhibits integrin αv activity at the transcriptional level and that the regulatory interaction typically occurs in the region between − bp and − bp of the itgav promoter. sp plays a critical role in imb-s -mediated integrin αv regulation to determine how the − /− bp promoter region is regulated by imb-s , we used the jaspar database (http:// jaspar.genereg.net/), which specializes in predicting transcription factor binding profiles. among the predicted factors, the klf and sp families score highest, especially the subunits of klf and sp , and acta (f) and western blot analysis for tgf-β , col a , α-sma, p-smad / and smad (g). *p < . vs the control group. (data not shown). furthermore, at the − /− bp region of the itgav promoter, we found the typical binding sites of klf and sp (fig. a , the red bases indicate klf binding sites, and the green bases indicate sp binding sites). to further distinguish which transcription factor is the critical factor, we overexpressed pcdna . -sp and pcdna . -klf in lx cells. elevated mrna levels of itgav were observed in the group that overexpressed sp but not klf (fig. b) . thus, we speculated that the transcription factor sp participated in imb-s -mediated regulation. to confirm the function of sp , we performed overexpression and knockdown experiments. the downregulation of imb-s on integrin αv expression was partially reversed by sp overexpression (fig. c) . at the same time, we used a specific sirna targeting sp to knockdown its expression. we observed that the downregulation of imb-s on integrin αv was further enhanced (fig. d) . furthermore, to examine how sp mediates integrin αv expression, we performed a dapa experiment to assess whether imb-s could influence the direct binding of sp to the itgav promoter. to simulate the binding as accurately as possible, we used biotin-labeled '-tgcgctgctgtccccgccccgcgcgctctg- ' (− /− bp) oligonucleotides (biotin-sp ), including the binding site '-tccccgccccg- ' (− /− bp) of sp . the biotin-labeled '-tgcgctgctgcgcgctctg- ' oligonucleotides (biotin-control), excluding the sp binding site, were used as a negative control. as expected, sp was pulled down together with the oligonucleotides in the biotin-sp group, indicating that sp could interact directly with the itgav promoter (fig. e) . furthermore, after the addition of imb-s , the binding effect was significantly repressed (fig. e ). our results indicated that imb-s functions as a fibrotic inhibitor by repressing sp binding to the itgav promoter. together, we first assessed the novel biphenyl compound imb-s in this article, and our study comprehensively confirmed its in vivo and in vitro antifibrotic activity. we reported that imb-s inhibits hsc activation and liver fibrosis through sp -integrin αv signaling. hepatic fibrosis, as an unavoidable process from liver injury to cirrhosis and even liver cancer, is a dynamic process that has an inherent capacity for recovery and remodeling [ ] , and much has been learned about the pathophysiology of hepatic fibrosis in recent decades. however, new and effective agents for the treatment of hepatic fibrosis are urgently needed. [ , ] . however, it cannot improve the pathological changes in chronic hepatitis, and its antifibrotic effect has seldom been reported. thus, we modified the structure of ddb to identify candidates for antifibrosis. from dozens of candidates, we identified imb-s as a potential chemical compound for the treatment of liver fibrosis by the highthroughput drug screening model based on col a promoter activity established in our laboratory [ ] . the following verification in vivo and in vitro confirmed its remarkable antifibrotic effect. in bdl rats, the elevated serum activities of ast and alt caused by bdl were significantly decreased after imb-s supplementation. imb-s significantly reduced necrosis and bile duct proliferation and markedly inhibited fibrogenesis as evidenced by h&e or sirius red staining and the hydroxyproline content assay (fig. ) . these observations suggest that imb-s has a protective role against bdl-induced liver injury. in addition, the elevated inflammation-associated cytokines such as nf-κb and tnf-α in bdl rats were reduced by imb-s , and reduced superoxide dismutase in bdl rat livers was elevated by imb-s (data not shown), which suggests a protective effect of imb-s on liver inflammation and oxidative stress [ , ] . however, imb-s seems to have no impact on bile acids or bilirubin, although a bile duct ligation model was employed. we then speculated that imb-s regulates liver fibrosis in other ways instead of impacting bile acids or bilirubin. tgf-β has been characterized as one of the key cytokines that mediate hsc activation and hepatic fibrogenesis [ ] . the physiological and pathological activities of tgf-β were propagated by the canonical tgf-β/smad pathway to modulate gene expression [ ] . thus, we then detected the effect of imb-s on tgf-β and the classic smad signaling pathway. in our study, imb-s dramatically reduced tgf-β expression and the tgf-β/smad signaling pathway activity both in vivo and in vitro (fig. ) . activation of tgf-β requires the binding of integrin αv to an rgd sequence in the prodomain [ ] . integrin αv was identified as a core molecular pathway that regulates fibrosis in several organs [ ] . these reports and our observations attracted our attention, and we examined whether imb-s affects integrin αv, which may result in the antihepatic fibrosis effect of imb-s . as expected, the mrna and protein levels of integrin αv were both reduced significantly by imb-s both in vitro and in vivo (fig. ) . interestingly, we also found that itgav knockdown dramatically suppressed tgf-β/smad signaling pathway activation, which was similar to the effects of imb-s , and the inhibitory effect of imb-s was neutralized by itgav overexpression. thus, we hypothesized that imb-s suppressed the tgf-β/ smad signaling pathway through integrin αv. how does imb-s regulate integrin αv mrna? does imb-s act on itgav mrna directly? to answer this question, we constructed a recombinant plasmid with the itgav promoter and a luciferase reporter gene. the luciferase reporter system showed that imb-s reduced itgav promoter activity in vitro, indicating that imb-s directly acts on itgav (fig. ) . the itgav promoter truncation assay finally focused our attention on the − /− bp fragment, and the jaspar database predicted that the sp and klf families are the main transcription factors that bind to this region. overexpression or knockdown of sp or klf confirmed the role of sp , but not klf , in the regulation of integrin αv (fig. ). sp has been implicated in the regulation of fibrosis in several previous studies. wu et al. proved that silencing of sp diminished the stimulation of integrin αv expression by sulfatide [ ] , which agrees with our results. garcía-ruiz et al. stated that mutation of sp reversed leptininduced col α gene expression in primary rat hscs [ ] . dapa analysis revealed that imb-s reduced the direct interaction of sp and the itgav promoter. however, we found that imb-s not only regulated the binding of sp to the itgav promoter but also decreased the sp protein level (fig. c, d) , and the detailed regulatory mechanism of imb-s on sp needs further study. in conclusion, our study demonstrated that imb-s reduced the binding of sp to the itgav − bp to − bp promoter region, contributing to the decreased integrin αv expression in lx cells. decreased expression of integrin αv in turn leads to repression of the tgf-β/smad pathway. therefore, our study not only identified a novel biphenyl compound, imb-s , as a great potential compound for the treatment of hepatic diseases but also elucidated the regulatory mechanism between sp and integrin αv in fibrosis. fig. imb-s suppresses the promoter activity of itgav. a lx cells were transfected with pgl . - plasmid for h, followed by incubation with imb-s ( . mm) for another h, and the itgav promoter activity was determined by the dual luciferase reporter assay. the data are expressed as the mean ± sd, **p < . compared with the pgl . group; # p < . compared with the pgl . - group. b lx cells were transfected with pgl . - and different truncations (pgl . - , pgl . - , pgl . - , and pgl . - ) for h, followed by imb-s ( . mm) incubation for h, and the activity of different promoters was determined by the dual luciferase reporter assay. data are shown as a fold-induction relative to the pgl . group. the data are expressed as the mean ± sd, *p < . compared with the corresponding dmso group. imb-s ameliorates hepatic fibrosis by sp -integrin αv n zhang et al. fig. the transcription factor sp regulates the promoter activity of itgav. a the representative binding site of klf (red, tctcctccca) and sp (green, tccccgccccg) on the − /− bp region of the itgav promoter. b lx cells were transiently overexpressed with pcdna . -sp (left) or pcdna . -klf (right) for h, and real-time pcr for itgav and sp or klf . the data are expressed as the mean ± sd, *p < . compared with the pcdna . group. lx cells were transiently transfected with pcdna . -sp (c) or sirna-sp (d) for h, followed by imb-s for an additional h. western blot analysis for sp and integrin αv. e biotin-labeled -mer gc-rich oligonucleotides (biotin-sp ) were incubated with lx cell lysis in the presence of imb-s or not, and western blot analysis was performed for sp that specifically bound to the biotin probes. liver fibrosis liver cirrhosis protective effect of zingiber officinale against ccl -induced liver fibrosis is mediated through downregulating the tgf-beta /smad and nf-κb/iκb pathways transforming growth factor-beta mediates psoriasis-like lesions via a smad -dependent mechanism in mice integrins: bidirectional, allosteric signaling machines liver fibrosis and repair: immune regulation of wound healing in a solid organ the integrin alpha v beta binds and activates latent tgf beta : a mechanism for regulating pulmonary inflammation and fibrosis role of alphavbeta integrin in acute biliary fibrosis alphav beta integrin regulates renal fibrosis and inflammation in alport mouse integrin alphavbeta is a marker of the progression of biliary and portal liver fibrosis and a novel target for antifibrotic therapies alphav integrins: key regulators of tissue fibrosis targeting of alphav integrin identifies a core molecular pathway that regulates fibrosis in several organs myofibroblast contraction activates latent tgf-beta from the extracellular matrix establishment and application of a high-throughput drug screening model based on col a promoter for antiliver fibrosis anti-hbv efficacy of bifendate in treatment of chronic hepatitis b, a primary study bicyclol for chronic hepatitis b high-yield and highpurity isolation of hepatic stellate cells from normal and fibrotic mouse livers butyrate, a histone deacetylase inhibitor, activates the human igf binding protein- promoter in breast cancer cells: molecular mechanism involves an sp /sp multiprotein complex clinical evidence for the regression of liver fibrosis the effect of dimethyl dimethoxy biphenyl dicarboxylate (ddb) against tamoxifen-induced liver injury in rats: ddb use is curative or protective inhibition of lipopolysaccharide-induced i-kappab degradation and tumor necrosis factor-alpha expression by dimethyl- , '-dimethoxy- , , ', '-dimethylene dioxybiphenyl- , '-dicarboxylate (ddb): minor role in hepatic detoxifying enzyme expression targeting the tgfbeta signalling pathway in disease pro-metastasis function of tgfbeta mediated by the smad pathway latent tgf-beta structure and activation regulation of integrin alphav subunit expression by sulfatide in hepatocellular carcinoma cells sp and sp transcription factors mediate leptin-induced collagen alpha (i) gene expression in primary culture of male rat hepatic stellate cells imb-s ameliorates hepatic fibrosis by sp -integrin αv hwh and rgs designed the experiments. ssz performed animal experiments. yxz and nz performed the experiments on the cell model. jxw and ycw acquired and analyzed all data. nz and ssz wrote and revised the paper. every author read and approved the paper. competing interests: the authors declare no competing interests. key: cord- -yr zuvw authors: zhang, lei; li, yan-ge; he, shen; zhang, yan; yu, yi-min; li, yan; wen, hui; qiao, ying; shen, yi-feng; li, hua-fang title: quantitative efficacy of three antipsychotic drugs for schizophrenia based on a real-world study in china date: - - journal: acta pharmacol sin doi: . /s - - -x sha: doc_id: cord_uid: yr zuvw atypical antipsychotics exert remarkable long-term efficacy on the personal and social functions of schizophrenic patients. however, quantitative information on the social function of schizophrenic patients treated with atypical antipsychotics is scarce in the current clinical guidelines. in this study, we established pharmacodynamic models to quantify the time–efficacy relationship of three antipsychotic drugs based on the data from a real-world study conducted in china. a total of schizophrenic patients who received antipsychotic monotherapy with olanzapine (n = ), risperidone (n = ), or aripiprazole (n = ) were selected from a three-year prospective, multicenter study. the follow-up times were , , , , , , and weeks after baseline. a time–efficacy model was developed with nonlinear mixed effect method based on changes in personal and social performance (psp) score compared with the baseline level. crucial pharmacodynamic parameters, including maximum efficacy and drug onset time, were used to distinguish the efficacy of the three drugs. we quantified the time course of psp improvement in patients after treatment with these three antipsychotics: olanzapine, risperidone, and aripiprazole reached an e(max) value of . %, . %, and . % at weeks . , . , and . , respectively. general psychotic symptoms, onset frequency, and illness course were identified as significant factors affecting the efficacy of these drugs. the newly constructed models provide an evidence of the benefit of long-term maintenance therapy with atypical antipsychotics in individualized schizophrenia treatment in china. schizophrenia is a serious mental illness with a complex set of debilitating and chronic symptoms. at present, second-generation (atypical) antipsychotics (sgas) are used as first-line treatments for schizophrenia in the clinical setting owing to their higher tolerability than first-generation (typical) antipsychotics (fgas) and because they are subjectively preferred by patients [ ] . the relative effectiveness of atypical antipsychotics compared with that of fga has been addressed, and studies examining the effectiveness of sga drugs have also been conducted [ ] [ ] [ ] [ ] . the advent of sgas offered a renewed promise of beneficial long-term outcomes, especially in the social functioning of patients, which is defined as the patients' involvement in social interactions and activities. social functioning of schizophrenic patients has been recognized as a key outcome indicating treatment success [ ] ; however, the remission of social functioning difficulties would be a more adequate endpoint for evaluating long-term outcome efficacy [ ] . many studies have indicated that continuous antipsychotic treatment, especially as a maintenance treatment, decreases the risk of relapse and improves social function [ , ] . the catie (clinical antipsychotic trials of intervention effectiveness) schizophrenia study conducted by the national institute of mental health (nimh) is the most wellknown comparative study of the effectiveness of antipsychotic drugs [ , ] . however, few real-world studies have evaluated the effectiveness of various atypical antipsychotics in china. in addition, quantitative information on the long-term social functioning of schizophrenic patients treated with sgas is scarce in current clinical practice [ , ] , and the available information does not reflect the differences in therapeutic efficacies between various drugs. using data from the study of long-term outcomes for schizophrenia by atypical antipsychotic treatment in china (salt-c) study, which is a multicenter, real-world clinical study, we examined the differences in efficacy between three antipsychotics (olanzapine, risperidone, and aripiprazole) to provide a guide for clinicians when choosing an antipsychotic for the individualized treatment of schizophrenia. in this study, we established pharmacodynamic models to quantify the time-efficacy relationship of each antipsychotic drug, identify the relevant influencing factors affecting antipsychotic efficacy, and directly reflect the therapeutic efficacy of each drug. these models will ultimately provide quantitative information to realize individualized schizophrenia treatment in china. the salt-c study evaluated the effectiveness and safety of antipsychotic drugs in real-world settings and populations; thus, the study results can be applied in the routine clinical setting in china. the salt-c study was registered at https://www.clinicaltrials.gov (identifier: nct ) and produced a large data set of real-world schizophrenia patients in china recruited in an open-label -year follow-up clinical trial of widely used atypical antipsychotics. in this prospective, randomized, flexible dose, open-label study conducted by shanghai mental health center, data were also obtained from the sixth hospital of peking university, beijing anding hospital, guangzhou psychiatric hospital, west china hospital of sichuan university, second xiangya hospital, first affiliated hospital of kunming medical college and shanghai luwan mental health center between july and december . schizophrenic patients treated with atypical antipsychotics were followed for years. demographic data, medication history, drug dosage, illness course, and efficacy assessments, including the positive and negative syndrome scale (panss) [ ] and the personal and social performance scale (psp) [ ] , were collected using a standardized data collection form. all the centers followed the same research protocol, and all psychiatrists received training pertaining to these scales. as a real-world study, the protocol did not limit the dosage of drugs, and we only recorded the actual drug usage in clinical practice. we collected the blood drug concentration from the patients to monitor compliance. the medical history provided by guardians was also taken as important evidence to determine medication compliance. eligible patients were over years of age; diagnosed with schizophrenia based on the diagnostic and statistical manual of mental disorders, fourth edition (dsm-iv) or the international classification of diseases (icd ) by two certificated psychiatrists; and able to take atypical antipsychotic medications. patients with childbearing potential were required to have a negative pregnancy test at the time of screening. the exclusion criteria included substance dependency, dementia, mental retardation, and axis i or ii significant physical illness. the salt-c study was approved by the institutional review board of the shanghai mental health center, shanghai jiao tong university school of medicine. written informed consent was obtained from all participants and the patients' guardians. in this study, patients who received olanzapine, risperidone or aripiprazole monotherapy at least weeks after the baseline visit in the salt-c database were included in the model foundation. an average medication compliance of > % in month was assumed to be good compliance. after signing written informed consent forms, the potential subjects went through the screening process. each eligible subject was assessed at , , , , , , and weeks after the start of the study. assessments included clinical and functional measures. the measures of clinical evolution, as scored by the panss, and social functioning, as scored by the psp, were used in our study. the primary efficacy criterion was the psp score, which was recently developed to specifically assess social functioning in schizophrenic patients [ ] . the ratings were based on the assessment of four objective indicators: (a) socially useful activities, (b) personal and social relationships, (c) self-care, and (d) disturbing and aggressive behaviors. the chinese version of the psp rating had good reliability, validity, and sensitivity [ ] . higher psp scores indicate better functioning. after passing a test on the use of the scales and obtaining high internal concordance (an internal concordance of . was the minimum required), the psychiatrists were qualified as scorers. we found that the distribution of the improved percentage of the psp score from the baseline varied with time and at some time points reached a plateau. thus, the classic pharmacodynamic (e max ) model [ ] was used as the base model for our data (formula ). the parameters in formula are e max , et , and γ. e max is the theoretical maximal drug effect; et is the time required to reach half of the maximal efficacy, which also represents the rate of drug onset; and γ is a shape parameter. if the estimate of γ was near , the γ value was fixed at to simplify the model. e typical is the typical drug efficacy, and time is the time of the observation point. the nonlinear mixed effects model was used to fit the function. owing to the influence of variations, the values of observed efficacy fluctuate around the values of typical efficacy. therefore, the measured efficacy values in each investigation consisted of typical efficacy values, interindividual variability, and the residual error (rse). the interindividual variability of the parameters was assigned a logarithmic normal distribution across the population, and the distribution of pharmacodynamic parameters was defined in formula . p i is the individual pharmacodynamic parameter, whereas p pop is the population typical value of the corresponding pharmacodynamic parameter; η i is assumed to be normally distributed, with a mean of and variance of ω . the rse was tested by the additive (formula ), proportional (formula ), and proportional plus additive (formula ) models. the minimum value of the nonlinear mixed effect method (nonmem) objective function value (ofv) was used as a statistic to choose the rse model. where e i,j is the efficacy value at the observation time point j of the individual i. the ε i,j, and ε i,j, variables are the additive and proportional rses in individual i at time point j, respectively; ε i,j, and ε i,j, are assumed to be generally distributed with a mean of and variance of σ and σ , respectively. once the aforementioned basic model was established, potential factors that might affect the model parameters were explored. the factors tested in the study were age, sex, dosage, age at initial onset, illness duration, illness course, schizophrenic episodes, onset frequency, family history, and panss score at the baseline. in the process of covariate model building, categorical covariates were modeled according to formula , whereas the continuous covariates were tested by formula or formula . in formulas - , p i is the pharmacodynamic parameter for a patient with a covariate value of cov. cov median is the median value of the covariable in the population. p pop is the typical value of the parameter when the categorical covariates are equal to or quantitative efficacy of atypical antipsychotics l zhang et al. continuous covariates are equal to cov median . θ cov is a correction coefficient of the covariate of the model parameter. a difference in the ofv of . (p = . , df = ) and . (p = . , df = ) was considered statistically significant in the covariate model-building process. all factors as covariates were analyzed in a stepwise manner with a forward selection step (p = . ) and then a stricter backward elimination step (p = . ) [ ] . diagnostic plots were assessed to confirm the model performance. the monte carlo method was performed times to generate the % confidence intervals (ci) of the effects of each medicine. then, the interval was compared with the observed values to assess the accuracy of the model. validation of the model was also performed by the bootstrap method [ ] . software and statistical methods statistical analysis was conducted with spss (version . , ibm corp, armonk, ny, usa). the demographic and clinical characteristics of all the study samples are described with descriptive statistics. group comparisons were performed with anova or the kruskal-wallis h test for continuous variables and the fisher exact test for categorical variables. the model estimation and simulation were performed with nonmem . (level . , icon development solutions, usa). diagnostic graphics and visual predictive checks were conducted with r software (version . . , the r foundation of statistical computing, austria). a value of p ≤ . indicated a statistically significant difference. a total of schizophrenic participants were included in our analysis, of which were on olanzapine, were on risperidone and were on aripiprazole. the characteristics of the patients are summarized in table , and the sample size of each center is presented in supplementary table s . the mean age was . years, and the percentage of male participants was . %. the psp score at the baseline was . ± . . we found that age, occupation, illness duration, course, family history, panss, and psp scores at the baseline were significantly different (p < . ) among the three treatment drug groups. model establishment in our study, the e max model described an increased percentage of the psp score and could describe the time-effect of the three atypical antipsychotic medications. the rse was best described by the additive error model. the estimated values of γ for olanzapine, risperidone and aripiprazole were . , . , and . , respectively, which are very close to ; thus, we fixed the γ values to in the final model. the parameter estimation for the final model is presented in table . the values of rse for the pharmacodynamic parameters of olanzapine and risperidone ranged from . % to . %, which were considered acceptable. however, the rse for the pharmacodynamic parameters of aripiprazole was > %, which was owing to the small sample size and few observation time points. after removing the outliers (above the mean ± standard deviations), the rse for the pharmacodynamic parameters of aripiprazole was < %, and detailed information about the outliers is presented in supplementary table s . therefore, the estimated values of the pharmacodynamic parameters after removing the outliers were compared with those of the other two drugs. the typical e max values of olanzapine, risperidone, and aripiprazole were . %, . %, and . %, respectively. the time to achieve half of the maximal effect (et ) for olanzapine, risperidone, and aripiprazole was . , . , and . weeks, respectively. moreover, we observed that the number of schizophrenic attacks and general psychopathology had a significant effect on the et of olanzapine (formula ). et Àolanzapine ¼ : general psychopathology ð Ä Þ : oneset frequency ð Ä Þ : ( ) formula ( ) shows et values at . weeks for the general psychopathology score of in first-episode patients and . weeks in first recurrence patients. for patients who had a relapse, the time to reach their half maximal social function increased exponentially. meanwhile, in first recurrence patients, the et values were . weeks for a general psychopathology score of and . weeks for a general psychopathology score of . all of these indicated that the number of relapses and the general psychopathology score were closely related to the et . for example, when the general psychopathology score is , and the onset frequency is , the et value of olanzapine is . weeks. when the general psychopathology score is , and the onset frequency is , the et value of olanzapine is . weeks. episodes of schizophrenia and the total panss total score had significant effects on the e max , and the illness course (months) had a significant effect on the et of risperidone (formulas and ). et Àrisperidone ¼ : þ courseÀ ð : Þ : formula ( ) showed e max values of . % for a panss score of in first-episode patients and . % in relapsing patients undergoing risperidone treatment. for example, when the panss score was , and the episode was , the e max value of risperidone was . %. when the panss score was , and the episode was , the e max value of risperidone was . %. formula ( ) showed et values of . , . and . weeks for illness courses of , , and months, respectively. figure depicts the goodness-of-fit of the final models. the observed (obs) effect was in good accordance with the population-predicted effect data (pred) effects and the individual predicted data (ipred) effects. the wres magnitude was located within ± from the center and distributed randomly around the overall range of the pred. the visual predictive check with monte carlo simulation showed that the mean psp score increase (%) values for most actual values were distributed within the predicted % and % boundaries for all three atypical antipsychotics (fig. ) . bootstrap sampling also indicated the stability of the model by showing the similarities between the medians and the estimated values of the final pharmacodynamics parameters (tables , ) . the % ci of θ did not contain , suggesting that the factors were the significant factors influencing the parameters et and e max . all of these evaluation results indicated good predictability of the final models. typical drug efficacy values according to the final model parameters, we examined the efficacy values of the three antipsychotics at , , , , , and weeks and simulated the typical efficacy-time curves (fig. a) . the results showed that olanzapine had the best efficacy, with an actual maximal effect value of . %. the effect of onset frequency on long-term functioning may be particularly pronounced in patients treated with olanzapine or risperidone. a significant correlation between the number of relapses and the general psychopathology score was observed for patients treated with olanzapine. a three-dimensional curved surface can predict the typical effect of olanzapine with different quantitative efficacy of atypical antipsychotics l zhang et al. onset frequencies for the general psychopathology score of (fig. a) . the course-time-effect model of risperidone shows a better effect in first-episode patients (fig. b ) than in relapsing patients (fig. c) . moreover, no factor was significantly correlated with e max or et in the aripiprazole group. treatment discontinuation rate the duration for which patients continue to use a drug is considered a good measure of drug effectiveness [ ] . therefore, we conducted a statistical analysis of the treatment discontinuation rate (tdr) for the three drugs at different time points ( table ). the results showed that risperidone had the highest allcause tdr. tdr in schizophrenia patients treated with olanzapine was substantially lower than that in patients receiving aripiprazole before months but was higher than that in patients taking aripiprazole after months (fig. b) . in this prospective study of real-world schizophrenic patients treated with long-term antipsychotic monotherapy, maintenance treatment generally improved the social functioning of patients. we chose these three antipsychotics because of their widespread global use and their different receptor profiles, as well as because their efficacy has not been quantitatively assessed and compared. these three drugs are used for schizophrenia treatment. as traditional clinical efficacy trials usually rely on carefully planned treatment-optimizing protocols with strict inclusion and exclusion criteria [ , ] , meta-analyses or conventional group comparison analyses cannot clearly illustrate the different characteristics of antipsychotic efficacy. we quantitatively analyzed the therapeutic effects of three antipsychotics on the social functioning of real-world schizophrenic patients using a pharmacodynamic model. we found that the schizophrenic episode and total panss total score had a significant effect on the e max of risperidone. when the firstepisode patients had a panss score of , the e max values of olanzapine, risperidone and aripiprazole were . %, . %, and . %, respectively. the typical efficacy-time curves showed that aripiprazole possessed the lowest efficacy, with olanzapine showing much greater therapeutic efficacy than aripiprazole. this result agrees with the findings of an open randomized clinical trial conducted in countries, which suggested that the effectiveness of olanzapine is superior to that of other antipsychotic drugs [ ] . a meta-analysis also indicated that olanzapine and risperidone are significantly superior to other drugs in terms of overall efficacy [ ] . our model was thus successful in quantitatively analyzing the difference in effects among the three drugs. the number of schizophrenic episodes and general psychopathology significantly affected the et of olanzapine, whereas the illness course (months) significantly affected the et of risperidone. the et values of olanzapine were . weeks in firstepisode patients with a general psychopathology score of , . weeks in first recurrence patients, and . weeks in first recurrence patients with a general psychopathology score of . the et values of risperidone were . , . , and . weeks in patients with an illness course of , , and months, respectively. in the final model, the rse of e max and et for aripiprazole was > %. possible explanations are the relatively large individual variation, small sample size and insufficient observation points. the outliers, which were defined as above the mean ± standard deviations, had an impact on the model. as the outliers were fewer than % of the total, we removed them. after removing the outliers, the precision of estimation was improved. therefore, the large individual variation was the reason for the poor precision of the original estimates. previous studies included many factors, such as sociodemographic factors, treatment adherence, illness duration, and cognitive function, to screen for potential outcome predictors [ ] [ ] [ ] . it is unclear whether the collinearity effect between the factors was excluded. our study used quantitative pharmacological methods to show that factors such as schizophrenic episodes, total panss score, the number of schizophrenic attacks and general psychopathology significantly affect the efficacy of olanzapine and risperidone, excluding the collinearity effect. there were some differences in results between our study and previous studies because of the use of different baseline characteristics and because we focused on real-world data. the effect of onset frequency on the social functioning of schizophrenic patients has been studied. a study showed that after acute treatment, almost half of first-episode schizophrenia patients achieved symptom remission and had adequate social functioning, showing substantial improvement compared to relapsing patients [ ] . every relapse of schizophrenic psychosis is accompanied by an immense burden on the patients themselves, their families and society at large. in our study, we observed that for each increase in the number of schizophrenic attacks, the social function change rate decreased by~ % in the olanzapine group, and the first episode was two times more influential than in patients with recurrence in the risperidone group. our results showed an association between high relapse frequency and poor long-term outcomes, which was consistent with the results of previous studies. a possible explanation is that repeated relapse is always associated with poor drug compliance. thus, the stability of neurotransmitters such as dopamine and hydroxytryptamine is influenced by poor drug compliance, which is consistent with the hypothesis of schizophrenia pathogenesis [ ] . aripiprazole, unlike olanzapine and risperidone, is a partial agonist of d and -ht a receptors, which is different from olanzapine and risperidone. moreover, neuroimaging studies have shown structural and functional changes in brain regions with extended periods of relapse [ ] . therefore, clinicians should emphasize the importance of compliance and the negative effect of relapse on social functioning in schizophrenia patients. in addition, a shorter duration of psychosis before study entry predicts both full recovery and symptom remission [ ] . emerging evidence emphasizes the association between early detection and the long-term outcome [ ] . our study supported the importance of patient-specific factors, including illness severity at the time of diagnosis, onset frequency, and illness course as determinants of long-term antipsychotic response. the predicted e max value was not significantly correlated with the dose of each drug. the ofv did not significantly decrease when the dose factor was included in the model parameters, indicating that there was no significant correlation between drug dosage and efficacy, although the dose varies in clinical practice (fig. ) . we observed that the drug dose decreased rapidly after years. dose reduction of atypical antipsychotics is more recommended by clinicians than acute doses in the schizophrenia maintenance phase [ , ] . some experts also proposed that maintenance doses should be slowly reduced; moreover, studies have recommended maintaining the dose of an antipsychotic drug for at least months [ ] . finally, dose was not included in our model. we analyzed the tdr of the three drugs at different time points because the tdr is considered an composite representative of drug efficacy, safety, and tolerability by both the clinicians and the patients [ , , ] . a previous study indicated that olanzapine is the most effective drug for chronic schizophrenia [ ] . in our study, of the three long-term sgas, risperidone showed the highest dropout rate within one year, indicating low patient adherence. the tdr in patients receiving olanzapine was lower than that in patients receiving aripiprazole before months but was higher than that in patients receiving aripiprazole after months. the reason for the superiority of aripiprazole as a longterm continuous treatment may be related to its unique pharmacological profile [ ] . aripiprazole is well tolerated with e max maximal effect of drug, et time to achieve % of e max , θ on e max or et effect of the parameter θ used to correct the effect on e max or et , η interstudy variability of pharmacodynamic parameter, ε residual error, na parameter is not estimated, rse relative standard error. θ , episode; θ , panss total score; θ , onset frequency; θ , course (month); θ , general psychopathology score. aripiprazole , original data set; aripiprazole , outliers remove quantitative efficacy of atypical antipsychotics l zhang et al. fewer adverse events, such as extrapyramidal adverse reactions, hyperprolactinemia, and metabolic disorders. there were some limitations in our study. first, the open-label, nonrandomized design of this study was likely to have introduced selection bias. although we used a set of statistical adjustments for demographic and clinical parameters, bias could not be completely eliminated. second, in another study, a population pharmacokinetic model was developed to assess the magnitude and variability of exposure to clozapine [ ] ; however, because we did not collect pharmacokinetic data, we could not build a pharmacokinetic-pharmacodynamic model in this study. third, because most patients in our study discontinued drug treatment at~ weeks, the drug effect trends after years were unclear. fourth, although we used different methods to verify the stability of the models, external validation in further studies is required. we established pharmacodynamic models of three atypical antipsychotics based on real-world data. among the three drugs studied, olanzapine showed the highest efficacy, whereas aripiprazole showed the lowest efficacy. our current study provided quantitative information on the efficacy of three longterm atypical antipsychotics with regard to the social functioning of real-world schizophrenic patients. quantitative efficacy of atypical antipsychotics l zhang et al. second-generation versus first-generation antipsychotic drugs for schizophrenia: a meta-analysis effectiveness of antipsychotic drugs in patients with chronic schizophrenia assessing clinical and functional outcomes in the clinical antipsychotic trials of intervention effectiveness (catie) schizophrenia trial effectiveness of clozapine versus olanzapine, quetiapine, and risperidone in patients with chronic schizophrenia who did not respond to prior atypical antipsychotic treatment effectiveness of antipsychotic drugs in first-episode schizophrenia and schizophreniform disorder: an open randomised clinical trial social functioning as an outcome measure in schizophrenia studies remission in schizophrenia: proposed criteria and rationale for consensus on the continued benefit of antipsychotics after the first episode of schizophrenia should psychiatrists be more cautious about the long-term prophylactic use of antipsychotics? effectiveness of antipsychotic drugs in patients with chronic schizophrenia: efficacy, safety and cost outcomes of catie and other trials effectiveness of olanzapine, quetiapine, and risperidone in patients with chronic schizophrenia after discontinuing perphenazine: a catie study royal australian and new zealand college of psychiatrists clinical practice guidelines for the management of schizophrenia and related disorders evidence-based guidelines for the pharmacological treatment of schizophrenia: recommendations from the british association for psychopharmacology the positive and negative syndrome scale (ppanss) for schizophrenia validation of the personal and social performance (psp) scale in a german sample of acutely ill patients with schizophrenia development, reliability and acceptability of a new version of the dsm-iv social and occupational functioning assessment scale. (sofas) to assess routine social functioning the chinese version of the personal and social performance scale (psp): validity and reliability basic concepts in pharmaco-dynamic modeling comparison of stepwise covariate model building strategies in population pharmacokinetic-pharmacodynamic analysis the relationship between the dose and the predicted e max value. a olanzapine; b risperidone; c aripiprazole quantitative efficacy of atypical antipsychotics a procedure for generating bootstrap samples for the validation of nonlinear mixed-effects population models effectiveness of antipsychotics in first-episode schizophrenia and schizophreniform disorder on response and remission: an open randomized clinical trial (eufest) real-world evidence: what it is and what it can tell us according to the international society for pharmacoepidemiology (ispe) comparative effectiveness research (cer) special interest group (sig) comparative effectiveness and safety of antipsychotic drugs in schizophrenia treatment: a real-world observational study comparative efficacy and tolerability of antipsychotic drugs in schizophrenia: a multipletreatments meta-analysis social competence versus negative symptoms as predictors of real world social functioning in schizophrenia duration of untreated psychosis and social function: -year follow-up study of first-episode schizophrenia the relationship of social function to depressive and negative symptoms in individuals at clinical high risk for psychosis symptomatic and functional recovery from a first episode of schizophrenia or schizoaffective disorder relapse after antipsychotic discontinuation in schizophrenia as a withdrawal phenomenon vs illness recurrence: a post hoc analysis of a randomized placebo-controlled study relapse duration, treatment intensity, and brain tissue loss in schizophrenia: a prospective longitudinal mri study the lambeth early onset (leo) team: randomised controlled trial of the effectiveness of specialised care for early psychosis antipsychotic treatment for schizophrenia in the maintenance phase: a systematic review of the guidelines and algorithms statistical approaches to effectiveness measurement and outcome-driven re-randomizations in the clinical antipsychotic trials of intervention effectiveness (catie) studies dopamine d and d receptor occupancy in normal humans treated with the antipsychotic drug aripiprazole (opc ): a study using positron emission tomography and [ c]raclopride population pharmacokinetics of clozapine and its primary metabolite norclozapine in chinese patients with schizophrenia lz contributed to designing the work, analyzing data, and writing the paper. ygl, yz and sh provided approval for publication of the content and searched for the relevant papers. ymy, yl, hw and yq performed the research. yfs participated in the conception and design of the work. hfl contributed to the design of the study and critically revised the paper for important intellectual content. the online version of this article (https://doi.org/ . /s - - -x) contains supplementary material, which is available to authorized users.competing interests: the authors declare no competing interests. key: cord- -goaokk authors: ren, si-yu; wang, zhen-zhen; zhang, yi; chen, nai-hong title: potential application of endocannabinoid system agents in neuropsychiatric and neurodegenerative diseases—focusing on faah/magl inhibitors date: - - journal: acta pharmacol sin doi: . /s - - - sha: doc_id: cord_uid: goaokk the endocannabinoid system (ecs) has received extensive attention for its neuroprotective effect on the brain. this system comprises endocannabinoids, endocannabinoid receptors, and the corresponding ligands and proteins. the molecular players involved in their regulation and metabolism are potential therapeutic targets for neuropsychiatric diseases including anxiety, depression and neurodegenerative diseases such as alzheimer’s disease (ad) and parkinson’s disease (pd). the inhibitors of two endocannabinoid hydrolases, i.e., fatty acid amide hydrolase (faah) and monoacylglycerol lipase (magl), have the capacity to increase the level of endocannabinoids indirectly, causing fewer side effects than those associated with direct supplementation of cannabinoids. their antidepressant and anxiolytic mechanisms are considered to modulate the hypothalamic-pituitary-adrenal axis and regulate synaptic and neural plasticity. in terms of ad/pd, treatment with faah/magl inhibitors leads to reduction in amyloid β-protein deposition and inhibition of the death of dopamine neurons, which are commonly accepted to underlie the pathogenesis of ad and pd, respectively. inflammation as the cause of depression/anxiety and pd/ad is also the target of faah/magl inhibitors. in this review, we summarize the application and involvement of faah/magl inhibitors in related neurological diseases. focus on the latest research progress using faah/magl inhibitors is expected to facilitate the development of novel approaches with therapeutic potential. cannabis has been used in medicine for thousands of years, and its main active ingredients are cannabinoids. owing to the interactions between these active chemicals and the body's homeostasis system, cannabinoids were initially widely used for anticonvulsive, anti-inflammatory, and analgesic therapies, especially to alter neuronal function in the brain [ ] . advances in medicine led to the discovery of the endocannabinoid system (ecs), and many diseases that involve this system are receiving increasing attention [ ] . initially discovered as a complex network of cannabinoid receptors, the ecs is widely distributed in the central and peripheral nervous systems. it is involved in the regulation of several functions, including emotion and motivational behavior. it has also been shown to play a role in the pathophysiology of several mental disorders [ ] . its potential role in the etiology of mental disorders merits further study. scientists are exploring this new field via the development of new drugs and treatment methods. therefore, the three currently recognized types of cannabinoids, plant cannabinoids, synthetic cannabinoids and endocannabinoids, as well three components of the ecs, endocannabinoids, endocannabinoid receptors, and their corresponding ligands and proteins, have been used as the main research subjects, and extensive mechanistic and pharmacodynamics studies have been carried out. the structural and functional analogs that interact with the ecs and play a role in the treatment of central nervous system (cns) disorders are regarded as potential new cns drugs. because the action of hydrolase on endocannabinoids increases endocannabinoid levels indirectly and thus causes fewer side effects than direct exogenous supplementation, two types of hydrolases, fatty acids amide hydrolase (faah) and monoacylglycerol lipase (magl), have been examined as potential new drug targets for cns disorders. most neurodegenerative diseases are characterized by impaired memory and cognitive and motor function [ ] . the pathogenesis of emotional disorders such as depression is controversial and has multiple explanations. for example, the neuroendocrine hypothesis involving the dysfunction of the hypothalamic-pituitary-adrenal axis (hpa) caused by stimuli such as stress is widely accepted [ ] . endocannabinoid pathways are widely believed to be involved in neurodegenerative diseases and neuropsychiatric diseases [ ] [ ] [ ] . the development of faah and magl hydrolytic enzyme inhibitors and their extensive applications in neurological diseases have been a focus of research. the review explains the endocannabinoid system and eventually discusses two key types of enzyme inhibitors of the ecs: faah inhibitors and magl inhibitors. the current use of these inhibitors and relevant research with regard to nervous system diseases are summarized; based on the findings, these inhibitors exhibit potential as therapeutic agents in preclinical models of cns diseases. the discussion on neurological diseases mainly focuses on depression/anxiety and alzheimer's disease (ad)/parkinson's disease (pd). the ecs consists of the following three components: cannabinoid receptor proteins, corresponding ligands (endocannabinoids), and proteins involved in endocannabinoid regulation and metabolism. the ecs has been and is currently being studied extensively. due to the presence of cannabinoid binding sites throughout the central and peripheral nervous systems and the potent effects of the ecs on neurotransmission, these neuroendocrine interactions are receiving extensive attention and have been elucidated in several studies. endocannabinoid signaling induces the inhibition of hpa activity. the experimental finding that the exposure of rats to min of restraint stress leads to increased activity of faah in rats suggests that endocannabinoid signaling within the amygdala could be an important determinant of neuroendocrine system function. moreover, stress decreases the endocannabinoid and cannabinoid receptor type receptor (cb ) ligand concentrations and disinhibits excitatory transmission in the basolateral amygdala [ ] . it should be emphasized that the excitation, inhibition, or initiation of second messenger cascades by endogenous cannabinoids is mediated by retrograde signaling based on the interaction between the transmitter and receptor. this implies that the local excitation of a postsynaptic neuron, which is prompted by the release of anandamide (aea) and -arachidonoyl-glycerol ( -ag) into the extracellular space and followed by binding with receptors, is localized to the presynaptic membrane [ ] (fig. ) . endocannabinoids: aea and -ag endocannabinoids are lipid signaling molecules that potently act as cannabinoid receptors. two representative endocannabinoids that are widely distributed throughout the central nervous system, n-arachidonoyl-ethanolamine, called anandamide (aea), and arachidonoyl-glycerol ( -ag), were identified in the pig brain and canine gut [ ] , respectively. the kidneys are known to be enriched in aea and enzymes that metabolize aea, which are associated with many different physiological and pathophysiological functions, including the regulation of sensory and autonomic nerve signals, the regulation of energy expenditure and balance, and the initiation and control of inflammation [ ] . the detection of -ag in the cns has garnered extensive attention from scholars. importantly, the levels of -ag are times higher than those of aea in the brain [ ] . the opposing effects of -ag and aea indicate that an increase in the aea level is related to the improvement of decision-making ability and cognitive flexibility, while an increase in the -ag level is related to the destruction of cognitive flexibility and inhibitory response ability [ ] . the difference in their effects is attributed to the fact that they are derived from different biosynthetic pathways. the two biosynthetic pathways of -ag are the signaling pathway that begins with phosphatidylinositol- , -diphosphate and the metabolic pathway that involves triglycerides containing sn -arachidonic esters [ ] . on the other hand, the sequential action of a ca + -dependent or ca + -independent n-acyltransferase followed by n-acyl-phosphatidylethanolamine-specific phospholipase d appears to be the most relevant biosynthetic pathway of aea [ ] . in addition, aea and -ag exert their biological effects by binding to receptors. aea and -ag molecules that are not fig. schematic diagram of the endocannabinoid system. endocannabinoid aea and -ag are released after postsynaptic synthesis to retroactively bind to endocannabinoid receptors (cb and cb ) at the presynaptic site. exogenous cannabinoids such as phytocannabinoids (cbd and Δ -thc) and synthetic cannabinoids (hu- and jwh- ) can also be added to the synapse to activate endocannabinoid receptors. faah and magl exist simultaneously in presynaptic and postsynaptic regions and hydrolyze endogenous cannabinoids. aea anandamide, -ag -arachidonoyl-glycerol, faah fatty acid amide hydrolase, magl monoacylglycerol lipase, cbd cannabidiol, Δ -thc Δ tetrahydrocannabinol involved in the mentioned pathways are degraded by two major hydrolases: faah and magl. other metabolic processes, such as oxidation by cyclooxygenase- (cox- ), lipoxygenases, and cytochrome p , have also been indicated [ ] . in addition, phytocannabinoids and synthetic cannabinoids are structural or functional analogs of endocannabinoids. even today, phytocannabinoids, encompassing approximately compounds isolated from cannabis sativa so far, are poorly defined or have unknown pharmacological profiles [ ] . the predominant compound in cannabis extracts is Δ -tetrahydrocannabinol (Δ -thc), which is paralleled by cannabidiol (cbd), the predominant component in natural cannabis [ ] . both compounds have medical uses and are used as recreational drugs to produce effects on perception, mood, emotion, and cognition [ ] . synthetic cannabinoids (scs) are a class of heterogeneous compounds developed to explore endogenous cannabinoid systems or for use as potential therapeutic agents [ ] . currently, the classification of scs into three generations is widely accepted; chronologically, the "jwh group", "cp group", and "hu group" are the pioneers, with the second group containing alkyl derivatives, n-methyl piperidine, and benzoylindole. molecules in which the carbonyl group or indole ring is replaced with other functional groups are considered third generation scs [ ] . most scs, as structural analogs of endogenous cannabinoids, can bind to receptors as agonists and elicit cannabimimetic effects similar to those of phytocannabinoids. due to their greater binding affinity for cb and cb receptors than thc, scs show more intense antioxidant, anti-inflammatory, and neuroprotective effects [ ] . cb /cb receptor the discovery and identification of cannabinoid receptor type (cb ) and cb were breakthroughs. their endocannabinoid ligands were discovered in subsequent research, thereby generating new frontiers for correlational research on the ecs. both cb and cb receptors are coupled to gi/o proteins, and they negatively regulate adenylate cyclase and positively regulate mitogen-activated protein kinase [ ] . evidence exists that cb can also stimulate adenylyl cyclase via gs, induce receptormediated ca + fluxes and stimulate phospholipases in some experimental models [ ] . the distribution of these receptors varies; cb receptors have been found to be ubiquitously distributed in the cns, being highly expressed in the hippocampus, amygdala, prefrontal cortex, hypothalamus, and basal ganglia and expressed at lower level in peripheral neural and other tissues that participate in the regulation of emotions, stress, and responsiveness. consequently, some psychiatric issues, as well as distress and dysfunction, may be related to the regulation of cb activity. for instance, cb receptor agonists may cause psychotic episodes and panic reactions, while antagonism may result in symptoms indicative of depression and anxiety-related disorders [ ] . in contrast, the cb receptor is mainly expressed in peripheral tissues and is mainly expressed in the immune system. recent studies have also revealed the presence of cb receptors in the cns. the generally accepted and established consensus was that brain cb receptors are expressed predominantly in activated microglia, astrocytes, and their subpopulations, with the inducible nature of the cb receptor in microglia during neuroinflammation being uncovered later [ ] . cb receptors also exist on ventral tegmental area dopaminergic neurons, modulating dopaminergic neuronal function and da-regulated behavior [ ] . in addition, there are other endocannabinoid target receptors, such as the transient receptor potential vanilloid type- channel (trpv ), which might be related to the axonal transport and excitability of retinal ganglion cells, release of cytokines from microglia, regulation of retinal blood vessels [ ] , and trpv -like receptors, which can inhibit the release of the excitatory neurotransmitter glutamate in brain areas such as the hippocampus [ ] . grp , which is expressed in regions within the brain, can also interact with cannabinoids, and it has been reported that cb may regulate signaling pathways mediated by grp [ , ] . in terms of the affinity of endocannabinoids for receptors, -ag is a complete agonist of cb and cb receptors, while aea has a lower affinity for cb receptors than for cb receptors [ , ] . faah/magl enzymes and their inhibitors faah and magl are the principal catabolic enzymes for a class of bioactive lipids called fatty acid amides and are the key enzymes for the hydrolysis of endogenous cannabinoids. based on the hydrolytic mechanism of faah and magl, the study of endocannabinoids and their receptor system, as well as their potential therapeutic applications in several nervous system disorders, cancers, and neuroinflammatory diseases, a large number of irreversible/reversible inhibitors have been used to explore the different selectivities of these two enzymes. the reported structures of most reversible faah inhibitors contains αketo heterocycles, oleoyls-like trifluoromethyl ketones, oxime carbamates, and selected β-lactams [ ] . piperidine and piperazine urea compounds are considered the most promising irreversible faah inhibitors owing to their high selectivity and potency [ ] . their related roles include reducing inflammation and regulating myocardial lipid metabolism [ ] . for example, in an in vitro experiment, the knockdown of faah suppressed prostaglandin e production and proinflammatory gene expression. treatment with faah inhibitors also had anti-inflammatory effects [ ] . however, it has been revealed that % of total hydrolyzation of -ag in the brain is mediated via another major inhibitor, magl [ ] , and the bulk of the development of magl inhibitors occurred in the early s. in comparison, the hydrolase activity of aea in brain issues can be ascribed to faah. magl inhibitors constitute three classes of chemical compounds: noncompetitive inhibitors, partially reversible inhibitors, serinereactive agents, and cysteine-reactive agents [ ] . their functions, such as the regulation of lipid metabolism and anti-inflammation, have been reported in several animal experiments. for instance, they have protective effects in lung ischemia-reperfusion injury [ ] and chronic liver injury [ ] . in addition, dual faah/magl inhibition has been the focus of a small proportion of the research, as the individual inhibition of either faah or magl enzymes is unable to induce a full spectrum of activities [ ] . furthermore, it is thought that some of the pharmacological effects of dual faah/ magl inhibitors are stronger than the complete inhibition of either faah or magl [ , ] . for now, the pharmacological effects of faah inhibitors are being observed and tolerance tests for their clinical usage are being performed in healthy volunteers [ , ] . however, the safety limitations of the usage of faah/ magl inhibitors in clinical trials should not be overlooked. for example, the faah inhibitor bia - leads to acute and rapidly progressive neurological symptoms, such as headache and altered consciousness. one patient even developed brain death [ ] . the underlying mechanism is still unknown. an improper excessive dose was regarded as the chief culprit [ ] . in addition, gender differences in the effects of faah and magl inhibitors in clinical trials need to be further studied, with a focus on females, especially women of reproductive age [ ] . depression is a mental illness of great concern, with core symptoms including but not limited to, low mood, anhedonia, and irregular diet and sleep patterns. clinically, patients with depression often have anxiety-like symptoms, implying that the comorbidity of these two mental diseases is a common phenomenon. serotonergic and noradrenergic functional maladjustment [ ] and hpa axis alterations often result in depression endocannabinoid system potential in nervous system disease sy ren et al. and anxiety. several clinical trials and animal experiments have demonstrated that the endocannabinoid content in the tissues and serum of patients with depression show marked variation compared with those in healthy individuals. cb receptor expression and -ag levels have been found to be significantly reduced in the hippocampus following chronic unpredictable stress [ ] , which is thought to mimic the behavioral and endocrinal changes that promote the development of human clinical depression [ ] . an interesting study of alterations in the ecs revealed that the serum level of -ag is significantly decreased in patients with major depression, while in patients with mild depression, serum aea and -ag content show an upward trend [ ] ; this implies that the ecs could be interpreted as a buffer and regulator. there is a strong negative correlation between the serum aea level and anxiety symptoms in major depressive disorder patients. these pieces of evidence support an interaction between ecs and depression/anxiety. several cb /cb receptor agonists and antagonists have been used to explore the possible mechanisms of ecs in depression and anxiety. the antidepressant and anxiolytic effects of these compounds in ameliorating the disorder of the hpa axis and reversing the decline in monoamine neurotransmitter levels have been demonstrated in animal models. for instance, the administration of a cb receptor antagonist/inverse agonist increases the concentration of serum corticosterone in a dose-dependent manner, and pretreatment with receptor agonists, transport inhibitors, or faah inhibitors significantly reduces or eliminates the inhibition of corticosterone-induced release [ ] . cb receptor antagonists can block stress-induced corticosterone oversecretion by inhibiting faah activity within the basolateral amygdala complex [ ] . the amygdala is believed to be a key component of the excitatory drive of the hpa axis [ ] . this provides evidence that the roles of the hpa axis and ec system in the regulation of physiological processes in depression highly overlap. furthermore, cb /cb receptors are stimulated to increase the activity of adrenergic, serotonergic, and dopaminergic neurons, as well as the synthesis and/or release of corresponding neurotransmitters in specific brain regions. some specific studies are discussed below. low doses of win , - , a selective cb receptor agonist, enhance dorsal raphe nucleus -ht neuronal activity through a cb r-dependent mechanism [ ] . numerous studies have focused on the role of cb rs in pathological pain of immune origin [ ] because cb receptors are thought to play a role in the peripheral immune system. the mode of action of cb receptor agonists and agonists in reversing monoamine neurotransmitter levels has been poorly characterized. based on the fact that cb receptors are expressed in dopaminergic neurons in the mouse brain, liu et al. used dopaminergic neuron-specific cb receptor conditional knockout mice to confirm that cb may play important roles in the modulation of psychomotor behaviors [ ] ; the mechanism may be related to decreased activity of cb mediated dopaminergic neurons in the ventral tegmental area [ ] . β-caryophyllene, a naturally available sesquiterpene, has also been reported to have effects mediated by cb receptors on depression and anxiety [ ] . the mechanism can be ascribed to an increase in the uptake levels or function of glutamate as a subchronic immobilization and acoustic stress-induced inflammatory regulator. the latter hypothesis was also confirmed by experiments by zoppi et al. in which cb receptor agonists inhibited the increase in stress-induced proinflammatory cytokines such as tumor necrosis factor-alpha (tnf-α), nitric oxide (no), and cox- and regulated depression caused by neuroinflammation. however, cb and the glucocorticoid mechanism regulated by the hpa axis were not found to be relevant in this study [ ] . furthermore, one study found that exogenous cannabinoids taken up can interact with cb receptors and promote neurogenesis in the hippocampal dentate gyrus in adult rats, which translates to anxiolytic and antidepressant effects [ ] . alzheimer's disease (ad) and parkinson's disease (pd) ad and pd are neurodegenerative diseases that share similar molecular mechanisms and pathological processes: protein mutation leading to erroneous sorting or misfolding, protein aggregation due to blocked degradation, and inclusion body formation at specific sites. a mutation in the amyloid precursor protein gene that causes the abnormal accumulation of amyloid-β protein (aβ) in the brain [ ] and phosphorylated tau protein [ ] is the focus of attention for ad. it has been observed that cb receptors and faah are selectively overexpressed in neuritic plaque-associated glia in alzheimer's disease brains [ ] , especially in reactive astrocytes and activated microglial cells [ ] . a cb agonist demonstrated the ability to stimulate natural aβ removal in frozen human tissue sections and inhibit the synthesis of pathogenic peptides [ ] . the expression patterns of the cb receptor and faah are closely related to the deposition of aβ, suggesting that cb and faah may play a regulatory role in the function of microglial cells in the pathological changes associated with ad. the expression of cb receptors in different brain regions of ad patients remains controversial; however, research has indicated that cb receptor activity is contingent on the clinical period of ad, with higher cb receptor activity found in the early stage of ad and decreased levels found in later stages [ ] . additionally, tau hyperphosphorylation may be affected by cb agonism [ ] and cannabidiol [ ] , with the former downregulating the expression of inducible nitric oxide synthase and the production of no in aβ-stimulated c cells and the latter exerting its effect through wnt/β-catenin pathway rescue in aβstimulated pc neuronal cells. pd, also known as tremor paralysis, is a slowly progressing neurodegenerative disease of largely unknown etiology. the main biochemical pathology is the significant degeneration and loss of dopaminergic neurons in the substantia nigra and a significant decrease in the dopamine concentration in the striatum accompanied by dysfunction of the basal ganglia [ ] , a region that is a crucial regulator of motor activity affected by pd. cb receptors are highly distributed in the basal ganglia [ ] and exert complex regulatory effects on some important neurotransmitters, playing a role in anti-excitatory neural toxicity [ ] and neuroprotection. clinical observations have reported that the degeneration of dopaminergic neurons is accompanied by increased endocannabinoid system activity, and endocannabinoid system dysfunction is observed in both pd patients and experimental animal models. the upregulation of the cb receptor in glial cells in postmortem tissues obtained from pd patients has also been investigated [ ] . mounsey et al utilized the stereotaxic injection of -methyl- -phenyl- , , , -tetrahydropyridine (mptp), a neurotoxin, to establish a pd rat model by stereotactic injection and observed an elevated level of -ag [ ] . in the same model, cb agonists improved the survival of nigrostriatal dopaminergic neurons [ ] , and cb receptor activation may have blocked blood-brain barrier leakage and the neuroinflammation caused by the activation of glial cells, leading to decreased production of proinflammatory chemokines and thus preventing damage to dopaminergic neurons [ ] . the use of hydrolase inhibitors has been mentioned as a new drug strategy with a strong potential for treating cns disorders. the direct stimulation of a receptor by an endocannabinoid agonist or antagonist directly increases the content of endocannabinoids; this makes the action of the agonist or antagonist on the receptor less robust than indirect stimulation and causes this strategy to be prone to side effects. therefore, inhibitors of faah and magl indirectly increase the excitability of the ecs by reducing the hydrolysis of endocannabinoids. several faah and magl endocannabinoid system potential in nervous system disease sy ren et al. inhibitors have been assessed in preclinical studies ( fig. and table ). to produce antidepressant or anxiolytic effects, faah inhibitors may alter the response of the hpa axis and modulate its function, while magl inhibitors may be beneficial in suppressing cns inflammation, thus ameliorating the depression or anxiety caused by different physiological mechanisms [ ] . as a dual blocker of faah and trpv , n-arachidonoyl serotonin reverses the prolongation of immobility time in the forced swimming test through a mechanism associated with the normalization of the hpa axis after restraint stress by reversing the increase in plasma corticosterone levels induced by stress [ ] . previous studies suggest that narachidonoyl serotonin increases the levels of anandamide and facilitates its action on the cb receptor [ ] , resulting in an anxiolytic-like effect [ ] . the above studies suggest that inhibitors of enzymes that hydrolyze endogenous cannabinoids may modulate monoaminergic signaling to play a role. similarly, some other faah inhibitors, such as urb , urb , and st , also have the same influence on blood corticosterone levels in stress paradigms and further ameliorate the conditions of induced stress, such as depression or anxiety [ , , [ ] [ ] [ ] . ssr , a newly synthesized and effective reversible faah inhibitor, has robust antidepressant-like activity in the forced swimming test and chronic mild stress model. in experimental anxiety models, anxiolytic-like effects are observed in highintensity traumatic events [ ] . in addition, an increase in time spent in a lit arena in the light/dark test [ ] and a decrease in marble burying behavior [ ] are observed following treatment with urb and pf- , respectively. the magl inhibitor jzl also shows similar results, revealing its anxiolytic effects. a recent study performed comparative profiling of faah, magl, and dual inhibitors, and the results demonstrated that acute selective faah or magl inhibitors prevent stress-induced anxietylike behavior at doses that do not impair cognitive function but are associated with anxiety-like behavior [ ] . the magl inhibitor jzl has been reported to increase corticosterone levels and is thought to participate in the regulation of circulating corticosterone [ ] . nevertheless, the antidepressant effect of magl inhibitors is significant, and several special mechanisms have been reported. for example, high or low doses of magl may have an antidepressant effect on depressive states associated with acute stress and chronic corticosteroneinduced stress. the mechanism has been postulated to involve the disinhibition of the gabaergic synapses or glutamatergic synapses mediated by astrocytes [ ] , reversing the downregulation of mtor and erk signals induced by chronic unpredictable stress [ ] and enhancing adult neurogenesis and long-term synaptic plasticity in the hippocampal dentate gyrus [ ] . the endogenous cannabinoid -ag, which is dominant in vivo, is a complete agonist of the cb receptor and appears to be associated with inflammation. as -ag is mainly hydrolyzed by magl, magl inhibitors can contribute to the improvement of neuroinflammation-related depression symptoms. for example, as a magl inhibitor, jzl inhibits magl activity and elevates -ag levels in the spleen but not in the frontal cortex. this can be explained by the absence of detectable jzl levels in the frontal cortex as opposed to the spleen. however, jzl attenuates lipopolysaccharide (lps)-induced interleukin- β (il- β), interleukin- (il- ), interleukin- (il- ) and tnf alpha levels in the frontal cortex, suggesting the indirect actions of jzl on cns neuroinflammation [ ] . this study also provides further evidence for the potential of magl inhibitors as antidepressants targeting inflammation. in addition, a study reported that urb decreases fig. the application of faah and magl inhibitors in depression/anxiety and parkinson's disease/alzheimer's disease and the related mechanism. both endocannabinoid hydrolytic enzyme inhibitors improve the symptoms of depression/anxiety and pd/ad by regulating neuroinflammation. in addition, faah inhibitors can coordinate the function of the hpa axis, and magl inhibitors can play a role against depression and anxiety by blocking the inhibitory effect of astrocyte-mediated gabaergic synapses or glutamatergic synapses, activating the mtor signaling pathway, and enhancing neurogenesis and synaptic plasticity. in pd/ad, faah and magl play a role by inhibiting the death of dopaminergic neurons, reducing the immunoreactivity of microglial cells, and increasing the expression of gdnf. this affects the formation of aβ. pd parkinson's disease, ad alzheimer's disease, aβ amyloid β-protein pge prostaglandin e , no nitric oxide, dopa dopamine, bdnf brainderived neurotrophic factor, gdnf glial cell line-derived neurotrophic factor, il- β interleukin- β, tnf-α tumor necrosis factor-α, cort corticosterone, cox- cyclooxygenase- endocannabinoid system potential in nervous system disease sy ren et al. the expression of cox- and inducible nitric oxide in lps-stimulated microglia and inhibits the release of inflammatory factors such as prostaglandin e and no, suggesting that its antidepressant effect may be related to the mediation of neuroinflammation [ ] . in terms of rapid acting antidepressant effects, ketamine and jzl- both reverse evolving depressive-like behavior during forced abstinence from alcohol drinking. according to a review by ogawa et al., faah and magl inhibitors may require chronic administration for weeks to show clear antidepressant-like effects [ ] . other neuropsychiatric disorders the faah inhibitor am lowers the percentage of trials with a correct nonmatching response in the delayed nonmatching-toposition procedure [ ] , signifying a possible role in learning and memory function. however, reports to the contrary are also available, as the most commonly used inhibitor of faah, urb , enhances memory acquisition when used in passive avoidance experiments in rats. this enhancement is impeded by a pparalpha antagonist, supporting the ability of faah inhibitors to modulate memory by activating ppar-alpha [ ] . in a model of ethanol-related neuroinflammation and memory decline, the effect of the faah inhibitor on hippocampal-dependent memory may involve the regulation of hippocampal microglial cell recruitment and activation [ ] . in addition, urb has a positive effect on cognitive function in male rats [ ] and prevents epileptic-induced changes in short-term plasticity and long-term potentiation (ltp) [ ] . alterations in learning performance induced by the magl inhibitor sar in multiple tests are related to episodic, working and spatial memory, particularly a reduction in ltp of hippocampal ca synaptic transmission and acetylcholine release, two features of memory function [ ] . in recent years, the potential effects of faah inhibitors in several other stress-related disorders, such as post-traumatic stress disorder (ptsd), have also been explored. chronic treatment with urb prevents poststress symptoms (extinction and startle response), while acute delivery ameliorates depressive-like symptoms (anhedonia and reduced locomotion) in addition to ptsd-like symptoms (impaired fear extinction and enhanced fear retrieval) through the upregulation of cb induced by the return of shock/situational reminders to the normal level and by improving performance in hippocampal-dependent memory impairment and amygdaladependent memory enhancement in ptsd [ ] [ ] [ ] . chronic traumatic encephalopathy, a disease that was recently regarded as a target for magl, has neuropathological features such as neurodegeneration, tar dna-binding protein protein aggregation, and tau phosphorylation, all of which may be significantly inhibited [ ] . pd/ad however, no reports specifically focused on the use of magl inhibitors for depression related to neuroinflammation are available, although the capacity of magl to reduce the neuroinflammatory response has been implicated in several other related nervous system diseases. for instance, rea et al. demonstrated that jzl treatment significantly reduces the levels of inflammationinduced iba -immunoreactive microglia and total aβ burden in the hippocampus and temporal and parietal cortices in appswe/ psen Δe mice, a model of alzheimer's disease. thus, neuroinflammatory responses produced by hyperactive glial cells can be alleviated, dramatically affecting the course of ad, including the formation of neurotoxic aβ [ , ] . in pd, some scholars found that urb can inhibit the death of dopaminergic neurons, reduce the immunoreactivity of microglial cells, and improve the protective effect of mptp-induced motor changes, thereby improving the symptoms of pd [ ] . johnston et al. reported that when l-dopa and urb were coadministered to determine if urb hinders the effects of l-dopa therapy on pd, urb reduced total l-dopa-induced activity and the magnitude of hyperactivity by % and %. the lack of effects of urb on the antiparkinsonian actions of l-dopa shows that faah inhibitors may play an effective role in treating the side effects of l-dopa without impeding its therapeutic benefits [ ] . comparative profiling of faah and magl inhibitors has also been performed. in this study, scientists selected kml and pf- as magl and faah inhibitors with the purpose of investigating the differential influence of dopamine and bdnf in a pd rat model. the final result demonstrated that treatment with kml for over five weeks attenuated striatal dopamine depletion in mptp/probenecid mice, preserved mptp-induced cb expression and elevated the expression of glial-derived neurotrophic factor. while pf- did not show some degree of protection, it reduced cb expression. thus, magl inhibition is a better choice for pd treatment [ ] . we comprehensively summarized cannabinoid compounds, the ecs, and their applications in diseases of the nervous system. we also reviewed reports on the two hydrolysis enzymes (faah and magl) and their effect on nervous system diseases and depression/anxiety, with a special focus on pd/ad. although the application of faah/magl inhibitors in learning and memory, ptsd and other processes has been assessed, the literature survey shows that limited studies on these aspects have been conducted, and further progress should be made. since magl is still widely accepted to be associated with peripheral inflammation, only limited studies have explored the therapeutic potential of magl inhibitors for the treatment of neurological diseases. for example, in depression, despite the availability of reports on the mechanism of magl inhibitors, the lack of concrete evidence in animal behavioral experiments remains a challenge to be overcome. currently, the two inhibitors are obtained by chemical synthesis or structural modification, and we suggest that the extraction of active compounds from natural products should be considered as a good source of the inhibitors; additionally a more general in vitro screening model should be established because active substances produced by the metabolism of plants and other organisms can be used for defense or other physiological functions. the chemical structure of many natural products is so complex that it is difficult or impossible to obtain drug candidates by synthetic methods; most natural products have naturally generated chirals, whereas the chiral synthesis of most compounds is more medicinal. the above characteristics indicate that natural products are the original biological "design" and that biological macromolecules/drug targets (enzymes, proteins, etc.) have a natural affinity. although the efficacy of existing inhibitors has been summarized, the inhibitors to evaluate efficacy at the experiment level in animals, such as urb and jzl , are similar. hence, the actual effects of other inhibitors need to be further verified in animals. additionally, comparative studies of the interactions and relationships between these inhibitors and current antidepressants are limited. n-arachidonoyl serotonin has an antidepressive effect as a faah inhibitor, and we expect drug combinations involving currently available drugs that increase monoamine release to treat depression and faah/magl inhibitors to be more effective. from the overall perspective of the endogenous cannabinoid system, the role of receptors besides cb /cb receptors, such as trpv , remains poorly understood. since aea and -ag are two endocannabinoids without any apparent specialty, we believe that compounds other than the enzymes faah and magl may modulate the metabolism of endocannabinoids in animals, and this should be further explored. for numerous proteins, extracting the substance that conforms to the characteristics of the enzyme can improve the limitations of the current studies conducted on the ecs, and this will serve as an interesting and challenging task for researchers. a new dawn in cannabinoid neurobiology: the road from molecules to therapeutic discoveries targeting the endocannabinoid system in psychiatric illness the endogenous cannabinoid system controls extinction of aversive memories endocannabinoid system in neurodegenerative disorders hpa axis in major depression: cortisol, clinical symptomatology and genetic variation predict cognition integrating endocannabinoid signaling in the regulation of anxiety and depression endocannabinoid modulation of dopamine neurotransmission endocannabinoid signaling and the hypothalamic-pituitary-adrenal axis suppression of amygdalar endocannabinoid signaling by stress contributes to activation of the hypothalamic-pituitary-adrenal axis role of the endocannabinoid system in depression and suicide endocannabinoids and endocannabinoidrelated mediators: targets, metabolism and role in neurological disorders anandamide and its metabolites: what are their roles in the kidney -arachidonoylglycerol: a signaling lipid with manifold actions in the brain modulation of the endocannabinoids n-achidonoylethanolamine (aea) and -arachidonoylglycerol ( -ag) on executive functions in humans metabolism of the endocannabinoid anandamide: open questions after years metabolism of endocannabinoids molecular pharmacology of phytocannabinoids potency of Δ -tetrahydrocannabinol and other cannabinoids in cannabis in england in : implications for public health and pharmacology known pharmacological actions of delta- -tetrahydrocannabinol and of four other chemical constituents of cannabis that activate cannabinoid receptors. handb cannabis synthesis of phytocannabinoids synthetic cannabinoids: the hidden side of spice drugs synthetic cannabinoids: epidemiology, pharmacodynamics, and clinical implications the pharmacology of cannabinoid receptors and their ligands: an overview signal transduction of the cb cannabinoid receptor central side-effects of therapies based on cb cannabinoid receptor agonists and antagonists: focus on anxiety and depression regulatory role of the cannabinoid cb receptor in stress-induced neuroinflammation in mice cannabinoid cb receptors modulate midbrain dopamine neuronal activity and dopamine-related behavior in mice trpv and endocannabinoids: emerging molecular signals that modulate mammalian vision the cannabinoid receptor cb modulates the signaling properties of the lysophosphatidylinositol receptor gpr targeting cb and gpr endocannabinoid receptors as a potential neuroprotective approach for parkinson's disease anandamide and -ag are endogenously present within the laterodorsal tegmental nucleus: functional implications for a role of ecbs in arousal evidence that -arachidonoylglycerol but not n-palmitoylethanolamine or anandamide is the physiological ligand for the cannabinoid cb receptor. comparison of the agonistic activities of various cannabinoid receptor ligands in hl- cells the discovery and development of inhibitors of fatty acid amide hydrolase (faah) piperazine and piperidine carboxamides and carbamates as inhibitors of fatty acid amide hydrolase (faah) and monoacylglycerol lipase (magl) fatty acid amide hydrolase inhibitor (urb ) as a regulator of myocardial lipid metabolism in spontaneously hypertensive rats anti-inflammatory effects by pharmacological inhibition or knockdown of fatty acid amide hydrolase in bv microglial cells a comprehensive profile of brain enzymes that hydrolyze the endocannabinoid -arachidonoylglycerol structural basis for human monoglyceride lipase inhibition effects of monoacylglycerol lipase inhibitor urb on lung ischemia-reperfusion injury in mice inhibition of monoacylglycerol lipase, an anti-inflammatory and antifibrogenic strategy in the liver dual blockade of faah and magl identifies behavioral processes regulated by endocannabinoid crosstalk in vivo the effect of faah, magl, and dual faah/magl inhibition on inflammatory and colorectal distension-induced visceral pain models in rodents dual faah and magl inhibition might play a key role in visceral pain benzylamides and piperazinoarylamides of ibuprofen as fatty acid amide hydrolase inhibitors fatty acid amide hydrolase inhibition by jnj- : a multipleascending dose and a positron emission tomography study in healthy volunteers acute neurologic disorder from an inhibitor of fatty acid amide hydrolase inhibitor of fatty acid amide hydrolase-learning from tragic failures efficacy and safety of a fatty acid amide hydrolase inhibitor (pf- ) in the treatment of cannabis withdrawal and dependence in men: a double-blind, placebo-controlled, parallel group, phase a single-site randomised controlled trial role of serotonergic and noradrenergic systems in the pathophysiology of depression and anxiety disorders downregulation of endocannabinoid signaling in the hippocampus following chronic unpredictable stress validity, reliability and utility of the chronic mild stress model of depression: a -year review and evaluation serum endocannabinoid content is altered in females with depressive disorders: a preliminary report endocannabinoid signaling negatively modulates stress-induced activation of the hypothalamicpituitary-adrenal axis limbic system mechanisms of stress regulation: hypothalamo-pituitary-adrenocortical axis cannabinoids elicit antidepressantlike behavior and activate serotonergic neurons through the medial prefrontal cortex celastrol attenuates inflammatory and neuropathic pain mediated by cannabinoid receptor type cannabinoid type receptors in dopamine neurons inhibits psychomotor behaviors, alters anxiety, depression and alcohol preference β-caryophyllene, a cb receptor agonist produces multiple behavioral changes relevant to anxiety and depression in mice cannabinoids promote embryonic and adult hippocampus neurogenesis and produce anxiolytic-and antidepressant-like effects amyloid cascade hypothesis: pathogenesis and therapeutic strategies in alzheimer's disease tau and neurodegenerative disease: the story so far cannabinoid cb receptors and fatty acid amide hydrolase are selectively overexpressed in neuritic plaque-associated glia in alzheimer's disease brains cannabinoid cb receptors are expressed by perivascular microglial cells in the human brain: an immunohistochemical study the activation of cannabinoid cb receptors stimulates in situ and in vitro betaamyloid removal by human macrophages type- cannabinoid receptor activity during alzheimer's disease progression cb receptor selective activation inhibits beta-amyloid-induced inos protein expression in c cells and subsequently blunts tau protein hyperphosphorylation in co-cultured neurons the marijuana component cannabidiol inhibits beta-amyloid-induced tau protein hyperphosphorylation through wnt/beta-catenin pathway rescue in pc cells parkinson's disease the endocannabinoid system in parkinson's disease cb cannabinoid receptors and on-demand defense against excitotoxicity potential of the cannabinoid cb( ) receptor as a pharmacological target against inflammation in parkinson's disease increasing levels of the endocannabinoid -ag is neuroprotective in the -methyl- -phenyl- , , , -tetrahydropyridine mouse model of parkinson's disease cannabinoid receptor type protects nigrostriatal dopaminergic neurons against mptp neurotoxicity by inhibiting microglial activation cb receptor activation prevents glial-derived neurotoxic mediator production, bbb leakage and peripheral immune cell infiltration and rescues dopamine neurons in the mptp model of parkinson's disease inhibitors of fatty acid amide hydrolase and monoacylglycerol lipase: new targets for future antidepressants the dual blocker of faah/trpv n-arachidonoylserotonin reverses the behavioral despair induced by stress in rats and modulates the hpa-axis narachidonoyl-serotonin, a dual faah and trpv blocker, inhibits the retrieval of contextual fear memory: role of the cannabinoid cb receptor in the dorsal hippocampus anxiolytic effects in mice of a dual blocker of fatty acid amide hydrolase and transient receptor potential vanilloid type- channels cardioprotective effects of fatty acid amide hydrolase inhibitor urb , in a rodent model of trait anxiety effects of fatty acid amide hydrolase inhibitor urb in a rat model of trauma-induced longterm anxiety potential therapeutic value of a novel faah inhibitor for the treatment of anxiety the selective reversible faah inhibitor, ssr , restores the development of maladaptive behaviors to acute and chronic stress in rodents the endogenous cannabinoid anandamide has effects on motivation and anxiety that are revealed by fatty acid amide hydrolase (faah) inhibition. neuropharmacology inhibition of endocannabinoid catabolic enzymes elicits anxiolytic-like effects in the marble burying assay therapeutic endocannabinoid augmentation for mood and anxiety disorders: comparative profiling of faah, magl and dual inhibitors monoacylglycerol lipase inhibition-induced changes in plasma corticosterone levels, anxiety and locomotor activity in male cd mice monoacylglycerol lipase inhibitors produce pro-or antidepressant responses via hippocampal ca gabaergic synapses monoacylglycerol lipase inhibition blocks chronic stress-induced depressive-like behaviors via activation of mtor signaling blockade of -arachidonoylglycerol hydrolysis produces antidepressant-like effects and enhances adult hippocampal neurogenesis and synaptic plasticity the monoacylglycerol lipase inhibitor jzl attenuates lps-induced increases in cytokine expression in the rat frontal cortex and plasma: differential mechanisms of action inhibition of microglial fatty acid amide hydrolase modulates lps stimulated release of inflammatory mediators effects of fatty acid amide hydrolase (faah) inhibitors on working memory in rats fatty acid amide hydrolase (faah) inhibition enhances memory acquisition through activation of ppar-alpha nuclear receptors pharmacological blockade of fatty acid amide hydrolase (faah) by ethanol exposure inhibition of fattyacid amide hydrolyse (faah) exerts cognitive improvements in male but not female rats the faah inhibitor urb suppresses hippocampal maximal dentate afterdischarges and restores seizure-induced impairment of short and long-term synaptic plasticity selective blockade of the hydrolysis of the endocannabinoid -arachidonoylglycerol impairs learning and memory performance while producing antinociceptive activity in rodents cannabinoids prevent depressive-like symptoms and alterations in bdnf expression in a rat model of ptsd chronic treatment with urb ameliorates post-stress symptoms in a rat model of ptsd cannabinoids prevent the differential long-term effects of exposure to severe stress on hippocampal-and amygdala-dependent memory and plasticity inhibition of monoacylglycerol lipase prevents chronic traumatic encephalopathy-like neuropathology in a mouse model of repetitive mild closed head injury endocannabinoid -arachidonoylglycerol protects neurons against β-amyloid insults monoacylglycerol lipase inhibitor jzl reduces neuroinflammatory response in apde mice and in adult mouse glial cells effect of inhibition of fatty acid amide hydrolase on mptp-induced dopaminergic neuronal damage fatty acid amide hydrolase (faah) inhibition reduces l- , -dihydroxyphenylalanineinduced hyperactivity in the -methyl- -phenyl- , , , -tetrahydropyridinelesioned non-human primate model of parkinson's disease contrasting effects of selective magl and faah inhibition on dopamine depletion and gdnf expression in a chronic mptp mouse model of parkinson's disease competing interests: the authors declare no competing interests. key: cord- -d m sgk authors: zheng, xu-yong; sun, chu-chu; liu, qian; lu, xiao-yao; fu, li-li; liang, guang; zhang, xiu-hua; chen, gao-zhi title: compound lm , a novel myd inhibitor, efficiently mitigates inflammatory responses and fibrosis in obesity-induced cardiomyopathy date: - - journal: acta pharmacol sin doi: . /s - - -x sha: doc_id: cord_uid: d m sgk mechanisms of cardiomyopathy caused by obesity/hyperlipidemia are complicated. obesity is usually associated with chronic low-grade inflammation and may lead to the onset and progression of myocardial fibrosis and remodeling. tlr /myd signaling pathway, as a key regulator of inflammation, plays an important role in the pathogenesis of obesity-induced cardiomyopathy. we previously demonstrated that lm , a novel myd inhibitor, attenuated inflammatory responses and fibrosis in obesity-induced cardiomyopathy by inhibiting the formation of tlr /myd complex. in this study, we investigated the protective effects of lm on obesity-induced cardiomyopathy in vitro and in vivo. we showed that lm ( , μm) significantly attenuates palmitic acid (pa)-induced inflammation in mouse peritoneal macrophages, evidenced by decreased expression of proinflammatory genes including tnf-α, il- , il- β, and icam- . in cardiac-derived h c cells, lm treatment suppressed pa-induced inflammation, lipid accumulation, and fibrotic responses. in addition, lm treatment also inhibited pa-activated tlr /myd /nf-κb signaling pathway. we further revealed in hek cells that lm treatment blocked the tlr /myd binding and myd homodimer formation. in hfd-fed mice, administration of lm ( , mg/kg, ig, every other days for weeks) dose-dependently alleviated inflammation and fibrosis in heart tissues and decreased serum lipid concentration. in conclusion, this study demonstrates that myd inhibitor lm exerts protective effects against obesity-induced cardiomyopathy, suggesting lm to be a promising therapeutic candidate drug for the obesity-related cardiac complications. obesity incidence has increased each year, and it has become a global public health problem [ ] . obesity leads to an inflammatory condition, causing elevated levels of inflammatory cytokines and disorders of immune function, and is directly involved in the etiology of cardiovascular diseases, type diabetes mellitus, and certain types of cancer [ , ] . moreover, a large number of studies have demonstrated that obesity is one of the independent risk factors for myocardial dysfunction and remodeling, leading to an increase in the incidence and mortality of cardiovascular disease [ ] [ ] [ ] . several mechanisms have been identified in obesity-induced cardiomyopathy, including oxidative stress, autophagy, and adrenergic and renin-angiotensin aldosterone overflow [ ] [ ] [ ] . chronic lowgrade inflammation has also been indicated as an important process in obesity-induced cardiomyopathy. there is increasing interest in the link between obesity and inflammation [ , ] . due to the obesity process, ongoing chronic inflammation causes infiltration of inflammatory cells and progressive changes in tissue destruction and remodeling [ , ] . moreover, the link between obesity and fibrosis of the myocardium has been well determined [ , ] . the levels of free fatty acids (ffas), especially saturated fatty acids (sfas), are significantly elevated in obese patients [ ] and cause an inflammatory response, which is an important factor in the development of obesity-related cardiovascular diseases [ , ] . in particular, our previous study reported that palmitic acid (pa), the most abundant circulating sfa, induces myocardial inflammatory injury through the toll-like receptor (tlr ) accessory protein md in vitro and in vivo [ ] . tlr is an essential modulator of innate immunity and links innate immunity and metabolic disorders, such as obesity. myeloid differentiation protein (myd ) is one of the major adapterdependent downstream signaling pathways of activated tlr . once bound to lps or pa, the md protein induces the dimerization of tlr and then recruits myd and produces many proinflammatory molecules. increasing evidence indicates that the tlr /myd signaling pathway not only plays an important role in the inflammatory response to infection but also participates in the development of metabolic-related diseases such as cardiovascular diseases [ , ] . targeting myd may be an effective strategy for the treatment of obesity-induced cardiomyopathy. thus, in the current study, we provide evidence that compound lm , a novel myd inhibitor from our previous study [ , ] , efficiently attenuates inflammatory responses and fibrosis in obesity-induced cardiomyopathy by inhibiting the formation of the tlr /myd complex. we suggest that lm may be a promising therapeutic candidate drug for obesity-related cardiac complications. chemicals and reagents lm was synthesized with a purity over % in our laboratory. pa was solubilized in low-endotoxin bovine serum albumin (bsa) ( %) at a final concentration of mm. pa and bsa were purchased from sigma-aldrich (st. louis, mo, usa). the mtt assay kit and oil red o staining kit (for cultured cells) were acquired from solarbio (beijing, china). antibodies were purchased from the following suppliers: anti-lamin b (ab ), anti-iκb alpha (ab ), anti-ly g (ab ), anti-tnf-α (ab ), anti-collagen i (ab ), and anti-collagen iv (ab ) were obtained from abcam (cambridge, ma, usa). anti-tlr (sc- ) and anti-tgf-β (sc- ) were obtained from santa cruz (dallas, tx, usa). anti-flag (sab ) and anti-ha (mfcd ) were purchased from sigma-aldrich (st. louis, mo, usa). anti-icam- (cat# - -ig) was obtained from proteintech (wuhan, china). two plasmids encoding pcmv-ha-myd and flag-myd were obtained from sino biological inc. (beijing, china). elisa kits for mouse tnf-α, il- β, and il- were purchased from invitrogen (carlsbad, ca, usa). nuclear and cytoplasmic protein extraction kits were from keygen biotech (nanjing, china). primescript™ rt reagent kit with gdna eraser (perfect real time) was purchased from takara bio (rr a, shiga, japan). compound solution preparation lm was dissolved in dmso to a final drug concentration of mm and stored at − °c. before cell experiments, the lm stock solution was diluted to mm and mm working solutions with dmso. the final concentration of dmso in the cell experiments did not exceed . % (v/v). in animal experiments, % sodium carboxymethylcellulose solution was used to formulate lm into a . or mg/l suspension. the control group and the model group were given equal doses of % sodium carboxymethylcellulose solution. primary macrophage isolation and cell culture c bl/ j mice were injected i.p. with ml of % thioglycollate solution ( . g beef extract, . g tryptone, and . g nacl dissolved in ml double-distilled h o and filtered through a . -μm filter). after h, the mice were sacrificed, and peritoneal cavities underwent lavage with ml rpmi- medium. samples were centrifuged and resuspended in rpmi- medium with % fbs, u/ml penicillin g and mg/ml streptomycin. non-adherent cells were removed h after seeding. h c myoblasts and hek t cells were purchased from the cell bank of the chinese academy of sciences (shanghai, china) and american type culture collection (atcc, manassas, va, usa), respectively. the cells were cultured in dmem supplemented with % (v/v) fbs, u/ml penicillin g, and mg/ml streptomycin. the cells were maintained at °c under a humidified atmosphere of % co in an incubator. male c bl/ j mice weighing - g were purchased from the wenzhou medical university animal center (wenzhou, china). the mice were housed in a standard, pathogen-free animal facility under a h light/dark cycle at - °c with unrestricted access to food and water for the duration of the experiment. all animal experimental protocols were reviewed and approved by the wenzhou medical university animal policy and welfare committee. obesity was induced by feeding the mice an hfd (d ) composed of % fat, % protein, and % carbohydrate (catalogue no. md ; mediscience diets, yangzhou, china). control mice were fed a standard control diet containing % fat, % protein, and % carbohydrate (catalogue no. d b, research diets, yangzhou, china). the mice in this experiment were randomly divided into four groups as follows: ( ) the control group (n = ); ( ) the hfd group (n = ); ( ) the mg/kg lm treated hfd group (n = ); and ( ) the mg/kg lm -treated hfd group (n = ). the mice were fed a high-fat diet or a standard diet for weeks, and then the lm groups were treated with lm every days at a dose of or mg/kg for weeks by intragastric injection. weekly monitoring of body weight beginning at the fourth week was performed. at the end of the study, the mice were killed by cervical dislocation after anesthetization with % chloral hydrate. then, blood and heart tissues were collected. cell viability cell viability was measured by using the mtt assay. cells were cultured in -well plates at a density of × cells per well overnight and were subsequently treated with different concentrations of the indicated compound for h. then, μl mtt solution ( mg/ml) was added to each well. after h, the medium was discarded, μl dmso was added, and the absorbance was measured at nm. cell viability was defined as the percentage of the absorbance compared with the control. after pretreatment with lm ( and μm) for h, mpms were then incubated with pa ( μm) for h. the samples were subsequently centrifuged at , rpm for min at °c. supernatants from cell cultures were assayed for mouse tumor necrosis factor alpha (tnf-α) and interleukin- (il- ) according to the manufacturer's instructions (invitrogen). the levels of the proteins were calculated using the standard curve method. analysis of serological markers colorimetric assays were performed using a colorimetric assay kit for non-esterified fatty acids and creatine kinase, including triglycerides (tgs, a - - ), total cholesterol (tch, f - - ), high-density lipoprotein (hdl, a - - ), and creatine kinase (ck, a - - ). creatine kinase mb isoenzyme (ck-mb, h ) was determined using a ck-mb elisa kit. all of the kits were purchased from nanjing jiancheng biotech (nanjing, china). heart tissues were fixed in % formalin for h at rt. after embedding in paraffin, the heart samples were cut into μm sections, which were prepared and stained with h&e or sirius red using standard procedures. the sections for immunohistochemistry were deparaffinized, rehydrated and then incubated with % hydrogen peroxide for min and blocked with % bsa for min at room temperature. the sections were then incubated with anti-ly g or anti-tnf-α antibodies at °c overnight. next, the sections were incubated with horseradish peroxidase-conjugated secondary antibodies. histochemical visualization was carried out with dab. images were captured using a nikon microscope equipped with a digital camera (tokyo, japan). coimmunoprecipitation assay cells were seeded in mm cell culture dishes at a density of × - × cells per well and cultured overnight for coimmunoprecipitation assays. the cells were collected and lysed in lysis buffer (ar / , boster biological technology co. ltd, pleasanton, ca, usa) and centrifuged at , rpm for min at °c. two microliters of antibody were added to μg of protein, and the samples were gently rotated at °c overnight. the immune complexes were collected with protein a + g agarose beads (p , beyotime institute of biotechnology, haimen, china), and the precipitates were washed five times with ice-cold pbs. prewashed agarose beads were boiled in sample buffer for min. western blot analysis proteins were extracted from the heart tissue or cells using lysis buffer (ar / , boster biological technology co. ltd, pleasanton, ca, usa) according to the manufacturer's instructions. the proteins were separated by sds-polyacrylamide gel electrophoresis, transferred to polyvinylidene difluoride membranes (pvdf, bio-rad, hercules, ca, usa), blocked with % skim milk (bd, franklin lakes, nj, usa) and exposed to primary antibody overnight ( °c), followed by horseradish peroxidase-labeled secondary antibodies for another h. the immune complexes were visualized by exposure in a chemidoc xrs + system (bio-rad, hercules, ca, usa). quantitative real-time pcr total rna from heart tissues or cells was extracted using trizol reagent (takara bio, shiga, japan). reverse transcription was performed using the primescript™ rt reagent kit. qrt-pcr was performed using sybr green supermix (bio-rad, hercules, ca, usa). primers including tnf-α, il- , il- β, icam- , bnp, tgf-β , collagen i, collagen iv, and β-actin were purchased from invitrogen. the primer sequences used are shown in supplementary table s . statistical analysis all data were obtained from at least three independent replicates and calculated as the means ± standard deviation (mean ± sd). the significance of the intergroup differences was analyzed with student's t tests or one-way analysis of variance , il- (c), and il- β (d) production. the concentrations of tnf-α and il- in supernatants from mpms that were treated with various doses (above the lanes) of lm followed by stimulation with μm pa for h were measured by elisa. the data are expressed as the mean ± sd (n = ). student's t test was utilized for the statistical analysis, and significant differences are indicated as * or # . *p < . , **p < . , and ***p < . vs. the bsa-treated group; # p < . , ## p < . , and ### p < . vs. the pa alone group. e the protein level of icam- in mpms was determined by western blot assay (n = ). f mpms were pretreated with lm ( and μm) for h and stimulated with μm pa for h, after which tnf-α, il- , il- β, and icam- mrna levels were determined by qrt-pcr. the data are expressed as the mean ± sd (n = ). student's t test was utilized for the statistical analysis, and significant differences are indicated as * or # . **p < . , and ***p < . vs. the control group; # p < . , ## p < . , and ### p < . vs. the pa alone group. (anova). a p value of < . was considered statistically significant. to provide a potential treatment for myd -driven inflammation, a new myd inhibitor was synthesized in our laboratory, and the chemical structure of lm is shown in fig. a . to determine whether lm attenuates pa-induced inflammation, we examined the secretion of tnf-α and il- and the expression of il- β in painduced mouse peritoneal macrophages by elisa (fig. b-d) . our results showed that lm reduced pa-induced tnf-α, il- , and il- β in mpms. in addition, we tested the expression of icam- , and the results indicated that the expression of icam- was inhibited by lm (fig. e ). then, we tested the mrna levels of proinflammatory genes in mpms after treatment with lm , and we found that lm exhibited a dose-dependent inhibitory effect on the mrna levels of proinflammatory genes, including tnf-α, il- , il- β, and icam- (fig. f) . these results indicate that lm restrains pa-induced inflammation. lm suppresses the inflammatory response and nf-κb signaling pathway activation in pa-stimulated h c cells the toxicity of lm is shown in fig. a . here, we examined whether lm protects against the inflammatory response in painduced h c cells. the qrt-pcr results showed that the mrna levels of tnf-α, il- , icam- , and bnp were significantly reduced in the lm -treated group (fig. b-e) . as the nf-κb signaling pathway ) . b-e h c cells were pretreated with lm ( and μm) for h and stimulated with μm pa for h, after which tnf-α, il- , icam- , and bnp mrna levels were determined by qrt-pcr. the data are expressed as the mean ± sd (n = ). the protein levels of iκb-α (f) and p-iκb-α (g) in pa-induced h c cells were determined by western blot assay. representative immunoblots are shown in the upper panel, and densitometric quantification is presented as the mean ± sd (n = ) in the lower panel. h, i western blot of cytosolic and nuclear protein levels of nf-κb p subunit in h c cells that were stimulated with μm pa for h. representative immunoblots are shown in the upper panel, and densitometric quantification is presented as the mean ± sd (n = ) in the lower panel. student's t test was utilized for the statistical analysis (b)-(i), and significant differences are indicated as * or # . *p < . , **p < . , and ***p < . vs. the control group; # p < . , ## p < . , and ### p < . vs. the pa alone group. compound lm mitigates obesity-induced cardiomyopathy xy zheng et al. is downstream of tlr /myd in the inflammatory response, we tested the expression levels of iκb-α and piκb-α, and the results indicated that the degradation and phosphorylation of iκb-α were markedly inhibited by lm (fig. f, g) . in addition, cytosolic and nuclear protein levels of the nf-κb p subunit were tested, and we found that lm decreased the accumulation of nf-κb p in the nucleus (fig. h, i) . lm inhibits tlr /nf-κb signaling pathway activation by blocking tlr /myd binding and myd homodimer formation we further explored the mechanism by which lm inhibits tlr / nf-κb activation in pa-induced cells. tlr /myd binding and myd homodimer formation are necessary for activation of the tlr /nf-κb signaling pathway [ , ] . we found that lm treatment significantly inhibited pa-induced tlr /myd binding based on coimmunoprecipitation assays (fig. a) . we next examined whether lm inhibited the formation of the myd homodimer. the transfection efficacy of ha-myd and flag-myd plasmids is shown in fig. b . then, we tested the effect of pa on the formation of the myd homodimer at , , and min compared with that of the control group, and we found that the myd homodimer apparently increased at min (fig. c) . the effects of lm on blocking myd dimerization were examined and are shown in fig. d . thus, lm inhibits the tlr /nf-κb signaling pathway by blocking tlr /myd binding and myd homodimer formation. lm alleviates pa-induced lipid accumulation and fibrosis in h c cells since cardiac steatosis is associated with impaired cardiac function during obesity, oil red o staining was utilized to investigate lipid accumulation in h c cells with different treatments. our results revealed that lm alleviated pa-induced lipid distribution in h c cells (fig. a, upper panel) . saturated pa induces md -dependent inflammatory injury in cardiomyocytes and causes myocardial morphological changes [ ] . we measured cellular morphology in h c cells that were pretreated with lm before exposure to pa by hematoxylin staining, and the results indicated that morphological changes in h c cells were alleviated by lm (fig. a, lower panel) . fibrotic change is a feature of obesity-induced cardiomyopathy. we detected the expression levels of profibrotic proteins by western blotting, and the data showed that the protein levels of collagen / and tgf-β were markedly decreased in h c cells in the presence of lm (fig. b, c) . similar effects were observed when the mrna levels of profibrotic genes in h c cells were analyzed by qrt-pcr (fig. d) . effects of lm on body weight, cardiac function, and blood lipid profile in hfd mice we used an hfd mouse model to investigate whether lm exhibits cardioprotective effects in obesity in vivo. hfd feeding gradually and significantly increased the body weights in the hfd group compared with those in the control group. neither low nor high dose lm treatment had effects on hfd feeding-induced body weight increases (fig. a) . the cardiac function of mice in four groups was measured. notably, both treatment groups showed decreased levels of ck-mb and ck compared with those in the hfd group (fig. b, c) . we further tested other serological markers that correlate with obesity and lipid metabolism. the levels of serum triglyceride (tg), tch, hdl, and low-density lipoprotein (ldl) were increased in groups that were fed an hfd but were alleviated in groups that were treated with lm ( fig. d-g) . moreover, cardiac function was assessed by student's t test was utilized for the statistical analysis. significant differences are indicated as * or # . **p < . vs. the control group; ## p < . vs. the pa alone group. table s ). to further investigate the effects of lm , we observed the morphology of the hearts in the four groups by hematoxylin-eosin staining. the results showed that there were significant changes in cardiac structure, as well as myocardial thickening and interstitial proliferation, in the hfd group, while lm administration improved the obesity-induced cardiac pathological changes (fig. a, first and second panel) . in addition, lm ameliorated tnf-α accumulation (fig. a, third panel) and decreased neutrophil infiltration (fig. a , last panel) compared with those in the hfd groups. we also found that lm exhibited an obvious inhibitory effect on the mrna levels of proinflammatory genes, including tnf-α, il- , and icam- (fig. b) . previous studies have suggested that cardiac fibrosis and inflammation are potential contributors to obesity-induced changes in cardiac structure and function [ ] . we analyzed the interstitial collagen distribution by picrosirius red staining. as expected, there was significant fibrogenesis in control and hfd mice, while collagen deposition was significantly decreased in both lm treatment groups (fig. c) . the protein level of iκb-α is shown in fig. d , and lm markedly inhibited the hfd-induced degradation of iκb-α (fig. d) . we also examined the expression levels of profibrotic proteins in heart tissue, and our results revealed that lm attenuated hfd-induced cardiac fibrosis (fig. d and supplementary fig. s ). similar effects were observed when the mrna levels of profibrotic genes in heart tissue were analyzed (fig. e) . myocardial inflammation has been widely accepted to play a pivotal role in the physiological and pathological mechanisms of cardiac function and dysfunction [ ] . recent studies have shown that obesity-induced chronic inflammation is associated with the pathophysiology of obesity-related cardiovascular disease [ ] [ ] [ ] . hyperlipidemia leads to increased production of inflammatory cytokines and increased inflammatory cell and cellular morphology (lower panel) of h c cells that were stimulated with μm pa (× magnification). the protein levels (b) and the densitometric quantification (c) of profibrotic proteins in cells that were pretreated with lm before stimulation with μm pa for h were determined by western blotting (representative of n = , mean ± sd). d h c cells were pretreated with lm ( and μm) for h and stimulated with μm pa for h, after which collagen i, collagen iv, and tgf-β mrna levels were determined by qrt-pcr. the data are presented as the mean ± sd (n = ). student's t test was utilized for the statistical analysis (c), (d), and significant differences are indicated as * or # . **p < . and ***p < . vs. the control group; # p < . , ## p < . , and ### p < . vs. the pa alone group. infiltration, impairing the normal function of cardiomyocytes and leading to myocardial fibrosis and cardiac remodeling [ ] . it has been reported that anti-inflammatory drugs alleviate obesityrelated cardiomyopathy in animal models [ ] [ ] [ ] . due to the potential role of inflammation in cardiovascular disease, compounds with anti-inflammatory activity may have unexpected effects in inflammatory-related cardiovascular diseases such as obesity cardiomyopathy. notably, numerous studies have demonstrated that tlr and myd are robustly expressed in various inflammatory cardiovascular diseases, such as myocarditis, myocardial infarction, ischemia-reperfusion injury, and obesity-associated cardiovascular disease, and tlr activates the expression of several proinflammatory cytokine genes through the myd -dependent pathway, which plays pivotal roles in myocardial inflammation [ , ] . therefore, we can mitigate obesity-induced cardiomyopathy by targeting tlr or its downstream signaling pathways. currently, the unresolved question is whether it is possible to attenuate deleterious myocardial inflammation and preserve the advantages of innate immunity while also regulating tlr signaling [ ] . however, directly blocking tlr or md is not a perfect strategy for improving myocardial inflammation. by activating the myd -independent pathway and further promoting the secretion of type i interferon, which possesses an antiviral effect, the md /tlr signaling pathway also plays a beneficial role in myocardial inflammation, especially viral myocarditis. directly inhibiting md /tlr completely blocks the function of innate immunity. thus, it is reasonable to find an inhibitor that targets the myd -dependent pathway. it has been reported that both heart-specific autoimmunity and myd signaling are critical for cardiac fibrosis and heart failure progression [ ] . however, the prevention or treatment of cardiac diseases with myd inhibitors or antagonists to date has not been evaluated in human clinical trials. the lack of favorable myd inhibitors may be a major cause. recent studies have shown that -amino- -phenylthiazole structures have a variety of pharmacological activities, such as antiinflammatory and antioxidant activities, which can inhibit the dimerization of the myd -tir domain. our team took this as a lead compound to develop more effective myd inhibitors for the treatment of inflammation, and lm is one of the most active compounds with myd binding ability. in lps-stimulated mouse primary macrophages, lm significantly inhibited the tlr -myd interaction and myd dimerization [ ] . based on the robust anti-inflammatory effects of lm , our team explored whether lm alleviates the low-grade inflammation caused by long-term hyperlipidemia. we found that lm inhibited the inflammatory response of mouse primary macrophages induced by sfas (fig. ) . the relationship between obesity and inflammation has been well demonstrated. obesity is now often associated with chronic low-grade inflammation, suggesting that inflammation may be a potential mechanism leading to obesity-related cardiovascular complications. moreover, obesity is often accompanied by dyslipoidosis and hyperlipidemia. in obese patients, the level of sfas is usually elevated, which is directly involved in the etiology of cardiovascular diseases. pa is the main sfa in plasma and stimulates the production of inflammatory cytokines in cultured aortic smooth muscle cells and endothelial cells [ ] . our team found that pa induced the production of inflammatory factors and caused lipid accumulation, morphological changes and fibrosis in h c cells. however, this pathological state was reversed by lm (figs. and ) . subsequently, animal experiments further validated the effect of lm on myocardial inflammation, morphological changes, lipid accumulation and fibrosis (figs. and ). lipid accumulation activates harmful signaling pathways, resulting in the production of inflammatory factors and tissue remodeling in cardiomyocytes, leading to obesity-induced cardiomyopathy. the tlr /myd /nf-κb signaling pathway plays a key role in regulating the inflammatory response and is directly related to the release of inflammatory factors and cell adhesion molecules. our team found that lm inhibited pa/ hfd-induced nf-κb signaling pathway activation in cardiomyocytes or animal models, and blocking the nf-κb signaling pathway may be associated with lm -mediated inhibition of tlr /myd binding and myd dimerization (figs. and ) . in conclusion, our study demonstrates the cardioprotective effects of lm on pa-or hfd-induced inflammation and fibrosis fig. effects of lm on body weight, cardiac function and blood lipid profile in hfd mice. c bl/ j mice were fed an hfd for months, and blood was collected for evaluation of myocardial inflammatory injury. a body weight gain over a -week period. b, c serological markers of myocardium injury were evaluated by ck-mb and ck. d-g the blood lipid profile contains triglycerides (tg), total cholesterol (tch), highdensity lipoprotein (hdl), and low-density lipoprotein (ldl). the data in (a)-(g) are shown as the mean ± sd, n = or . student's t test was utilized for the statistical analysis, and significant differences are indicated as * or # . *p < . , **p < . , and ***p < . vs. the control group; # p < . , ## p < . vs. the pa alone group. compound lm mitigates obesity-induced cardiomyopathy xy zheng et al. in vitro and in vivo and provides a promising therapeutic drug for the treatment of obesity-related cardiac complications. inhibition of tlr /myd may be an effective strategy for the treatment of pathological changes in obesity-induced cardiomyopathy. fig. lm mitigates myocardial inflammatory injury and ameliorates fibrosis in hfd mice. c bl/ j mice were fed an hfd for months, and heart tissue was collected to evaluate myocardial inflammatory injury and amelioration of fibrosis. a the upper panel shows representative myocardium histological changes by hematoxylin and eosin staining; the middle panel shows representative images of ihc analysis of tnf-α accumulation in myocardial tissues; the lower panel shows ihc analysis of ly g in myocardial tissues as a marker of inflammatory cell infiltration. representative n = . b effects of lm on the mrna levels of proinflammatory genes and adhesion molecules in heart tissue were determined by qrt-pcr. the data are presented as the mean ± sd. n ≥ . c representative images of sirius red staining were used to assess the accumulation of collagen fibers. d the protein levels of iκb-α and profibrotic proteins in heart tissue were detected by western blotting (representative of n = ). e effects of lm on the mrna levels of profibrotic genes in heart tissue. the data are presented as the mean ± sd. n ≥ . student's t test was utilized for the statistical analysis (b), (e), and significant differences are indicated as * or # . **p < . , and ***p < . vs. the control group; # p < . , ## p < . , and ### p < . vs. the hfd mice group. obesity: trends in underweight and obesity -scale of the problem understanding obesity-related cardiovascular disease: it's all about balance obesity, inflammation, toll-like receptor and fatty acids obesity as a disease, not a behavior obesity and cardiovascular risk: a call for action from the european society of hypertension working group of obesity, diabetes and the high-risk patient and european association for the study of obesity: part a: mechanisms of obesity induced hypertension obesity-induced changes in adipose tissue microenvironment and their impact on cardiovascular disease cardiolipotoxicity, inflammation, and arrhythmias: role for interleukin- molecular mechanisms targeting autophagy in obesity-associated heart disease inflammatory mechanisms in obesity cardiac remodeling in obesity: time for a new paradigm adapting to obesity with adipose tissue inflammation oxidative stress and inflammation interactions in human obesity extracellular matrix remodeling in the heart of the homocysteinemic obese rabbit epicardial adipose tissue may mediate deleterious effects of obesity and inflammation on the myocardium changes in the serum composition of free-fatty acids during an intravenous glucose tolerance test free fatty acids are independently associated with all-cause and cardiovascular mortality in subjects with coronary artery disease free fatty acids as a cardiovascular risk factor saturated palmitic acid induces myocardial inflammatory injuries through direct binding to tlr accessory protein md toll-like receptor- mediates vascular inflammation and insulin resistance in diet-induced obesity saturated fatty acids activate tlr-mediated proinflammatory signaling pathways development of -amino- -phenylthiazole analogues to disrupt myeloid differentiation factor and prevent inflammatory responses in acute lung injury a novel myd inhibitor lm prevents atherosclerosis by regulating inflammatory responses and oxidative stress in macrophages identification of binding sites for myeloid differentiation primary response gene (myd ) and toll-like receptor in myd adapter-like (mal) the emerging role of toll-like receptor in myocardial inflammation inflammation and metabolic cardiomyopathy cardiac remodeling in obesity chronic inflammation in fat plays a crucial role in the development of obesity-related insulin resistance cardiac function and obesity manifestations and mechanisms of myocardial lipotoxicity in obesity inhibition of inflammation and oxidative stress by an imidazopyridine derivative x prevents heart injury from obesity endoplasmic reticulum stressinduced irhom up-regulation promotes macrophage-regulated cardiac inflammation and lipid deposition in high fat diet (hfd)-challenged mice: intervention of fisetin and metformin eet intervention on wnt , nov, and ho- signaling prevents obesity-induced cardiomyopathy in obese mice dual activation of trif and myd adaptor proteins by angiotensin ii evokes opposing effects on pressure, cardiac hypertrophy, and inflammatory gene expression myeloid differentiation factor- /interleukin- signaling controls cardiac fibrosis and heart failure progression in inflammatory dilated cardiomyopathy compound lm mitigates obesity-induced cardiomyopathy xy zheng et al. xyz, xhz, and gzc designed the research. xyz, ccs, ql, and xyl performed the research. xyz and gzc wrote the paper. llf and gl provided the tested compound. gzc, xhz, and gl reviewed and modified the paper. the online version of this article (https://doi.org/ . /s - - -x) contains supplementary material, which is available to authorized users.competing interests: the authors declare no competing interests. key: cord- - ukffdmq authors: xu, deng-qiu; li, chun-jie; jiang, zhen-zhou; wang, lu; huang, hong-fei; li, zhi-jian; sun, li-xin; fan, si-si; zhang, lu-yong; wang, tao title: the hypoglycemic mechanism of catalpol involves increased ampk-mediated mitochondrial biogenesis date: - - journal: acta pharmacol sin doi: . /s - - - sha: doc_id: cord_uid: ukffdmq mitochondria serve as sensors of energy regulation and glucose levels, which are impaired by diabetes progression. catalpol is an iridoid glycoside that exerts a hypoglycemic effect by improving mitochondrial function, but the underlying mechanism has not been fully elucidated. in the current study we explored the effects of catalpol on mitochondrial function in db/db mice and c c myotubes in vitro. after oral administration of catalpol ( mg·kg(− )·d(− )) for weeks, db/db mice exhibited a decreased fasting blood glucose level and restored mitochondrial function in skeletal muscle. catalpol increased mitochondrial biogenesis, evidenced by significant elevations in the number of mitochondria, mitochondrial dna levels, and the expression of three genes associated with mitochondrial biogenesis: peroxisome proliferator-activated receptor gammaco-activator (pgc- α), mitochondrial transcription factor a (tfam) and nuclear respiratory factor (nrf ). in c c myotubes, catalpol significantly increased glucose uptake and atp production. these effects depended on activation of amp-activated protein kinase (ampk)-mediated mitochondrial biogenesis. thus, catalpol improves skeletal muscle mitochondrial function by activating ampk-mediated mitochondrial biogenesis. these findings may guide the development of a new therapeutic approach for type diabetes. diabetes mellitus (dm) is a serious endocrine and metabolic disease. the vast majority of dm cases fall into two broad etiopathogenetic categories [ ] . type diabetes mellitus (t dm) accounts for only %- % of all dm cases and is caused by β-cell destruction, usually leading to complete insulin deficiency. type dm (t dm) accounts for %- % of all dm cases and is caused by insulin resistance (ir), sometimes accompanied by defects in insulin secretion. most anti-diabetic drugs exert unwanted side effects (hypoglycemia and gastrointestinal problems) [ ] . there is an urgent need to develop safer and more effective drugs to treat t dm and insulin resistance. changes in cell energy metabolism and nutrient partitioning are responsible for the development of insulin resistance [ ] . mitochondria generate most of the cell's energy via aerobic respiration and are indispensable for life [ ] . mitochondrial dysfunction may affect energy production, thereby impairing cell function, and has been implicated in many acute and chronic diseases [ ] [ ] [ ] . a strong association of insulin resistance and t dm development with mitochondrial dysfunction is evident, and mitochondrial dysfunction causes insulin resistance [ ] [ ] [ ] . however, the details of this association remain unclear. some studies have shown that lifestyle interventions (exercise or caloric restriction) increase insulin sensitivity, delaying or preventing diabetes onset, and insulin sensitivity is closely related to increases in mitochondrial mass and oxidative phosphorylation [ , ] . a defect in oxidative phosphorylation caused by a reduced number of mitochondria may affect fatty acid metabolism, ultimately causing insulin resistance [ , ] . in addition, one study found that a reduction in mitochondrial content was caused by a reduced type i:ii muscle fiber ratio [ ] . a few studies on diabetics have reported substantial decreases in mitochondrial volume and obvious abnormalities in mitochondrial morphology, consistent with the mitochondrial ultrastructural changes and decreased oxidative phosphorylation activity evident in t dm patients [ , ] . together, these data suggest that the mitochondrion may be an important therapeutic target for the treatment of t dm. mitochondrial biogenesis is dynamically regulated; new mitochondria are constantly produced to maintain the appropriate number of morphologically normal active organelles to meet the constantly changing bioenergetic needs of the cell via oxidative metabolism [ ] . therefore, compounds that stimulate mitochondrial biogenesis may improve mitochondrial dysfunction. mitochondrial biogenesis is coordinately controlled by transcription factors, nuclear hormone receptors, and transcriptional coactivators. peroxisome proliferator-activated receptor gamma coactivator , a member of the pgc- family of transcriptional coactivators, is the master regulator of mitochondrial biogenesis in many tissues [ ] . pgc- α controls the expression levels of mitochondrial genes and induces mitochondrial biogenesis by interacting with transcription factors such as nuclear respiratory factors (nrfs), peroxisome proliferator-activated receptors, and estrogen receptor-related receptors (errs) [ ] . mitochondrial transcription factor a, a dna-binding stimulatory protein, regulates mitochondrial dna transcription and replication to maintain the mitochondrial dna (mtdna) content [ ] . pgc- α interacts with nrf to activate mitochondrial transcription factor a (tfam) expression, thereby stimulating mitochondrial biogenesis [ ] . furthermore, t dm development is associated with decreased pgc- α expression in the liver and skeletal muscle caused by a reduction in pgc- α-dependent gene expression. this compromises mitochondrial biogenesis and further impairs mitochondrial atp synthesis [ ] . overall, mitochondrial biogenesis regulated by pgc- α may be critical for t dm treatment. catalpol is an iridoid glucoside of rehmannia glutinosa that exerts significant hypoglycemic effects on different diabetic models [ , ] . a recent study indicated that orally administered catalpol has beneficial effects on insulin resistance thus lowering blood glucose [ ] . we recently showed that catalpol improved hfd/stz-induced muscle mitochondrial dysfunction [ ] . insulin resistance and mitochondrial dysfunction are closely related. moreover, it is still debated whether catalpol exerts direct mitochondrial protection or insulin-sensitizing effects on skeletal muscles. although the hypoglycemic effect of catalpol is related to increased mitochondrial function, the detailed molecular mechanism remains unknown. here, we explored how catalpol improves mitochondrial function in db/db mice and c c myotubes. animal studies all experimental procedures were conducted in accordance with institutional guidelines for the care and use of laboratory animals in china. male c bl ks/j-db/db mice ( - weeks old, - g in body weight, n = animals) and their normoglycemic littermates ( - weeks old, - g in body weight, n = animals) were obtained from the nanjing university animal model research center (nanjing, china). sixteen c blks/j-db/ db mice were randomly allocated to the db/db mouse group or the mg/kg catalpol-treated group (orally administered for consecutive weeks). all animals were housed in an airconditioned room maintained at a temperature of - °c with a / -h light/dark cycle and were provided with standard mouse chow and water ad libitum. at the end of the experimental period, all mice were anesthetized using isoflurane following h of fasting. blood was collected from the inferior vena cava for the determination of glucose, hba c, triglyceride (tg), total cholesterol (tc), high-density lipoprotein cholesterol (hdl-c) and low-density lipoprotein cholesterol (ldl-c) levels. then, the mice were killed, and tibialis anterior (ta) muscle sections were collected. the ta muscle sections were used for western blotting, rt-qpcr and histological analysis. all dissected tissues were immediately frozen in liquid nitrogen and stored at − °c until analysis. the glucose tolerance test ( . g/kg i.p.) was performed in the animals following h of fasting. serum glucose concentrations were detected by using a glucometer (bayer corporation, mishawaka, in, usa) at , , , , and min after glucose injection. the insulin tolerance test ( . iu/kg i.p.) was performed in the animals following h fasting. serum glucose concentrations were determined using a glucometer at , , , and min after insulin injection. the areas under the curves (aucs) of the insulin tolerance test results were calculated [ ] . mitochondrial assays mitochondria were separated from fresh ta muscles as previously described [ ] . the relative mitochondrial dna content was measured by quantitative polymerase chain reaction (pcr) as described previously [ ] . nuclear β-actin and nd were used to represent ndna and mtdna. the primer pairs used for pcr are listed in table . determination of mitochondrial membrane potential and atp content the mitochondrial membrane potential (Δψm) in the ta muscles of db/db mice was evaluated using a jc- mitochondrial membrane potential detection kit, as previously described [ ] . the atp content of the isolated mitochondria was determined using a cell titer-glo® . assay kit (promega, madison, wi, usa) according to the manufacturer's instructions. electron microscopy fresh ta muscles were immediately fixed with cold . % glutaraldehyde in phosphate-buffered saline (pbs, ph = . ). all samples were processed, as previously described [ ] . images were acquired using a jem- electron microscope (jeol usa inc., peabody, ma, usa). for each group, the number of mitochondria was assayed from images from animals using image-pro plus software (media cybernetics, rockville, md, usa). cell culture c c cells were purchased from the american tissue culture collection (atcc) and grown in dmem growth medium (dulbecco's modified eagle's medium supplemented with % fetal bovine serum and % penicillin/streptomycin) in a % co incubator at °c as previously described [ ] . confluent c c myoblasts were differentiated into myotubes by switching the medium to medium containing % horse serum and % penicillin/ streptomycin when the cells reached %- % confluency. differentiated myotubes were serum-starved overnight and then incubated in serum-free media containing , , or μm catalpol or mm metformin. stimulated myotubes were collected for western blotting or real-time pcr. differentiated myotubes were incubated in serum-free medium containing μm compound c for h and then incubated in new serum-free medium containing , , or μm catalpol or mm metformin for h as described in our previous report [ ] . glucose uptake activity measurement using -nbdg glucose uptake activity in myotubes was measured using -nbdg, as described previously [ ] . briefly, myotubes were washed with cold pbs and incubated in glucose-free dmem with μm -nbdg for h. then, the myotubes were washed twice with cold pbs to remove free -nbdg. finally, the myotubes were suspended in cold pbs and transferred to the wells of a -well fluorescence microplate. mitochondrial staining following incubation with mm d-glucose for h and treatment with catalpol, cells were stained with nm mito-tracker red for min at °c, as described in our previous report [ ] . mitochondria were visualized under a confocal microscope, and the images were analyzed using image-pro plus software (media cybernetics, rockville, md, usa). real-time quantitative polymerase chain reaction (pcr) total rna was extracted from the ta muscle using trizol reagent and an rneasy kit (vazyme biotech, nanjing, china) according to the manufacturer's instructions. complementary dna (cdna) was synthesized from total rna using a cdna synthesis kit (vazyme biotech, nanjing, china). the resulting cdnas were amplified using a sybr green master mix kit in an iq™ real-time pcr detection system (bio-rad laboratories, hercules, ca, usa), as reported previously [ ] . gene expression was evaluated via the ΔΔct method using glyceraldehyde -phosphate dehydrogenase (gapdh) as a reference gene. the primer pairs used for pcr are listed in table . western blot analysis excised ta muscles were dissolved in total protein extraction buffer (keygen biotech, nanjing, china) with protease and phosphatase inhibitors. the total protein ( μg) was fractionated on - % sodium dodecyl sulfate (sds) polyacrylamide gels and transferred to nitrocellulose membranes (millipore, billerica, ma, usa) by electroblotting. the membranes were blocked in tris-buffered saline containing % milk and then incubated overnight at °c with one of the following primary antibodies: rabbit anti-tfam ( for immunohistochemistry, ta muscle samples were fixed in % paraformaldehyde (pfa), and paraffin-embedded sections were incubated overnight at °c with a primary antibody against myosin heavy chain ii (my- ) ( : ; ab , abcam, cambridge, ma, usa) [ ] . stained tissues were examined under a light microscope (bx , olympus, japan). all obtained data were analyzed with one-way analysis of variance (anova) followed by tukey's multiple comparison posttest using graphpad . (graph-pad software inc., san diego, ca, usa). data are presented as the mean ± sd. values with p < . were considered statistically significant. catalpol lowers fasting blood glucose levels and ameliorates mitochondrial dysfunction in the skeletal muscle of db/db mice body weight and the serum levels of glucose, hba c, tg, tc, and ldl-c were increased in the db/db mice compared with the control mice, whereas, the serum level of hdl-c was decreased in the db/db mice. catalpol treatment decreased the serum levels of glucose, hba c, tc, and ldl-c (table ) . moreover, catalpol treatment increased insulin sensitivity (fig. a, b) but had no effect on glucose tolerance (fig. c) . as we reported previously, catalpol improved skeletal muscle mitochondrial function [ ] . the mitochondrial membrane potential of the skeletal muscle of db/db mice was % lower than that of control mice but increased after catalpol treatment (fig. d) . as glucose utilization and mitochondrial activity are associated with changes in atp production [ ] , we performed an atp assay to evaluate mitochondrial function. the catalpol treatment of db/db mice increased atp production (fig. e) . although the mtdna copy number was only slightly reduced in the skeletal muscle of db/db mice, catalpol markedly increased the mtdna level (fig. f) . to explore whether catalpol increases the number of mitochondria in mouse skeletal muscle, we assessed both mitochondrion numbers and morphology via electron microscopy. the number of mitochondria was lower in db/db mice than in control mice. after the catalpol treatment of db/db mice, the mitochondrion number increased, but mitochondrial morphology was abnormal. the skeletal muscles of the db/db mice contained abundant lipid droplets, reflecting ectopic lipid deposition (fig. g, h) . mitochondrial dynamics are usually associated with quality control and the functional maintenance of bioenergetics [ ] . the expression levels of three genes associated with mitochondrial biogenesis (pgc- α, nrf , and tfam) were markedly decreased in the skeletal muscle of db/db mice, but these decreases were reversed by catalpol (fig. i) , as revealed by western blotting (fig. j, k) . catalpol ameliorates the abnormal muscle fiber ratio in db/db mice recent evidence has shown that a shift in the muscle fiber type from oxidative type i to the more glycolytic type ii damages the muscles of obese subjects with impaired glucose tolerance [ ] . we quantified the muscle fiber types by immunohistochemical staining of ta muscle sections and assessed the findings. the proportion of myosin heavy chain i (mhc i) fibers was decreased, whereas the proportion of mhc ii fibers was significantly increased in db/db mice compared with control mice (fig. a) . however, the proportions of mhc i and ii fibers did not differ between the catalpol treatment and control groups (fig. b, c) . catalpol increased the proportion of mhc i fibers and decreased that of mhc ii fibers in db/db mice. in addition, the p-ampk/t-ampk and p-acc/t-acc protein ratios were notably increased in db/db mice after catalpol treatment. (fig. d, e) . to further demonstrate that catalpol could cause a switch from glycolysis back to oxidative phosphorylation in db/db mice, we examined these two types of metabolism in the ta muscle. regarding glycolysis, the protein level of pfkp (fig. f, g) and the lactate level (fig. h) in the ta muscle did not differ between the db/db group and the control group. to examine oxidative phosphorylation, we detected the protein expression of the oxphos subunits, including ndufs (complex i), sdha (complex ii), uqcrc (complex iii), cox (complex iv), and atp a (complex v). the levels of these proteins were markedly decreased in the ta muscle of db/db mice, but these decreases were reversed by catalpol (fig. i, j) . to further examine the effects of catalpol on mitochondrial activity, we performed an atp assay. catalpol increased atp production in a dose-dependent manner (fig. a) and markedly increased the mtdna copy number in c c myotubes (fig. b) . thus, catalpol may increase mitochondrial biogenesis in skeletal muscle. moreover, consistent with the in vivo results (fig. d, e) , catalpol significantly increased the p-ampk/t-ampk and p-acc/t-acc protein ratios in c c myotubes (fig. c) . pgc- α and tfam protein levels were also increased after catalpol treatment in c c myotubes (fig. d) . c the glucose tolerance results from the glucose tolerance test are shown. d mitochondrial membrane potential, e atp production, and f mtdna copy number were determined in the ta muscle of db/db mice. g representative electron micrographs of mitochondria in the ta muscle. red arrowheads: mitochondria; blue arrowheads: lipid droplets. scale bar, nm. h the number of mitochondria calculated using innerview . software (lumenera). i the relative mrna levels of pgc- α, tfam, err-α, and nrf measured by rt-qpcr. gapdh served as the control housekeeping gene. j, k pgc- α and tfam protein levels in mouse ta muscle measured by western blot analysis. the data are expressed as the means ± sd (n = animals), *p < . , ***p < . vs. control mice; # p < . , ## p < . vs. db/db mice. catalpol ameliorates the mitochondrial dysfunction induced by high glucose levels in c c myotubes although catalpol treatment increased mitochondrial biogenesis in c c myotubes, whether catalpol protects c c myotubes against high-glucose (hg)-induced mitochondrial dysfunction remained unknown. we used mm glucose to simulate an hg environment in c c myotubes. to explore whether catalpol inhibits hg-induced mitochondrial fragmentation, we used mito-tracker red staining to monitor mitochondrial morphology. in c c myotubes cultured with a normal level of glucose ( mg/l), the mitochondria were intact and elongated. in contrast, the mitochondria were shorter and fragmented following hg treatment (fig. a) . the p-ampk/t-ampk and p-acc/t-acc protein ratios were significantly decreased in hg-treated c c myotubes, and this effect was reversed by catalpol or metformin treatment (fig. b) . additionally, catalpol significantly increased the protein levels of pgc- α and tfam compared with their levels in the hg group (fig. c) . prior studies have suggested that ampk is important for the maintenance of mitochondrial function [ , ] . we found that the p-ampk/t-ampk protein ratio increased notably after catalpol treatment both in vivo (fig. d , e) and in vitro (fig. c) . to explore the role of ampk in the hypoglycemic effect of catalpol, compound c, an ampk inhibitor, was used to block ampk phosphorylation in c c myotubes (fig. a) . as expected, both glucose consumption and atp production decreased following the inhibition of ampk-mediated phosphorylation. both catalpol and metformin increased glucose consumption and atp production via the activation of ampk signaling (fig. c) . notably, the catalpol-induced increases in pgc- α and tfam protein levels decreased after ampk inhibition (fig. b) . strict regulation of ampk phosphorylation is essential for optimal mitochondrial function [ ] . thus, ampk may play an important role in the hypoglycemic effect of catalpol. previous studies have shown that catalpol effectively lowers fasting blood glucose levels in rodent models of type dm and t dm [ , ] . consistent with these works, we also found that catalpol significantly reduced blood glucose levels in db/db mice. moreover, we identified a novel molecular mechanism by which catalpol lowers blood glucose levels. using db/db mice and c c cells, we showed that catalpol increases mitochondrial biogenesis by activating the ampk/pgc- α/tfam signaling pathway, which improves both mitochondrial function and glucose homeostasis in skeletal muscle (fig. ) . overall, ampk/pgc- α/tfam signaling plays a vital role in the hypoglycemic effect of catalpol. mitochondrial dysfunction and insulin resistance are closely related; insulin resistance can trigger mitochondrial dysfunction in the liver and skeletal muscle [ ] , and mitochondrial injury can develop after insulin resistance is established [ ] . therefore, increased mitochondrial function is critical for improving insulin resistance. we found that mitochondrial function was impaired in the skeletal muscle of db/db mice. after catalpol treatment, mitochondrial biogenesis was markedly increased. catalpol also ameliorated mitochondrial dysfunction induced by hg in skeletal muscle cells. together, the results show that catalpol increased mitochondrial biogenesis, thereby ameliorating mitochondrial impairment in skeletal muscle. thus, catalpol increased glucose uptake and atp production by skeletal muscle, reducing the blood glucose level. it is generally acknowledged that ampk, a sensor of cellular energy status, serves as a key regulator of glucose uptake, fatty acid oxidation, and mitochondrial biogenesis [ ] . ampk is a well-known upstream regulator of pgc- α expression in skeletal muscle. ampk activation increases the expression and phosphorylation of pgc- α, fig. catalpol increases mitochondrial biogenesis in c c myotubes. a atp production and b the mtdna copy number were measured in c c myotubes exposed to catalpol for h. c western blot analysis using anti-phospho-thr -ampk, anti-ampk, anti-phospho-ser -acc and anti-acc antibodies to determine protein levels and the ratios of p-ampk/t-ampk and p-acc/t-acc. d western blot analysis of pgc- α and tfam (con: control group; met: metformin mm). values are expressed as the means ± sd (n = ), *p < . , **p < . , ***p < . vs. control. fig. catalpol protects against hg-induced mitochondrial dysfunction. a c c myotubes stained with mito-tracker (red) and visualized by fluorescence microscopy after exposure to hg ( mm), as described in the methods (magnification, × ). cell nuclei were stained with dapi (blue). b western blot analysis using anti-phospho-thr -ampk, anti-ampk, anti-phospho-ser -acc and anti-acc antibodies to determine protein levels and the ratios of p-ampk/t-ampk and p-acc/t-acc. c western blot analysis of pgc- α and tfam (con: control group; met: metformin mm). values are expressed as the means ± sd (n = ), *p < . , **p < . vs. control; # p < . , ## p < . vs. hg. catalpol enhances mitochondrial biogenesis dq xu et al. further stimulating mitochondrial biogenesis [ ] . as mentioned above, the activation of mitochondrial biogenesis may be useful to treat t dm and/or insulin resistance. previous studies have suggested that ampk stimulates mitochondrial biogenesis and βoxidation via pgc- α upregulation in skeletal muscle [ , ] . upregulated expression of ampk and pgc- α increased mitochondrial function and muscle glucose metabolism, as shown using both glucose uptake and atp assays, suggesting a strong association between ampk activity and the hypoglycemic effects of catalpol. additionally, the hypoglycemic effect was reduced when catalpol hypoglycemic mechanism of catalpol in db/db mouse skeletal muscle. catalpol increased ampk/pgc- α/tfam-mediated mitochondrial biogenesis in skeletal muscle cells. increased mitochondrial biogenesis resulted in increased glucose uptake and atp production in skeletal muscle cells. catalpol enhances mitochondrial biogenesis dq xu et al. was coadministered with compound c (an ampk inhibitor). thus, ampk-mediated mitochondrial biogenesis may be crucial for the hypoglycemic effect of catalpol. the regulation of mitochondrial replication and biogenesis increases mitochondrial function. mitochondria play vital roles in diverse metabolic pathways [ , , ] . pgc- α stimulates oxidative metabolism and increases the number of mitochondria. pgc- α is strongly expressed in the skeletal muscle, heart, white adipose tissue, and brain, all of which are highly oxidative tissues [ ] . we found that db/db mice exhibited decreased pgc- α and tfam levels in skeletal muscle, consequently, reductions in mitochondrial biogenesis and the number of mitochondria. additionally, db/db mice exhibited a lower mitochondrial membrane potential and atp production in skeletal muscle. our results are consistent with earlier data showing mitochondrial dysfunction and reduced mitochondrial biogenesis in the skeletal muscles of db/db mice. catalpol increased the protein levels of both pgc- α and tfam in the skeletal muscle of db/db mice and c c myotubes, consistent with the observed increases in mtdna copy number. we found that pgc- α/tfam-mediated mitochondrial biogenesis may play a vital role in the hypoglycemic effect of catalpol. in summary, mitochondrial dysfunction was evident in the skeletal muscle of db/db mice. the catalpol-induced activation of ampk/pgc- α/tfam signaling increased mitochondrial biogenesis in skeletal muscle, thereby increasing glucose uptake and atp production. hence, skeletal muscle mitochondria may be a useful target for the treatment of t dm. american diabetes a. diagnosis and classification of diabetes mellitus prep controls insulin glucoregulatory function in liver by transcriptional targeting of shp tyrosine phosphatase pharmacological induction of mitochondrial biogenesis as a therapeutic strategy for the treatment of type diabetes mitochondrial dysfunction in diabetes and the regulatory roles of antidiabetic agents on the mitochondrial function mitochondrial dysfunction precedes insulin resistance and hepatic steatosis and contributes to the natural history of non-alcoholic fatty liver disease in an obese rodent model skeletal muscle mitochondria as a target to prevent or treat type diabetes mellitus benzoylaconine induces mitochondrial biogenesis in mice via activating ampk signaling cascade insulin receptor associates with promoters genome-wide and regulates gene expression impaired in vivo mitochondrial function but similar intramyocellular lipid content in patients with type diabetes mellitus and bmi-matched control subjects mitochondrial dysfunction and inhibition of myoblast differentiation in mice with high-fat-dietinduced pre-diabetes therapeutic approaches in mitochondrial dysfunction, proteolysis, and structural alterations of diaphragm and gastrocnemius in rats with chronic heart failure pharmacological approaches to restore mitochondrial function impaired mitochondrial substrate oxidation in muscle of insulin-resistant offspring of type diabetic patients deficiency of subsarcolemmal mitochondria in obesity and type diabetes resveratrol alleviates diabetic cardiomyopathy in rats by improving mitochondrial function through pgc α deacetylation impaired mitochondrial biogenesis due to dysfunctional adiponectin-ampk-pgc- α signaling contributing to increased vulnerability in diabetic heart mitochondrial biogenesis as a pharmacological target: a new approach to acute and chronic diseases new insights into pgc- coactivators: redefining their role in the regulation of mitochondrial function and beyond control of mitochondrial transcription specificity factors (tfb m and tfb m) by nuclear respiratory factors (nrf- and nrf- ) and pgc- family coactivators pgc- α controls mitochondrial biogenesis in drugresistant colorectal cancer cells by regulating endoplasmic reticulum stress pgc- α regulates mitochondrial properties beyond biogenesis with aging and exercise training a new hypoglycemic mechanism of catalpol revealed by enhancing myod/myog-mediated myogenesis effect of catalpol on diabetic nephropathy in rats catalpol ameliorates high-fat dietinduced insulin resistance and adipose tissue inflammation by suppressing the jnk and nf-kappab pathways hypoglycemic effect of catalpol on high-fat diet/streptozotocin-induced diabetic mice by increasing skeletal muscle mitochondrial biogenesis mitochondrial fusion/ fission process involved in the improvement of catalpol on high glucose-induced hepatic mitochondrial dysfunction mg -induced irs- ubiquitination negatively regulates skeletal myogenesis and insulin signalling the stimulatory effect of essential fatty acids on glucose uptake involves both akt and ampk activation in c c skeletal muscle cells -methoxypsoralen disrupts mdr -mediated phospholipids efflux and bile acid homeostasis and its relevance to hepatotoxicity the preventive effects of weeks of resistance training on glucose tolerance and muscle fiber type composition in zucker rats mitochondrial diseases and genetic defects of atp synthase mitochondria in the pathogenesis of diabetes: a proteomic view role of skeletal muscle-fibre type in regulation of glucose metabolism in middle-aged subjects with impaired glucose tolerance during a long-term exercise and dietary intervention amp-activated protein kinase (ampk) action in skeletal muscle via direct phosphorylation of pgc- α direct substrate delivery into mitochondrial fission-deficient pancreatic islets rescues insulin secretion phosphorylation of the insulin receptor by amp-activated protein kinase (ampk) promotes ligand-independent activation of the insulin signalling pathway in rodent muscle anti-diabetic activities of catalpol in db/db mice mitochondrial involvement in skeletal muscle insulin resistance: a case of imbalanced bioenergetics mitochondrial dysfunction in the elderly: possible role in insulin resistance ampk signalling in health and disease ampk promotes mitochondrial biogenesis and function by phosphorylating the epigenetic factors dnmt , rbbp , and hat deficiency of the hepatokine selenoprotein p increases responsiveness to exercise in mice through upregulation of reactive oxygen species and amp-activated protein kinase in muscle mitochondrial function and dysfunction in exercise and insulin resistance mitochondrial quality control in insulin resistance and diabetes pgc- α, glucose metabolism and type diabetes mellitus dqx and lw analyzed the data, contributed to discussions and wrote the manuscript. dqx, cjl, and ssf collected the data. zjl and hfh analyzed the data. lxs reviewed the manuscript. tw and zzj conceived the experiments, analyzed the data and edited/reviewed the manuscript. lyz is the guarantor of this work, had full access to all the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis. conflict of interest: the authors declare that they have no conflict of interest. key: cord- -djjtgbh authors: zhou, bei-xian; li, jing; liang, xiao-li; pan, xi-ping; hao, yan-bing; xie, pei-fang; jiang, hai-ming; yang, zi-feng; zhong, nan-shan title: β-sitosterol ameliorates influenza a virus-induced proinflammatory response and acute lung injury in mice by disrupting the cross-talk between rig-i and ifn/stat signaling date: - - journal: acta pharmacol sin doi: . /s - - - sha: doc_id: cord_uid: djjtgbh β-sitosterol ( -ethyl- -cholestene- -ol) is a common phytosterol chinese medical plants that has been shown to possess antioxidant and anti-inflammatory activity. in this study we investigated the effects of β-sitosterol on influenza virus-induced inflammation and acute lung injury and the molecular mechanisms. we demonstrate that β-sitosterol ( – μg/ml) dose-dependently suppresses inflammatory response through nf-κb and p mitogen-activated protein kinase (mapk) signaling in influenza a virus (iav)-infected cells, which was accompanied by decreased induction of interferons (ifns) (including type i and iii ifn). furthermore, we revealed that the anti-inflammatory effect of β-sitosterol resulted from its inhibitory effect on retinoic acid-inducible gene i (rig-i) signaling, led to decreased stat signaling, thus affecting the transcriptional activity of isgf (interferon-stimulated gene factor ) complexes and resulting in abrogation of the iav-induced proinflammatory amplification effect in ifn-sensitized cells. moreover, β-sitosterol treatment attenuated rig-i-mediated apoptotic injury of alveolar epithelial cells (aec) via downregulation of pro-apoptotic factors. in a mouse model of influenza, pre-administration of β-sitosterol ( , mg·kg(− )·d(− ), i.g., for days) dose-dependently ameliorated iav-mediated recruitment of pathogenic cytotoxic t cells and immune dysregulation. in addition, pre-administration of β-sitosterol protected mice from lethal iav infection. our data suggest that β-sitosterol blocks the immune response mediated by rig-i signaling and deleterious ifn production, providing a potential benefit for the treatment of influenza. the annual spread of seasonal influenza a virus (iav), particularly the sporadic transmission of highly pathogenic avian influenza (hpai) viruses (e.g., the h n and h n subtypes) continues to constitute a major threat to public health. as of december , avian influenza a h n viruses have caused an estimated individual infections, with an overall mortality rate of . % (http://www.fao.org/ag/againfo/programmes/en/empres/h n / situation_update.html). recently, the na r k and ns g a substitutions in the h n virus have been reported to be associated with increased resistance to oseltamivir and to confer enhanced viral replication in mammalian cells [ , ] . this raises concerns about a potential human pandemic. iav initiates infection in humans via entry through the upper respiratory tract, eliciting symptoms that can be mild and self-limited, but in some patients, it can lead to acute respiratory distress syndrome (ards), which is characterized by the impairment of gas exchange resulting in a fatal outcome [ , ] . a series of reports have indicated that a robust and dysregulated innate immune response is primarily responsible for the high mortality rate associated with influenza [ , ] . however, there are few alternative medicine strategies available for the treatment of influenza virus-infected patients who have an intense inflammatory response. the innate immune system is armed with an array of pattern recognition receptors (prrs) that provide the principal barrier defense against infectious agents [ ] . during the process of viral replication, influenza virus synthesizes viral rna containing a ′triphosphate end and is then transported to the cytoplasm [ ] . this pathogen-associated molecule (pam) is detected by the cytosolic sensor retinoic acid-induced gene i (rig-i) and subsequently activates multiple cellular signal cascades [ ] . ultimately, these signaling pathways lead to the activation of transcription factors including nf-κb, ap- (atf :c-jun), and irf , which together bind to specific sites of the ifn-β promoter and initiate ifn-β synthesis for the antiviral response [ ] . targeting p map kinase with a specific inhibitor affects the induction of ifn-β [ ] , which inhibits the downstream phosphorylation of atf by p map kinase [ ] . studies have demonstrated the crucial role of ifn production mediated by rig-i signaling in protecting against lethal iav challenge [ , ] . in vivo studies have shown that the increased susceptibility of both rig-i-and ifn-deficient mice to several rna viruses [ ] [ ] [ ] , including vesicular stomatitis virus (vsv), newcastle disease virus (ndv), and iav, is associated with the failure of ifns levels to increase or a lack of ifn signaling. there is evidence that infection with hpai h n viruses triggers the hyperinduction of proinflammation via rig-i signaling cascades despite the crucial role of rig-i signaling in defense against iav [ ] . the induction of downstream genes of rig-i signaling, such as il- , tnf-α, il- , and il- β, is significantly upregulated in the bronchoalveolar lavage fluid (balf) of patients with sustained ards [ , , ] . type i and type iii ifns secreted by infected cells bind to distinct receptors but trigger similar signal transduction through the jak/ stat cascades [ , ] . upon ifn stimulation, the phosphorylation of the stat :stat heterodimer leads to its interaction with irf and the formation of the interferon-stimulated gene factor (isgf ) complex [ ] . then, the complex moves from the cytoplasm to the nucleus where it binds to the interferonstimulated response element (isre) and drives the expression of interferon-stimulated genes (isgs). these genes possess direct antiviral and immunomodulatory activity [ ] . for example, the mx protein, which was the first isg identified to restrict influenza virus infection to be identified, has been demonstrated to impair vrnp nuclear import [ ] . however, despite the abundant production of ifns and hundreds of isgs in response to viral infection, the ifnmediated antiviral response is blocked by various viral products. the non-structural (ns ) protein of iav is a typical multifunctional protein involved in the blockade of the antiviral effect of isgs, such as ′, ′-oligoadenylate synthetase (oas) and dsrnadependent protein kinase r (pkr) [ , ] . in addition, iav infection has also been shown to suppress the antiviral response mediated by type i and iii ifns by upregulating the negative regulators socs and socs [ , ] . given that viruses can overcome the ifn-mediated antiviral response through various mechanisms, it is possible that excessive ifn production during the antiviral response may contribute to the harmful effects. surprisingly, studies have found that increased host susceptibility to secondary bacterial co-infections correlates with ifn induction during iav infection [ ] . moreover, a deficiency in ifn-α/β signaling increases survival through a reduction in excessive inflammation and apoptotic injury to lung epithelial cells [ ] . similarly, high levels of trail in balf collected from patients with influenza-associated ards may be a result of increased trail expression induced by macrophage-derived ifn-β [ ] , which contributes to lung alveolar epithelial cell injury. in addition, recent evidence has also revealed that ifn-mediated signaling drives immunopathologic injury in response to various viral infections, including respiratory syncytial virus infection (rsv) [ ] and severe acute respiratory syndrome cov (sars-cov) [ ] . given these findings, it is clear that the disease-promoting effects of ifn contribute to deleterious outcomes, and should be limited by pharmacological intervention. the identification of agents derived from medicinal plants that can prevent influenza is valuable. chinese medicinal plants, including lonicera japonica [ ] , chrysanthemum morifolium [ ] , taraxacum mongolicum [ ] , forsythia suspense [ ] , and isatis indigotica [ ] have been prescribed for the common cold, heatclearing, and detoxication for thousands of years, but the bioactive ingredients of these plants that mediate these pharmacological effects is unknown. phytosterols contain structural features that resemble those of cholesterol and are abundant in vegetables, fruits, and medicinal plants [ , ] . among phytosterols, β-sitosterol ( -ethyl- -cholestene- -ol) is the most common sterol and has been shown to possess antioxidant, antiinflammatory, antitumor, and antiasthmatic effects [ ] [ ] [ ] [ ] . in the present study, we hypothesized that β-sitosterol is the bioactive component of five types of medicinal plants. to test this hypothesis, we investigated the effects of β-sitosterol and the underlying mechanisms by which it may exert a therapeutic effect against influenza-mediated injury and dysregulated inflammation. preparation of extracts and quantitative analysis of β-sitosterol samples of four kinds of different heat-clearing and detoxifying traditional chinese medicines samples (l. japonica, c. morifolium, t. mongolicum, and f. suspense) were purchased from local markets in bozhou, china. i. indigotica was supplied by hutchison whampoa guangzhou baiyunshan chinese medicine co., ltd (guangzhou, china). a β-sitosterol standard was purchased from sigma (san francisco, usa), and hplc-grade methanol was purchased from fisher scientific (fisher, usa). a sample of each of the five medicinal materials was crushed into a coarse powder, and . g was placed in a -ml flask. extraction was performed using ultrasonic waves for min and the addition of ml of chloroform and was repeated three times. the samples were then centrifuged at × g for min. the supernatants were combined and condensed to a proper volume under reduced pressure, and then the concentrates were dissolved with chloroform. the samples were transferred to -ml volumetric flasks, diluted with chloroform to ml, and mixed. a total of . mg of the β-sitosterol standard was accurately weighed and dissolved in ml of chloroform to produce individual stock solutions. hplc analysis of β-sitosterol was performed at °c on an hplc instrument (shimadzu a, japan) with a dad detector at nm. chromatographic separation was performed on a shimadzu ods column ( . × mm, μm, tokyo, japan). the mobile phase was methanol, and the injection volume was μl. the samples were subjected to quantitative analysis, which was performed using the external standard method. the results are expressed as mg/g, and all analyses were performed in triplicate. influenza a/puerto rico/ / (h n ) and a/fm/ / (h n ) mouse-adapted viruses were stored in our laboratory and propagated in the allantoic cavities of -day-old specific pathogen-free embryonated chicken eggs at °c. freshly collected allantoic fluids were clarified by low-speed centrifugation at h postinoculation and then stored in small aliquots at − °c. the virus titers were determined using a plaque forming assay in monolayers of madin-darby canine kidney (mdck) cells as previously described. mouse experiments and viral challenge four-to six-week-old female balb/c mice (weighing - g) were purchased from guangdong medical laboratory animal center. all mice were housed and cared for under specific pathogen-free conditions at the state key laboratory of respiratory disease or guangdong laboratory animal monitoring institute. all animal experimental procedures in this study were approved by the ethics committee of the first affiliated hospital of guangzhou medical university and conducted in strict accordance with the approved guidelines. the % lethal dose (ld ) of the mouse-adapted h n virus was estimated in mice after the stock virus was serially diluted. the mice were treated intragastrically with β-sitosterol ( mg·kg − ·d − , mg·kg − ·d − ) or pbs (vehicle group) days prior to viral challenge. the mice were anesthetized ( % isoflurane inhalation) and challenged intranasally with ld of mouse-adapted h n virus. cell culture and viral infection human alveolar epithelial a cells and t human embryonic kidney cells were grown in dulbecco's modified eagleʼs medium (dmem/f , : mixture) (gibco) supplemented with % fetal bovine serum (fbs) (gibco) in a humidified incubator at °c and % co . for the viral infection, a cells ( × cells/ml) grown in well tissue culture plates (guangzhou jet bio-filtration co., ltd, tcp- - ) were inoculated with a/pr/ / (h n ) in serumfree dmem/f medium at an indicated multiplicity of infection (moi). after h absorption, the inoculum was discarded and replaced with fresh serum-free dmem/f medium containing diluted compounds. antibodies and recombinant protein the following primary antibodies were used in the current study: anti-rig-i, anti-nf-κb p , anti-phospho-nf-κb p (ser ), anti-p mapk, anti-phospho-p mapk ( protein preparation and immunoblot analysis excised lungs and cells were lysed using ice-cold ripa buffer ( mm tris·hcl ph . , mm nacl, % np- , % sodium deoxycholate, and . % sds) supplemented with protease inhibitors (sigma). the crude lysates were centrifuged at , rpm ( , × g) for min at °c, and the supernatant was collected. equal amounts of cell or tissue extracts were loaded onto % sds-polyacrylamide gels for separation. then, proteins were transferred to . μm pvdf membranes (bio-rad), which allowed subsequent probing with primary antibodies. after overnight incubation at °c, the membranes were washed with . % tbst (tbs/ . % tween ) and incubated with corresponding horseradish peroxidase (hrp)conjugated secondary antibodies (multisciences). the signals were visualized using enhanced chemiluminescence (ecl) reagents (perkin-elmer). quantification of the relative band intensities was performed using imagej software version . . ppprna generation, rna isolation, and real-time rt-pcr viral rna ( ′-triphosphate rna, ′ppp-rna) and cellular rna were generated as previously described [ ] . briefly, total rna was isolated from a lung epithelial cells infected with a/pr/ / (h n ) for h (viral rna) and uninfected a lung epithelial cells (cellular rna) using trizol reagent (takara, usa). then, the '-phosphate group was dephosphorylated by treatment with calf-intestinal alkaline phosphatase (ciap) (takara). a lung epithelial cells were transfected with the indicated rna using lp (thermo, usa). for qpcr, total rna ( μg) was reverse transcribed into cdna and subjected to real-time quantitative pcr with gene-specific primers and probes on an abi real-time pcr system. relative gene expression was calculated using the −ΔΔct method [ ] . the specific primer and probe sets are shown in supplementary table s . small interfering rna (sirna), plasmid transfection, and reporter gene assays transient transfection of plasmids into cells, including an isre luciferase reporter plasmid (beyotime) and a flag-rig-i overexpression plasmid (geneppl), was performed by using lipofectamine (invitrogen). after isre luciferase reporter plasmid ( . μg) and flag-rig-i ( . μg) overexpression plasmid were transfected into a cells for h, the transfected cells were stimulated with ifns or iav (moi = . ). in the other experiment, hek cells stably co-transfected with pnf-κb-tata-f-luci and pqcxip-egfp plasmid were stimulated with tnf-α ( ng/ml) or iav for the indicated times. firefly luciferase activity was assayed using the luciferase reporter system (promega, usa) and normalized to renilla luciferase activity or the levels of gfp expression. rig-i-specific sirnas (rig-i # sirna and, rig-i # sirna) were purchased from guangzhou ribobio co., ltd. and transfected into cells with lipofectamine according to the manufacturer's instructions. histology and immunohistology lung tissue was excised, fixed in % formalin and embedded in paraffin using routine procedures. tissue sections ( μm) were stained with hematoxylin-eosin for histological examination. for immunohistochemical staining, the sections were deparaffinized with xylene and rehydrated in a graded alcohol series. endogenous peroxidase was blocked with % hydrogen peroxide (h o ) in methanol for min at room temperature and antigen retrieval was performed in . mm citrate buffer (ph . ). afterward, the sections were blocked with % normal serum and incubated with primary antibody at °c overnight. the sections were then incubated for h in hrp-labeled secondary antibody solution and then visualized using a dab reagent kit (maixin, china). the sections were counterstained with mayer's hematoxylin for min before mounting. apoptosis detection by annexin v and flow cytometry cells collected from both suspension and adherent were washed twice with cold pbs, and subsequently resuspended in × annexin binding buffer (bioscience, usa) at a concentration of × cells/ml. one hundred microliters of cell suspension was stained with μl of annexin v-fitc and μl of propidium iodide (pi) for min at room temperature in the dark. the cells were analyzed using a novocyte flow cytometer within h. bronchoalveolar lavage and flow cytometry mice were euthanized intraperitoneally (i.p.) with an overdose of sodium pentobarbital at the indicated time point. the trachea was cannulated via ventral middle incision. the lungs were lavaged three times with μl of sterile saline. bronchoalveolar lavage fluid (balf) was centrifuged at × g for min at °c and the supernatants were collected and stored at − °c for biochemical analysis. the balf pellets were resuspended in pbs with . % bsa and stained with fluorochrome-conjugated monoclonal antibodies (mabs) (bioscience, usa) that bound to surface molecules for min at °c. all samples were analyzed on a novocyte flow cytometer. statistical analysis all data are presented as the mean ± sem. statistical analysis was performed using spss software version . . one-way anova followed by the student-newman-keuls (snk) test was carried out to assess differences between the study groups, and p values less than . were considered significant. quantitative analysis of β-sitosterol in five medicinal materials medicinal plants including l. japonica, c. morifolium, t. mongolicum, f. suspense, and i. indigotica are traditionally used for heatclearing and detoxification. we hypothesized that β-sitosterol is the common substance in these medicinal plants that mediates their pharmacological actions against respiratory diseases, such as β-sitosterol ameliorates iav-induced inflammation and ali bx zhou influenza. first, we quantified the content of β-sitosterol in these plants. as shown in table , β-sitosterol was detected in all samples at concentrations ranging from . to . mg/g. β-sitosterol treatment inhibits the activation of the cellular signaling pathway in iav-infected cells to clarify whether β-sitosterol possesses antiviral activity for the treatment of iav infection, cytopathic effect (cpe) inhibition assays and plaque reduction assays were performed to investigate the antiviral effects of β-sitosterol. as shown in supplementary table s , β-sitosterol showed no activity against a/gz/gird / (h n ), a/pr/ / (h n ), a/hk/ / (h n ), a/hk/y / (h n ), or b/lee/ (flub). these findings were further confirmed by plaque reduction assays ( supplementary fig. s ). the activation of multiple cellular signaling events has been implicated in the molecular pathogenesis of iav infection [ ] . to identify the pharmacological effects exerted by β-sitosterol during iav infection, we assessed the impact of β-sitosterol on the activation of cellular signaling pathway in cells infected with iav. hek cells in which an nf-κb-luc reporter was stably expressed were stimulated with either tnf-α ( ng/ml) (fig. a) or a/pr/ / (h n ) (fig. b) , and then incubated with β-sitosterol for h. we observed that hek cells stimulated with tnf-α or a/pr/ / (h n ) exhibited robust nf-κb activation, on which β-sitosterol treatment had a dose-dependent suppressive effect ( fig. a, b) . furthermore, to test whether β-sitosterol possesses other pharmacological properties, we assessed the activation of signaling pathways in iav-infected a cells in the presence or absence of β-sitosterol by immunoblotting. nf-κb and mapk (p , erk / , and jnk/spak) signaling was activated by iav infection (fig. c) . as expected, β-sitosterol inhibited the iav-induced p mapk activation and p phosphorylation, which is consistent with the data gathered using the nf-κb-luc reporter cell line. however, βsitosterol had no inhibitory effect on the erk / or jnk mapk pathway. together, these data demonstrate that β-sitosterol has the potential to inhibit the activation of nf-κb and p mapk signaling in response to iav infection. β-sitosterol treatment decreases the expression of iav-induced proinflammatory mediators both the nf-κb and p mapk signaling cascades have been implicated as major contributors to hypercytokinemia during human hpaiv h n infection [ , ] . therefore, we next investigated the effect of β-sitosterol on the expression of proinflammatory mediators in iav-infected cells. a cells were infected with the pr /h n virus (moi = . ) in the presence of increasing concentrations of β-sitosterol ( - μg/ml) for h. subsequently, the transcript levels of proinflammatory mediators were measured by qpcr. our data showed that the gene expression of an array of cytokines and chemokines, including il- , tnf-α, ip- , il- , mcp- , mip- β, and rantes, was elevated dramatically following iav infection, and that this elevation was attenuated by β-sitosterol treatment (fig. a) . the kinetics of iavinduced proinflammatory mediator release showed that the protein levels of these proinflammatory mediators (including il- , tnf-α, il- , ip- , rantes, and mcp- ) reached their expression peak at h after viral infection (fig. b) , and that the increases in the levels of these mediators were reversed by β-sitosterol treatment (fig. c) . the production of cyclooxygenase- (cox- ) and its derivative prostaglandin e (pge ), has been shown to occur via nf-κb and p mapk signaling and play a pathogenic role during iav infection [ , ] . indeed, increasing the expression of iav-induced cox- at both the mrna and protein levels at h p.i., while treatment with β-sitosterol profoundly reduced cox- production (fig. d, e) . furthermore, we measured the effect of β-sitosterol on the iav-induced expression of iav-induced cox- and carried out elisa to quantify cox- -derived pge in the culture supernatants. as expected, β-sitosterol treatment of iav-infected cells led to a significant dose-dependent reduction in pge levels (fig. f) . these results indicate that β-sitosterol treatment decreases the expression of iav-induced proinflammatory mediators through the inactivation of the nf-κb and p mapk signaling pathways. β-sitosterol treatment suppresses the iav-mediated induction of interferon expression and signal transduction by targeting rig-i in response to iav infection, transcription factors including nf-κb, atf /c-jun, and irf / bind cooperatively to the promoter regions of ifn genes, which are secreted to establish an antiviral state, to initiate their expression [ ] . the activation of p , which mediates the activation of its downstream target atf , is a prerequisite for optimal ifn-β induction [ ] . since β-sitosterol inhibits the iav-induced activation of p , we hypothesized that β-sitosterol treatment might reduce the expression of ifns. to test this hypothesis, culture supernatants from iav-infected a cells treated with or without β-sitosterol were collected at h p.i. and transferred to uninfected a cells. after min of incubation, we assessed ifn levels in the culture supernatant by monitoring the phosphorylation and downstream signaling of ifns by immunoblot analysis. as shown in fig. a , supernatants from infected a cells stimulated the phosphorylation of stat tyr and stat tyr , while supernatants from donor cells treated with β-sitosterol attenuated the phosphorylation of stat tyr . our results indicated that the suppression of stat tyr phosphorylation by β-sitosterol was associated with reduced ifns production. to further confirm that β-sitosterol inhibits the iav-induced expression of ifns, ifn concentrations in supernatants collected at h p.i. were measured using luminex. as demonstrated in fig. b , the iav-induced elevation of ifns, including type i ifn (ifnβ), type ii ifn (ifn-γ), and type iii ifn (ifn-λ ), was significantly decreased in the supernatants of β-sitosterol-treated cells. these observations led us to ask whether β-sitosterol directly affects the ifn signaling transduction pathway. to address this question, we tested whether signal transduction induced by exogenous ifn-β is altered by β-sitosterol treatment. then, we pretreated iav-infected a cells with β-sitosterol for h before stimulating them with ifn-β for min. the phosphorylation of stat tyr , but not stat , was markedly inhibited by β-sitosterol treatment (fig. c) . however, at later time points ( h p.i.), the ifnβ-mediated phosphorylation of stat tyr was reduced in iavinfected a cells regardless of whether they were pretreated with β-sitosterol or not (fig. d) . our data demonstrated that β-sitosterol reduced ifn production through the inhibition of nf-κb and p map kinase and that it blocks ifn-mediated signal transduction. we next sought to elucidate the mechanism by which β-sitosterol disrupts ifn production and signaling. the intracellular prr rig-i senses iav ′ppp-rna, resulting in the activation of nf-κb and p map kinase which preferentially promote the initiation of ifn transcription [ ] . it is worth mentioning that rig-i, an interferon-stimulated gene (isg), has been reported to augment stat activation and inhibit leukemia cell proliferation [ ] . thus, it is reasonable to speculate that cross-talk between the rig-i and ifn signaling pathways is affected by β-sitosterol. to confirm this assumption, we investigated the effect of β-sitosterol on the iavinduced expression of rig-i. our results show that β-sitosterol decreased the induction of rig-i in infected a cells (fig. e, f) . furthermore, cells transfected with the flag-rig-i overexpression plasmid transfection were found to have elevated p-stat levels, which were diminished by β-sitosterol treatment (fig. g) . the tyrosine kinases jaks are well recognized to be involved in the activation of stat . therefore, it is possible that β-sitosterol affected stat activation by inhibiting jaks. interestingly, we found that a cells pretreated with β-sitosterol for h prior to ifn-β ( ng/ml) stimulation did not exhibit decreased ifn-βmediated activation of jak , stat , and stat levels (fig. h) . together, these data indicate that β-sitosterol can exert an inhibitory effect on rig-i, which leads to decreased iav-induced ifn production and ifn-β signal transduction. β-sitosterol treatment attenuates the ifn-mediated amplification of the proinflammatory response during iav infection via the inhibition of rig-i it is widely recognized that ifn signaling plays a vital role in host antiviral immunity [ ] . furthermore, ifns possess immunomodulatory activities on the induction of both chemokines and cytokines and on the recruitment of various immune cells [ ] . to examine the effect of rig-i inhibition by β-sitosterol on the transcription of ifn signaling-related molecules, we analyzed iav-mediated ifn-stimulated response element (isre) activation with an assay that utilized a transient isre reporter plasmid. the results presented in fig. a show that iav-mediated isre-dependent transcription was significantly inhibited by βsitosterol treatment. rig-i knockdown in iav-infected cells by specific sirnas in iav-infected cells was confirmed to significantly inhibit the iav-mediated isre transcriptional activity (fig. b, c) . to further determine the effects of β-sitosterol on ifn-induced proinflammatory responses during iav infection, isre reporter plasmid-transfected cells were stimulated with ifn-β ( ng/ml) h prior to iav infection. prestimulation with ifn-β led to much higher isre activity than iav infection alone (fig. d) . similarly, ifn-β stimulation following iav infection also resulted in increased isre activity, but to a lesser extent than what was seen after ifn-β prestimulation (fig. d) . this indicates that ifn-β signaling contributes to the amplification of isre activity during iav infection. nevertheless, this increase in isre activity was diminished in a dose-dependent manner by β-sitosterol treatment representative results from at least three independent experiments are shown. d the band intensities of p-p , p-p , p-erk / , and p-jnk were semiquantified using imagej (normalized to the loading control gapdh). the data are presented as the mean ± sem (n = - ). **p < . , ***p < . versus the group treated with tnf-α or iav. β-sitosterol ameliorates iav-induced inflammation and ali bx zhou immunoblot analysis was performed to evaluate the protein expression of cox- . gapdh was used as the internal control (e). f quantification of pge in the culture supernatants using elisa. *p < . , **p < . , ***p < . versus the iav control group. ( fig. d) . we next assessed the effects of β-sitosterol on the expression of proinflammatory genes in iav-infected cells pretreated with or without ifn-β. consistent with the previously observed increase in isre activity, the mrna and protein levels of cytokines and chemokines, including il- , ip- , tnf-α, il- , mcp- and gm-csf, were robustly increased in iav-infected cells after stimulation with ifn-β (fig. e, f) . a similar cytokine and chemokine expression pattern was observed in response to stimulation with ifn-λ (data not shown), which signals through the jak/stat pathway leading to isre activation. as expected, the elevation in cytokine and chemokine levels induced by ifn stimulation was decreased by β-sitosterol treatment (fig. e, f) . to understand whether the attenuation of the amplified proinflammatory response in ifn-β pretreated cells was solely due to a reduction in ifn-β levels, cells with ifn-β prestimulated for h were treated with an ifn-β neutralizing antibody ( . μg/ml) prior to virus infection. however, we observed that the amplification effects of proinflammatory mediators (il- and ip- ) in cells prestimulated with ifn-β were not abrogated by ifn-β neutralizing antibody treatment (fig. g) . this indicates that cells prestimulated with ifn-β become sensitized and thereby amplify proinflammatory responses. to understand whether the observed inhibitory effect of βsitosterol was due to stat inhibition, we measured the phosphorylation of stat tyr in a cells stimulated with ifn-β ( ng/ml). stat was significantly activated in cells pretreated with ifn-β (lane ), but not in cells infected with iav prior to ifn-β stimulation (lane ). treatment with β-sitosterol abrogated stat activation in a dose-dependent manner (lanes - ) (fig. h) , indicating that the inhibitory effect of β-sitosterol on stat reduced the ifn-mediated amplification of cytokine and chemokine expression. notably, pretreatment with ifn-β significantly increased the iav-triggered expression of rig-i in a cells, which was inhibited by β-sitosterol treatment (fig. i, j) . together, these data demonstrate that β-sitosterol blocks the iav-induced amplification of the proinflammatory response in ifn-β-activated a cells, which is due to inhibition of rig-i levels by β-sitosterol, leading to the inactivation of stat , and thereby diminishes the transcriptional activity of interferon-stimulated gene factor (isgf ). in addition to playing a role in ifn induction, rig-i signaling has been demonstrated to be involved in apoptosis [ ] , which is implicated in iav-induced lung epithelial cell damage and injury. therefore, we investigated whether β-sitosterol affects rig-imediated apoptosis using intracellular viral rna (vrna, ′ppp-rna) stimulation. stimulation with cellular rna (crna) or vrna and treatment with calfintestine alkaline phosphatase (ciap) to fig. effect of β-sitosterol treatment on iav-induced ifn production and ifn signal transduction. a after h, iav-infected cells with or without the indicated concentration of β-sitosterol treatment were harvested, and the supernatants were transferred to uninfected cells. after min of incubation, the cells were lysed, and the expression of phosphorylated stat and stat was analyzed by immunoblotting. equal loading of protein was verified by immunoblotting for gapdh. b ifns (ifn-β, ifn-γ, and ifn-λ ) secretion into the culture media was measured at h p.i. using a multiplex luminex assay. c, d after allowing h for iav absorption, a cells were incubated with the indicated concentration of β-sitosterol for h (c) or h (d). then, the cells were stimulated for an additional min with human ifn-β ( ng/ml). the cells were lysed, and total extracts were processed for immunoblotting. e, f effect of β-sitosterol on the expression of rig-i. rig-i mrna expression levels were assayed by quantitative real-time pcr (e). rig-i protein levels were assessed by western blotting (f). g a cells were transfected with a flag-rig-i overexpression plasmid, and then treated with β-sitosterol. after h, the cell lysates were collected for immunoblotting. h a cells were pretreated with β-sitosterol for h, stimulated with ifn-β ( ng/ml) for min, and immunoblotted for the indicated proteins. *p < . , **p < . , ***p < . versus the iav control group. dephosphorylate ′-triphosphate did not induce apoptosis, excluding the possibility that crna or the non-phosphate at the ′ end of rna has a pro-apoptotic effect (fig. a) . strikingly, the apoptosis of vrna-transfected cells was reduced by β-sitosterol treatment (fig. a) . we confirmed the anti-apoptotic effect of βsitosterol by measuring the active caspase- and its substrate parp, observing that these products were found in cells transfected with vrna but not in those treated with β-sitosterol fig. (continued) β-sitosterol ameliorates iav-induced inflammation and ali bx zhou (fig. b) . to determine whether the inhibitory effect of β-sitosterol on rig-i-mediated apoptosis is related to alterations in the expression of pro-apoptotic factors, we quantified trail and sfas ligand levels in the supernatants of viral rna-transfected cells. trail and sfas ligand levels were significantly reduced by βsitosterol treatment (fig. c ). next, we determined the effect of βsitosterol on the apoptosis of iav-infected cell. β-sitosterol treatment reduced iav-mediated apoptosis and active caspase- and parp (fig. d, e) . last, we observed that β-sitosterol treatment blocked the increase in the release of trail and sfas ligand in iavinfected cells (fig. f) . furthermore, rig-i knockdown by specific sirnas was demonstrated to significantly decrease iav-mediated apoptosis and the release of trail (fig. g, h) . however, the combination of rig-i sirnas and β-sitosterol did not have an additive effect on the inhibition of iav-mediated apoptosis and trail release, which indicated that β-sitosterol decreased iavmediated apoptosis via the inhibition of rig-i. given that vrna ( ′ ppp-rna) recognition is associated with the rapid induction of ifns, we wondered whether β-sitosterol treatment affects ifn induction in the context of vrna transfection. as shown in fig. i , the upregulated expression of both type i ifn (ifn-β) and type iii ifn (ifn-λ ) in response to vrna was reversed by β-sitosterol treatment in a dose-dependent manner. to further explore whether the signaling cascade underlying rig-i-mediated apoptosis and ifn induction is affected by βsitosterol, we performed immunoblot analysis h after the fig. effect of β-sitosterol on the ifn-β-mediated amplification of iav-induced proinflammatory mediators. a the effect of β-sitosterol on isre luciferase reporter activity in iav-infected cells. a cells were co-transfected with . μg of pisre-ta-luc reporter plasmid and . μg of prl-tk plasmid as described in the materials and methods. at h posttransfection, the cells were infected with iav and treated with βsitosterol. after h, the cells were lysed, and luciferase activity was measured. ## p < . versus the control group. *p < . , **p < . versus the iav control group. b rig-i knockdown by specific sirnas in iav-infected cells was confirmed by immunoblotting. c the effect of rig-i knockdown by specific sirnas on isre luciferase reporter activity in iav-infected cells. ### p < . versus the control group. ***p < . versus the iav control group. d the effect of β-sitosterol on isre luciferase reporter activity induced by stimulation with a combination of ifn-β and iav. after h of transfection, a cells were pretreated with ifn-β ( ng/ml) (columns - ) for h or infected with iav prior to ifnβ stimulation (columns - ) for h. ifn-β-pretreated cells were infected with iav in the presence or absence of β-sitosterol ( - μg/ml). iav-infected cells were stimulated with ifn-β ( ng/ml) in the presence or absence of β-sitosterol ( - μg/ml). the cells were harvested and subjected to a luciferase assay at h p.i. # p < . versus the iav control group (column ). *p < . versus the ifn-β-pretreated group (column ). § p < . versus the group infected with iav before being stimulated with ifn-β ( ng/ml) stimulation group (column ). e the effect of β-sitosterol on the ifn-β-mediated amplification of iav-induced proinflammatory cytokines and chemokines at the mrna levels was determined by real-time pcr. ### p < . versus the iav control group (column ). *p < . , **p < . , ***p < . versus ifn-β-pretreated group (column ). § p < . , § § p < . versus the group infected with iav before being stimulated with ifn-β ( ng/ml) (column ). f the effect of β-sitosterol on the ifn-β-mediated amplification of iav-induced proinflammatory cytokines and chemokines at the protein level was determined by a multiplex luminex assay. # p < . , ## p < . versus the iav control group (column ). *p < . , **p < . , ***p < . versus ifn-β-pretreated group (column ). § p < . , § § p < . , § § § p < . versus the group infected with iav before being stimulated with ifn-β ( ng/ml) (column ). g the effect of ifn-β neutralization on the ifn-β-mediated amplification of the iav-induced proinflammatory response. a cells prestimulated with ifn-β ( ng/ml) for h were treated with an ifn-β neutralizing antibody ( . μg/ml) prior to infection with iav. after h, the culture supernatants were collected to measure proinflammatory mediator levels by a multiplex luminex assay. ***p < . versus iav control group. ns, not significant. h the effect of β-sitosterol on the ifn-β-mediated activation of stat . lanes - : a cells were treated with either ifn-β ( ng/ml) or with iav for h. lanes - : a cells were pretreated with ifn-β ( ng/ml) for h prior to iav infection. lanes - : a cells were infected with iav for h and then stimulated with ng/ml ifn-β. after the indicated treatments, the cells were incubated with or without β-sitosterol for h. the cell lysates were analyzed by immunoblotting for the expression of phospho-stat and phospho-stat . i, j the effect of β-sitosterol on the expression of rig-i. a cells were pretreated with ifnβ ( ng/ml) for h and infected with iav in the presence or absence of β-sitosterol ( - μg/ml) (columns - , lanes - ) . meanwhile, a cells were infected with iav for h prior to ifn-β ( ng/ml) stimulation (columns - , lanes [ ] [ ] [ ] [ ] . i the expression of rig-i was determined by quantitative real-time pcr. ## p < . versus the iav control group (column ). *p < . , **p < . , ***p < . versus ifn-βpretreated group (column ). j the expression of rig-i was determined by immunoblotting at h p.i. the band intensities of rig-i were semiquantified using imagej (normalized to the loading control gapdh). *p < . versus ifn-β-pretreated group (column ). § p < . versus the group infected with iav before being stimulated with ifn-β ( ng/ml) (column ). (fig. j) (lane ) . notably, the dephosphorylation of the ʹtriphosphate of rna led to the inactivation of p nf-κb and p mapk but not stat (lane ). moreover, the vrna-triggered activation of p nf-κb, p mapk, and stat was inhibited by βsitosterol (lanes - ). the stimulation of rig-i with vrna initiated signaling events that ultimately led to the expression of proinflammatory cytokines. we detected increased expression of proinflammatory mediators after h of vrna stimulation, and observed that β-sitosterol treatment blocked this effect (fig. k) . meanwhile, these proinflammatory mediators induced by iav were effectively reduced by specific rig-i sirnas (fig. l) . the levels of il- , mcp- , rantes, and gm-csf were further reduced by the combination of rig-i sirnas and β-sitosterol (fig. l) . furthermore, the further increased levels of il- , tnf-α, and ip- in flag-rig-i overexpression plasmid-transfected cells with iav infection is dose-dependently decreased by β-sitosterol treatment (fig. m) . interestingly, the viral rna-induced upregulation of cox- expression was decreased following β-sitosterol treatment (fig. n) . to further confirm that β-sitosterol suppresses viral rna-mediated cox- upregulation, we quantified the level of its downstream product pge in the culture medium using elisa. our results showed that treatment with β-sitosterol dosedependently suppressed the viral rna-induced production of pge (fig. o) . collectively, these data suggest that β-sitosterol inhibits the activation of rig-i signaling and downstream apoptosis in response to vrna. considering that β-sitosterol exhibited immunomodulatory properties in vitro, we next sought to investigate whether β-sitosterol has a protective effect in a mouse model of influenza. balb/c mice were pretreated with β-sitosterol for days prior to intranasal challenge with ld of iav. at days p.i., histological analysis of lung sections showed that infection with iav resulted in extensive inflammation characterized by massive leukocyte recruitment to the lung parenchyma (fig. a, upper panel) . pre-treatment with mg·kg − ·d − β-sitosterol decreased leukocyte infiltration, and as a result, relatively few inflammatory cells were observed surrounding the bronchioles (fig. a, upper panel) . we next performed immunohistochemical staining for cd (pan t lymphocytes) and observed that the infiltration of cd + t lymphocytes was more pronounced in the lungs of mice with iav analysis of active caspase- and parp cleavage in a cells transfected with vrna at h. c luminex assay for trail and sfas ligand levels in the supernatants of a cells transfected with vrna. *p < . , **p < . versus the vrna transfection group (column ). d flow cytometry analysis of the effect of β-sitosterol on apoptotic a cells infected with iav at h. *p < . , **p < . versus iav control group. e analysis of active caspase- and parp cleavage in a cells infected with iav at h. f luminex assay for trail and sfas ligand levels in the supernatants of a cells infected with iav at h. *p < . versus the iav control group. g flow cytometry analysis of the effect of rig-i knockdown by specific sirnas on iav-induced apoptosis. h luminex assay for trail in the supernatants of a cells with rig-i knockdown. ***p < . versus iav control group (column ). i luminex assay for ifn-β and ifn-λ levels in the supernatants of a cells transfected with vrna at h. *p < . , **p < . , ***p < . versus the vrna-transfected group (column ). j a cells were transfected with vrna in the presence or absence of the indicated concentration of β-sitosterol; h later, the cells were lysed and analyzed by immunoblotting with the indicated antibodies. gapdh was used as a control for equal loading. k the effect of β-sitosterol on the protein expression of cytokines and chemokines in the supernatants of vrna-transfected cells. *p < . , **p < . , ***p < . versus the vrna transfection group (column ). l luminex assay for proinflammatory mediators in the supernatants of a cells with knockdown of rig-i or in combination with β-sitosterol treatment. ***p < . versus the iav control group (column ). # p < . , ### p < . versus the rig-i # sirna transfection group (column ). § p < . , § § p < . , § § § p < . versus the rig-i # sirna transfection group (column ). m luminex assay for cytokines and chemokines in the culture supernatants of iav-infected a cells transfected with flag-rig-i overexpression plasmid with or without β-sitosterol treatment. ## p < . versus the iav control group (column ). *p < . , ***p < . versus the flag-rig-i overexpression plasmid transfection control group (column ). n the effect of β-sitosterol on the expression of cox- in vrna-transfected cells. the cells were transfected with vrna in the presence or absence of the indicated concentration of β-sitosterol; h later, the cells were lysed and analyzed by immunoblotting using a specific antibody to cox- . o quantification of pge in the culture supernatants of vrna-transfected cells in the presence or absence of the indicated concentrations of β-sitosterol at h. ***p < . versus the vrna-transfected group (column ). β-sitosterol ameliorates iav-induced inflammation and ali bx zhou infection alone compared with those that received β-sitosterol treatment (fig. a, lower panel) . consistent with the immunohistochemical results, a significantly greater percentage of cd + cd + cytotoxic t lymphocytes (ctls) was detected in balf from iav-infected mice compared with balf from mice treated with β-sitosterol (fig. b) . moreover, the quantification of granzyme b, a pro-apoptotic enzyme secreted by ctl, in lung homogenates revealed a significant increase in its expression in iav-infected mice that was significantly reduced by β-sitosterol administration (fig. c) . similar results were obtained for the levels β-sitosterol ameliorates iav-induced inflammation and ali bx zhou of active caspase- measured by immunoblotting (fig. c) . although influenza antigen-specific cd + t cells are recruited to the sites of infection and contribute to viral clearance, immunemediated lung injury can be elicited by aberrant t-cell responses. lung index and total protein levels in balf can be used as an assessment of lung damage. our results showed that compared with iav infection alone, β-sitosterol treatment produced a significantly lower lung index and protein levels in balf (fig. d , e), suggesting that β-sitosterol treatment alleviated lung injury likely through the suppression of cd + t-cell recruitment. last, iav infection resulted in % ( / ) mortality by days p.i. and rapid and continuous weight loss (fig. f, g) . remarkably, the survival rate of mice that were treated with and mg/kg βsitosterol was significantly increased to . % ( / ) and . % ( / ), respectively. furthermore, β-sitosterol-treated mice exhibited less initial weight loss after viral challenge and a gradual recovery of body weight (fig. g) . together, these data reveal that β-sitosterol attenuates iav-induced lung injury and reduces mortality. β-sitosterol protects against iav by abrogating the iav-mediated activation of multiple signaling cascades to investigate the mechanisms underlying the protective effect of β-sitosterol against iav in vivo, we focused on inflammationassociated signal transduction in the lung. the phosphorylation levels of stat , stat , p , and erk / in lung homogenates were significantly increased in mice challenged with iav relative to uninfected mice (figs. a), whereas the phosphorylation levels of these molecules were decreased in mice treated with β-sitosterol. to address whether β-sitosterol inhibits the signaling events mediating the iav-induced expression of proinflammatory cytokines, we quantified cytokine and chemokine levels using luminex. the expression of cytokines and chemokines in balf (il- , tnf-α, rantes, kc, mcp- and mip- α) and lung homogenates (il- , tnf-α, ip- , and rantes) was reduced in mice treated with β-sitosterol (fig. b, c) . the iav-induced elevation of serum cytokines including ifn-γ, ip- , and rantes, was blocked in β-sitosterol-treated mice (fig. d) . given that β-sitosterol inhibited iav-mediated stat / activation in vivo and decreased the expression of rig-i and ifn in vitro (fig. b, f) , it was necessary to examine the impact of β-sitosterol treatment on the expression of rig-i and ifns in vivo. as expected, iav-induced expression of rig-i in lung homogenates was decreased by β-sitosterol treatment (fig. e) . similarly, the expression of ifns in balf (ifn-α, ifn-β, and ifn-γ) (fig. f) and lung homogenates (ifn-β) (fig. g) was also decreased. collectively, these data provide evidence regarding the mechanism by which β-sitosterol modulates dysregulated signaling cascades and proinflammatory responses linked to severe influenza. patients with influenza are frequently afflicted with severe pneumonia characterized by excessive infiltration of leukocytes and proinflammatory cytokine production [ ] [ ] [ ] , leading to a high risk of death. recent studies have revealed that iav-mediated ifns play a disease-promoting role in the pathogenesis of influenza [ ] . chinese herbal medicines, including l. japonica [ ] , c. morifolium [ ] , t. mongolicum [ ] , f. suspense [ ] , and i. indigotica [ ] , have a long history of being used for the treatment of the common cold and for heat-clearing. in the current study, we demonstrated that β-sitosterol derived from these herbal medicines has the ability to block iav-mediated ifn and proinflammatory mediator production through the inhibition of rig-i signaling. furthermore, our data showed that β-sitosterol attenuates the amplification of the iav-mediated proinflammatory response in ifn-sensitized cells by disrupting rig-i-mediated stat activation. furthermore, we showed that β-sitosterol abrogates the recruitment of cytotoxic t lymphocytes (ctls) in the lung, thereby significantly improving lung injury and survival in mice challenged with iav (fig. ) . rig-i detects viral rna ( ′ppp-rna) in the cytoplasm, which is then ubiquitinated by trim (tripartite motif-containing protein ) and subsequently interacts with ips- (ifn-β promotor stimulator ) to initiate the activation of nf-κb, p , and irf / [ , ] . we asked whether rig-i signaling is affected by βsitosterol during iav infection or in cells transfected with viral rna. we found that rig-i expression was increased following iav infection or prestimulation with ifn-β prior to iav infection, and that this increase in expression was significantly reduced in the presence of β-sitosterol. furthermore, the activation of nf-κb and p in both iav-infected and viral rna-transfected cells was inhibited by β-sitosterol treatment. these results suggest that βsitosterol treatment antagonizes the rig-i signaling cascades. the activation of rig-i and its downstream targets nf-κb and p contributes to the induction of proinflammatory cytokines in response to influenza virus infection [ ] . in previous studies, the upregulation of rig-i during fatal h n infection was shown to cause an amplification of inflammatory responses [ ] . the hyperinduction of proinflammatory cytokines via rig-i, nf-κb, and p signaling has been suggested to contribute to severity of symptoms in patients with h n infection [ , ] . we measured nf-κb activation using an nf-κb luciferase reporter system and found that β-sitosterol treatment downregulated the transcriptional activation of nf-κb following the administration of tnf-α and iav infection. consistent with these findings, the inhibitory effects of β-sitosterol on rig-i signaling led to reduced production of cytokines, such as il- and tnf-α, and chemokines such as il- and ip- in iav-infected and viral rna-transfected cells. rig-i or nf-κb and p activation in response to viral or bacterial infection, respectively, has been reported to induce cox- expression [ , ] . furthermore, it has been shown that decreased expression of cox- and its derivative pge has beneficial effects during influenza virus infection that lead to reduced hypothermia and enhanced type i ifn antiviral immunity [ ] . we observed that iav infection and viral rna stimulation were associated with increased expression of cox- and pge and that β-sitosterol treatment reversed the increase in a dosedependent manner. fig. β-sitosterol prevents iav-induced lung pathology in mice. two days prior to infection with ld of a/fm /h n virus, pbs or β-sitosterol ( mg·kg − ·d − or mg·kg − ·d − ) was intragastrically administered to mice for consecutive days. a on day p.i., the lungs were harvested and subsequently subjected to histological analysis by h&e staining (original magnification, × ) or cd antigen (t-cell marker) staining (original magnification, × ). b representative flow cytometry quantification of cd + cd + t cells in balf on day p.i. the histograms represent the percentage of cd + cd + t cells in balf (right panel). the data are representative of three independent experiments using - mice per group. *p < . , **p < . versus the iav-infected group. c on day p.i., the lungs were removed and homogenized. lung homogenates were subjected to immunoblot analysis of granzyme b and active caspase- . the ratios of the relative band intensities of granzyme b and active caspase- normalized to gapdh are shown (n = - mice per group) (right panel). *p < . , ***p < . versus the iav-infected mice group. d the lung index (lung/body weight ratios) of mice treated with pbs (n = ) or β-sitosterol (n = ) on day p.i. *p < . , **p < . versus the iav-infected group. e on day p.i., the total protein concentrations in balf was measured by bca assay (n = - mice per group). *p < . , **p < . versus the iav-infected group. f, g survival rate (f) and weight curves (g) of iav-infected mice treated with or without β-sitosterol (n = mice per group). β-sitosterol ameliorates iav-induced inflammation and ali bx zhou nf-κb, atf (a downstream target of p ), and irf form a transcriptional complex that drives the expression of the antiviral factor ifn-β [ ] . in addition, the viral-induced expression of type iii ifn requires the involvement of rig-i, ips- , tbk , and p signaling [ ] [ ] [ ] , suggesting that the expression of type i and iii ifns is promoted via a common mechanism. interestingly, some studies have indicated that nf-κb is crucial for ifn-β production when irf activation is weak but not when irf activation is strong [ ] . although we did not detect significant activation of irf at h, we were able to detect an inhibitory effect of β-sitosterol on the expression of ifns, including ifn-β and ifn-λ , in cells infected with iav or in those subjected to viral rna transfection. these findings may be attributable to the inactivation of rig-i, nf-κb, and p signaling. a clear link between rig-i expression and stat activation has been established by previous studies. experiments involving rig-i overexpression or knockdown have suggested that rig-i is essential for stat activation in leukemia cell lines [ , ] . in accordance with these findings, our results show that the augmentation of stat activation by rig-i overexpression was suppressed by β-sitosterol or the inhibition of iav-mediated isre transcriptional activity by specific rig-i sirnas. in addition, we observed that the activation of jaks was not affected by β-sitosterol. therefore, the inhibition of stat phosphorylation in response to ifn-β treatment for min or h can be attributed to the downregulation of rig-i by βsitosterol. moreover, the activation of rig-i, but not of mda- , has been shown to involve double-stranded rna (dsrna)-induced stat phosphorylation [ ] . our data showed that β-sitosterol treatment abrogated stat phosphorylation in cells stimulated with vrna ( ′ppp-rna), which binds to and activates rig-i. however, the dephosphorylation of viral rna with ciap did not reduce stat phosphorylation. a possible explanation for these findings is that the dsrna that is generated following viral rna dephosphorylation is also a ligand for rig-i and induces stat phosphorylation in an ifn-dependent or ifn-independent manner, as described previously [ ] . the important role of ifns in the defense against viral infection is widely recognized [ , ] . however, ifn receptor deficiency does not lead to a detrimental outcome, which is perhaps due to decreased ifn-induced immune injury [ , , ] . recent studies have clearly revealed the pathogenic potential of ifn-β-and ifn-λ -mediated immunopathology in viral infectious diseases and autoimmune diseases [ , , , ] . here, we have proposed a model in which ifns (including type i and iii ifn) secreted from iav-infected cells bind to their receptors and sensitize uninfected neighboring cells, leading to the amplification of proinflammatory responses driven by isgf following infection by progeny viruses. β-sitosterol treatment blocked the amplification of this proinflammatory response and the concomitant expression of proinflammatory cytokines through the inhibition of isgf complexes. the inhibitory effect on isgf complexes was due to the failure of downregulated rig-i to exert a converse effect on stat activation in β-sitosterol-treated cells (fig. ) . the activation of rig-i-mediated apoptosis via type i ifn-dependent and type i ifnindependent mechanisms has been considered a promising strategy for cancer therapeutics [ , ] . the ifn-induced activation of isgf leads to trail expression, resulting in substantial alveolar epithelial cell (aec) apoptosis and lung injury [ , ] . aec apoptosis has been found to play a critical role in the pathogenesis of h n and pandemic h n in patients with ards [ , ] . our data suggest that β-sitosterol prevents iav-induced apoptosis associated with decreased ifn-driven expression of trail. the loss of rig-i signaling has been correlated with a reduction in antigen presentation in bone marrow derived dendritic cells (bmdcs), and in the antiviral function of cd + cytotoxic t cells [ ] . thus, it is clear that the rig-i pathway is important for mediating the production of ifns during antiviral responses. in contrast, the rig-i-mediated expression of inflammatory mediators has been shown to induce the recruitment of monocyte-derived dcs (modcs) to support viral replication [ ] . antiviral effector cd + t cells and nk cells eliminate invading pathogens through fig. β-sitosterol effectively abrogates iav-triggered signaling in vivo. a mice were infected with ld of a/fm /h n virus and treated with pbs or β-sitosterol (intragastrically administered for consecutive days beginning days prior to viral infection). the lungs were harvested and homogenized on day p.i. and the processed for immunoblotting with the indicated antibodies. the relative band intensities of the indicated proteins were normalized to that of gapdh (n = - mice per group) (right panel). *p < . , **p < . versus the iav-infected group. b-d detection of cytokine and chemokine production in balf (b), lung homogenates (c), and serum (d) by luminex analysis. *p < . , **p < . versus iav-infected group. e on day p.i., lung homogenates were subjected to immunoblot analysis of rig-i. the rig-i band intensity normalized to that of gapdh is shown (n = - mice per group) (right panel). *p < . versus iav-infected group. f, g detection of ifns (ifn-α, ifn-β, and ifn-γ) production in balf (f) and lung homogenates (g) by luminex analysis. *p < . , **p < . versus the iavinfected group. β-sitosterol ameliorates iav-induced inflammation and ali bx zhou several mechanisms, including the expression of pro-apoptotic proteins such as trail and fas and the secretion of granzyme b (grb) and perforin. through these mechanisms, the apoptotic caspase cascade is activated in virus-infected cells [ ] [ ] [ ] . studies have reported that ifn-γ plays an important role in modulating cd + t-cell recruitment and that the production of granzyme b during the recruitment of cd + t cells is dependent on the ifn-βinduced activation of stat [ , , ] . accordingly, our data show that β-sitosterol administration decreases the levels of ifn-γ and ifn-β and concomitantly induces a low level of cd + t-cell recruitment and granzyme b secretion in the lungs. cytotoxic cd + t lymphocytes (ctls) seem to be essential for viral clearance. however, severe pneumonia in patients with pandemic influenza a (h n ) virus infection is apparently related to high levels of cd + t cells [ ] . interestingly, one study showed that ha-transgenic mice develop lethal lung injury following treatment with influenza ha-specific cd + cytotoxic t cells [ ] . deficiency of a (tnf alpha-induced protein , tnfaip ), which is a negative feedback ubiquitin-editing protein that inhibits nf-κb signaling, protects mice against viral challenge by reducing the population of grb + cd + t cells [ ] . consistent with these studies, our data showed that iav-induced acute lung injury and mortality are attenuated in mice treated with β-sitosterol and that this attenuation is associated with decreased cd + t-cell recruitment and granzyme b secretion in the lung. furthermore, we observed the inhibition of stat / and p phosphorylation by β-sitosterol in mouse lung tissue and made a similar observation in iavinfected a cells. the expression of cytokines driven by stat / and p , which exacerbate iav-induced immunopathology, in balf and lung tissue was decreased following β-sitosterol administration in balf and lung tissue. in contrast to our in vitro results, the phosphorylation of erk / was attenuated in the lung tissues of β-sitosterol-treated mice. the inhibition of erk / signaling is involved in the retention of viral rnp in the nucleus, but its activation also correlates with cytokine expression [ ] . it is likely that the decrease in phosphorylated erk / expression in vivo was a result of the immunoregulatory effects of β-sitosterol. fig. schematic diagram showing the mechanism by which βsitosterol attenuates iav-induced proinflammatory responses and injury. invading viruses are sensed by rig-i, leading to the activation and that of rig-i, nf-κb, and p , which initiates the expression of proinflammatory mediators and ifns. secreted ifns, including type i and iii ifns, bind to their receptors via an autocrine or paracrine mechanism and then exert their antiviral effects. subsequently, previously uninfected ifn-sensitized neighboring cells become infected by progeny viruses, which triggers the amplification of the inflammatory response. the inhibition of rig-i signaling by βsitosterol attenuates rig-i-linked proinflammatory ifn production, which results in a reduction in stat activation and thus a decrease in the amplification of proinflammatory responses driven by isg complexes in ifn-sensitized cells. furthermore, the inhibition of rig-i signaling by β-sitosterol also suppresses the recruitment of cd + t cells and granzyme b release in vivo, thereby blocking lung immune injury during influenza virus infection. influenza a(h n ) virus gains neuraminidase inhibitor resistance without loss of in vivo virulence or transmissibility an ns-segment exonic splicing enhancer regulates influenza a virus replication in mammalian cells clinical aspects and cytokine response in severe h n influenza a virus infection clinical aspects and cytokine response in adults with seasonal influenza infection fatal outcome of human influenza a (h n ) is associated with high viral load and hypercytokinemia cytokine and chemokine levels in patients infected with the novel avian influenza a (h n ) virus in china pathogen recognition and inflammatory signaling in innate immune defenses de novo replication of the influenza virus rna genome is regulated by dna replicative helicase recognition of viruses by cytoplasmic sensors an atomic model of the interferon-beta enhanceosome inhibition of p mitogen-activated protein kinase impairs influenza virus-induced primary and secondary host gene responses and protects mice from lethal h n infection the kinetic mechanism of the dual phosphorylation of the atf transcription factor by p mitogen-activated protein (map) kinase alpha. implications for signal/response profiles of map kinase pathways early control of h n influenza virus replication by the type i interferon response in mice protective role of beta interferon in host defense against influenza a virus differential roles of mda and rig-i helicases in the recognition of rna viruses distinct rig-i and mda signaling by rna viruses in innate immunity cell typespecific involvement of rig-i in antiviral response h n influenza virus-induced mediators upregulate rig-i in uninfected cells by paracrine effects contributing to amplified cytokine cascades inflammatory cytokines in the bal of patients with ards. persistent elevation over time predicts poor outcome bronchoalveolar and systemic cytokine profiles in patients with ards, severe pneumonia and cardiogenic pulmonary oedema ifnlambdas mediate antiviral protection through a distinct class ii cytokine receptor complex cloning and expression of a long form of the beta subunit of the interferon alpha beta receptor that is required for signaling signaling pathways activated by interferons innate immune modulation by rna viruses: emerging insights from functional genomics the human interferon-induced mxa protein inhibits early stages of influenza a virus infection by retaining the incoming viral genome in the cytoplasm binding of the influenza a virus ns protein to pkr mediates the inhibition of its activation by either pact or double-stranded rna the primary function of rna binding by the influenza a virus ns protein in infected cells: inhibiting the '- ' oligo (a) synthetase/rnase l pathway influenza a virus inhibits type i ifn signaling via nf-kappab-dependent induction of socs- expression suppression of interferon lambda signaling by socs- results in their excessive production during influenza virus infection influenza-induced type i interferon enhances susceptibility to gram-negative and gram-positive bacterial pneumonia in mice pathogenic potential of interferon alphabeta in acute influenza infection macrophageexpressed ifn-beta contributes to apoptotic alveolar epithelial cell injury in severe influenza virus pneumonia alpha/beta interferon receptor signaling amplifies early proinflammatory cytokine production in the lung during respiratory syncytial virus infection dysregulated type i interferon and inflammatory monocyte-macrophage responses cause lethal pneumonia in sars-cov-infected mice honeysuckle-encoded atypical microrna directly targets influenza a viruses antioxidant and anti-inflammatory flavonoids from the flowers of chuju, a medical cultivar of chrysanthemum morifolim ramat taraxacum mongolicum extract exhibits antimicrobial activity against respiratory tract bacterial strains in vitro and in neonatal rats by enhancing systemic th immunity antiviral effect of forsythoside a from forsythia suspensa (thunb.) vahl fruit against influenza a virus through reduction of viral m protein lariciresinol- -o-beta-d-glucopyranoside from the root of isatis indigotica inhibits influenza a virus-induced proinflammatory response sterol content of foods of plant origin natural sources of dietary plant sterols antioxidant effects of phytosterol and its components protective role of plant sterol and stanol esters in liver inflammation: insights from mice and humans beta-sitosterol attenuates high-fat dietinduced intestinal inflammation in mice by inhibiting the binding of lipopolysaccharide to toll-like receptor in the nf-kappab pathway beta-sitosterol-induced-apoptosis is mediated by the activation of erk and the downregulation of akt in mca- murine fibrosarcoma cells influenza a viruses suppress cyclooxygenase- expression by affecting its mrna stability analysis of relative gene expression data using real-time quantitative pcr and the (-delta delta c(t)) method influenza virus and cell signaling pathways induction of proinflammatory cytokines in primary human macrophages by influenza a virus (h n ) is selectively regulated by ifn regulatory factor and p mapk role of hypercytokinemia in nf-kappab p -deficient mice after h n influenza a virus infection targeted prostaglandin e inhibition enhances antiviral immunity through induction of type i interferon and apoptosis in macrophages hyperinduction of cyclooxygenase- -mediated proinflammatory cascade: a mechanism for the pathogenesis of avian influenza h n infection rig-i detects viral genomic rna during negative-strand rna virus infection ra-inducible gene-i induction augments stat activation to inhibit leukemia cell proliferation interferons at age : past, current and future impact on biomedicine disease-promoting effects of type i interferons in viral, bacterial, and coinfections proapoptotic signaling induced by rig-i and mda- results in type i interferon-independent apoptosis in human melanoma cells cytokine response patterns in severe pandemic h n and seasonal influenza among hospitalized adults pathological and ultrastructural analysis of surgical lung biopsies in patients with swine-origin influenza type a/h n and acute respiratory failure a question of self-preservation: immunopathology in influenza virus infection trim ring-finger e ubiquitin ligase is essential for rig-i-mediated antiviral activity type i inteferon gene induction by the interferon regulatory factor family of transcription factors antiviral innate immunity and stress granule responses p mitogen-activated protein kinase-dependent hyperinduction of tumor necrosis factor alpha expression in response to avian influenza virus h n essential impact of nf-kappab signaling on the h n influenza a virus-induced transcriptome streptococcus pneumoniae induced p mapk-and nf-kappab-dependent cox- expression in human lung epithelium viral infections activate types i and iii interferon genes through a common mechanism map kinase p alpha regulates type iii interferon (ifn-lambda ) gene expression in human monocyte-derived dendritic cells in response to rna stimulation rig-i-mediated activation of p mapk is essential for viral induction of interferon and activation of dendritic cells: dependence on traf and tak nf-kappa b rela subunit is crucial for early ifn-beta expression and resistance to rna virus replication involvement of retinoic acid-inducible gene-i in the ifn-{gamma}/stat signalling pathway in beas- b cells doublestranded rna induces biphasic stat phosphorylation by both type i interferon (ifn)-dependent and type i ifn-independent pathways interferons and viruses: an interplay between induction, signalling, antiviral responses and virus countermeasures interferon-inducible antiviral effectors interleukin- modulates proinflammatory cytokine production in synovial inflammation of rheumatoid arthritis synergy between ra and tlr promotes type i ifn-dependent apoptosis through upregulation of trail pathway in breast cancer cells antiviral response by natural killer cells through trail gene induction by ifn-alpha/beta molecular biology, and pathogenesis of avian influenza a (h n ) infection in humans rig-i signaling is critical for efficient polyfunctional t cell responses during influenza virus infection efficient influenza a virus replication in the respiratory tract requires signals from tlr and rig-i type i interferons regulate cytolytic activity of memory cd + t cells in the lung airways during respiratory virus challenge role of tumor necrosis factorrelated apoptosis-inducing ligand in immune response to influenza virus infection in mice distinct roles of nk cells in viral immunity during different phases of acute friend retrovirus infection intracellular ifn-gamma expression in natural killer cells precedes lung cd + t cell recruitment during respiratory syncytial virus infection production of interferon-gamma by influenza hemagglutinin-specific cd effector t cells influences the development of pulmonary immunopathology cd + /cd + t lymphocytes imbalance in children with severe pandemic influenza a (h n ) pneumonia structural and functional consequences of alveolar cell recognition by cd + t lymphocytes in experimental lung disease a deficiency in lung epithelial cells protects against influenza a virus infection inhibition of influenza virus-induced nf-kappab and raf/mek/erk activation can reduce both virus titers and cytokine expression simultaneously in vitro and in vivo zfy and nsz conceived the study; bxz, zfy, and nsz designed the study; bxz and xll conducted the in vitro experiments; jl and xpp isolated and analyzed the compound β-sitosterol; bxz, xll, hmj, ybh, and pfx performed animal experiments; and bxz and jl wrote the paper. the online version of this article (https://doi.org/ . /s - - - ) contains supplementary material, which is available to authorized users.competing interests: the authors declare no competing interests. key: cord- -wtgk d authors: xu, ming-ming; ryan, philip; rudrawar, santosh; quinn, ronald j; zhang, hai-yan; mellick, george d title: advances in the development of imaging probes and aggregation inhibitors for alpha-synuclein date: - - journal: acta pharmacol sin doi: . /s - - -y sha: doc_id: cord_uid: wtgk d abnormal protein aggregation has been linked to many neurodegenerative diseases, including parkinson’s disease (pd). the main pathological hallmark of pd is the formation of lewy bodies (lbs) and lewy neurites, both of which contain the presynaptic protein alpha-synuclein (α-syn). under normal conditions, native α-syn exists in a soluble unfolded state but undergoes misfolding and aggregation into toxic aggregates under pathological conditions. toxic α-syn species, especially oligomers, can cause oxidative stress, membrane penetration, synaptic and mitochondrial dysfunction, as well as other damage, leading to neuronal death and eventually neurodegeneration. early diagnosis and treatments targeting pd pathogenesis are urgently needed. given its critical role in pd, α-syn is an attractive target for the development of both diagnostic tools and effective therapeutics. this review summarizes the progress toward discovering imaging probes and aggregation inhibitors for α-syn. relevant strategies and techniques in the discovery of α-syn-targeted drugs are also discussed. parkinson's disease (pd) is a progressive neurodegenerative disorder that causes severe motor deficits. common symptoms include rigidity, bradykinesia, tremor and postural instability [ ] . with disease progression, nonmotor symptoms, such as depression, psychosis, falls, and sleep disturbance, also emerge [ ] . globally, . % of the population over years of age [ ] and more than million people [ ] are affected by this devastating disease. pd is pathologically characterized by the substantial loss of dopamine (da)-containing neurons in the midbrain [ ] and the presence of intraneuronal cytoplasmic inclusions [ ] , known as lewy bodies and lewy neurites [ ] , both of which comprise alphasynuclein (α-syn) aggregates. a definitive diagnosis of pd has to rely on histopathological postmortem analysis and requires the detection of dopaminergic cell loss and the presence of lewy bodies and lewy neurites. for living patients, several clinical diagnostic criteria have been formulated, including the uk parkinson's disease society brain bank (ukpdsbb) [ ] , the national institute of neurological disorders and stroke (ninds) criteria [ ] and the movement disorder society clinical diagnostic criteria for pd [ ] . however, the diagnostic accuracy obtained by using these criteria is only between % and % [ , ] . compared with the observation of clinical symptoms, neuroimaging may help to increase diagnostic precision. clinically available imaging approaches include positron emission tomography (pet), single-photon emission computed tomography (spect) and magnetic resonance imaging (mri). pet and spect use a variety of radiotracers to quantitatively measure the metabolic and neurochemical changes in the brains. for example, fluorine- -l-dihydroxyphenylalanine ( f-dopa) pet can mark dopaminergic deficiencies in the brains of patients with pd [ ] . mri uses different sequences and contrasts to study brain structure and function [ ] . although substantial progress has been made in pd neuroimaging, currently, it can only be used as a supplementary tool to clinical examination [ ] . increasing evidence suggests that prior to the motor phase of classical pd there is a prodromal period that lasts for several years [ ] . after the appearance of typical motor features, the disease can continue to progress for many years or even decades, although it is impossible to predict the trajectory of this progression at diagnosis [ ] . the pathological changes in the central nervous system during the prodromal phase, such as the formation of α-syn aggregates, appear to mirror the occurrence of motor and nonmotor symptoms. a timeline of pd from onset to death has been proposed [ ] , which not only promotes a comprehensive understanding of the overall disease process but is also a reminder that before a patient shows any pd-related clinical symptoms, the disease may have progressed severely and possibly irreversibly as a result of neuronal dysfunction and cell loss [ , ] . in the future, the traditional symptom-based clinical examination may be pre-empted by the use of biomarkers that may assist in identifying early disease-specific changes by early prodromal diagnosis, namely detecting specific changes in biomarkers during the early development of pd [ ] . since diagnosis informs treatment, novel therapeutics targeting the potential pathological culprits need to be developed. until now, no neuroprotective or neurorestorative therapy has been found for the treatment of pd. existing treatments predominantly target dopamine-related symptoms. few, if any, target nondopaminergic symptoms, which cause a severe burden for patients in the advanced stages of the disease. in addition, current pd treatments provide little or no relief for disease progression because they do not alter the rate or extent of neuronal cell loss [ , ] . therefore, for the future direction of pd medications, the priority is the development of neuroprotective or neurorestorative drugs that can stop or at least relieve disease progression and the relevant nonmotor symptoms [ ] . to realize this goal, it is necessary to identify and target the key culprit underlying the pathogenesis of pd. native α-syn is a small protein encoded by the gene snca, with a molecular mass of . kda ( amino acids) [ ] . its primary structure ( fig. ) shows three different regions: the n-terminal region (residues − ) that is characterized by repetitions of a highly conserved lysine-rich motif ktk(e/q)gv, the hydrophobic central part (residues − , known as the non-amyloidogenic component (nac) region), and the acidic c-terminal region (residues − ) . the amphipathic n-terminal region has a structural alpha-helix propensity similar to apolipoprotein-binding domains, suggesting that α-syn is a membrane-bound protein [ ] . the nac region is believed to be involved in protein aggregation by mediating the conformational changes of α-syn from random coil to a beta-sheet structure [ ] . the c-terminal region, having no distinct secondary structure, has been reported to interact with the n-terminal region or the nac region to maintain the natively unfolded state of α-syn [ ] . although it is widely believed that α-syn exists mainly as monomer, studies have also shown that endogenous α-syn can form tetramers [ , ] , which comprise an alpha-helical secondary structure and show little tendency to form aggregates [ ] . all these findings suggest that there may be a dynamic equilibrium between different α-syn conformational states [ ] . the exact normal functions of α-syn are not well understood. although some animal studies suggest that an α-syn-knockout is not fatal, deficiencies in synaptic transmissions were observed in some knockout animal lines [ , ] . in neurons α-syn is mainly localized at the presynaptic terminals [ , ] and is associated with the reserve pool of synaptic vesicles [ ] [ ] [ ] . α-syn can promote the assembly of the presynaptic soluble n-ethylmaleimide-sensitive factor activating protein receptor (snare) protein complex, that mediates vesicle fusion, which is a vital step for the release of neurotransmitters, including dopamine [ ] . in addition to its role in synaptic transmission, α-syn is also involved in the suppression of apoptosis [ ] , antioxidation [ ] and even regulation of dopamine biosynthesis [ , ] . the first connection between α-syn and pd was made in when point mutations in the snca gene were identified in familial pd cases [ ] . to date, six missense mutations in snca are known to lead to autosomal dominant pd: a t [ ] , a p [ ] , e k [ ] , h q [ ] , g d [ ] , and a e [ ] . meanwhile, the link between α-syn and pd was further elucidated with the discovery of duplications and triplications of the snca gene in pd families, which also suggested that increased expression of α-syn was a causative factor of pd [ , ] . moreover, certain polymorphisms in snca, such as the dinucleotide repeat rep located in the snca promoter (snca-rep ), a − and − base-pair (bp) singlenucleotide polymorphism, are major risk factors for sporadic pd, and are thought to result from altered expression of the gene product [ , ] . genome-wide association studies have also consistently revealed highly significant regions of genetic variation around the snca gene as contributors to the risk of pd. a meta-analysis of genome-wide association studies has identified new pd risk loci [ ] . as mentioned earlier, the nac region of monomeric α-syn mediates the misfolding from a random coil to a beta-sheet structure, leading to the formation of oligomers, protofibrils and eventually mature fibrils [ ] . this pathway is also shared by other amyloid-forming proteins such as beta-amyloid (aβ), tau and human islet amyloid polypeptide (hiapp). the fluorescent dye thioflavin-t (tht) is a widely used "gold standard" for staining and identifying fibrils of these amyloid proteins [ ] . kinetics studies of α-syn aggregation using tht [ , ] revealed a sigmoidal curve of fibril growth, which is composed of the lag phase representing the nucleation stage, an exponential phase representing the elongation stage and a plateau phase corresponding to the completion of fibril formation [ ] . it is natural to deduce that α-syn can cause toxicity to neurons, as cell death is a major hallmark of pd and α-syn plays a causal role in the disease. given that all known clinical mutations are thought to be linked to increased α-syn aggregation and that the main components of lewy bodies and lewy neurites are aggregated α-syn fibrils, it was initially believed that α-syn fibrils were the toxic species. later, increasing evidence from both in vitro and in vivo studies has supported the proposal that oligomeric species are the most pathogenically relevant [ ] [ ] [ ] [ ] [ ] . as a result, the oligomers may be a better therapeutic target, as the aggregated lewy bodies themselves might be protective and represent a form of an aggresome [ , ] . α-syn oligomers are believed to exert toxicity both intracellularly and extracellularly. within the cytoplasm, α-syn oligomers can inhibit the hsp chaperone system which is important for protein refolding [ ] . α-syn oligomers can also bind to and inhibit the activities of proteasomes [ ] , thus affecting protein degradation. consequently, proteostasis is impaired, leading to endoplasmic reticulum (er) stress and finally cell death. in vivo, α-syn oligomers have been detected within the er lumen, and treatment with an inhibitor of er stress, dramatically delayed the onset of motoric symptoms and decreased the accumulation of α-syn oligomers [ ] , suggesting that er stress is involved in the toxicity of α-syn oligomers. in addition, α-syn plays an important role in the assembly of the presynaptic snare protein complex. the formation of oligomeric α-syn can inhibit snare-mediated vesicle docking and consequently reduce exocytosis, suggesting that inhibition of dopamine release is a potential mechanism in pd [ ] . α-syn oligomers can also induce the dysfunction of synapses by compromising the axonal transport of critical presynaptic proteins [ ] . other potential intracellular targets of α-syn oligomers include mitochondria [ ] , lysosomes [ ] and microtubules [ ] . extracellular sources of α-syn oligomers can form pores in cell membranes, causing an increase in intracellular calcium levels that leads to cell death [ ] . exposure of neurons to exogenous α-syn oligomers can activate glutamatergic receptors, resulting in longterm potentiation (ltp) and excitotoxicity that leads to neuronal death [ , ] . moreover, there is significant evidence that α-syn oligomers can transfer from neuron to neuron or to glial cells via a prion-like process [ , ] , which may explain the spread of lewy pathology throughout the brain in pd [ ] . overall, the accumulation of α-syn aggregates is a major pathological hallmark of pd and a priority target for drug development given its hypothesized contribution to neurodegeneration. in vivo imaging of α-syn pathology could be useful as a biomarker of the presence of the disease, disease progression and as a pharmacodynamic tool for drug development. therefore, αsyn imaging is a critical need for pd research [ ] . moreover, since α-syn aggregation is regarded as a major pathogenic process in pd, several strategies exist for the prevention of α-syn toxicity [ ] . among them, inhibition of α-syn aggregation remains an extremely attractive target for drug development [ ] . the representative progress in the development of imaging probes and aggregation inhibitors over the past decade will be discussed further. although several pet/spect tracers targeting the dopamine system have been used clinically, as reviewed by politis [ ] , braak staging of the pd brain suggests that the severe loss of dopaminergic neurons occurs at stage , while lewy body pathology appears as early as stage . as such, imaging α-syn pathology, rather than dopaminergic changes, would be more suitable for early diagnosis in the prodromal period of pd. while great progress has been made toward developing imaging probes for other amyloid-forming proteins [ ] (and notably three aβ imaging probes have gained fda approval), the development of α-syn imaging probes is still at an early stage. despite the fact that there are no selective α-syn pet or spect probes for clinical pd diagnosis, several α-syn imaging probes have been developed and tested over the past decade (fig. , table ). amyloid proteins, such as aβ and α-syn, tend to form similar beta-sheet structures upon aggregation. as a result, probes that can interact with aβ aggregates also have the potential to bind to aggregated α-syn. following this rationale, some established aβ pet probes have been evaluated against α-syn. one well-known example is c-pittsburgh compound-b (pib, ), a derivative of thioflavin-t (tht) which is the gold standard for staining all types of amyloid proteins. probe exhibited a similar binding affinity (k d = nm) for in vitro generated α-syn fibrils compared to aβ [ ] , but failed to bind to brain homogenates containing lewy bodies [ ] . additionally, probe displayed poor selectivity for α-syn in the staining of brain sections where aβ was also present [ ] . benzoxazole bf is another established aβ-binding probe. f-bf ( ) was proven to bind to α-syn fibrils at an equimolar concentration compared to aβ - fibrils, but the binding affinity for α-syn fibrils (k d = . nm) was approximately sevenfold lower [ ] . however, like c-pib, probe also failed to bind to brain homogenates containing lewy bodies [ ] . a follow-up in vivo study using an accelerated mouse model of synucleinopathy failed to observe a significant difference in the brains of α-syn transgenic mice compared with α-syn-ko mice after treatment with probe [ ] . these results suggest that it is necessary to develop specific α-syn pet probes. to discover selective ligands for α-syn aggregates, yu et al. synthesized a series of phenothiazine derivatives and evaluated their binding affinities for α-syn fibrils with a tht competition assay. compounds b, a and b exhibited k i values of less than nm ( . nm for b, . nm for a and . nm for b) and were considered for further study [ ] . later, i labeled b ( ), denoted i-sil , was synthesized and tested by the same research group. probe can bind to recombinant α-syn fibrils and brain homogenates from pd patients and α-syn transgenic mice. probe exhibited a relatively high binding affinity for α-syn fibrils compared with aβ − fibrils (fivefold lower than α-syn) and tau fibrils (twofold lower than α-syn) but the selectivity was not high enough for in vivo imaging [ ] . using a [ ] . in addition, c- a ( ) and f- b ( ) were synthesized and proven to cross the blood−brain barrier (bbb) in sprague−dawley (sd) rats. both radiotracers exhibited high initial uptake ( . % id/g for , . % id/g for ), homogeneous distribution and rapid washout kinetics. the authors believed that these two radiotracers could be good leads for the discovery of selective imaging probes for α-syn [ ] . identifying new chemical scaffolds is necessary for developing specific radiotracers with high selectivity for α-syn fibrils versus other amyloid proteins such as aβ and tau. recently, a series of compounds based on the -(benzylidene)-indolin- -one scaffold were synthesized and the most potent compound was a, having a high affinity (k i = nm) and very good selectivity for α-syn versus aβ (k i = . nm) and tau fibrils (k i = . nm). the binding affinity of f-labeled a ( ) was determined from a saturation binding assay. the k d of probe for α-syn fibrils was . nm while the values for aβ and tau fibrils were and nm, respectively. however, probe is not believed to be a suitable pet tracer for in vivo imaging due to poor druggability [ ] . flavonoids are a common source for the discovery of inhibitors of aβ aggregation as well as α-syn. chalcone is a good example and ono et al. synthesized a series of chalcone derivatives in the hope of discovering new scaffolds for α-syn imaging. of all four synthesized compounds, idp- showed the most selective binding to α-syn aggregates (k d = . nm, k d = . nm for aβ). fluorescent staining of pd brain sections confirmed the affinity of idp- for lewy bodies. unfortunately, i-idp- ( ) displayed the lowest initial uptake of the four compounds synthesized ( . % id/g) in normal mice, making it difficult to use for in vivo imaging [ ] . more recently, the same group also synthesized three novel radioiodinated benzoimidazole (bi) derivatives based on the previous findings: ( ) the benzoimidazole scaffold could bind to α-syn aggregates; ( ) the diene moiety helps to increase the affinity for α-syn aggregates; and ( ) introduction of large substituents increases the selectivity for α-syn. compound bi- showed the highest selectivity and binding affinity for α-syn aggregates (k d = . nm, k d = nm for aβ). fluorescent staining of pd and ad brain sections using bi- was conducted and bi- clearly stained lewy bodies but failed to label aβ aggregates, further confirming its selectivity for α-syn. however, the promising compound i-bi- ( ) exhibited low initial uptake ( . % id/g) and poor washout kinetics in normal mouse brains, suggesting that the introduction of bulky substitution groups may affect penetration into the bbb [ ] . the authors also mentioned that criteria exist for ideal aβ and tau imaging probes according to previous reports [ , ] : ( ) an initial brain uptake up to % id/g at -min postinjection in mice; and ( ) a remaining amount of less than % id/g at -min postinjection in normal mouse brains. clearly, none of the reported probes for α-syn met these criteria, including probe . in the past decade, significant progress toward discovering useful α-syn radiotracers has been made; however, an ideal, druggable probe possessing high affinity and selectivity for α-syn has yet to be identified [ ] . fluorescence imaging presents a promising alternative technique to radiotracers. in contrast to pet/spect techniques, fluorescence imaging is monitored in real time, is inexpensive, is nonradioactive and is of high-resolution when using near-infrared fluorescence (nirf) imaging probes. as a technique, fluorescence imaging is progressively being explored for the neuroimaging of aβ aggregates [ ] . the ideal fluorescent probe will not only possess high selectivity and binding affinity, penetrate rapidly into the bbb and be cleared quickly from normal brain regions, but will also have low background fluorescence (ideal emission wavelength of greater than nm) in addition to producing a significant increase in fluorescence upon binding to target proteins. given that aggregated α-syn is far less abundant in the brain than aβ, αsyn fluorescent probes will require very high selectivity for α-syn over aβ and tau. since the majority of α-syn is found intracellularly, in addition to penetrating the bbb, such a probe has to cross the cell membrane [ ] . tht is a widely used fluorescent dye for the nonselective staining of protein aggregates. its in vivo utility is limited due to its positive charge which affects its penetration through the bbb and its emission wavelength is not suitable for in vivo imaging. to search for improved fluorescent probes for α-syn, celej et al. evaluated the abilities of several n-arylaminonaphthalene sulfonate (nas) derivatives to be used as fluorescent markers for α-syn aggregates since the nas scaffold has a long history of being applied to study the molecular microenvironment and conformation of proteins. compounds , -ans (fig. , ) , , -tns ( ), bis-ans ( ), and bis-tns ( ) exhibited slightly improved binding affinity (k d values: . , . , . and . μm, respectively) for αsyn fibrils compared with tht (k d = . μm) [ ] . although these molecules also displayed advantages over tht in terms of providing structural information during α-syn fibrillation, they are still not useful as imaging probes as a result of their charged properties and unideal emission wavelengths. in an endeavor to search for novel specific fluorescent probes for α-syn, volkova and coworkers studied the potential of a series of monomethine and trimethinecyanines to detect the formation of α-syn fibrils. the most potent dyes, t- ( ) and sh- ( ), had similar binding affinities to tht but showed large increases in fluorescence (~ . -fold for and~ . -fold for ) upon binding to α-syn fibrils relative to tht ( . -fold). a notable improvement was that the emission wavelengths of probes and after binding to α-syn fibrils were determined to be and nm, respectively, compared with tht ( nm) [ ] . the specificity of these two dyes was not evaluated by assays against other amyloid proteins. later, carbocyanine compound jc- ( ) was introduced to conduct real-time analyses of α-syn fibril formation. probe could bind to α-syn monomers as well as fibrils. interestingly, the maximum emission wavelengths of probe after binding to monomers and fibrils were different ( nm for monomers, nm for fibrils), indicating that probe is able to distinguish between monomeric and fibrillar α-syn. furthermore, probe did not interact with either monomeric or fibrillary aβ. the high selectivity of probe might be explained by its interaction with the acidic c-terminal region of α-syn [ ] . the results also suggested that promising selective probes for α-syn may be discovered by searching for compounds that can interact with the monomeric form of α-syn, since different amyloid proteins have different primary structures but share similar beta-sheet structures once aggregated. some fluorescent probes previously used as sensors for other biomolecules or microenvironments were tested to monitor α-syn aggregation. one of the compounds, coumarin ( ), displayed a similar sigmoidal curve as tht when monitoring the aggregation process of α-syn. the lag phase ( h) was shorter than that of tht ( . h) and the concentration required ( nm) was much lower than that of tht ( μm), suggesting that probe is more sensitive than tht. the other compound , -diphenyl- , , hexatriene (dph, ), failed to show a sigmoidal curve and was believed to interact with hydrophobic environments without selectivity, while probe could bind to the beta-sheet structure more specifically [ ] . emerging evidence indicates that α-syn oligomers are more toxic and more pathologically connected to pd than fibrils. consequently, recent research has focused on developing [ ] . the emission wavelength of probe after binding to α-syn oligomers was determined to be nm, showing its potential to be used in vivo as a nirf imaging probe. however, it remains questionable whether the bulky probe could penetrate the bbb. another novel probe aimed at detecting α-syn oligomers is tetraphenylethene tethered with triphenylphosphonium (tpe-tpp, ) . unlike sl- , probe is able to detect the monomeric, oligomeric and also fibrillar forms of α-syn. at the same concentration, probe emitted over four times more fluorescence than tht after incubation with α-syn fibrils. the calculated k d for probe was . μm, lower than that of tht ( . μm) [ ] . compound selectivity among different amyloids has yet to be determined. huge challenges still exist in the discovery of probes targeting α-syn oligomers and major breakthroughs in terms of the d structure of oligomeric α-syn and the synthesis of high contrasting and selective imaging probes are urgently needed. a known modulator of α-syn aggregation, anle b ( ) , was found to exhibit a significant increase in fluorescence upon binding to α-syn fibrils with high affinity (k d = nm) [ ] . the study of probe indicates not only the feasibility of discovering fluorescent probes from known inhibitors of α-syn aggregation but also the possibility of developing drugs with both therapeutic and diagnostic functions. until now there have not been any useful α-syn fluorescent probe reported for in vivo imaging (table ). in fact, the great progress made in the development of aβ fluorescent probes might negatively affect the discovery of a specific imaging probe for α-syn because naturally the same strategies to design aβ probes have been applied to the development of α-syn probes [ ] . since most of the existing aβ fluorescent probes share the classical push−pull structure [ ] , which shows little selectivity for the different amyloid protein aggregates, it has been recommended to work on novel strategies to search for or design specific ligands for α-syn. the idea of inhibiting α-syn aggregation, especially oligomerization, using small molecules to combat α-syn toxicity and prevent neurodegeneration has been gaining increasing attention. it has been proposed that the identification of aggregation inhibitors from screening compound libraries should be a priority [ ] . until now, a variety of small molecules have been discovered to inhibit α-syn aggregation (table ). in many cases, however, the inhibitory effects have also been translated to other amyloid proteins. in addition to their roles as antimicrobial agents to fight infectious diseases, antibiotics have demonstrated other properties, such as neuroprotective activity. rifampicin (fig. , ) stabilized α-syn as a monomer and blocked the fibrillation process. moreover, rifampicin was also able to disaggregate existing fibrils [ ] . another study confirmed the efficacy of compound by discovering that rifampicin could prevent -methyl- -phenylpyridinium (mpp + )induced toxicity in pc cells, increase their survival and reduce α-syn oligomer formation [ ] . another molecule tetracycline ( ) is also able to inhibit the formation of fibrils and destabilize preformed fibrils. compound exhibited moderate effective concentrations (ec values) for the formation ( . μm) and destabilization ( . μm) of α-syn fibrils while the values for aβ − and aβ − were higher ( and μm respectively for fibril formation; and μm respectively, for fibril destabilization), indicating a slight selectivity for α-syn over aβ [ ] . dopamine and its analogs dopamine ( ) is believed to react with α-syn covalently to form αsyn-quinone adducts which are primarily large molecular mass oligomers. these oligomeric intermediates can cause cytotoxicity, implying a potential role for the interaction of compound with αsyn in pd pathogenesis [ ] . however, other evidence suggests that compound inhibits α-syn fibrillation via binding noncovalently to α-syn [ ] . based on this fact, latawiec et al. screened analogs of compound and selected five potent compounds ( − ) by molecular dynamics (md) simulations. atomic force microscopy (afm) and transmission electron microscopy (tem) analysis confirmed the in silico simulation predictions that the selected compounds may affect the aggregation process of α-syn [ ] . the combination of in silico approaches with in vitro assays to discover ligands that interact with target proteins may emerge as a novel and efficient way to identify aggregation inhibitors for α-syn. some common dyes, such as lacmoid ( ) , congo red ( ) and phthalocyanine tetrasulfonate ( ) , have been reported to inhibit α-syn fibrillation [ ] . compounds and were found to be nonspecific inhibitors; however, their effects were mediated by the formation of aggregates of these compounds which can interact with different regions of α-syn monomer [ ] . unlike these two nonspecific inhibitors, the inhibition of α-syn fibrillation by compound is believed to be mediated by specific interactions with the n-terminus of α-syn [ ] . this molecule has also been tested in animal models for the treatment of scrapie disease and exhibited anti-prion activity and showed low toxicity [ ] . therefore, compound could be a potential therapeutic candidate for amyloid protein-related diseases. recently, another dye, coomassie brilliant blue r ( ) not only exhibited significant inhibition of α-syn fibrillation but also prevented the formation of oligomers that caused notable neurotoxicity in cells, making it a potentially useful candidate for future in vivo studies [ ] . the red blood pigment hemin ( ) is a well-known inhibitor of aβ aggregation [ ] . until recently, compound was also thought to interfere with α-syn aggregation [ , ] . the effects of compound on α-syn remain controversial; however, it has not been confirmed whether compound inhibits tht fluorescence via inhibition of α-syn aggregation or by obstructing the interaction between tht and the proteins [ ] . polyphenols are the largest group of inhibitors of α-syn aggregation and many of them can also inhibit the aggregation of other amyloid proteins such as aβ. we have categorized representative polyphenols into the following groups. curcuminoids. the interaction of curcumin with α-syn seems complicated in that different mechanisms of anti-aggregation by curcumin ( ) have been reported. early studies from the tht assay and tem showed that compound not only inhibited the formation of α-syn fibrils but also destabilized the preformed fibrils [ ] . later, the anti-aggregation ability of compound was confirmed by western blot analysis and in a cell model of αsyn aggregation [ ] . interestingly, another report stated that compound does not interact with α-syn monomers but binds to oligomers and fibrils, causing morphological changes and consequently reducing their toxicity. moreover, the interaction of compound with early oligomers could impact the toxicity by converting the preformed oligomers into the less toxic fibrils [ ] . although compound has been widely studied, its instability and poor bioavailability limits its medical use. with the aim of discovering more druggable molecules with a scaffold similar to that of compound , ahsan et al. μm) . surprisingly, unlike compound , compound failed to reduce the cytotoxicity caused by α-syn oligomers [ ] . while compound is a promising therapeutic agent for the potential treatment of pd, the case of compound strengthens the importance of evaluating the anti-oligomerization ability of any molecules that are expected to show neuroprotection in cells and/ or animals. another group focused on modifications of the two aromatic rings of curcumin to increase the hydrophobicity. of the nine reported analogs, only two (c and c ) showed lower stability than curcumin, and one (c ) exhibited significant cytotoxicity. among the remaining compounds, c ( ) , which was synthesized by replacing all the hydroxyl groups of compound with −och ph groups, showed the highest reduction in cytotoxicity caused by the preformed oligomers and fibrils. consistent with previous study [ ] , curcumin and its analog compound were found to accelerate the process of α-syn aggregation into less toxic fibrils [ ] . flavanols. the green tea polyphenol egcg (fig. , ) is perhaps the most studied inhibitor of aggregation of different amyloid proteins. it is also a common positive control in many studies aiming to develop new anti-amyloidogenic molecules. ehrnhoefer et al. demonstrated that compound can directly bind to monomeric α-syn and promote the formation of unstructured, nontoxic α-syn oligomers. nmr results suggested that the compound binds randomly to the backbone of α-syn [ ] , which, along with another report using ms to study the binding of compound with α-syn [ ] , may explain why the effects of compound on α-syn can also be seen on aβ. additionally, compound is able to bind to preformed α-syn fibrils (k d = nm) and transform them into smaller amorphous aggregates that are less toxic [ ] . however, the mechanism by which compound reduces the toxicity of α-syn oligomers is not the same as in the case of fibrils. one group found that treatment with compound does not cause notable changes in the structure or size of α-syn oligomers. instead, the binding of compound to α-syn oligomers prevents permeabilization of the α-syn oligomers with cell membranes, thus reducing the toxicity [ ] . in animal studies, tea polyphenols mainly containing compound were reported to reduce the level of α-syn oligomers in a pd monkey model [ ] . theaflavins, which are abundant in black tea, exert antiamyloidogenic effects in the same way as compound , promoting the assembly of monomeric α-syn and aβ into nontoxic aggregates and converting the preformed fibrils into nontoxic aggregates [ ] . nevertheless, the tendency of compound and theaflavins to be oxidized could compromise their ability to inhibit amyloidogenesis. although preoxidized compound is still able to significantly inhibit the aggregation process, a reduction in efficacy can be seen after long-term preoxidation in the case of compound but not in the case of another theaflavin, tf ( ) , which is less rapidly oxidized than egcg [ ] . these results suggest that the anti-amyloidogenic abilities of common polyphenols may partially depend on the antioxidant properties. developing polyphenols that are resistant to oxidation, such as compound , may help to discover promising therapeutic agents for amyloid protein-related disease. stilbenes. previous evidence has demonstrated that some stilbenes can inhibit aβ aggregation [ ] . to explore the potential application of stilbenes in the discovery of inhibitors for α-syn aggregation, temsamani et al. studied three wine stilbenes, piceatannol ( ) , ampelopsin a ( ) and isoheopeaphenol ( ) . although all three stilbenes inhibited fibrillation, only compound showed notable protection of cells treated with α-syn aggregates [ ] . an explanation for this may be that compound is also able to disaggregate preformed fibrils, and its relatively small size could enable compound to easily penetrate the cell membrane to exert its protective effects. other phenolic compounds. using confocal single-particle fluorescence techniques, caruana et al. investigated the effects of different polyphenolic compounds. in addition to the abovementioned theaflavins and compound , they also studied flavones (apigenin, baicalein and scutellarein), flavonols (myricetin and quercetin), phenolic acids (rosmarinic acid and tannic acid), a stilbene (resveratrol) and others (ginkgolide b, nordihydroguaiaretic acid). the most potent compounds, baicalein ( ) , scutellarein ( ) , myricetin ( ) , compound , nordihydroguaiaretic acid ( ) and black tea extract (mainly theaflavins), were selected because they exhibited significant inhibition and disaggregation of α-syn at low concentrations (ic < μm). the structure activity relationship (sar) study suggested that aggregation inhibitors should contain aromatic rings for interactions with monomeric and oligomeric α-syn and adjacent hydroxyl groups on the same ring [ ] . moreover, the authors proposed that the selected compound would be promising for tests in pd animal models. in addition to compound , compound has also been studied in vivo and should be considered a potential drug candidate for clinical trials. in mpp + -treated sd rats, compound reduced the increase of α-syn aggregates and protected the nigrostriatal dopaminergic system in the brain [ ] . similar effects from compound were observed in the rotenone mouse model, where α-syn oligomers greatly decreased the striatal neurotransmitters, including dopamine, and rotenone-associated behavioral dysfunction was improved [ ] . as a main metabolite of green tea polyphenols, protocatechuic acid ( ) is able to inhibit α-syn aggregation, disaggregate preformed α-syn fibrils and protect pc cells from toxicity caused by α-syn aggregates [ ] . however, this phenolic acid can also exert similar effects on aβ. oleuropein aglycone ( ) , which is commonly found in olive oil, was found to protect sh-sy y cell viability via stabilization of α-syn in the monomeric state, directing α-syn to form nontoxic aggregates and obstructing the binding of α-syn to cell membranes [ ] . another α-syn aggregation inhibitor discovered from olives is hydroxytyrosol ( ) . similar to compound , compound can also inhibit α-syn aggregation and destabilize preformed α-syn fibrils. notably, compound at a concentration of μm almost completely reversed the toxicity caused by α-syn aggregates [ ] while compound only rescued the cell viability to approximately % at the same concentration [ ] . the discovery of active anti-amyloidogenic compounds from olive indicates another good dietary source that may help to prevent neurodegenerative diseases, in addition to green tea. glucosylation of common organic compounds may help to increase their bioavailability [ ] . curcumin-glucoside ( ), which was synthesized by replacing the two hydroxyl groups on the aromatic rings with glucose moieties, showed similar effects to inhibit α-syn oligomerization as well as to destabilize preformed fibrils compared with compound but was believed to be more potent. importantly, the introduction of glucose increased the solubility [ ] . there are also natural glycosides that have been reported to be anti-amyloidogenic agents. one common group is ginsenosides, the main biologically active extract from ginseng. rb ( ) is one of the most studied ginsenosides and can inhibit oligomerization and fibrillation as well as disaggregate preformed fibrils. administration of compound in cells can significantly attenuate the toxicity induced by α-syn aggregates, thus making it worthwhile for compound to be further tested in animal models [ ] . another glucoside, liquiritin ( ), comes from glycyrrhizauralensis, a common traditional chinese medicine for the treatment of pd. in vitro studies have demonstrated that compound can inhibit both oligomerization and fibrillation of α-syn. in a transgenic c. elegans expressing human α-syn, compound significantly inhibited α-syn aggregation and extended the life span of the treated worms [ ] . it has been reported that several vitamins k, unlike many promiscuous inhibitors that bind to α-syn via nonspecific hydrophobic interactions, can inhibit α-syn fibrillation and disassemble preformed fibrils by specifically binding to the nterminal region of α-syn. these vitamins (fig. , phylloquinone, , menaquinone, and menadione, ) are actually derivatives of , -naphthoquinone ( , ) , indicating a potential role for the scaffold of compound in the design of novel specific inhibitors for α-syn [ ] . interactions of these vitamins k with other amyloid proteins have yet to be explored. tanshinone i ( ) and tanshinone ii a ( ) are the main active components in the traditional chinese medicine danshen. similar to egcg, these two phenanthrenequinones have been demonstrated to reduce the formation of α-syn oligomers and fibrils as well as destabilize the preformed fibrils. the benefits of these two molecules were further confirmed in a dye leakage assay where they managed to prevent the membrane damage caused by α-syn aggregates. the good performance in vitro paved the way for later in vivo assays conducted in a c. elegans model expressing α-syn. compounds and both significantly prevented α-syn aggregation and extended the life span of treated c. elegans [ ] . since other neuroprotective effects of these compounds have already been studied in different rodent models, compounds and are expected to target α-syn aggregation in pd rodent models. hybrid molecules in light of the complex pathogenesis of pd, designing molecules that are capable of targeting different factors related with the disease is an attractive approach. compounds ( ) and ( ) , which are -arylcoumarin-tetracyclic tacrine derivatives, were synthesized and selected as promising leads for the further development of potential pd therapeutics. consistent with the design strategy, these two compounds exhibited multitargeted properties, including inhibition of α-syn aggregation, antioxidation and enhancement of the content of dopamine [ ] . the concept of designing hybrid compounds for multitargeted functions may contribute to discovering novel therapeutics for future pd treatment. it has been found that lipid vesicles can assist the nucleation of monomeric α-syn, which is a key step in α-syn aggregation [ ] . the potential effects of the aminosterol compound squalamine ( ) on α-syn aggregation was studied as this compound is able to translocate proteins from the cell membrane to the cytoplasm [ ] . in an α-syn aggregation system containing lipid vesicles, compound intervened in the interaction between α-syn and the surface of the vesicles, thereby inhibiting α-syn aggregation [ ] . this mechanism of compound also counteracted the toxicity of oligomeric α-syn in sh-sy y cells, reduced α-syn aggregation and alleviated the immobility caused by α-syn aggregates in c. elegans overexpressing α-syn [ ] . later, the same group studied another structurally similar compound, trodusquemine ( ) . the side chain of compound is spermine rather than spermidine as in compound . the introduction of additional positive charges was attributed to the increased ability of compound to displace α-syn from both lipid vesicles and preformed α-syn fibrils, thus making this aminosterol able to inhibit not only the nucleation of α-syn monomers but also fibrilinduced aggregation [ ] . like compound , compound also protected sh-sy y cells from the toxicity of oligomeric α-syn and improved the fitness of c. elegans overexpressing α-syn [ ] . future investigations of these two aminosterols in rodent pd models are anticipated. pyrimido pyrazine targeting the interaction between α-syn and membranes has become an important therapeutic strategy for synucleinopathies [ ] . while the aforementioned aminosterols are molecules isolated from nature, neuropore therapies, inc. along with its collaborators developed a promising compound, npt - a ( ), a de novo synthesized molecule with a pyrimido pyrazine scaffold. compound was designed to target the − domain of α-syn that is believed to mediate the dimerization of α-syn on membranes [ ] . by inhibiting the interaction between α-synuclein imaging probes and aggregation inhibitors mm xu et al. α-syn and lipid membranes, compound reduced the formation of toxic α-syn oligomers in primary rat neurons. the long-term effects of compound in a wild-type α-syn transgenic mouse model improved the performance of treated mice in motor behavioral assessments [ ] . in the brains of these treated mice, the accumulation of α-syn was significantly reduced in different regions (neocortex, hippocampus, and striatum). a reduction in α-syn was also seen in the substantia nigra but this result was not significant. sodium dodecyl sulfate polyacrylamide gel electrophoresis (sds-page) demonstrated that compound decreased the amount of α-syn dimers and oligomers, a phenomenon that was also seen in an oligomer-prone e k α-syn transgenic mouse model [ ] . pathologically, neuronal death, synapto-dendritic damage and astrogliosis were notably improved, indicating an association between inhibition of α-syn accumulation, recovered neurodegeneration, and ameliorated motor function [ ] . the short-term effects of compound were evaluated in real time in α-syn-gfp transgenic mice. the reduction of the α-syn-gfp signal at synapses could be seen between and min after administration, indicating the efficiency of compound to inhibit α-syn accumulation at synaptic terminals [ ] . compound suffered from poor oral bioavailability and poor brain penetration; therefore, this compound was not advanced further for clinical studies [ ] . based on the structure of compound , a new compound, npt - ( ) , with improved pharmacokinetic properties, was introduced. compound maintained similar efficacies compared to compound , including a decrease in α-syn accumulation, amelioration of neurodegenerative pathology and improvements in behavioral performance [ ] . according to the authoritative database of clinical trials (clinicaltrials.gov), the phase study of compound in healthy subjects was successfully completed in . a phase b clinical study in both healthy subjects and pd patients is expected to be conducted in europe. without a doubt, compound is the most promising small molecule drug candidate targeting α-syn aggregation. there are still many other molecules that have been reported to be inhibitors of α-syn aggregation at the molecular level, such as aldehyde -hydroxy- -nonenal (hne, ) [ ] and scyllo-inositol ( ) [ ] . compound has perhaps the simplest structures among all the reported inhibitors since it has no aromatic ring and the molecular mass is only da. compound is already well known for its efficacy to inhibit aβ aggregation in vitro and in vivo. in cell models, compounds such as dieckol ( ) [ ] , melatonin ( ) [ ] , selegiline ( ) [ ] , and synuclean-d ( ) [ ] can exert neuroprotection against toxicity induced by α-syn aggregates. the discovery of compound resulted from drug screening based on the tht assay [ ] . the ameliorative effects of compound were also observed in pd c. elegans models. treatment with compound remarkably reduced the formation of α-syn aggregates and protected dopaminergic cells from death in c. elegans, attributing to the improved motility [ ] . the following three molecules are promising compounds to be further developed into useful pd disease-modifying drugs for clinical applications. the first one, clr ( ), also termed molecular tweezer, was first studied systematically by prabhudesai et al. in vitro assays including tht assays and tem demonstrated that compound can inhibit α-syn aggregation as well as disassemble preformed fibrils. compound also exhibited significant protection in both hek cells expressing α-syn endogenously and pc cells treated with aggregated α-syn. excitingly, compound managed to reduce apoptosis induced by α-syn aggregation in zebra fish embryos expressing α-syn, which contributed to increased survival of the treated embryos [ ] . both intracerebroventricular and subcutaneous administration of compound to mice overexpressing α-syn can notably alleviate the motor deficits caused by α-syn pathology and is accompanied by a decrease in the soluble α-syn fraction. the long-term efficacy of compound could be explained by its stable kinetics in vivo [ ] . modifications of this molecule to increase its ability to penetrate the bbb would be required before moving to human trials. anle b ( ) was discovered by a high-throughput screening system against the aggregation of the prion protein. interestingly, compound is also able to reduce the formation of α-syn oligomers. administration of compound in the rotenone mouse model successfully ameliorated motor dysfunction. in another transgenic mouse model expressing human a p mutated α-syn, compound was shown to improve motor performance, prevent the spread of deposited α-syn in the brain and reduce the level of α-syn oligomers [ ] . since compound possesses ideal pharmacokinetic properties, it would not take long for it to be tested clinically to treat pd and prion diseases. the identification of fasudil ( ) as a potent inhibitor of α-syn aggregation is quite exciting, as it is already an approved drug for cerebral vasospasm. therefore, it would not be difficult for compound to be used clinically in the future for treatment of pd given that this molecule can effectively inhibit α-syn aggregation via specific binding to the c-terminal region and more importantly, treatment with compound can improve motor performance and recognition memory in α-syn a t mutated mice [ ] . lewy bodies were first identified over years ago and their main component, α-syn, though seemingly discovered much later, has been investigated for over years. it is believed that the aggregation process of α-syn plays a central role in pd pathogenesis and as a result, the past decade has seen a large number of studies focusing on α-syn aggregation and its role as a biomarker. a variety of small molecule probes and inhibitors of α-syn have been developed over the past decade. some of these inhibitors have been tested in vivo and therefore are promising candidates for clinical trials. many of the discovered molecules are also able to affect the aggregation of other amyloid proteins, indicating their roles as nonspecific amyloid inhibitors. the reason for this may be explained by discussing the common techniques for the discovery of molecules capable of interacting with α-syn. similar to other biophysical techniques, tht assays are not able to sensitively detect and individually characterize unique protein subspecies with which a small molecule ligand interacts [ ] . a tht assay can also be compromised if the small molecule can competitively bind to the dye-binding site on the protein or quench the emission of fluorescence via interaction with the dye [ ] . the advantages of mass spectrometry (ms) over other screening assays include the rapid discovery of candidate inhibitors and, more importantly, the ability to identify binding modes and the particular protein conformations responsible for the interactions with the small molecules [ , , ] . based on previous studies, we have recently established an automated screening system for screening scaffolds that can bind to monomeric α-syn. by screening over pure compounds, we identified a new αsyn aggregation inhibitor ( ) with a pyrazolo [ , -a] pyrimidine- -carboxylic acid scaffold. this molecule exhibited similar effects in the tht assay and circular dichroism (cd) and tem experiments with the positive controls egcg and hemin. coincubation of α-syn with compound protected sh-sy y cells from the toxicity of α-syn aggregates. ms techniques also allow the electron capture dissociation (ecd) fragmentation of the protein−ligand complexes, which can offer information about the binding regions of the ligands [ , ] . compound displayed a more specific binding region on the primary sequence of monomeric α-syn and a clear binding preference for low-charge states, suggesting its higher selectivity for monomeric α-syn than egcg and hemin [ ] . in an endeavor to discover specific α-syn aggregation inhibitors, research has started to focus on developing molecules that target specific regions of monomeric α-syn, such as vitamins ( , and ) [ ] and the two de novo synthesized pyrimido pyrazine derivatives ( and ) [ , ] . the ability to detect the interactions between small molecules and monomeric α-syn α-synuclein imaging probes and aggregation inhibitors mm xu et al. makes ms a powerful technique to discover specific α-syn binding compounds and identify the specific regions to which the compounds bind. we also propose that the α-syn binding scaffolds screened by ms have the potential to be further developed into both fluorescent and pet imaging probes. this means that a single molecule may have both therapeutic and diagnostic functions. these types of theranostic reagents have already been investigated for cancer [ ] and ad [ ] and we believe that developing molecules with theranostic functions for pd will become a hot topic in the near future. parkinson's disease: clinical features and diagnosis non-motor symptoms of parkinson's disease: diagnosis and management priorities in parkinson's disease research the many faces of alpha-synuclein: from structure and toxicity to therapeutic target midbrain dopaminergic cell loss in parkinson's disease: computer visualization paralysis agitans. i. pathologische anatomie. handb der neurologie contribution à l'étude de l'anatomie pathologique du locus niger de soemmering avec quelques déductions relatives à la pathogénie des troubles du tonus musculaire et de la maladie de parkinson accuracy of clinical diagnosis of idiopathic parkinson's disease: a clinico-pathological study of cases diagnostic criteria for parkinson disease mds clinical diagnostic criteria for parkinson's disease improved accuracy of clinical diagnosis of lewy body parkinson's disease accuracy of clinical diagnosis of parkinson disease: a systematic review and meta-analysis separating parkinson's disease from normality: discriminant function analysis of fluorodopa f positron emission tomography data changes in substantia nigra and locus coeruleus in patients with early-stage parkinson's disease using neuromelanin-sensitive mr imaging neuroimaging in parkinson disease: from research setting to clinical practice the prodromal phase of sporadic parkinson's disease: does it exist and if so how long is it? a timeline for parkinson's disease. parkinsonism relat disord invited article: nervous system pathology in sporadic parkinson disease a not entirely benign procedure: progression of parkinson's disease staging of brain pathology related to sporadic parkinson's disease van der schyf cj. why should we use multifunctional neuroprotective and neurorestorative drugs for parkinson's disease? multimodal drugs and their future for alzheimer's and parkinson's disease rational drug discovery design approaches for treating parkinson's disease molecular cloning of cdna encoding an unrecognized component of amyloid in alzheimer disease the synucleins: a family of proteins involved in synaptic function, plasticity, neurodegeneration and disease the role of the c-terminus of human αsynuclein: intra-disulfide bonds between the c-terminus and other regions stabilize non-fibrillar monomeric isomers in vivo cross-linking reveals principally oligomeric forms of α-synuclein and β-synuclein in neurons and nonneural cells purification of α-synuclein from human brain reveals an instability of endogenous multimers as the protein approaches purity α-synuclein occurs physiologically as a helically folded tetramer that resists aggregation targeting αsynuclein for treatment of parkinson's disease: mechanistic and therapeutic considerations mice lacking α-synuclein display functional deficits in the nigrostriatal dopamine system synaptic vesicle depletion correlates with attenuated synaptic responses to prolonged repetitive stimulation in mice lacking α-synuclein the precursor protein of non-aβ component of alzheimer's disease amyloid is a presynaptic protein of the central nervous system delayed localization of synelfin (synuclein, nacp) to presynaptic terminals in cultured rat hippocampal neurons subcellular localization of wild-type and parkinson's disease-associated mutant α-synuclein in human and transgenic mouse brain semi-quantitative analysis of α-synuclein in subcellular pools of rat brain neurons: an immunogold electron microscopic study using a c-terminal specific monoclonal antibody alpha-synuclein is localized in a subpopulation of rat brain synaptic vesicles α-synuclein promotes snare-complex assembly in vivo and in vitro α-synuclein negatively regulates protein kinase cδ expression to suppress apoptosis in dopaminergic neurons by reducing p histone acetyltransferase activity α-synuclein can function as an antioxidant preventing oxidation of unsaturated lipid in vesicles α-synuclein activation of protein phosphatase a reduces tyrosine hydroxylase phosphorylation in dopaminergic cells alpha-synuclein overexpression increases phospho-protein phosphatase a levels via formation of calmodulin/ src complex mutation in the α-synuclein gene identified in families with parkinson's disease synuclein imaging probes and aggregation inhibitors mm alasopro mutation in the gene encoding α-synuclein in parkinson's disease the new mutation, e k, of α-synuclein causes parkinson and lewy body dementia alpha-synuclein p. h q, a novel pathogenic mutation for parkinson's disease g d αsynuclein mutation causes a novel parkinsonian-pyramidal syndrome a novel α-synuclein missense mutation in parkinson disease synuclein locus triplication causes parkinson's disease α-synuclein locus duplication as a cause of familial parkinson's disease australian data and meta-analysis lend support for alpha-synuclein (nacp-rep ) as a risk factor for parkinson's disease collaborative analysis of α-synuclein gene promoter variability and parkinson disease a meta-analysis of genome-wide association studies identifies new parkinson's disease risk loci α-synuclein: normal function and role in neurodegenerative diseases molecular mechanism of thioflavin-t binding to amyloid fibrils nmr of α-synuclein-polyamine complexes elucidates the mechanism and kinetics of induced aggregation cellular polyamines promote the aggregation of α-synuclein α-synuclein fibrillogenesis is nucleation-dependent implications for the pathogenesis of parkinson′ s disease different species of α-synuclein oligomers induce calcium influx and seeding pre-fibrillar α-synuclein variants with impaired β-structure increase neurotoxicity in parkinson's disease models in vivo demonstration that α-synuclein oligomers are toxic direct observation of the interconversion of normal and toxic forms of α-synuclein large α-synuclein oligomers inhibit neuronal snare-mediated vesicle docking lewy-body formation is an aggresome-related process: a hypothesis aggresomes formed by α-synuclein and synphilin- are cytoprotective stable α-synuclein oligomers strongly inhibit chaperone activity of the hsp system by weak interactions with j-domain co-chaperones proteasomal inhibition by α-synuclein filaments and oligomers endoplasmic reticulum stress is important for the manifestations of α-synucleinopathy in vivo a pathologic cascade leading to synaptic dysfunction in α-synuclein-induced neurodegeneration α-synuclein promotes mitochondrial deficit and oxidative stress the role of α-synuclein assembly and metabolism in the pathogenesis of lewy body disease demonstration of a role for α-synuclein as a functional microtubule-associated protein ampareceptor-mediated excitatory synaptic transmission is enhanced by ironinduced α-synuclein oligomers extracellular alpha-synuclein oligomers modulate synaptic transmission and impair ltp via nmda-receptor activation seeding induced by αsynuclein oligomers provides evidence for spreading of α-synuclein pathology direct transfer of αsynuclein from neuron to astroglia causes inflammatory responses in synucleinopathies spreading of pathology in neurodegenerative diseases: a focus on human studies α-synuclein imaging: a critical need for parkinson's disease research slowing of neurodegeneration in parkinson's disease and huntington's disease: future therapeutic perspectives past and recent progress of molecular imaging probes for β-amyloid plaques in the brain in vitro high affinity α-synuclein binding sites for the amyloid imaging agent pib are not matched by binding to lewy bodies in postmortem human brain in vitro characterization of pittsburgh compound-b binding to lewy bodies in vitro characterisation of bf binding to alpha-synuclein/lewy bodies binding of the pet radiotracer [ f] bf does not reflect the presence of alpha-synuclein aggregates in transgenic mice synthesis and in vitro evaluation of alpha-synuclein ligands binding of the radioligand sil to alpha-synuclein fibrils in parkinson disease brain tissue establishes feasibility and screening approaches for developing a parkinson disease imaging agent radiosynthesis and in vivo evaluation of two pet radioligands for imaging alpha-synuclein design, synthesis, and characterization of -(benzylidene)indolin- -one derivatives as ligands for alphasynuclein fibrils structure activity relationships of radioiodinated diphenyl derivatives synthesis and biological evaluation of novel radioiodinated benzimidazole derivatives for imaging alphasynuclein aggregates positron emission tomography radioligands for in vivo imaging of aβ plaques tau pet imaging in alzheimer's disease current status of the development of pet radiotracers for imaging alpha synuclein aggregates in lewy bodies and lewy neurites advances in development of fluorescent probes for detecting amyloid-β aggregates fluorescent n-arylaminonaphthalene sulfonate probes for amyloid aggregation of alpha-synuclein synuclein imaging probes and aggregation inhibitors mm specific fluorescent detection of fibrillar alpha-synuclein using mono-and trimethine cyanine dyes real-time analysis of amyloid fibril formation of alpha-synuclein using a fibrillation-state-specific fluorescent probe of jc- coumarin and , -diphenyl- , , -hexatriene (dph) as fluorescent probes to monitor protein aggregation tri-and pentamethine cyanine dyes for fluorescent detection of alpha-synuclein oligomeric aggregates detection of oligomers and fibrils of α-synuclein by aiegen with strong fluorescence anle b and related compounds are aggregation specific fluorescence markers and reveal high affinity binding to alpha-synuclein aggregates novel benzothiazole derivatives as fluorescent probes for detection of beta-amyloid and alphasynuclein aggregates rifampicin inhibits alpha-synuclein fibrillation and disaggregates fibrils rifampicin protects pc cells against mpp + -induced apoptosis and inhibits the expression of an α-synuclein multimer antioxidant compounds have potent anti-fibrillogenic and fibril-destabilizing effects for alpha-synuclein fibrils in vitro inhibition of alpha-synuclein fibrillization by dopamine analogs via reaction with the amino groups of alphasynuclein. implication for dopaminergic neurodegeneration inhibition of α-synuclein fibrillization by dopamine is mediated by interactions with five c-terminal residues and with e in the nac region modulation of alpha-synuclein aggregation by dopamine analogs small molecule inhibitors of alpha-synuclein filament assembly on the mechanism of nonspecific inhibitors of protein aggregation: dissecting the interactions of alpha-synuclein with congo red and lacmoid structural and mechanistic basis behind the inhibitory interaction of pcts on alpha-synuclein amyloid fibril formation phthalocyanines as molecular scaffolds to block disease-associated protein aggregation a routinely used protein staining dye acts as an inhibitor of wild type and mutant alpha-synuclein aggregation and modulator of neurotoxicity hemin and related porphyrins inhibit β-amyloid aggregation hemin as a generic and potent protein misfolding inhibitor ion mobility-mass spectrometry-based screening for inhibition of alpha-synuclein aggregation screening and classifying small-molecule inhibitors of amyloid formation using ion mobility spectrometry-mass spectrometry curcumin inhibits aggregation of alpha-synuclein curcumin modulates alpha-synuclein aggregation and toxicity curcumin pyrazole and its derivative (n-( -nitrophenylpyrazole) curcumin inhibit aggregation, disrupt fibrils and modulate toxicity of wild type and mutant alpha-synuclein effect of curcumin analogs on α-synuclein aggregation and cytotoxicity egcg redirects amyloidogenic polypeptides into unstructured, off-pathway oligomers opposite structural effects of epigallocatechin- -gallate and dopamine binding to alpha-synuclein egcg remodels mature alpha-synuclein and amyloid-beta fibrils and reduces cellular toxicity how epigallocatechin gallate can inhibit alpha-synuclein oligomer toxicity in vitro tea polyphenols alleviate motor impairments, dopaminergic neuronal injury, and cerebral α-synuclein aggregation in mptp-intoxicated parkinsonian monkeys black tea theaflavins inhibit formation of toxic amyloid-beta and alpha-synuclein fibrils new stilbene dimers against amyloid fibril formation piceatannol and other wine stilbenes: a pool of inhibitors against alphasynuclein aggregation and cytotoxicity inhibition and disaggregation of alpha-synuclein oligomers by natural polyphenolic compounds baicalein attenuates alpha-synuclein aggregation, inflammasome activation and autophagy in the mpp + -treated nigrostriatal dopaminergic system in vivo baicalein inhibits alphasynuclein oligomer formation and prevents progression of alpha-synuclein accumulation in a rotenone mouse model of parkinson's disease protocatechuic acid: inhibition of fibril formation, destabilization of preformed fibrils of amyloid-beta and alpha-synuclein, and neuroprotection oleuropein aglycone stabilizes the monomeric alpha-synuclein and favours the growth of non-toxic aggregates protective effects of hydroxytyrosol against alpha-synuclein toxicity on pc cells and fibril formation bioavailabilities of quercetin- -glucoside and quercetin- ′-glucoside do not differ in humans curcuminglucoside, a novel synthetic derivative of curcumin, inhibits α-synuclein oligomer formation relevance to parkinson's disease ginsenoside rb inhibits fibrillation and toxicity of alpha-synuclein and disaggregates preformed fibrils isoliquiritigenin and liquiritin from glycyrrhiza uralensis inhibit α-synuclein amyloid formation vitamins k interact with n-terminus alpha-synuclein and modulate the protein fibrillization in vitro. exploring the interaction between quinones and alphasynuclein inhibition effects of tanshinone on the aggregation of alpha-synuclein discovery of -arylcoumarin-tetracyclic tacrine hybrids as multifunctional agents against parkinson's disease lipid vesicles trigger α-synuclein aggregation by stimulating primary nucleation synuclein imaging probes and aggregation inhibitors mm membrane phosphatidylserine regulates surface charge and protein localization a natural product inhibits the initiation of alpha-synuclein aggregation and suppresses its toxicity multistep inhibition of alpha-synuclein aggregation and toxicity in vitro and in vivo by trodusquemine modulating membrane binding of α-synuclein as a therapeutic strategy a de novo compound targeting alpha-synuclein improves deficits in models of parkinson's disease the small molecule alpha-synuclein misfolding inhibitor, npt - , produces multiple benefits in an animal model of parkinson's disease effect of -hydroxy- -nonenal modification on alpha-synuclein aggregation alpha-synuclein aggregation, seeding and inhibition by scyllo-inositol dieckol, an edible seaweed polyphenol, retards rotenone-induced neurotoxicity and α-synuclein aggregation in human dopaminergic neuronal cells effect of melatonin on α-synuclein self-assembly and cytotoxicity the anti-parkinsonian drug selegiline delays the nucleation phase of alpha-synuclein aggregation leading to the formation of nontoxic species small molecule inhibits alpha-synuclein aggregation, disrupts amyloid fibrils, and prevents degeneration of dopaminergic neurons high-throughput screening methodology to identify alpha-synuclein aggregation inhibitors a novel "molecular tweezer" inhibitor of alpha-synuclein neurotoxicity in vitro and in vivo a molecular tweezer ameliorates motor deficits in mice overexpressing alpha-synuclein anle b: a novel oligomer modulator for disease-modifying therapy of neurodegenerative diseases such as prion and parkinson's disease fasudil attenuates aggregation of alpha-synuclein in models of parkinson's disease proteins behaving badly: emerging technologies in profiling biopharmaceutical aggregation dye-binding assays for evaluation of the effects of small molecule inhibitors on amyloid (aβ) self-assembly mass spectrometry-based screening for inhibitors of β-amyloid protein aggregation top-down esi-ecd-ft-icr mass spectrometry localizes noncovalent protein-ligand binding sites molecular basis for preventing α-synuclein aggregation by a molecular tweezer identification of a new α-synuclein aggregation inhibitor via mass spectrometry based screening in vivo covalent cross-linking of photon-converted rare-earth nanostructures for tumour localization and theranostics a bifunctional curcumin analogue for two-photon imaging and inhibiting crosslinking of amyloid beta in alzheimer's disease this work was supported by the griffith university international postgraduate research scholarship (guiprs) and the griffith university postgraduate research scholarship (guprs). the authors also want to thank dr. stephen wood and dr. alex sykes for their helpful discussions and proofreading of this paper. author contributions mmx wrote the paper; pr, sr, rjq, hyz and gdm read and revised the paper. competing interests: the authors declare no competing interests. key: cord- -zr xw ph authors: zhang, ying-ying; zhao, zi-de; kong, peng-yun; gao, lin; yu, ya-nan; liu, jun; wang, peng-qian; li, bing; zhang, xiao-xu; yang, li-qiang; wang, zhong title: a comparative pharmacogenomic analysis of three classic tcm prescriptions for coronary heart disease based on molecular network modeling date: - - journal: acta pharmacol sin doi: . /s - - - sha: doc_id: cord_uid: zr xw ph traditional chinese medicine (tcm) has evolved over several thousands of years, which has been shown to be efficacious in the treatment of ischemic heart disease. three classical tcm prescriptions, namely xuefu zhuyu decoction, zhishi xiebai guizhi decoction, and gualou xiebai banxia decoction, have been extensively used in the treatment of coronary heart disease (chd). based on molecular network modeling, we performed a comparative pharmacogenomic analysis to systematically determine the drug-targeting spectrum of the three prescriptions at molecular level. wide-area target molecules of chd were covered, which was a common feature of the three decoctions, demonstrating their therapeutic functions. meanwhile, collective signaling involved metabolic/pro-metabolic pathways, driving and transferring pathways, neuropsychiatric pathways, and exocrine or endocrine pathways. these organized pharmacological disturbance was mainly focused on almost all stages of chd intervention, such as anti-atherosclerosis, lipid metabolism, inflammation, vascular wall function, foam cells formation, platelets aggregation, thrombosis, arrhythmia, and ischemia-reperfusion injury. in addition, heterogeneity analysis of the global pharmacological molecular spectrum revealed that signaling crosstalk, cascade convergence, and key targets were tendentious among the three decoctions. after all, it is unadvisable to rank the findings on targeting advantages of the three decoctions. comparative pharmacological evidence may provide an appropriate decoction scheme for individualized intervention of chd. coronary heart disease (chd) remains a major health burden worldwide, although the mortality rate has been declining over the past few decades [ , ] . optimal pharmacological therapy for ischemic heart disease, includes an angiotensin converting enzyme (ace) inhibitor, beta-blocker, statin, aspirin, etc. however, it is troubling that before giving prescriptions, clinicians need to carefully screen dozens of contraindications for patients such as pregnancy or lactation or liver or kidney dysfunction. adverse reactions and toxicities have limited the clinical use of drugs. recently, systems biology studies have shown that for complex diseases, chinese medicine formulas have the advantages of multitarget interventions and minimal side effects [ , ] . in china, decoctions including xuefu zhuyu (xfzy), zhishi xiebai guizhi (zsxbgz), and gualou xiebai banxia (glxbbx) have contributed to fighting against coronary artery disease for a long time. however, the critical issue must be "how to do?" and "what is it?" for distinguishing the three decoctions at the molecular level, which involves the "different treatments for the same disease" concept of tcm. network simulation of the multitarget action of drugs is a fascinating work because it may provide an opportunity to observe a particular angle of the pharmacological nature of a drug. the development of network pharmacology provides an effective solution for decoding drug combinations, such as triggering divergence/convergence pathways or concentrated/ integrated regulation [ , ] . here, we use the known pharmacogenomic data and molecular network modeling to distinguish the target spectrum of the three formulas from easily confused pharmacodynamics and then determine the principle of formula screening for precise interventions for coronary heart disease. related molecules of xfzy decoction, zsxbgz decoction, and glxbbx decoction all drugs included in the xfzy decoction, zsxbgz decoction, and glxbbx decoction were searched in the compound reference database, chinese academy of sciences (http://www.chemcpd.csdb. cn/) and the national scientific data sharing platform for population and health (http://www.ncmi.cn/) to identify all of the known chemical compositions of each decoction. then, these decoctionrelated compound data were entered into the stitch database (http://stitch.embl.de/) [ ] . species were set to "homo sapiens", and thus, the related molecular information could be obtained. chd-related gene acquisition and network construction in the omim database (https://www.ncbi.nlm.nih.gov/omim/) search interface, "coronary heart disease" was entered to gain access to chd-related genes. the related genes were uploaded to the search tool for recurring instances of neighboring genes (string) database version . (http://string .embl.de/) to characterize the associations between these molecules and to generate molecular interaction networks using homo sapiens as the background. the chd-related network was obtained. comparison of the three decoctions in treating chd the related molecules of the xfzy decoction, zsxbgz decoction, and glxbbx decoction were mapped to the chd-related network, and the similarities and differences of the three decoctions were compared. the functional enrichment analyses of the three decoctions were performed by the database for annotation, visualization, and integrated discovery (david; https://david-d.ncifcrf.gov/) version . (parameters, count = , expression analysis systematic explorer = . , background and species = homo sapiens). the p values of the kyoto encyclopedia of genes and genomes (kegg) pathway were ranked. a fisher exact p value of represents perfect enrichment. a p < . was considered to indicate a statistically significant difference in the annotation categories. chd-related signaling pathways were obtained from the comparative toxicogenomics database (ctd; http://ctdbase.org/). the pathological mechanism of chd based on the published literature (pubmed, https://www.ncbi.nlm. nih.gov/pubmed/), information about the pathological mechanism of chd was extracted to outline the pathology of chd. tables s -s ). after the names of these compounds were searched in the stitch database and only high quality (higher than . ) results were extracted, we obtained related molecules of xfzy decoction, related molecules of zsxbgz decoction, and related molecules of glxbbx decoction (supplementary tables s -s ). there were overlapping molecules among the three decoctions, unique molecules in the xfzy decoction, unique molecules in the zsxbgz decoction, and unique molecules in the glxbbx decoction (fig. a) . related genes and networks of chd based on the omim database, we obtained chd-related genes (supplementary table s ). among them, there were direct interactions between genes (score ≥ . ). when the three decoction-related molecules were compared with chd-related molecules, overlapping molecules were identified (fig. a) . when the genes were compared with chd-related molecules, ( . % of ) overlapping molecules were noted (fig. b) , and the other nonoverlapping molecules were gla, ldlr, hmgcr, cdkn a, tnf, and hgd. therefore, we might focus on the network consisting of the molecules and these direct interactions and then compare the relationship between the decoctions and the network. of the overlapping molecules, were markers or targets for chd (fig. c) , and of the nonoverlapping molecules (ldlr and tnf) were markers or targets for chd. among the overlapping molecules between the decoctions and the network of chd, (nos , pla g , apoa , angptl , apob, cd , apoe, mthfr, apoc , apoa , and gnb ) were markers of chd. when the molecules of the three decoctions were compared with those of chd network, overlapping molecules were found, including only overlapping molecule between the xfzy decoction and the zsxbgz decoction and chd, between the xfzy decoction and the glxbbx decoction and chd, between the xfzy decoction and chd, and between the glxbbx decoction and chd (fig. ) . the top hub nodes with higher degrees in the chd network model were listed as candidate hubs, the details that the three decoction groups targeting hub proteins (apoe, nfkb , esr , serpine , ifng, acta , apob, ttr, apoa , ccl , pon ) were listed in fig. . functional enrichment analysis of the three decoctions after the related molecules were entered into the divid software based on a p < . indicating a statistically significant singling pathway, a total of , , and kegg pathways were identified from the xfzy decoction, glxbbx decoction, and zsxbgz decoction, respectively (supplementary table s -s ). based on the ctd database, we obtained related kegg pathways of chd (supplementary table s ). thirty-six kegg pathways from the three decoctions were related to chd, and kegg pathways overlapped with those of chd (fig. , supplementary table s ). based on these findings, we could compare the similarities and differences in the pharmaceutical mechanisms of the three decoctions. taking the ppar signaling pathway as an example, which was one of the chd-related pathways, all three decoctions had an impact on this pathway (fig. ) . moreover, it could be seen that the three decoctions had both similar and different effects on this signaling pathway. diagram of the pathological mechanism of chd based on the pubmed database, we extracted the triggers and pathological mechanism of chd ( fig. ) , which might help us better understand the mechanism of drug intervention and mutual links among individual drugs. network modeling-based drug-targeting spectrum tcm decoctions are characterized by the inclusion of a large family of effective components. the multicomponent nature of tcm decoctions can often confuse people because of the nontraceability of multitarget disturbances. therefore, the concept of network pharmacology has been presented, emphasizing that the actions of multitarget drugs often surpass the actions of single-target drugs [ , ] . in this study, we analyzed hundreds of compounds and thousands of their well-founded acting molecules from each decoction. in addition, pathology-related molecules of chd were analyzed for tracing the drug-targeting spectrum and deleting redundant disturbance information. networks were constructed with interactive molecules of chd, and the validity of the network model was proved by wayne analysis based on compound coverage of the marker-target of chd ( fig. ) . the drug targets of the xfzy, zsxbgz, and glxbbx decoctions were mapped onto the chd network ( fig. a-c) , and details of the hit range were recognizable. the node color switch of the network model seemed to imply that the three decoction groups were biased with homogeneity and heterogeneity of the targeting spectrum. to improve the resolution of spectrum recognition, we fused the network with colorful nodes for labeling overlapping or isolated targets (fig. d) . then, we came to a few interesting conclusions regarding the three decoctions: ( ) the node hit rate was significant in the three decoction groups; ( ) numerous shared targets were an essential feature; ( ) the targeting spectrum was more similar between zsxbgz and glxbbx; ( ) the drug targets from zsxbgz were absolutely covered by those from xfzy; and ( ) xfzy and glxbbx had their own unique drug targets. regardless of whether the target spectrum was similar or identical, the shared molecular background might drive directed pharmacological attacks to the marker-target network of chd. this similarity was considered to be possible to construct a drug prediction model via the interaction between chemical space and genomic space [ ] . another work that we have discussed was a similar target spectrum of different types of diseases for developing copathology and shared drugs [ ] . the specificity of the target spectrum is the motive driving drug development. this specificity is traceable in terms of network topology and functional annotations. thus, the network molecular spectrum may be helpful for systematically discerning pharmacological characteristics due to the advantages of visualization, perspective, comparability, and flexibility. shared signaling pathways of pharmacogenomics the response of biological cells to drugs depends more on signaling pathways. it is important to decide whether the molecule node in the network could be validated as a member of the pathway to eliminate "path orphans" [ ] . comfortingly, the kegg pathway database has been constantly updating new perspectives on genomes, pathways, diseases, and drugs [ ] . after the three groups of decoction network models were annotated with pathways, shared and several unshared signaling pathways associated with chd were labeled (fig. ) . the evidence-based pathology cascade of chd was mapped [ ] , and then efforts were devoted to find the response site of the pathway triggered by drugs (fig. ) . in this case, the high-frequency shared signaling pathway might be involved in the routine mechanism of reversing the pathology of chd (supplementary table s ), which could be integrated into several main categories, and part of the core signaling pathways are listed below. metabolic/pro-metabolic pathways. in terms of lipid metabolism, an adipocytokine signaling pathway participates in almost all pathological links of chd [ ] and can mediate the crosstalk between adipose tissues, the heart and the vasculature. peroxisome proliferator-activated receptors (ppars) can be widely expressed in adipose tissue, vascular smooth muscle tissue and myocardial tissue. the ppar signaling pathway is also a regulator with the potential to reverse several key pathological mechanisms of chd (fig. ) , such as vascular wall function, foam cell formation, platelet aggregation, and thrombosis [ ] [ ] [ ] [ ] . fat digestion and absorption, fig. the related molecules of the three decoctions and chd. a the overlapping molecules among the three decoctions and chd. b the overlapping molecules among the three decoctions and the network of chd. c the markers and targets of chd in comparison with the overlapping molecules between the chd network (both in and out) and the three decoctions. arachidonic acid metabolism, and ether lipid metabolism are commonly implicated in chd or other secondary-level etiology [ , , ] , such as arteriosclerosis [ ] , the pro-inflammatory process [ ] , and vascular endothelial dysfunction [ ] . driving and transferring pathways. there are members of the chemokine family involved in all stages of cardiovascular disease [ ] . these cytokines may contribute to arteriosclerosis formation in the early stage, arrhythmia, or ischemia-reperfusion injury in the moderate-advanced stage, and dilated cardiomyopathy or heart failure in the late stage. the calcium signaling pathway is a hub induction switch in chd that is involved in the inflammatory process of atherogenesis [ ] and affects coronary endothelial function [ ] , foam cell formation [ ] , vascular tension adjustment [ ] , platelet aggregation [ ] , and vascular smooth muscle cell (vsmc) and fibroblast proliferation [ , ] . the relationship between calcium signaling and arrhythmia has been deeply analyzed [ ] ; this association can be caused by calcium-channelrelated heritable mutations or acquired diseases of the myocardium. the gap junction allows intercellular communication in the cardiovascular system through connexons for the maintenance of the normal cardiac rhythm, endothelial function and the regulation of vascular tone as well as thrombosis control [ , ] . histological research has also shown that atheroma formation might be due to the loss of gap junction proteins, leading to increased permeability of the coronary artery [ ] . neuropsychiatric pathways. in this category, long-term depression is more prospectively associated with chd [ ] . under the premise of confirmed relevance via high-level clinical evidence, two types of mechanisms are hypothesized [ ] : one is the biological mechanism, including cardiac autonomic dysfunction, inflammatory reactions, endothelial dysfunction, and platelet dysfunction; the other is the behavioral mechanism, involving sedentary behavior and poor adherence to medication, diet, exercise, and smoking cessation. exocrine or endocrine pathways. our understanding of the relationship between chd and exocrine/endocrine function is still limited. dietary fat digestion and absorption is initially exposed to oral saliva secretions, which may be the starting factor of abnormal lipid metabolism and obesity [ ] . in addition, saliva components are increasingly deemed biomarkers that are used to evaluate the inflammation level and endothelial function of chd patients [ , ] . mass spectrometry analysis has further identified the salivary peptidome, a novel biomarker for the early diagnosis of cardiovascular disease patients with obstructive sleep apnea [ ] . bile acid is an important component of bile secretion, which reduces cardiovascular risk by lowering cholesterol levels [ ] . compared with patients without coronary artery disease (cad), cad patients have significantly decreased bile acid excretion levels, which may be a risk factor in the process of atherosclerosis [ ] ; in addition, bile acid may modulate endothelial function as vascular endothelial cells express the g protein-coupled bile acid receptor [ ] . other shared core signaling pathways. vascular smooth muscle contraction is the organizational behavior in response to modulation. the normal function of vsmcs is beneficial and protective for making atherosclerotic plaques stable [ ] . this benefit may exist throughout the process, from the initial formation of foam cells to metalloproteinase-mediated plaque degradation. as a paradigm, shared pathogenic crosstalk also links cancer to chd [ ] . common risk factors for both diseases have been explored to highlight potential biological mechanisms, such as atherosclerosis, vascular endothelial function, vascular tension, smooth muscle cell function, inflammation, abnormal lipid metabolism, and obesity. comparable signaling pathways of pharmacogenomics different from shared signaling pathways, comparability emphasizes the individualization of signaling navigation after drug use (fig. ) , just like a drug signature that can help us to make a choice to fit a special indication. . out of the several discriminating signaling pathways of the xfzy decoction molecular network, inositol phosphate metabolism and phosphatidylinositol signaling are important transmembrane signaling pathways that can release the second messenger inositol , , -trisphosphate (ip ). ip /ip receptor signaling has been confirmed to be responsible for the transmission of apoptotic information following crosstalk with the gap junction pathway and calcium signaling pathway [ ] . as a main regulator, the ip r participates in ca + release and cx phosphorylation, thereby affecting gap junction intercellular communication in cardiomyocytes [ ] . cardiovascular actions of insulin contribute to physiological hemodynamic homeostasis [ ] because these phosphatidylinositol -kinase (pi k)-dependent actions can stimulate the release of nitric oxide from the endothelium. blocking the pi k-dependent pathways can enhance the effects of insulin or vascular endothelial growth factor (vegf) to increase the expression of those adhesion molecules, including intercellular adhesion molecule- , vascular cell adhesion molecule (vcam- ), and e-selectin [ ] . these adhesion molecules may be extremely important for the regulation of circulating inflammation and vascular endothelial function. in the heart, insulin can dilate coronary arteries to improve myocardial perfusion and affect lipid metabolism [ , ] . in addition, vsmc contractility would be decreased because of insulin regulating agonist-induced increases in cytosolic calcium through voltage-sensitive calcium channels [ ] , while it can also be increased when the sympathetic nervous system is involved [ ] . the atp-binding cassette (abc) transporters form one of the largest known protein families, whose members abca and abcg can act to transfer cholesterol to mature high-density lipoproteins (hdl) particles [ ] . this feed-forward-like transfer process of mobilization and excretion of cholesterol and other lipids can prevent atherosclerosis, which is termed reverse cholesterol transport. . three unique signaling pathways were enriched from the molecular network of glxbbx decoction. glycerolipid metabolism in genome-wide association studies of chd has been analyzed [ ] , and its metabolizing enzymes are widely expressed in the ventricle and cardiac myocytes, most likely implicating ischemia. lipolysis determines the process of inflammation during the development of atherosclerosis [ ] , even fatal obesity [ ] . glycerolipid metabolism can protect the heart from lipid accumulation under conditions that normalize ppar-α expression [ ] . vitamin digestion and absorption is another pathway that is strongly involved in preventing cardiovascular disease. in particular, vitamin e, a fat-soluble antioxidant vitamin, could be a key to preventing atherosclerosis [ ] , specifically, blocking the inflammatory response, foam cell formation, endothelial cytotoxicity, macrophage motility, and nitric oxide-induced vasodilatation. randomized controlled trials showed that vitamin d supplementation is beneficial for the protection of heart failure [ ] . vitamin d receptors are found in almost all cardiovascular cell types, and vitamin d metabolites in pathways have been shown to regulate a number of pathways related to cardiovascular disease, including inflammation, thrombosis, and vasoconstriction [ ] . the relationship between serum vitamin c levels and cardiovascular outcomes remains unknown. the improvements in lipid profiles, arterial stiffness, and endothelial function seem to support the use of vitamin c supplements to reduce the risk and mortality of cardiovascular disease [ ] , and further evidence needs to be provided. moreover, vitamin b ( ) together with folic acid are involved in the pathogenesis of cardiovascular disease [ ] , mainly depending on higher levels of serum homocysteine. . one overlapping pathway between the molecular network of the xfzy decoction and the glxbbx decoction is glycerophospholipid metabolism. in recent studies, this previously unreported pathway was shown to be associated with atherosclerosis based on the pathway analysis of plasma metabolites at the pathological stage [ ] . renal cell carcinoma is not a routine pathway for chd pathology signaling clusters. it has been reported that local atherosclerosis is more distinct in tumor-positive than in tumornegative renal specimens [ ] . conversely, high-frequency correlations are shown between type diabetes and chd [ ] , such as high levels of postprandial plasma glucose, total cholesterol, low-density lipoproteins (ldls), and triglycerides and low levels of hdls. furthermore, a wideranging prevention strategy is able to improve insulin sensitivity and reduce insulin resistance as well as cardiovascular risk factors [ ] . it now appears that combined with these particular pathways, most pathways regulated by the xfzy decoction exhibit strong and frequent interpathway crosstalk, highlighting robust convergence of the signaling cascade. pathway cooperation in nested or multilayer hierarchical forms can perform biofunctions more integrally [ ] . for example, intercellular communication in the cardiovascular system through the gap junction pathway, which dominates multi-pathway combinations including the gap junction-calcium-glutamatergicinositol phosphate-phosphatidylinositol pathway, inflammation and vascular endothelial function and lipid metabolism regulated by the insulin signaling pathway, plays a leading role in a wide range of interpathway crosstalk involving the type diabetes mellitus-calcium-phosphatidylinositol-vegf-vascular smooth muscle contraction-adipocytokine pathway. similar to the above crosstalk paradigm-like gap junction community that also responded in the glxbbx decoction according to data enrichment, only some of the pathway components involved are reduced. however, the pathway still has one noteworthy trigger-vitamin digestion and absorption in the glxbbx decoction, whose family member metabolism is significantly associated with many pathologies of chd. in addition, compared with the other two decoctions, the glxbbx decoction did not have any individualized pathway response. decoding hub nodes of the network model in the three decoctions the power-law degree distribution is used to characterize conventional biomolecular networks, which indicates a few hubs holding together numerous small nodes [ ] . dynamic information coupling of critical hub radiation-like links makes these nodes occupy the top of the high-level biological behavior, such as being responsible for cognition in the brain function network or immune response and lipid homeostasis in the cardiovascular disease network [ , ] . in this study, the top nodes with higher degrees in the chd network model were listed as candidate hubs (fig. ) , and the details that the decoction groups targeting hub proteins were clear. if the hub node can be in charge of key regulation, it is worth looking forward to the prospect of the three decoctions for the treatment of chd. similar to pathway analysis as a whole, quantitative superiority for targeting hubs in xfzy decoction is outstanding, including of the hubs, i.e., apoe, serpine , ifng, acta , apob, apoa , ccl , and pon . degree calculation results in the addition of a candidate hub node. according to the three colors of arrows in fig. , cotargeted hubs of the three decoctions (apoa , apob, apoe, and serpine ) are also exactly unique in the zsxbgz decoction. here, apolipoprotein family members are very active target hubs in response. apoa , apob, and apoe are the main component proteins of hdl, ldl, and very low-density lipoprotein, respectively. the protection of atherosclerosis by hdl has already gone from hypothesis to validation. the preclinical experiment showing that the overexpression of major hdl protein apoa matched the epidemiological data for confirming the hypothesis [ ] , thereby making hdl a novel therapeutic target for preventing atherosclerosis. the malfunction of tissue cholesterol efflux has been proven in patients with a loss-of-function mutation in apoa by experimental evidence [ ] . anti-inflammatory, antioxidant, antiapoptotic, and no-promoting effects could make apoa have the ability to be atheroprotective [ ] . as better predictors of cardiovascular events, apob, apoa , and the apob/apoa ratio have been reported to be able to improve cardiovascular risk prediction [ ] . because ldl particles can increase the risk of atherothrombotic events via being easily internalized into the subintimal space where they adhere to matrix proteoglycans [ ] , and each particle has only one apob molecule, the total apob value indicates the total number of potentially atherogenic lipoproteins. compared with other risk predictors, high apob levels and a high apob/apoa ratio showed stronger predictive performance in multivariate analyses. apoe, as a hub with the top degrees in this study, was initially described as a major ligand for ldl receptors with a role in cholesterol metabolism and the progression of atherosclerosis and cardiovascular disease. analysis shows that the anti-atherosclerosis effect of apoe is mainly dependent on the clearance of circulating cholesterol [ ] . in parallel, as subtypes of apoe, apoe increases atherogenic lipoprotein levels and apoe increases ldl levels, both enhancing the risk of heart disease together [ ] . however, the association between apoe gene polymorphism and chd is still not completely clear, which may require evidence from a large sample of epidemiological studies. serpine is a major physiological inhibitor of endogenous plasminogen activator, which can inhibit fibrin degradation, promote fibrin deposition in the vessel wall and stimulate smooth muscle cell proliferation. there is sufficient evidence to confirm the causal relationship between high plasma serpine concentrations and various thrombotic disorders [ , ] . endothelial cell synthesis with platelet surface release increases the circulating concentration of serpine . when this incremental mechanism is inhibited, the thrombolytic outcome may be altered [ ] . a recent systematic review has indicated the causal effect of elevated serpine levels on chd risk involving glucose dysfunction [ ] . as one of the two shared target hubs between the xfzy decoction and glxbbx decoction, ccl , also called monocyte chemoattractant protein (mcp- ), is considered to be involved in the pathogenesis of human atherosclerosis and myocardial infarction. atherosclerotic plaque formation can be attributed to the chemotactic activity of mcp- to promote foam cell development [ ] . a myocardial infarction cohort study further revealed that in this context, the significant clinical relevance of mcp- levels were age, cigarette smoking, triglycerides, body mass index, and waist-to-hip ratio [ ] . ccl also has a powerful modulation of fibroblasts and endothelial cell phenotypes and thus may promote myocardial infarction healing [ ] ; otherwise, ccl deficit may lead to weakened tissue remodeling postinfarction at the expense of a prolonged inflammatory phase. pon is another shared hub node between the two decoctions and is a calcium-dependent hdl-associated lactonase. studies have shown that pon can both significantly reduce lipid peroxide generation and provide hdl-associated protection against atherosclerosis [ ] . these anti-atherosclerosis processes mainly depend on reducing macrophage oxidation, foam cell formation and homocysteine levels as well as promoting hdl cholesterol efflux. interestingly, pon and mcp- seem to be coincidentally packaged into a pair of hubs with up-/downstream regulatory relationships. it has been reported that oxidized fatty acids have anti-atherogenic effects only after being hydrated by pon . otherwise, unhydrated oxidized fatty acids with strong pro-inflammatory properties can mediate mcp- production and attract monocytes into the endarterium [ ] . therefore, the expression of pon and mcp- regulated by the two decoctions may be an important way to fight against the early stage of chd pathology. ifng and acta are the exclusive hubs of xfzy decoction. the gene ifng encodes a soluble cytokine that is a member of the type ii interferon class. studies have shown that increased ifng during infection can regulate the production of matrix metalloproteinases and their inhibitors, which are important in plaque stability [ ] . another clinical study revealed that serum ifng levels were significantly positively correlated with tg concentration in chd patients, which could be involved in the development of atherosclerosis of the coronary artery [ ] . acta , the human aortic smooth muscle actin (sma) gene typically expressed in vsmcs, contributes to vascular motility and contraction and may cause a variety of vascular diseases in the event of mutation, such as coronary artery disease and multisystemic smooth muscle dysfunction syndrome [ , ] . in experimental homozygous α-sma knockout mice, decreased vascular contractility and reduced basal blood pressure were notable, suggesting the important role of α-sma in maintaining vascular tone [ ] . the historical view of vsmcs in atherosclerotic plaques presents a double-edge sword. both the harm and the benefit of vsmcs depend on the stage of plaque development [ ] . hence, drug-affected acta expression is crucial in the prevention and treatment of atherosclerosis. conclusion this is a pioneering study based on a global molecular simulation between pharmacology and pathology to identify how three decoctions directionally fight against chd. shared signaling pathways and hub nodes as carriers of basic biological functions make us aware of why these prescriptions have long been active in first-line clinical practice in tcm. dense and organized pharmacological disturbances mainly focused on almost all stages of chd intervention, such as the prevention of atherosclerosis, lipid metabolism, inflammation, vascular wall function, foam cell formation, platelet aggregation, thrombosis, arrhythmia, and ischemia-reperfusion injury. in addition to these aforementioned shared presumptive pharmacological characteristics, highfrequency crosstalk, and strong convergence are the macroscopic features of the signaling pathway triggered by the xfzy decoction. in particular, inositol phosphate metabolism and phosphatidylinositol signaling are considered to be intermediate modulators of multiple signaling pathway linkages, which can be associated with intercellular communication in cardiomyocytes, circulating inflammation, and vascular endothelial function. in addition, for the xfzy decoction, reversing cholesterol transport by abc transporters, plaque stability, and vascular activity improvement are still independent potential preventive targets of cardiovascular circulatory disorders. the glxbbx decoction is concentrated in the regulation of glyceride metabolism and the digestion and absorption of vitamins individually. in particular, multiple vitamins may respond to resistance to pathological processes such as inflammation, foam cell formation, endothelial cell toxicity, and thrombosis. in this study, the glxbbx decoction did not have any independent targeting features; however, a larger scale of molecular mining and analysis is still needed. in summary, the method of comparative pharmacogenomic analysis provides us with an opportunity to gain insights into the large-scale molecular target differential spectrum characteristics of the three decoctions. by using the molecular network model, drug target tracing is clear, and then molecular data can be further discussed in an integrated manner at different dimensions comparatively. the high-frequency and similar target spectral features among the three decoctions in treating chd were highlighted. although it is unadvisable to rank the findings on targeting the advantages of the three decoctions, xfzy seems to have better pharmacological heterogeneity both in signaling pathways and in hub nodes. comparative pharmacological evidence may provide an appropriate decoction scheme for individualized interventions for chd. heart disease and stroke statistics- update: a report from the cardiovascular disease in europe: epidemiological update fangjiomics: in search of effective and safe combination therapies fangjiomics: revealing adaptive omics pharmacological mechanisms of the myriad combination therapies to achieve personalized medicine convergent and divergent pathways decoding hierarchical additive mechanisms in treating cerebral ischemia-reperfusion injury topdown lipidomics reveals ether lipid deficiency in blood plasma of hypertensive patients network pharmacology of cancer: from understanding of complex interactomes to the design of multitarget specific therapeutics from nature the efficiency of multi-target drugs: the network approach might help drug design similarity-based machine learning methods for predicting drug-target interactions: a brief review significant overlapping modules and biological processes between stroke and coronary heart disease pathdip: an annotated resource for known and predicted human gene-pathway associations and pathway enrichment analysis kegg: new perspectives on genomes, pathways, diseases and drugs andreoli and carpenter's cecil essentials of medicine adipocytokines in relation to cardiovascular disease the dual pparalpha/gamma agonist aleglitazar increases the number and function of endothelial progenitor cells: implications for vascular function and atherogenesis ppar-alpha and ppar-gamma activators induce cholesterol removal from human macrophage foam cells through stimulation of the abca pathway nongenomic effects of ppargamma ligands: inhibition of gpvi-stimulated platelet activation endothelial ppar-gamma protects against vascular thrombosis by downregulating p-selectin expression adipocytokines in obesity and metabolic disease adipose tissue arachidonic acid content is associated with the risk of myocardial infarction: a danish case-cohort study the role of adipocytokines in coronary atherosclerosis obesity-induced changes in adipose tissue microenvironment and their impact on cardiovascular disease endothelial dysfunction in obesity: role of inflammation chemokines and heart disease: a network connecting cardiovascular biology to immune and autonomic nervous systems microarray analysis of gene expression in mouse aorta reveals role of the calcium signaling pathway in control of atherosclerosis susceptibility upregulation of sk and ik channels contributes to the enhanced endothelial calcium signaling and the preserved coronary relaxation in obese zucker rats inhibition of orai store-operated calcium channel prevents foam cell formation and atherosclerosis modulatory role of endothelial calcium level in vascular tension of canine depolarized coronary arteries store-operated calcium entry and non-capacitative calcium entry have distinct roles in thrombin-induced calcium signalling in human platelets the intermediate conductance calcium-activated potassium channel kca . regulates vascular smooth muscle cell proliferation via controlling calcium-dependent signaling calcium sensing receptor promotes cardiac fibroblast proliferation and extracellular matrix secretion calcium signaling and cardiac arrhythmias role of connexin in cardiovascular diseases gap junctions and connexin hemichannels in the regulation of haemostasis and thrombosis tight junction proteins and gap junction proteins play important roles in high fat dietary atherosclerosis pathogenesis depression, social support, and long-term risk for coronary heart disease in a -year longitudinal epidemiological study depression and coronary heart disease. nat rev cardiol salivary composition in obese vs normal-weight subjects: towards a role in postprandial lipid metabolism? salivary lysozyme and prevalent coronary heart disease: possible effects of oral health on endothelial dysfunction assessing salivary c-reactive protein: longitudinal associations with systemic inflammation and cardiovascular disease risk in women exposed to intimate partner violence salivary biomarkers indicate obstructive sleep apnea patients with cardiovascular diseases effect of bile acid sequestrants on the risk of cardiovascular events: a mendelian randomization analysis the association of bile acid excretion and atherosclerotic coronary artery disease stimulation of g protein-coupled bile acid receptor enhances vascular endothelial barrier function via activation of protein kinase a and rac emerging regulators of vascular smooth muscle cell function in the development and progression of atherosclerosis shared risk factors in cardiovascular disease and cancer transfer of ip( ) through gap junctions is critical, but not sufficient, for the spread of apoptosis cx phosphorylation on s / and intercellular communication are regulated by ip /ip receptor signaling cardiovascular actions of insulin inhibition of phosphatidylinositol -kinase enhances mitogenic actions of insulin in endothelial cells insulin and myocardial blood flow cardiac dysfunction induced by high-fat diet is associated with altered myocardial insulin signalling in rats insulin attenuates agonistmediated calcium mobilization in cultured rat vascular smooth muscle cells insulin as a vascular and sympathoexcitatory hormone: implications for blood pressure regulation, insulin sensitivity, and cardiovascular morbidity abc transporters, atherosclerosis and inflammation pathway-based genome-wide association analysis of coronary heart disease identifies biologically important gene sets vascular lipases, inflammation and atherosclerosis glycerolipid metabolism and signaling in health and disease caloric restriction in leptin deficiency does not correct myocardial steatosis: failure to normalize ppar{alpha}/pgc {alpha} and thermogenic glycerolipid/fatty acid cycling cardiovascular disease and vitamin d supplementation: trial analysis, systematic review, and meta-analysis vitamin d and cardiovascular disease vitamin c and heart health: a review based on findings from epidemiologic studies homocysteine, folate and vitamin b in patients with coronary heart disease comprehensive plasma metabolomic analyses of atherosclerotic progression reveal alterations in glycerophospholipid and sphingolipid metabolism in apolipoprotein e-deficient mice association between local atherosclerosis and renal cell carcinomas risk factors for coronary heart disease in type ii diabetes mellitus insulin resistance implications for type ii diabetes mellitus and coronary heart disease multi-hierarchical profiling: an emerging and quantitative approach to characterizing diverse biological networks network biology: understanding the cell's functional organization a resilient, low-frequency, small-world human brain functional network with highly connected association cortical hubs the use of network analyses for elucidating mechanisms in cardiovascular disease hdl and cardiovascular disease in vivo tissue cholesterol efflux is reduced in carriers of a mutation in apoa high-density lipoprotein: vascular protective effects, dysfunction, and potential as therapeutic target apolipoprotein b and apolipoprotein a-i: risk indicators of coronary heart disease and targets for lipid-modifying therapy differential uptake of proteoglycan-selected subfractions of low density lipoprotein by human macrophages review: apolipoprotein e (apo e) gene polymorphism and coronary heart disease in asian populations high plasminogen activator inhibitor and tissue plasminogen activator levels in plasma precede a first acute myocardial infarction in both men and women: evidence for the fibrinolytic system as an independent primary risk factor abnormally high circulation levels of tissue plasminogen activator and plasminogen activator inhibitor- in patients with a history of ischemic stroke plasminogen-activator inhibitor type and coronary artery disease causal effect of plasminogen activator inhibitor type on coronary heart disease role of mcp- in cardiovascular disease: molecular mechanisms and clinical implications ccl polymorphisms are associated with serum monocyte chemoattractant protein- levels and myocardial infarction in the framingham heart study ccl /monocyte chemoattractant protein- regulates inflammatory responses critical to healing myocardial infarcts paraoxonase and coronary heart disease paraoxonase- inhibits oxidised ldl-induced mcp- production by endothelial cells interferon-gamma increases the ratio of matrix metalloproteinase- /tissue inhibitor of metalloproteinase- in peripheral monocytes from patients with coronary artery disease serum levels and clinical significance of ifn-gamma and il- in patients with coronary heart disease alpha-smooth muscle actin and acta gene expressions in vasculopathies two patients with the heterozygous r h mutation in acta and complex congenital heart defects expands the cardiac phenotype of multisystemic smooth muscle dysfunction syndrome impaired vascular contractility and blood pressure homeostasis in the smooth muscle alpha-actin null mouse vascular smooth muscle cells in atherosclerosis yyz and zdz wrote the paper and drew the figures; pyk sorted the data and created the tables; lg, xxz, pqw, and bl modified the tables and revised the paper; yny and jl revised the paper; and lqy and zw directed the research and revised the paper. the online version of this article (https://doi.org/ . /s - - - ) contains supplementary material, which is available to authorized users.competing interests: the authors declare no competing interests. key: cord- -qnp w authors: cheng, qiao-qiao; wan, yu-wei; yang, wei-min; tian, meng-hua; wang, yu-chuan; he, hai-yan; zhang, wei-dong; liu, xuan title: gastrodin protects h c cardiomyocytes against oxidative injury by ameliorating imbalanced mitochondrial dynamics and mitochondrial dysfunction date: - - journal: acta pharmacol sin doi: . /s - - -x sha: doc_id: cord_uid: qnp w gastrodin (gas) is the main bioactive component of tianma, a traditional chinese medicine widely used to treat neurological disorders as well as cardio- and cerebrovascular diseases. in the present study, the protective effects of gas on h c cells against ischemia–reperfusion (ir)-like injury were found to be related to decreasing of oxidative stress. furthermore, gas could protect h c cells against oxidative injury induced by h( )o( ). pretreatment of gas at , , and μm for h significantly ameliorated the decrease in cell viability and increase in apoptosis of h c cells treated with μm h( )o( ) for h. furthermore, we showed that h( )o( ) treatment induced fragmentation of mitochondria and significant reduction in networks, footprint, and tubular length of mitochondria; h( )o( ) treatment strongly inhibited mitochondrial respiration; h( )o( ) treatment induced a decrease in the expression of mitochondrial fusion factors mfn and opa , and increase in the expression of mitochondrial fission factor fis . all these alterations in h( )o( )-treated h c cells could be ameliorated by gas pretreatment. moreover, we revealed that gas pretreatment enhanced the nuclear translocation of nrf under h( )o( ) treatment. knockdown of nrf expression abolished the protective effects of gas on h( )o( )-treated h c cells. our results suggest that gas may protect h c cardiomycytes against oxidative injury via increasing the nuclear translocation of nrf , regulating mitochondrial dynamics, and maintaining the structure and functions of mitochondria. introduction gastrodin (gas) (p-hydroxymethylphenyl-b-d-glucopyranoside) is the main bioactive component of rhizoma gastrodiae (the dried rhizome of gastrodia elata blume, also known as tianma), a popular traditional chinese medicine. in shennong's classic of materia medica, the first herbal monograph in chinese history, written during the han dynasty, tianma was described as a "top grade medicine" that can rejuvenate the body, enhance health, and extend life without toxicity, and can be used long term without harm [ ] . although tianma is traditionally used in the treatment of nervous diseases, tianma has been reported to have protective activities against convulsion, oxidation, depression, epilepsy, obesity, asthma, and inflammation, in addition to the effects on analgesia, sedation, learning and memory improvement, and neuroprotection [ ] . tianma is currently used clinically to treat various cardiovascular and cerebrovascular diseases, as well as nervous diseases, including hypertension, headache, convulsion, epilepsy, and coronary heart diseases [ ] [ ] [ ] [ ] . however, the mechanisms of tianma and gas have not been fully clarified. furthermore, most of the previous studies on gas focused on its effects on neurons, and little is known about the effects of gas on cardiomyocytes. previous studies showed that gas ameliorated myocardial ischemia-reperfusion (ir) injury in rats [ ] . gas pretreatment ameliorated myocardial ir injury by decreasing calcium overload [ ] . gas also protected h c cardiomyocytes against injuries induced by serum deprivation [ ] or lps treatment [ ] . in the present study, we examined the protective effects of gas on h c cells that underwent ischemia-like injury (deprivation of oxygen, glucose, and serum for h) or ir-like injury (deprivation of oxygen, glucose, and serum for h, and then culture under normal conditions in complete medium for an additional h). based on the observation of the antioxidant activity of gas in protecting h c cells against ir-like injury, we focused on studying the effects and mechanisms of gas on h c cells that underwent oxidative injury induced by h o treatment. recent studies have suggested that imbalanced mitochondrial dynamics and related mitochondrial dysfunction play important roles in cardiac ir injury [ ] [ ] [ ] [ ] . mitochondria are highly dynamic organelles that undergo coordinated cycles of fission (to generate discrete fragmented mitochondria) and fusion (to form an interconnected elongated phenotype), referred to as "mitochondrial dynamics", to maintain their shape, distribution, and size [ , ] . the balance of mitochondrial fusion and fission determines mitochondrial morphology, and is necessary to support mitochondrial functions. mitochondrial dynamics are coupled to the bioenergetics and signaling functions of mitochondria. therefore, imbalanced mitochondrial dynamics cause mitochondrial dysfunctions such as changes in mitochondrial respiration and the induction of apoptosis [ ] [ ] [ ] . mitochondrial dynamics may be a therapeutic target for treating cardiac diseases [ ] . in the present study, we observed the morphology and functions of mitochondria in h c cells under h o -induced oxidative injury with or without gas pretreatment, and attempted to clarify the mechanisms by which gas protects h c cells against oxidative injury. chemicals and materials gas with a purity > % was purchased from nature standard company (shanghai, china). srb (sulforhodamine b), tca (trichloroacetic acid), oligomycin, fccp ( -[ -[ -(trifluoromethoxy)phenyl]hydrazinylidene]-propanedinitrile), and rotenone were purchased from sigma-aldrich (st. louis, mo, usa). mitotracker green fm and the alexa fluor annexin v/dead cell apoptosis kit were purchased from thermo fisher scientific (waltham, ma, usa). dcfh-da ( ′, ′-dichloro-fluorescin diacetate) and the bicinchoninic acid (bca) protein assay kit were obtained from yeasen (shanghai, china). antimycin a was purchased from abcam biotechnology (cambridge, ma, usa). the reversetranscription kit was obtained from takara (shiga, japan). the faststart essential dna green master for pcr was purchased from roche (basel, switzerland). antibodies, except where specifically noted, were purchased from cell signaling technology (danvers, ma, usa). cell culture and treatments h c cells were obtained from the cell bank of the institute of biochemistry and cell biology (shanghai, china), and cultured in dulbecco's modified eagle's medium (dmem) containing . g/l d-glucose supplemented with % fetal bovine serum, mg/ml streptomycin, and u/ml penicillin, all bought from hyclone (logan, utah, usa), at °c in a humidified incubator with % co . the cells were passaged every or days. for treatments, h c cells were seeded in -well plates at a density of × cells per well. after culturing for h and reaching~ %- % confluence, the cells were pretreated with the indicated concentrations of gas (dissolved in culture medium) for h before induction of ischemialike injury or ir-like injury, or treatment with h o at the indicated concentrations. ischemia-like or ir-like injury was induced in h c cells using methods similar to those in a previous report [ ] . for ischemia-like injury, the cells were deprived of oxygen, glucose, and serum for h; briefly, the culture medium was replaced with serum-and glucose-free medium, and the plates were placed in an incubator with % o , % n , and % co air for h. for ir-like injury, the cells were deprived of oxygen, glucose, and serum for h, and then cultured under normal conditions in complete medium for an additional h. for h o treatment, the cells were treated with the indicated concentrations of h o for different time periods. cell viability assay cell viability was determined using the sulforhodamine b (srb) assay [ ] . briefly, after treatments, the cells were fixed with cold % trichloroacetic acid at °c for min. the plates were washed five times with tap water and then air dried. srb solution ( μl) at . % (w/v) in % acetic acid was added to each well and incubated at room temperature for min. after staining, the plates were washed five times with % acetic acid to remove unbound dye and then air dried. bound dye in the cells was subsequently solubilized with mm trizma base, and the absorbance was read using an automated plate reader at a wavelength of nm. three independent experiments (each with at least triplicate samples) were conducted, and the statistical results are shown. after treatments, the cells were incubated with μm dcfh-da in serum-free and phenol red-free medium for min. then, after two phosphate-buffered saline (pbs) washes, the fluorescence intensity of each well was detected using a cytation cell imaging multi-mode reader (biotek, winooski, vt, usa) at an excitation wavelength of nm and an emission wavelength of nm. three independent experiments (each with triplicate samples) were conducted, and the statistical results are shown. images of the cells were also captured with an inverted fluorescence microscope (leica, wetzlar, germany). cell morphology observation and flow cytometric analysis of apoptosis the morphological changes in h c cells after treatments were observed with an optical microscope (dmil, leica). for flow cytometric analysis, cells (both adherent and detached) were collected, washed with pbs, and stained by using an alexa fluor annexin v/dead cell apoptosis kit (thermo fisher scientific) according to the manufacturer's instructions. then, flow cytometric analysis was conducted using a facscalibur flow cytometer, and data analysis were performed with cellquest software (bd biosciences, sparks glencoe, md, usa). three independent experiments (each with triplicate samples) were conducted, and the results of one representative experiment are shown. mitochondrial morphology observation and network quantification mitochondria in living h c cells were stained with nm mitotracker green fm (invitrogen, carlsbad, ca, usa) for min. after washing twice with pbs and transfer to phenol red-free dmem, mitochondrial morphology was observed using a deltavision omx sr imaging system with super resolution (ge healthcare bio-sciences, pittsburgh, pa, usa). the mitochondrial morphology in individual cells was quantified as described in previous reports [ , ] . three independent experiments were conducted, and the results of one representative experiment are shown. briefly, random fields of cells ( cells per condition) in each group were evaluated. the mitochondrial morphology types included long tubular, short tubular, and fragmented. the percentage of different mitochondrial morphology types in cells was quantified. furthermore, the imagej macro tool mina was used to analyze mitochondrial network morphology. mitochondrial respiration assay a mitochondrial respiration assay in living cells was conducted [ ] using a seahorse xfe extracellular flux analyzer (seahorse bioscience, north billerica, ma, usa). the real-time oxygen consumption rate (ocr), which indicated the mitochondrial respiration potential of the cells, was measured. briefly, h c cells were seeded into xfe cell culture microplates (seahorse bioscience) at a density of - cells/well. after treatments, the culture medium was changed to unbuffered dmem (ph . ) supplemented with mm pyruvate, mm glutamine, and mm d-glucose at h before the assay. after baseline rate measurement, oligomycin ( μm), fccp ( μm), and rotenone ( . μm) combined with antimycin a ( . μm) were injected sequentially through ports in the seahorse flux park cartridges. the ocr was recorded and normalized to the number of cells per well, and then the data were analyzed by using wave desktop software provided by seahorse bioscience. three independent experiments (each with at least triplicate samples) were conducted, and the results of one representative experiment are shown. transfection of sirnas h c cells were transfected with sirna for nrf ( ′-caaaca gaatggacctaaa- ′) or a nontargeting sirna, both purchased from genepharma (shanghai, china) by using lipofectamine (invitrogen) according to the manufacturer's instructions. briefly, h c cells were plated in -or -well plates and incubated until they reached % confluence. lipofectamine and sirna were mixed and incubated for min at room temperature before being added to the cells. the cells were incubated with sirna at a final concentration of nm for h. the expression of nrf in negative control cells (cells treated with nontargeting sirna) and nrf -sirna-treated cells was evaluated using both rt-pcr and western blot analysis. rna extraction and rt-pcr total cellular rna was extracted using rnaiso plus reagent (takara, shiga, japan) dissolved in rnase-free water. the quantification of total rna was conducted by detecting ultraviolet absorption (optical density, / nm) using a nanodrop (thermo scientific, usa). an equal amount of total rna ( ng) was reverse transcribed to cdna using primescript tm rt master mix (takara) according to the manufacturer's instructions. rt-pcr amplifications were performed using the faststart essential dna green master and lightcycler® instrument (roche, basel, switzerland). the thermal profile was °c for min, followed by cycles at °c for s and °c for s. each sample was analyzed in triplicate, and the mean cycle threshold (ct) value was calculated. the relative expression level was calculated using the ΔΔct method. the mrna expression of actin was used as an internal control. primer sequences for rt-pcr analysis are listed in supplementary table s . three independent experiments (each with at least triplicate samples) were conducted, and the statistical results are shown. immunofluorescence staining after treatments, the cells were fixed in % paraformaldehyde for min at room temperature, and then washed three times with pbs. the cells were permeabilized in . % triton x- (sigma-aldrich) for min, and then blocked in % bovine serum albumin for h at room temperature. after an overnight incubation with mouse monoclonal anti-nrf antibody (#ab , : dilution, abcam, cambridge, cambridgeshire, uk) at °c, the cells were washed three times with pbs, and then incubated with secondary antibody (#a , fitc-labeled goat anti-mouse igg, : dilution, beyotime biotechnology) for h at room temperature. afterward, the cells were washed three times with pbs, and stained with dapi for min at room temperature. the cells were then observed using a deltavision omx sr imaging system. three independent experiments were conducted, and the results of one representative experiment are shown. western blotting assay h c cells were harvested and lysed in ripa buffer containing the protease inhibitor phenylmethane-sulfonyl fluoride. the total protein concentrations were determined by using a bca protein assay kit. equal amounts of total proteins ( μg) from each treatment group were separated by % sodium dodecyl sulfate polyacrylamide gel electrophoresis at v for h, and then electrophoretically transferred to polyvinylidene fluoride membranes. the membranes were blocked using tbst ( × tbs and . % tween- ) containing % skim milk for h, and then incubated with rabbit polyclonal anti-nrf (c- ) (#sc , : dilution, santa cruz biotechnology, dallas, tx, usa) or rabbit polyclonal anti-actin (# s, : dilution, cell signaling technology, danvers, ma, usa) overnight at °c. after washing for min in tbst ( × tbs and . % tween- ) three times, the membranes were incubated with secondary polyclonal antibody (goat anti-rabbit hrp-conjugated igg, : dilution, absin, shanghai, china) for h at room temperature. after the membranes were washed again in tbst, the immune complexes were detected by using ecl-plus tm chemiluminescent detection reagent (p a, beyotime biotechnology), and visualized with a molecular imagerr chemidoc tm xrs + system (bio-rad laboratories, hercules, ca, usa). three independent experiments were conducted, and the results of one representative experiment are shown. values are expressed as the mean ± standard deviation. all statistical analyses were performed using graphpad prism . software, and comparisons between two groups were analyzed using two-tailed student's t test. a p value less than . was considered statistically significant. gas protected h c cells against ischemia-like injury, ir-like injury, and h o -induced injury as shown in fig. a , treatment with gas at concentrations ranging from to μm exhibited no significant influence on the viability of h c cells. as shown in supplementary fig s , pretreatment with gas significantly protected h c cells against ischemia-like injury (fig s a) , as well as ir-like injury (fig s b) . intracellular reactive oxygen species (ros)-level measurements indicated that the increase in intracellular ros induced by ir-like injury was significantly ameliorated by gas pretreatment (fig s c) . these results suggest that gas-mediated protection of h c cells against ir-like injury is related to a decrease in oxidative stress. therefore, the present study focused on studying the effects of gas on h c cells that underwent oxidative injury induced by first, the viability of h c cells treated with different concentrations of h o for h (fig. b) was observed. as shown in fig. b , h o at , , , and μm induced~ %, %, %, and % decreases in the viability of h c cells, respectively. to determine the conditions in which cell viability would decrease by~ % or % in less than h, the effect of μm gas treatment for different times ( , , , and h) on cell viability was further examined. as shown in fig. c , treatment with μm h o for h induced an approximately % decrease in cell viability, and thus was used as a condition for h o treatment in the subsequent experiments. as shown in fig. d , pretreatment with gas at , , and μm significantly ameliorated the decrease in viability in h o -treated h c cells. intracellular roslevel measurements indicated that the h o -induced increase in ros was significantly ameliorated by gas pretreatment (fig. e) . representative photographs of the intracellular ros signal in h c cells treated with μm h o for h with or without gas pretreatment (fig. f ) also show that gas pretreatment inhibited the h o -induced increase in intracellular ros. these results suggest that gas protects h c cells against h o -induced oxidative injury. gas attenuated h o -induced apoptosis of h c cells representative photographs of cultured h c cells treated with μm h o with or without gas pretreatment are shown in fig. a . treatment with h o induced considerable cell death in h c cells, while gas pretreatment ameliorated h o -induced cell death (fig. a) . flow cytometry results showed that h o -induced cell death included both early and late apoptosis (fig. b) . the cells that were annexin v-fitc + /pi − were defined as early apoptotic cells, while the cells that were annexin v-fitc + /pi + were defined as late apoptotic cells. as shown in the quantification of the flow cytometry results, gas significantly decreased the percentage of apoptotic cells (fig. c) . these results suggest that gas attenuates h o -induced apoptosis in h c cells. notably, no obvious dose-response effect of gas was observed in either the apoptosis or cell viability analysis (as shown in fig. d ). the reason for this is unclear. it is possible that the protective effects of gas on h c cells were mainly due to decreasing ros levels, and the response to ros elimination might plateau. (fig. a) . quantification of random cells in each group confirmed that gas significantly ameliorated the decrease in the percentage of cells with long tubular mitochondria, and the increase in the percentage of cells with short tubular mitochondria or fragmented mitochondria induced by h o (fig. b) . mitochondrial network morphology characteristic analyses using the imagej macro tool mina are shown in fig. c -g. gas significantly attenuated the h o -induced change in networks (fig. c) , mitochondrial footprint (fig. e) , and mean branch length (fig. f) . these results suggest that h o treatment induces considerable imbalance in mitochondrial dynamics in h c cells, while gas pretreatment partly attenuates the h o -induced change in mitochondrial morphology, and might be helpful in maintaining mitochondrial function. the mitochondrial respiration potency of h c cells treated with h o with or without gas pretreatment was measured. as shown in fig. a , h o treatment induced a considerable decrease in mitochondrial respiration potency. treatment with gas alone exhibited no influence on mitochondrial respiration, while pretreatment with gas partly ameliorated the h o -induced inhibition of mitochondrial respiration in h c cells (fig. a) . analysis of the mitochondrial respiration parameters, such as basal respiration, maximal respiration, and atp production, are shown in fig. b-d , respectively. gas pretreatment significantly ameliorated the h o -induced decrease in basal respiration (fig. b) , maximal respiration (fig. c) , and atp production (fig. d) in h c cells. these results suggest that h o -induced mitochondrial dysfunction in h c cells was partly attenuated by gas. gas induced nuclear translocation of the nrf protein in h c cells as shown in fig. a , the western blotting results showed that h o treatment with or without gas pretreatment did not cause a significant change in the protein level of nrf . the nrf protein only showed a slight increase in cells that were treated with gas. interestingly, as shown in fig. b , the distribution of nrf protein in h c cells exhibited great differences in the different groups of cells. in control cells, nrf protein was almost exclusively observed in the cytoplasm. treatment with h o induced translocation of nrf protein from the cytoplasm into the nucleus, which indicated activation of nrf ; therefore, the yellow signal (merge of the red signal of nrf and the green signal of nuclear dapi staining) quantitative results of the percentage of cells with long tubular mitochondria, short tubular mitochondria, or fragmented mitochondria. c-g quantitative results of mitochondrial network characteristics, including networks (c), individuals (d), mitochondrial footprint (e), mean branch length (f), and median branch length (g). the data presented are the mean ± sd and # p < . vs. the control group, *p < . vs. the h o -induced group increased in these cells. importantly, pretreatment with gas enhanced the nuclear translocation of nrf , and the cells exhibited a strong yellow signal, which suggested that gas induced activation of nrf in h c cells (fig. b) . nrf was involved in the protective effects of gas on h o -treated h c cells to examine whether nrf was involved in the protective effects of gas on h c cells, nrf expression in h c cells was knocked down using sirna transfection, and then the effects of gas on these nrf -sirna-treated cells were compared with the effects on negative control cells (cells treated with nontargeting sirna). as shown in fig. a (rt-pcr results) and fig. b (western blotting results), nrf -sirna transfection successfully decreased both the mrna and protein expression of nrf in h c cells. in negative control cells, h o treatment induced a significant decrease in cell viability, while gas pretreatment significantly ameliorated the h o -induced decrease (fig. c) . in contrast, in nrf -sirna-treated cells, h o treatment also induced a significant decrease in cell viability, but gas pretreatment did not ameliorate the h o induced decrease in cell viability (fig. c) . analysis of intracellular ros levels showed similar results: in nrf -sirna-treated cells, gas pretreatment did not ameliorate the h o -induced increase in ros levels (fig. d) . these results suggest that nrf knockdown attenuated the protective effects of gas against oxidative stress, and that nrf is involved in the mechanisms of gas. knockdown of nrf expression attenuated the effects of gas on h o -induced imbalanced mitochondrial dynamics as shown in fig. a , in negative control cells, h o treatment induced imbalanced mitochondrial dynamics, while gas pretreatment ameliorated the imbalanced mitochondrial dynamics induced by h o . in contrast, in nrf -sirna-treated cells, gas pretreatment did not successfully ameliorate the imbalanced mitochondrial dynamics induced by h o (fig. b) . quantitative analysis of mitochondrial network characteristics showed similar results: in nrf -sirna-treated cells, gas pretreatment did not ameliorate the decrease in mitochondrial footprint, mean branch length, or median branch length induced by h o (fig. c) . these results suggest that nrf is involved in the effects of gas in maintaining the mitochondrial morphology of h c cells that underwent h o treatment. gas inhibited h o -induced changes in expression levels of genes related to mitochondrial fusion and fission as shown in fig. a , in negative control cells, h o treatment induced significant changes in the expression levels of genes related to mitochondrial fusion (mfn , mfn , and opa ) and mitochondrial fission (fis and drp ). gas pretreatment significantly ameliorated the changes in the expression levels of mfn , opa , and fis , but did not influence those of mfn and drp (fig. a) . these results suggest that mfn , opa , and fis play roles in the protective effects of gas on the mitochondria of h c cells. furthermore, as shown in fig. b , nrf knockdown attenuated the regulatory effects of gas on the expression levels of mfn , opa , and fis . these results confirmed the involvement of nrf in the mechanisms of gas. to determine whether nrf knockdown directly affects the expression levels of these genes related to mitochondrial fusion and fission, the expression levels of the genes in negative control cells and nrf -sirna-treated cells were also compared. as shown in fig. c , nrf knockdown significantly decreased the expression level of mfn but not that of opa or fis . these results suggest that nrf has direct regulatory effects on mitochondrial fusion. knockdown of nrf expression attenuated the effects of gas on h o -induced mitochondrial dysfunction as shown in fig. a , in negative control cells, h o treatment induced a considerable decrease in mitochondrial respiration potency, while gas pretreatment partly ameliorates the h o induced mitochondrial dysfunction. treatment with nrf -sirna induced a slight decrease in mitochondrial respiration potency. importantly, in nrf -sirna-treated cells, h o treatment also induced a considerable decrease in mitochondrial respiration potency, but gas pretreatment did not ameliorate the h o induced mitochondrial dysfunction (fig. a) . analysis of the parameters of mitochondrial respiration, such as basal respiration, maximal respiration, and atp production, are shown in fig. b-d, fig. gas attenuated mitochondrial dysfunction in h c cells induced by h o . a mitochondrial respiration potency of control cells, μm gas-treated cells, and μm h o -treated cells, pretreated with μm gas, was measured using a seahorse metabolic analyzer. b results of quantification analysis of basal respiration. c results of quantification analysis of maximal respiration. d results of quantification analysis of atp production. the data presented are the mean ± sd and # p < . vs. the control group, *p < . vs. the h o -induced group respectively. in nrf -sirna-treated cells, gas pretreatment did not ameliorate the decrease in basal respiration (fig. b) , maximal respiration (fig. c) , or atp production (fig. d) induced by h o . these results suggest that nrf is involved in the effects of gas in maintaining the mitochondrial functions of h c cells that underwent h o treatment. in the present study, gas protected h c cells against ischemialike and ir-like injury. the results were consistent with previous reports about the cardioprotective effects of gas [ ] [ ] [ ] ] . importantly, the protective effects of gas against ir-like injury were found to be related to its ros-decreasing activity, and gas directly protected h c cells against oxidative injury (h o treatment). the antioxidant effects of gas have also been observed in other types of cells, such as neurons [ , ] , astrocytes [ ] , osteoblasts [ ] , bone marrow mesenchymal stem cells, and macrophages [ ] . these results suggest that antioxidant activity plays important roles in the various effects of gas. mitochondria are especially important in cardiomyocytes, since the heart is an organ with high bioenergetic demands, and more than % of atp is supplied by mitochondria [ ] . interestingly, mitochondria are highly dynamic organelles with constant movement and morphological changes. mitochondrial dynamics are involved in fundamental biological processes such as cell metabolism, cell survival, and death [ ] . our results in observing the morphology of mitochondria by using super resolution microscopy showed that h o treatment induced fragmentation of mitochondria, and a significant decrease in networks, footprints, and tubular lengths of mitochondria in h c cells. disruption of mitochondrial dynamics has been implicated in various human diseases, including developmental defects, neurodegenerative diseases, metabolic diseases, and cardiovascular diseases [ ] . previous reports have shown that mitochondrial morphological defects, such as increased mitochondrial fission and decreased fusion, are important events in cardiac ischemia and reperfusion [ ] [ ] [ ] [ ] . for example, fragmented mitochondria and a decrease in mitochondrial fusion were detected in cardiomyocytes from both heart failure patients and cardiac ischemia animal models [ ] . the results of the present study clearly showed imbalanced mitochondrial dynamics in h o -treated h c cells, as indicated by a decrease in mitochondrial fusion and an increase in mitochondrial fission. furthermore, gas ameliorated the imbalanced mitochondrial dynamics induced by h o . the effects of gas in maintaining the normal structure of mitochondria might be the basis for protecting h c cells against oxidative injury. cell apoptosis is closely related to mitochondrial dynamics. during apoptosis, mitochondria undergo extensive fragmentation, which precedes caspase activation, to release proapoptotic factors [ ] . excessive fission or decreased fusion contributes to cell apoptosis, while inhibition of mitochondrial fission blocks or delays cell death [ ] [ ] [ ] [ ] . the gas-mediated protection of mitochondria from fragmentation might contribute to the decrease in apoptosis in h o -treated h c cells that were pretreated with gas. mitochondrial dynamics and bioenergetics are reciprocally coupled [ ] ; thus, imbalanced mitochondrial dynamics might result in dysfunctional mitochondrial respiration and atp production. measurement of real-time mitochondrial respiration in living h c cells using a seahorse xfe extracellular flux analyzer showed that mitochondrial respiration in h c cells, including basal respiration, maximal respiration, and atp production, was strongly inhibited by h o treatment. gas treatment alone did not affect mitochondrial respiration, while pretreatment with gas partly ameliorated the h o -induced inhibition of mitochondrial respiration. previous reports have shown that elongated mitochondria are linked to more efficient atp production and distribution, and better sustained stress-induced cell damage, while fragmented (through fission) mitochondria are associated with decreased atp production and increased susceptibility to injury [ , ] . the protective effects of gas on mitochondrial a representative photographs of mitochondrial morphology in negative control h c cells treated with μm h o for h with or without gas pretreatment. bar = or µm. b representative photographs of mitochondrial morphology in nrf -sirna-treated h c cells treated with μm h o for h with or without gas pretreatment. bar = or µm. c quantitative results of the mitochondrial network characteristics, including networks, mitochondrial footprint, mean branch length, and median branch length. the data presented are the mean ± sd and # p < . vs. the control group, *p < . vs. the h o -induced group respiration and atp production might be related to its regulatory effects on mitochondrial dynamics and mitochondrial fragmentation. notably, accumulating evidence supports that mitochondrial dynamics and bioenergetics may reciprocally influence each other, depending on the experimental condition or initial stimulation [ ] . the interaction and feedback regulation between mitochondrial dynamics and mitochondrial respiration might be involved in the mechanisms of gas. mitochondrial dynamics are controlled by a group of proteins, such as the fusion proteins mitofusin and (mfn / ) and optic atrophy (opa ), and the fission proteins fission protein (fis ) and dynamin-related protein (drp ). mitochondrial fusion is an essential mechanism by which damaged mitochondria mitigate stresses by exchanging proteins, lipids, and mitochondrial dna with healthy mitochondria [ ] . mfn and mfn are the primary regulators of outer mitochondrial membrane fusion, while opa mediates inner mitochondrial membrane fusion [ , ] . the present study showed that h o treatment induced a significant decrease in the expression of mfn , mfn , and opa . these results suggest that both outer and inner mitochondrial membrane fusion were inhibited by h o treatment. gas pretreatment significantly ameliorated the decrease in the expression levels of mfn and opa , but not mfn in h o -treated h c cells. mitochondrial fission is also critical in mitochondrial quality control by separating damaged mitochondria and subsequently removing them through mitophagy [ ] . in yeast, mitochondrial fission is regulated by cytosolic dynamin-like gtpase (dnm p) and a c-tail-anchored outer membrane protein, fis p. in mammals, drp and fis are involved in mitochondrial fission as dnm and fis orthologs, respectively [ ] . fis , located on the mitochondrial outer membrane, recruits drp from the cytosol to the outer mitochondrial membrane, and then the gtpase activity of drp provides a driving force through hydrolyzing gtp to mediate outer and inner mitochondrial membrane fission [ , ] . the present study showed that h o treatment induced a significant increase in the expression of fis and a decrease in drp . the increase in fis , a mitochondrial fission-stimulating protein [ ] , indicates activation of the mitochondrial fission process in h o treated h c cells. the decrease in drp is interesting, although the mechanism for its decrease is unknown. a previous report , and fis in nrf -sirna-treated cells that underwent h o treatment with or without gas pretreatment. c comparison of the expression levels of mfn , opa , and fis in negative control cells and nrf -sirna-treated cells. the data presented are the mean ± sd of at least three independent experiments. *p < . between the indicated two groups examining the expression of drp in h c cells and b osteosarcoma cells also showed that under h o treatment, drp expression significantly decreases in h c cells and slightly decreased in b cells [ ] . the roles of drp in oxidative stressinduced cells need further study. gas pretreatment ameliorated the increase in fis , but exhibited no influence on the expression of drp . these results suggest that gas ameliorated the activation of mitochondrial fission induced by h o . the present study confirmed that nuclear factor erythroid related factor (nrf ) is an important target of gas. nrf is a transcription factor that is activated by increased ros production, and reduces ros levels by upregulating antioxidant/detoxification genes [ ] . activation of nrf , including an increase in expression and/or nuclear translocation, has been observed in various types of cells treated with h o in time-and dose-dependent manners [ , ] . in the present study, treatment with h o at µm for h induced the nuclear translocation of nrf without a significant increase in the nrf expression level. importantly, gas pretreatment resulted in a slight increase in nrf expression, and strongly enhanced the nuclear translocation of nrf under h o treatment. the roles of nrf in the mechanisms of gas have been reported in neurons [ , , [ ] [ ] [ ] [ ] [ ] [ ] , osteoblasts [ , ] , liver hl- cells [ ] , and endothelial cells [ ] . in the present study, knockdown of nrf expression attenuated the protective effects of gas on h c cells, as indicated by increasing cell viability, decreasing ros levels, ameliorating mitochondrial fragmentation, and maintaining mitochondrial respiration potency. these results suggest that nrf is also an important target of gas in h c cardiomyocytes, and plays critical roles in the protective effects of gas against oxidative injury. furthermore, nrf knockdown also attenuated the effects of gas on the expression of mitochondrial fusion-and fission-related genes such as mfn , opa , and fis . nrf knockdown also directly influenced the expression of mfn in h c cells. these results suggest that nrf contributes to the control of mitochondrial dynamics. in summary, gas induces the activation of nrf , regulates mitochondrial dynamics, maintains the structure and functions of mitochondria, and thus protects h c cells against oxidative fig. knockdown of nrf expression attenuated the effects of gas on h o -induced mitochondrial dysfunction in h c cells. a mitochondrial respiration potency in negative control cells or nrf -sirna-treated cells that underwent h o treatment with or without pretreatment with μm gas. b results of quantification analysis of basal respiration. c results of quantification analysis of maximal respiration. d results of quantification analysis of atp production. the data presented are the mean ± sd and *p < . between the indicated two groups injury. there has been a renaissance in mitochondrial research in recent years, featuring exciting developments in understanding the structure and organization of mitochondria, and the roles of mitochondria in cellular-signaling mechanisms, in addition to their traditional role as a cell powerhouse in eukaryotes [ ] . the canonical and noncanonical roles of mitochondrial dynamics factors, such as mfn / , opa , fis , and drp , suggested by recent discoveries, have added another dimension to the integration of mitochondrial functions [ ] . in addition to the canonical roles of these factors in controlling the fusion/fission of mitochondria, they might also bear noncanonical roles, such as regulating mitochondrial bioenergetics and cell death, including apoptosis and autophagy [ , , ] . through dynamic changes in structure, mitochondria play important and pleiotropic roles in cells by regulating atp production, ros levels, ion channels, cell survival, and death under physiological conditions, as well as under injury. the present study suggests that the regulation of mitochondria is involved in gas-mediated protection of h c cardiomyocytes, and confirmed the critical role of mitochondria in cell regulation under oxidative injury. however, the mechanisms of mitochondrial dynamics regulation have not been fully clarified, and further study in this area is necessary and important. a review on central nervous system effects of gastrodin analytical techniques and pharmacokinetics of gastrodia elata blume and its constituents tianma modulates blood vessel tonicity research progress on pharmacological mechanism of gastrodiae rhizoma in cardiovascular and metabolic diseases heart protection by herb formula banxia baizhu tianma decoction in spontaneously hypertensive rats tianma gouteng decoction for essential hypertension: protocol for a systematic review and meta-analysis. evid based complement altern med protective effect of gastrodin pretreatment on myocardial ischemia reperfusion injury gastrodin pretreatment impact on sarcoplasmic reticulum calcium transport atpase (serca) and calcium phosphate (plb) expression in rats with myocardial ischemia reperfusion anti-apoptotic effect of gastrodin through inhibiting serum deprivation-induced autophagy in rat h c cardiomyocytes gastrodin attenuation of the inflammatory response in h c cardiomyocytes involves inhibition of nf-kappab and mapks activation via the phosphatidylinositol -kinase signaling mitophagy and mitochondrial integrity in cardiac ischemia-reperfusion injury balancing mitochondrial dynamics via increasing mitochondrial fusion attenuates infarct size and left ventricular dysfunction in rats with cardiac ischemia/reperfusion injury the role of mitochondria in the mechanisms of cardiac ischemia-reperfusion injury mitochondrial dynamics as a therapeutic target for treating cardiac diseases penehyclidine hydrochloride preconditioning provides cardiac protection in a rat model of myocardial ischemia/reperfusion injury via the mechanism of mitochondrial dynamics mechanism mitochondrial dynamics: overview of molecular mechanisms regulation of mitochondrial bioenergetics by the non-canonical roles of mitochondrial dynamics proteins in the heart mitochondrial bioenergetics and cardiolipin alterations in myocardial ischemia-reperfusion injury: implications for pharmacological cardioprotection mitochondrial dynamics and apoptosis compound k inhibits autophagy-mediated apoptosis through activation of the pi k-akt signaling pathway thus protecting against ischemia/reperfusion injury new colorimetric cytotoxicity assay for anticancer-drug screening bif- interacts with prohibitin- to regulate mitochondrial inner membrane during cell stress and apoptosis proteolytic cleavage of opa stimulates mitochondrial inner membrane fusion and couples fusion to oxidative phosphorylation fgf- transcriptionally down-regulates the expression of bnip l via pi k/akt/foxo a signaling and inhibits necrosis and mitochondrial dysfunction induced by high concentrations of hydrogen peroxide in h c cells gastrodin protects myocardial cells against hypoxia/reoxygenation injury in neonatal rats by inhibiting cell autophagy through the activation of mtor signals in pi k-akt pathway gastrodin improves cognitive dysfunction and decreases oxidative stress in vascular dementia rats induced by chronic ischemia gastrodin protects against mpp + -induced oxidative stress by up regulates heme oxygenase- expression through p mapk/nrf pathway in human dopaminergic cells gastrodin exerts robust neuroprotection in the postischemic brain via its protective effect against zn( +)-toxicity and its anti-oxidative effects in astrocytes gastrodin protects mc t -e osteoblasts from dexamethasone-induced cellular dysfunction and promotes bone formation via induction of the nrf signaling pathway gastrodin: an ancient chinese herbal medicine as a source for anti-osteoporosis agents via reducing reactive oxygen species mitochondrial dynamics and inter-mitochondrial communication in the heart mitochondrial fusion is essential for organelle function and cardiac homeostasis mitochondrial dynamics in cardiovascular health and disease inhibiting mitochondrial fission protects the heart against ischemia/reperfusion injury mitochondrial opa , apoptosis, and heart failure mitochondria on guard: role of mitochondrial fusion and fission in the regulation of apoptosis the role of dynamin-related protein , a mediator of mitochondrial fission, in apoptosis mitochondria as playmakers of apoptosis, autophagy and senescence mitochondrial fission, fusion, and stress novel insights into the role of mitochondrial fusion and fission in cardiomyocyte apoptosis induced by ischemia/reperfusion mitochondrial dynamics in the regulation of nutrient utilization and energy expenditure mitofusins, from mitochondria to metabolism mitochondrial 'kiss-and-run': interplay between mitochondrial motility and fusion-fission dynamics the i-aaa protease yme l and oma cleave opa to balance mitochondrial fusion and fission analysis of functional domains of rat mitochondrial fis , the mitochondrial fission-stimulating protein structural basis for recruitment of mitochondrial fission complexes by fis fis , mff, mid , and mid mediate drp recruitment in mitochondrial fission oxidative insults disrupt opa -mediated mitochondrial dynamics in cultured mammalian cells role of nrf /ho- system in development, oxidative stress response and diseases: an evolutionarily conserved mechanism activation of nrf by h o : de novo synthesis versus nuclear translocation hydrogen peroxide sensing, signaling and regulation of transcription factors evaluation of the mitochondria-related redox and bioenergetics effects of gastrodin in sh-sy y cells exposed to hydrogen peroxide gastrodin and isorhynchophylline synergistically inhibit mpp + -induced oxidative stress in sh-sy y cells by targeting erk / and gsk- beta pathways: involvement of nrf nuclear translocation gastrodin protect primary cultured rat hippocampal neurons against amyloid-beta peptide-induced neurotoxicity via erk / -nrf pathway gastrodin attenuates neuronal apoptosis and neurological deficits after experimental intracerebral hemorrhage inhibition of the nrf /ho- axis suppresses the mitochondria-related protection promoted by gastrodin in human neuroblastoma cells exposed to paraquat nrf mediates the anti-apoptotic and antiinflammatory effects induced by gastrodin in hydrogen peroxide-treated sh-sy y cells gastrodin alleviates glucocorticoid induced osteoporosis in rats via activating the nrf signaling pathways gastrodin ameliorates oxidative stress and proinflammatory response in nonalcoholic fatty liver disease through the ampk/nrf pathway gastrodin induced ho- and nrf up-regulation to alleviate h o -induced oxidative stress in mouse liver sinusoidal endothelial cells through p mapk phosphorylation mitochondrial-shaping proteins in cardiac health and disease-the long and the short of it! cardiovasc drugs ther qqc conducted the experiments, analyzed the data, and drafted the paper. yww participated in the experiments. wmy, mht, ycw, and hyh helped to design the experiments. wdz and xl supervised the research and revised the paper. all authors reviewed and approved the paper. the online version of this article (https://doi.org/ . /s - - -x) contains supplementary material, which is available to authorized users.competing interests: the authors declare no competing interests. key: cord- -ehxxvenb authors: pang, xiaocong; cui, yimin; zhu, yizhun title: recombinant human ace : potential therapeutics of sars-cov- infection and its complication date: - - journal: acta pharmacol sin doi: . /s - - - sha: doc_id: cord_uid: ehxxvenb nan cardiovascular disease through modulating ace activation and expression to increase ang - production and improve vascular function [ ] . owing to the role of ace in the entry of sars-cov- , the upregulated expression of ace had an unwanted effect. therefore, dize is not suggested to be applied in the treatment of sars-cov- infection. however, the addition of exogenous ace could be a potential treatment for sars-cov- infection, which might not only restrain the spread of sars-cov- by blocking its interaction with ace on the host cell, but also modulate ras to treat sars-cov- -related underlying comorbidities and protect the lung from developing ards. given that ace is generated mainly in clara cells and type ii alveolar epithelial cells, the production of ace is severely impaired after epithelial injury in the development of ards [ ] . in addition, the expression of ace is also severely decreased in patients with pulmonary fibrosis [ ] . therefore, injection of recombinant human ace (rhace ) is currently considered for treating ards and pulmonary arterial hypertension [ ] . circulatory levels of ace activity were markedly increased by rhace , which further effectively lowered ang ii levels and generated ang - from ang ii (fig. ) . although ang ii receptor and ace blockage were also effective in lung failure in animal models, this treatment could cause potential adverse effects, causing systemic hypotension in humans [ ] . as shown in fig. , rhace also acts as a potential therapy for hypertension, heart failure, kidney injury, and liver fibrosis [ ] [ ] [ ] . currently, phase i (nct ) and phase ii (nct ) clinical studies with a recombinant version of the catalytic ectodomain of human ace (gsk ) have been successfully completed, providing safety and efficacy for ards treatment [ , ] . the administration of rhace was well tolerated without clinically significant hemodynamic changes in healthy subjects and patients with ards [ ] . during the administration period, no antibodies to rhace were detected, and no serious adverse events were reported [ ] . the twice-daily doses of gsk treatment-regulated angiotensin system peptide, leading to a significant reduction in the concentration of ang ii, accompanied by a rapid rise in ang - and ang - concentrations, and caused a reduction in il- concentration [ ] . however, given the small cohort of critically ill patients, infusion of gsk did not contribute to ameliorated ards through physiological or clinical measures, and a clear role of gsk in the increased reports of adverse events referring to hypernatremia, pneumonia, dysphagia, and rash was difficult to establish. therefore, to assess clinical outcomes powerfully, further clinical trials need a larger sample size. recently, monteil et al. [ ] reported that hrace could significantly inhibit sars-cov- infection of vero-e cells, and of human capillary and kidney organoids, providing an evidence that rhace might not only reduce lung injury but also block early entry of sars-cov- infections in target cells. further studies are needed to illuminate the effect of hrace in sars-cov- infections from bench to clinic. to ensure the quality of the data and clinical success of rhace , the trials for using rhace in patients with sars-cov- infection or ards should consider the patient's stratification and continuous infusion dose. first, various plasma ang ii levels may pose some difficulties in identifying responders. hence, before gsk infusion, the ang ii concentrations and the ratio of ace /ace activity of enrolled patients were evaluated for improved risk stratification. ace gene insertion/deletion (i/d) polymorphisms play an important role in the development of hypertension, nephritis, and cardiovascular diseases in different ethnic populations by influencing ace and ang ii activities [ , ] . identifying the specific population that is most likely to benefit from rhace represents a bright prospect. second, due to the short half-life of soluble ace in vivo, a continuous infusion of rhace may enhance efficacy. in addition, an excess of rhace is likely to influence the balance of the ras; therefore, it is important to identify the effective infusion dose to prevent underlying ras-related adverse events. recently, it was reported that a chimeric fusion of rhace and igg fc fragments could improve rhace plasma stability [ ] . this rhace -fc fusion protein retained full peptidase activity and had extended plasma half-life in mice [ ] . the strategy for rhace -fc will be expected to provide patients with added convenience, largely reducing administration frequency and greatly improving treatment effectiveness [ ] . taken together, these findings indicate that rhace would represent a potential therapeutic strategy for sars-cov- infection and its complications. fig. the mechanism and functions of rhace . rhace is able to lower ang ii levels and increase ang - levels effectively and exert protective effects in the heart, lung, liver, and kidney. a novel coronavirus from patients with pneumonia in china coronaviridae study group of the international committee on taxonomy of viruses. the species severe acute respiratory syndrome-related coronavirus: classifying -ncov and naming it sars-cov- a pneumonia outbreak associated with a new coronavirus of probable bat origin identification of critical determinants on ace for sars-cov entry and development of a potent entry inhibitor cryo-em structure of the -ncov spike in the prefusion conformation structure of dimeric full-length human ace in complex with b at sars-cov- cell entry depends on ace and tmprss and is blocked by a clinically-proven protease inhibitor ace revisited: a new target for structure-based drug design angiotensin receptors ace , a promising therapeutic target for pulmonary hypertension effects of losartan on haemodynamic parameters and angiotensin receptor mrna levels in rat heart after myocardial infarction time-dependent apoptotic development and pro-apoptotic genes expression in rat heart after myocardial infarction the angiotensin-converting enzyme gene family: genomics and pharmacology angiotensin-converting enzyme protects from lethal avian influenza a h n infections angiotensin-converting enzyme (ace ) mediates influenza h n virus-induced acute lung injury pathological findings of covid- associated with acute respiratory distress syndrome the potential actions of angiotensin-converting enzyme ii (ace ) activator diminazene aceturate (dize) in various diseases structure-based identification of small-molecule angiotensin-converting enzyme activators as novel antihypertensive agents angiotensin converting enzyme is primarily epithelial and is developmentally regulated in the mouse lung angiotensin converting enzyme- is protective but downregulated in human and experimental lung fibrosis recombinant human ace : acing out angiotensin ii in ards therapy recombinant human ace and the angiotensin - axis as potential new therapies for heart failure ace therapy using adeno-associated viral vector inhibits liver fibrosis in mice novel ace -fc chimeric fusion provides long-lasting hypertension control and organ protection in mouse models of systemic renin angiotensin system activation pharmacokinetics and pharmacodynamics of recombinant human angiotensinconverting enzyme in healthy human subjects a pilot clinical trial of recombinant human angiotensin-converting enzyme in acute respiratory distress syndrome inhibition of sars-cov- infections in engineered human tissues using clinical-grade soluble human ace association between genetic polymorphisms and angiotensin-converting enzyme inhibitor-induced cough: a systematic review and meta-analysis therapeutic strategies in an outbreak scenario to treat the novel coronavirus originating in wuhan, china key: cord- - mz w authors: xu, zhen-dong; wang, yong; liang, ge; liu, zhi-qiang; ma, wu-hua; chu, charleen t; wei, hua-feng title: propofol affects mouse embryonic fibroblast survival and proliferation in vitro via atg - and calcium-dependent regulation of autophagy date: - - journal: acta pharmacol sin doi: . /s - - -z sha: doc_id: cord_uid: mz w propofol is a commonly used intravenous anesthetic agent, which has been found to affect cell survival and proliferation especially in early life. our previous studies show that propofol-induced neurodegeneration and neurogenesis are closely associated with cell autophagy. in the present study we explored the roles of autophagy-related gene (atg ) in propofol-induced autophagy in mouse embryonic fibroblasts (mef) in vitro. we showed that atg was functionally related to propofol-induced cell survival and damage: propofol significantly enhanced cell survival and proliferation at a clinically relevant dose ( µm), but caused cell death at an extremely high concentration ( µm) in atg (−/−) mef, but not in wt cells. the dual effects found in atg (−/−) mef could be blocked by intracellular ca( +) channel antagonists. we also found that propofol evoked a moderate (promote cell growth) and extremely high (cause apoptosis) cytosolic ca( +) elevation at the concentrations of µm and µm, respectively, only in atg (−/−) mef. in addition, atg (−/−) mef themselves released more ca( +) in cytosolic space and endoplasmic reticulum compared with wt cells, suggesting that autophagy deficiency made intracellular calcium signaling more vulnerable to external stimuli (propofol). altogether, our results reveal that atg plays a crucial role in propofol regulation of cell survival and proliferation by affecting intracellular ca( +) homeostasis. autophagy-related gene (atg ) is one of the key proteins regulating autophagy activity [ ] [ ] [ ] , which is also associated with intracellular ca + homeostasis [ ] [ ] [ ] . atg has been demonstrated to affect cell survival and proliferation via its effects on autophagy [ ] [ ] [ ] . similar to other important autophagy regulators, such as mtor and beclin- , atg can be targeted to change autophagy activity and therefore affect the pathology of some diseases, such as parkinson's disease [ ] , myeloid leukemia [ ] , and some tumors [ , ] . atg knockout cells are generated [ , ] and available so that the effects of drugs on cell physiology/pathology via the function of atg and associated autophagy activity can be investigated conveniently. our previous studies suggested that propofol can regulate cell autophagy via activation of insp (insp r) or ryanodine receptors (ryr) [ , ] , with unclear mechanisms. calcium may also be involved in autophagy by affecting atg . elevation of intracellular ca + levels can activate atg , which initiates autophagy [ ] . interestingly, reducing intracellular ca + prevents the cleavage of atg , which in turn increases the levels of full-length atg and atg -atg conjugate [ ] . considering the essential role of atg in autophagy and calcium regulation [ ] , it is plausible that propofol may affect cell survival or proliferation by varying the function of atg . although propofol at high concentrations has been demonstrated to cause neurotoxicity and impairment of neurogenesis [ ] [ ] [ ] , the effects of propofol at clinically relevant low concentrations on autophagy and cell function need to be investigated. here, we studied the effects of propofol on cell survival, growth and autophagy activity in both wild-type (wt) and atg knockout cells (atg −/− ). we demonstrated that atg plays an important role in propofol effects on autophagy, cell survival, and growth due to its effects on the regulation of intracellular ca + homeostasis. cell culture wt and atg −/− mouse embryonic fibroblast (mef) cell lines purchased from riken bio resource center (tsukuba, ibaraki - , japan) were maintained in dulbecco's modified eagle's medium, supplemented with % fetal bovine serum and % penicillin/streptomycin (both from life technologies, carlsbad, ca, usa) and were cultured in a % co humidified atmosphere at °c. the culture medium was changed every two days. cells with %- % confluence were obtained from t-flask cultures using trypsinization and incubated onto the disk samples. the cell viability/proliferation was determined using the mtt ( -( , dimethylthiazol- -yl)- , -diphenyltetrazolium bromide, sigma-aldrich, st. louis, mo, usa) assay at , , and h as previously described [ ] . after washing with pbs, the cells were incubated with fresh culture medium containing mtt ( . mg/ml in the medium) at °c for h in the dark. the medium was then removed, and formazan was solubilized with dimethyl sulfoxide. the absorbance of this solution in each well was quantified by spectrophotometry at nm using a synergy™ h microplate reader (biotek, winooski, vt, usa). cell viability was further confirmed by the trypan blue assay. then, × cells per well were seeded onto -well plates. following propofol treatment, cell viability was determined by trypan blue exclusion assay. cells were mixed with . % trypan blue solution for min at room temperature. the cell number was countered with a hemocytometer in a bright field under a microscope as described previously [ ] . cells without trypan blue entry were regarded as viable. cytotoxicity was determined by lactate-dehydrogenase (ldh) detection with an ldh-release assay kit (thermo scientific, rockford, il, usa) according to the manufacturer's instructions, as described previously [ ] . briefly, μl of supernatant was collected and transferred to each well of a new -well plate. then, μl of reaction mixture was added to each well. after min of incubation at room temperature, μl of stop solution was added to terminate the reaction. the nm absorbance of the samples was measured along with background absorbance at nm using a synergy™ h microplate reader (biotek, winooski, vt, usa). cell proliferation assays wt and atg −/− mefs were plated onto cover glasses in culture medium. -bromodeoxyuridine (brdu, invitrogen, eugene, or, usa) was added to the culture medium for h before the end of treatment with a final concentration of μm. the cells were then fixed in % paraformaldehyde and permeabilized with . % triton x- . for brdu detection, acid treatment ( n hcl min on ice followed by n hcl min at room temperature) separated dna into single strands so that the primary antibody could access the incorporated brdu. after incubation with blocking solution ( % normal goat serum in pbs containing . % triton x- ), the cells were incubated with rat monoclonal anti-brdu primary antibody ( : ; santa cruz biotechnology, dallas, tx, usa) overnight at °c. after a subsequent wash with pbs containing . % triton x- , the cells were incubated with fluorescently labeled secondary antibody conjugated with anti-rat igg ( : ; invitrogen, eugene, or) for h at room temperature. cell nuclei were counterstained with ′, diamidino- -phenylindole (dapi, invitrogen, eugene, or, usa) for min at room temperature. the immunostained cells were covered and then mounted on an olympus bx tf fluorescence microscope ( ×; olympus usa, center valley, pa, usa). images were acquired using ivision . . software (biovision technologies, exton, pa, usa). five sets of images were acquired at random locations on the cover glass and were subsequently merged using imagej . v software (national institutes of health, bethesda, md, usa). the percentage of brdu-positive cells over the total number of cells was calculated and compared across different groups from at least three different cultures. the level of cytosolic ca + concentration ([ca + ] c ) of mefs with or without propofol treatment was measured by fura- /am fluorescence (molecular probe, eugene, or, usa) using a previously described method [ , ] . the assays were carried out on an olympus ix inverted microscope (olympus america inc., center valley, pa, usa) and iplab v . software (scanalytics, milwaukee wi, usa). briefly, mefs were plated onto a mm culture dish. after the cells were washed three times in hank's buffered salt solution (hbss) containing mg + and ca + and loaded with . μm fura- /am in the same buffer for min at °c, the cells were then washed twice and incubated with hbss for another min at °c. fura- am was measured by recording alternate at and nm excitation, and emission at nm was detected for up to min for each treatment. the evoked changes were recorded in response to treatment with μm versus μm propofol with or without dantrolene (dan), xestospongin c (xc) or bapta-am. the results are presented as a ratio of f /f nm and averaged from at least three separate experiments. basal calcium in the cytoplasm and er in atg −/− and wt mefs mef cells were plated on -mm round coverslips in -well plates and then cultured overnight. the next day, plasmid pcmvr-cepia er ( ; addgene, cambridge, ma, usa) was transfected with lipofectamine (l ; invitrogen, carlsbad, ca, usa) according to the manufacturer's instructions. at h post transfection, the transfection reagent was replaced by the normal culture medium and the treatment was applied. images were taken at the end of the treatment. for labeling intracellular ca + , cells were incubated with fluo- am reagent (f ; molecular probes, eugene, or, usa) at μm for min at °c. western blotting was performed according to the standard procedure and as we described before [ ] . total protein extracts from mefs cells were obtained by lysing the cells in ice-cold lysis buffer ( mm tris-hcl, mm nacl and % triton x- ) in the presence of a cocktail of protease inhibitors [ ] . after centrifugation, the supernatant was collected, and the total protein was quantified using a bicinchoninic acid protein assay kit (thermo scientific, rockford, il, usa). equal amounts of protein for each lane were loaded and separated on % sodium dodecyl sulfatepolyacrylamide gel electrophoresis (sds-page). after electrophoresis, the proteins were transferred onto a polyvinylidene fluoride (pvdf) membrane. the membranes were blocked with % fat-free milk dissolved in pbs-t for h at room temperature and then stained with primary antibody at °c overnight. after washing with pbs-t, the membranes were incubated with secondary antibodies (hrp conjugated anti-rabbit and anti-mouse igg) at : dilutions, and β-actin served as a loading control. signals were detected with an enhanced chemiluminescence detection system (millipore, billerica, ma, usa) and quantified by scanning densitometry. data analysis and statistics parametric variables were expressed as the mean ± sd and analyzed using the student's unpaired two-tailed t test, one-way or two-way anova, followed by tukey's post hoc analysis. graphpad prism software (graphpad software, inc., san diego, ca, usa) was used for statistical analyses and graph creation. p values less than . were considered statistically significant. propofol affects cell survival in an atg -dependent manner and is associated with intracellular ca + channels we first determined whether propofol affects cell viability in atg −/− and wt cells by mtt and ldh detection. after -h exposure to propofol, the mtt signal of atg −/− cells was significantly enhanced at clinically relevant concentrations ( µm); however, this effect was inverted at high pharmacological general anesthetics and autophagy zd xu et al. concentrations ( µm, fig. c ). toxic effects of atg deficiency at high doses were further confirmed by ldh detection (fig. e) . in contrast, propofol did not significantly affect cell survival at any tested concentration in wt cells. according to our prior study [ , ] , we hypothesized that intracellular calcium channels, such as inositol- , , -trisphosphate receptor (insp r) and ryr located in the sarcoplasmic/endoplasmic reticulum membrane, may be involved in propofol-induced cell survival. indeed, the ability of µm propofol to increase viability in atg −/− cells was blocked by the antagonists of insp r and ryr (fig. d) , which also blocked the toxic effects at the high µm dose (fig. d, f) . in contrast, treatment with µm propofol became harmful to wt cells after the inhibition of insp r and ryr (fig. d, f) . these results indicate that autophagy deficiency may facilitate intracellular ca + channel opening, which triggers the dual functional effects of propofol on survival in atg −/− cells. atg plays a key role in propofol effects on cell growth we next determined whether the elevation of mtt by propofol at clinically relevant concentrations in atg −/− cells (fig. c) was caused by changes in mitochondrial reductase activity (early cell damage) or an increase in cell numbers (proliferation). propofol at a low concentration ( µm) significantly increased both total viable cells (fig. c) as determined by the trypan blue assay and newly derived cells (fig. a, b) as determined by the brdu incorporation assay. in contrast, propofol at a high concentration . data in a-c are given as the mean ± sem, and d-f are given as the mean ± sd. all data are from at least three separate experiments and analyzed by two-way anova followed by the tukey's multiple comparison tests. *p < . , **p < . , or ***p < . , respectively general anesthetics and autophagy zd xu et al. ( µm) decreased the total cell numbers more significantly in atg −/− cells than in wt cells (fig. a-c) . atg is necessary for propofol-induced functional effects on autophagy activity atg plays an essential role in the induction and regulation of autophagy activity, but it is still unclear how propofol mediates functional effects by autophagy. we first confirmed that propofol up to µm did not affect atg expression and that no atg existed in atg −/− cells using western blotting (fig. a ). in addition, propofol did not produce a significant change in atg in wt cells (fig. c) . since autophagy flux, which is a dynamic process, consists of several sequential steps-sequestration, transport to lysosomes, degradation, and utilization of degradation products, it is possible that autophagy activity is altered despite unchanged atg protein expression. to further study this possibility, we applied bafilomycin a (baf a ), which can prevent fusion of the autophagosome and lysosomes. we found that µm but not µm propofol significantly increased the expression of lc ii, a biomarker to detect autophagy, by pretreatment with baf a in wt cells (fig. b, d) , indicating that high concentrations of propofol can induce autophagy. as expected, there was no lc ii protein bands formed in atg −/− cells (fig. b) . these findings suggest that the enhanced cell death in atg −/− cells treated with µm could be related to inability to activate autophagy. propofol facilitates cytosolic ca + release in atg -deficient cells our previous studies have shown that propofol increases intracellular ca + release in different types of cells [ , ] . to further confirm the change in basal ca + release in both the cytoplasm and er, we measured these effects by fluo- am dye on an inverted microscope and er ca + transfected with cepia er (fig. a) . we found that basal cytosolic and er ca + were significantly increased in atg −/− cells compared with wt cells (fig. b ). in addition, propofol-mediated ca + release could be inhibited by xc or dantrolene (fig. e , f, xc, nm, dantrolene µm), confirming the involvement of intracellular calcium channels. it is possible that the dual functional effects of propofol on atg −/− cells (fig. ) may be due to various degrees of intracellular ca + release at different concentrations. figure shows that propofol at extremely high concentrations ( µm) increased [ca + ] c more robustly than at clinically relevant concentrations ( µm) in atg −/− cells, compared with wt cells. these results suggest that autophagy-deficient cells are primed for greater lability in response to ca + mobilizing stimuli. moderate intracellular ca + release enhances cell survival/ proliferation, but over release causes cell death, which could be supported by the results shown in fig. . we have demonstrated that atg is essential for propofol effects on cell survival and growth. the above effects may be related to propofol effects on insp r/ryr activity and changes in intracellular ca + homeostasis. this study provides a detailed mechanism of propofol regulation of autophagy and associated cell survival or growth. although commonly used inhalational anesthetics (sevoflurane, isoflurane, or desflurane) [ , ] and intravenous anesthetics (propofol) [ ] all have been shown to cause ca + release from the er, their potency to affect intracellular ca + homeostasis is quite different. inhalational anesthetics at clinically relevant . all data are presented as the mean ± sd from three separate experiments and analyzed by one-way anova followed by the tukey's multiple comparison tests. *p < . , ***p < . , or ****p < . , respectively concentrations ( - minimal alveolar concentration, mac) can induce ca + release from the er via activation of insp r [ , ] , with maximum activation at mac [ ] . inhalational anesthetics at clinically relevant concentrations can also induce cell damage in different types of cells [ , , ] . however, propofol at clinically relevant concentrations ( - µm) rarely induces elevation of [ca + ] c or induced cell damage, and very high pharmacological concentrations (> µm) are usually needed for propofol to induce the elevation of [ca + ] c and cell damage [ ] . similar to previous research, the results of this study suggest that a high concentration of propofol (> µm) is needed to cause cell death in atg −/− cells or cells with dysfunctional autophagy. in addition, propofol at a clinically relevant concentration ( µm) increased cell proliferation only in atg −/− but not wt cells, suggesting that the moderately enhanced ca + release in autophagy-deficient cells crosses a threshold necessary to trigger proliferation. in combination with previous studies [ , ] , the results from this study suggest that propofol at clinically relevant concentrations (< µm) typically does not cause cell damage in the presence of normal autophagy function. however, on the basis of impaired autophagy function, such as in alzheimer's disease [ , ] , cells are more vulnerable to propofol-mediated cell cycle re-entry at low clinically relevant concentrations ( µm) and cell damage at high pharmacological concentrations (> µm). overall, these results suggest that propofol regulates cell survival and growth signaling by activating insp r/ryr, but autophagy deficiency predisposes to calcium overload at high doses of propofol. previous studies have suggested that anesthetics elevated [ca + ] c by the activation of insp r/ryr [ , , ] , but the effects of the autophagy regulatory factor atg on the propofol-mediated ca + response are not clear. in this study, we demonstrated that propofol induced a significantly higher [ca + ] c in atg −/− cells than in wt cells, and this effect can be inhibited by antagonists for insp r (xc) or ryr (dantrolene). interestingly, propofol, even at clinically relevant concentrations ( µm), increased [ca + ] c , especially in atg −/− cells. these results suggested that atg might typically buffer the activation of insp r/ryr and affect intracellular ca + homeostasis either directly or indirectly via its effects on autophagy activity [ ] . autophagy generally regulates the intracellular homeostasis of organelles such as the er by sequestrating organelles and membrane compartments. damaged or redundant er can be removed by autophagy. er is expanded when the autophagy pathway is impaired, and the ca + stores in er are also increased, consistent with the expansion of er contents in autophagy-deficient cells [ , , ] , which is in accordance with our results in the current study. however, it is not clear why cytosolic ca + is also significantly increased in autophagy-deficient cells. nevertheless, . all data are given as the mean ± sd from four separate experiments and analyzed by one-way anova followed by the dunnett's multiple comparisons test. *p < . fig. basal level ca + concentriation in both cytosolic and er significantly increased in atg −/− compared with wild type cells. the cytoplasmic ca + concentration was measured using fluo- am dye on an inverted microscope and er ca + was measured in cells transfected with cepia er (a). the ca + concentrations in both the cytoplasm and er were significantly increased in atg −/− mefs compared with wt mefs (b). all data are presented as the mean ± sd from three separate experiments and analyzed by the t test. *p < . , **p < . . all data are given as the mean ± sd from three separate experiments and analyzed by the t test. *p < . , **p < . . pfl-mediated [ca + ] c elevation was significantly inhibited by xc, dantrolene and bapta-am in both wt (g) and atg −/− cells (h), more significantly in the latter. all data are given as the mean ± sd from at least three separate experiments and analyzed by one-way anova followed by the tukey's multiple comparison tests. *p < . , **p < . , ***p < . , or ****p < . , respectively autophagy deficiency has been demonstrated to lead to an imbalance between ca + release/influx or reuptake/extrusion, resulting in higher basal [ca + ] c [ ] . in vascular smooth muscle cells (vsmcs) with defective autophagy, cytosolic ca + levels increased under basal conditions, which may be related to decreased plasma membrane ca + -atpase expression in atg −/− vsmcs, thus hampering ca + extrusion to the extracellular environment [ ] . although anesthetics have been proposed to affect autophagy and cell survival in a dose-dependent manner, with low doses stimulating autophagy and cell survival [ , ] , and high concentrations impairing autophagy flux and causing cell damage [ , ] , this may be oversimplified. in the current study, we demonstrated that high doses of propofol induces autophagy in wild type cells, but not in atg −/− cells. we propose an alternative model in which the er is expanded and ca + stores are increased in autophagy-deficient mef cells. while this enhances vulnerability to cell death triggered by calcium overload at high propofol doses, propofol at clinically relevant concentrations ( μm) induces a moderate increase in ca + release from the er into the cytosol, favoring cell survival and proliferation of autophagy-deficient cells (fig. ) . in this study, we demonstrated that the effects of propofol on calcium release are modulated by atg . furthermore, propofol at clinically relevant concentrations ( µm) promoted the proliferation of atg −/− cells through moderately elevated [ca + ] c , while at high concentrations ( µm), propofol increased the large outflow of ca + from the er, inducing the death of atg −/− cells. it is not clear whether atg has direct effects on the activation of insp r/ryr or indirect effects via the regulation of autophagy. in addition, it is not clear whether the effect of propofol on intracellular ca + homeostasis is associated with ca + influx from the extracellular space, which requires further study. our study has the following limitations: ( ) . although these two types of cells provide good tools to investigate the role of atg on the propofol effect on autophagy and its association with cell survival and growth, these cells are fibroblasts and not closely related to neurons. ( ) . whether or not atg regulates activation of insp r/ryr by either directly interacting with receptor activation or indirectly through changes in lysosome or autophagy function requires further study. ( ) . this study relied upon the conversion of lc -i (cytosolic form) to lc -ii (membrane-bound lipidated form) by immunoblotting with and without lysosomal inhibitor treatment, which is a conventional method to monitor cellular autophagic activity [ ] . now with a novel tandem construct (mrfp-gfp-lc ), autophagic flux can also be morphologically traced, although we did not perform this experiment. in summary, atg plays an essential role in modulating dosedependent propofol effects on autophagy, cell proliferation and cell death related to changes in intracellular ca + homeostasis. fig. propofol at clinically relevant concentrations promotes cell survival and proliferation in autophagy-deficient cells. the er is expanded, and the basal ca + stores are increased in autophagy-deficient mef cells. propofol at clinical concentrations ( µm) significantly induces the release of more ca + from the er into the cytosol in atg −/− cells than in wt cells via the activation of inositol- , , -trisphosphate receptor (insp r) and/or ryanodine receptor (ryr) calcium channels, favoring cell survival and proliferation the role of autophagy during the early neonatal starvation period atg is essential for the development and survival of innate lymphocytes involvement of autophagy in nk cell development and function atg and bak play important roles in the crosstalk between apoptosis and autophagy induced by influx of extracellular calcium calcium homeostasis and er stress in control of autophagy in cancer cells lysosomal calcium signalling regulates autophagy through calcineurin and tfeb the crucial role of atg in cortical neurogenesis during early brain development a critical role for the autophagy gene atg in t cell survival and proliferation autophagy limits proliferation and glycolytic metabolism in acute myeloid leukemia a novel and functional variant within the atg gene promoter in sporadic parkinson's disease atg -dependent autophagy contributes to the development of acute myeloid leukemia in an mll-af -driven mouse model protective role of autophagy and autophagy-related protein in early tumorigenesis upregulation of autophagyrelated gene- (atg- ) is associated with chemoresistance in human gastric cancer discovery of atg /atg -independent alternative macroautophagy propofol affects neurodegeneration and neurogenesis by regulation of autophagy via effects on intracellular calcium homeostasis general anesthetics regulate autophagy via modulating the inositol , , -trisphosphate receptor: implications for dual effects of cytoprotection and cytotoxicity control of basal autophagy by calpain mediated cleavage of atg autophagy, a novel pathway to regulate calcium mobilization in t lymphocytes protective effect of acetyl-l-carnitine on propofol-induced toxicity in embryonic neural stem cells down-regulation of microrna- is involved in the propofol-induced neurotoxicity observed in human stem cell-derived neurons propofol induces apoptosis and inhibits the proliferation of rat embryonic neural stem cells via gamma-aminobutyric acid type a receptor impaired autophagy in mouse embryonic fibroblasts null for kruppel-like factor promotes dna damage and increases apoptosis upon serum starvation knockdown of autophagy-related protein , atg , decreases oxidative stress and has an opposing effect on camptothecin-induced cytotoxicity in osteosarcoma cells inhalational anesthetics induce cell damage by disruption of intracellular calcium homeostasis with different potencies the common inhaled anesthetic isoflurane increases aggregation of huntingtin and alters calcium homeostasis in a cell model of huntington's disease general anesthetic isoflurane modulates inositol , , -trisphosphate receptor calcium channel opening the common inhalation anesthetic isoflurane induces apoptosis and increases amyloid beta protein levels the effect of endoplasmic reticulum stress on neurotoxicity caused by inhaled anesthetics prolonged treatment with propofol transiently impairs proliferation but not survival of rat neural progenitor cells in vitro the effects of intravenous anesthetics on mouse embryonic fibroblast viability and proliferation autophagy and alzheimer's disease endo-lysosomal and autophagic dysfunction: a driving factor in alzheimer's disease? autophagy regulates endoplasmic reticulum homeostasis and calcium mobilization in t lymphocytes defective autophagy in vascular smooth muscle cells alters contractility and ca + homeostasis in mice propofol-enhanced autophagy increases motility and angiogenic capacity of cultured human umbilical vascular endothelial cells mitigation of h o -induced autophagic cell death by propofol in h c cardiomyocytes methods in mammalian autophagy research we appreciate the valuable discussion from maryellen eckenhoff, roderic eckenhoff and editing assistance from divakara gouda at the department of anesthesiology, university of pennsylvania, philadelphia, usa. supported by grants to hw from the nih (r gm , r gm - s , and r gm - a ) and r ag - s to ctc. supported by grants to zx from shanghai municipal health bureau ( ). supported by grants to zx from the national natural science foundation of china ( ). supported by grants to yw from the national natural science foundation of china ( ). hfw designed research; zdx, yw, and gl performed research; zdx, hfw, and ctc analyze and interpret data; zdx, hfw, ctc, zql, and whm wrote the paper. competing interests: hua-feng wei is a member of advisory board on march , at eagle pharmaceutical company, new jersey, usa, which produce and sale ryanodex, a new formula of dantrolene. the dantrolene used in this study was purchased from a different company. no other authors state conflict of interest. key: cord- -d k ow authors: zhang, shi-rong; zhang, xiao-chen; liang, jia-feng; fang, hong-ming; huang, hai-xiu; zhao, yan-yan; chen, xue-qin; ma, sheng-lin title: chalcomoracin inhibits cell proliferation and increases sensitivity to radiotherapy in human non-small cell lung cancer cells via inducing endoplasmic reticulum stress-mediated paraptosis date: - - journal: acta pharmacol sin doi: . /s - - - sha: doc_id: cord_uid: d k ow chalcomoracin (cmr) is a kind of diels–alder adduct extracted from the mulberry leaves. recent studies showed that cmr has a broad spectrum of anticancer activities and induces paraptosis in breast cancer and prostate cancer cells. in this study, we investigated the effects of cmr against human non-small cell lung cancer cells and the underlying mechanisms. we found that cmr dose-dependently inhibited the proliferation of human lung cancer h , a and pc- cells. furthermore, exposure to low and median doses of cmr induced paraptosis but not apoptosis, which was presented as the formation of extensive cytoplasmic vacuolation with increased expression of endoplasmic reticulum stress markers, bip and chop, as well as activation of mapk pathway in the lung cancer cells. knockdown of bip with sirna not only reduced the cell-killing effect of cmr, but also decreased the percentage of cytoplasmic vacuoles in h cells. moreover, cmr also increased the sensitivity of lung cancer cells to radiotherapy through enhanced endoplasmic reticulum stress. in lung cancer h cell xenograft nude mice, combined treatment of cmr and radiation caused greatly enhanced tumor growth inhibition with upregulation of endoplasmic reticulum stress proteins and activation of perk in xenograft tumor tissue. these data demonstrate that the anticancer activity and radiosensitization effect of cmr result from inducing paraptosis, suggesting that cmr could be considered as a potential anticancer agent and radiation sensitizer in the future cancer therapeutics. lung cancer is the leading cause of cancer death in the world [ ] . despite the rapid development of cancer treatments, including targeting therapy, checkpoint-based immunotherapy and precise radiotherapy, unavoidable drug resistance and rapid distant metastasis are still major obstacles in the clinical management of this disease. radiation therapy is one of the standard therapeutic strategies in the treatment of most common nonsmall cell lung cancer (nsclc), especially in patients with unresectable tumors. for patients with local advanced nsclc, the current multimodal chemotherapy and radiotherapy do not reach satisfactory treatment outcomes for most patients, and the median progression-free survival is only - months [ ] [ ] [ ] . ionizing radiation (ir) can trigger many types of cell death, such as apoptosis, in targeted cancer cells. apoptosis is induced by surface death receptors, through mitochondrial release of cytochrome c or by cellular stress induced by drug/radiation exposure. the most common signaling pathways that regulate apoptosis include cell death receptor signaling, the caspase signaling cascade and mitochondrial signaling [ ] [ ] [ ] [ ] [ ] . by acting in parallel or sequentially, autophagy can facilitate and cooperate with apoptosis to drive cell death. blockage of autophagy by pharmacological inhibitors or knockdown of beclin and atg expression significantly inhibits apoptosis in cells. when a cell is unable to undergo apoptosis, it may undergo necrotic cell death, which does not involve caspase activation [ , ] . chalcomoracin (cmr) is a diels-alder adduct derived from mulberry leaves. it has been reported that cmr inhibits fatty acid synthesis and can serve as an anti-staphylococcal agent [ ] [ ] [ ] . recent studies have shown that cmr has a broad spectrum of biological activities against human cancers and induces paraptosis in breast cancer and prostate cancer cells. paraptosis is a type of nonapoptotic cell death characterized by dilation of the endoplasmic reticulum (er) and/or mitochondria. although the molecular mechanism of paraptosis is not well elucidated, studies have revealed that paraptosis is independent of the caspase signaling cascade and may involve activation of the mitogenactivated protein kinase (mapk) pathway [ , ] . the er stress markers bip and chop were found to be elevated in paraptosis [ , ] . in this study, we tested the potential antitumor and radiosensitizing activities of cmr in nsclc cells. our results showed that cmr increased clonogenic cell death by inducing paraptosis in nsclc cells in response to radiotherapy, with characterized cytoplasmic vacuolation and dilated er triggering er stress. chemicals and cell lines cmr was provided by jingkui tian's lab [ ] at the key laboratory of biomedical engineering of zhejiang university; it had a purity ≥ % as determined by high-performance liquid chromatography (hplc). cmr stock solutions were dissolved in dimethyl sulfoxide (dmso) and diluted to the required concentrations before use. u (phz , thermo fisher, ma, usa) was used to inhibit the mapk pathway. the h (human large cell lung cancer), a and pc- cell lines (human lung adenocarcinoma) were obtained from american type culture collection. cells were grown in rpmi- medium supplemented with % fetal bovine serum (fbs), iu/ml penicillin and μg/ml streptomycin and were maintained at °c in % co in a humidified incubator. the cells were passaged twice a week and used in the exponential growth phase. cell viability assays cell viability was determined by the cck- assay. briefly, cells were plated into -well plates at~ cells per well until cell adhesion. cmr was added to the medium at the indicated concentration. after incubation with cmr for , and h, each well was incubated with μl of cck- solution per µl of medium for h away from light. the absorbance of each well was measured at nm by using a multimode detection platform (molecular devices, ca, usa) according to the instructions of the cck- assay kit. clonogenic survival assays cells in log phase were plated in six-well plates and pretreated with cmr at the indicated concentrations or with dmso as controls. ir treatment was delivered h later. irradiated cells were then maintained in culture medium for days. colonies greater than cells were counted as positive surviving colonies, and the number of colonies was normalized to that in the controls. mean inactivation doses were determined as previously reported by the fertil method [ ] , and the sensitizer enhancement ratio (ser) of cmr treatment was calculated as the ratio of mean inactivation dose control /mean inactivation dose cmr-treated . median effect analysis cells were treated with a constant ratio of doses of ir and cmr based on the corresponding lethal doses (ld) ld , and cell survival was determined by clonogenic survival assay. a plot of the log of the total dose versus the log of the reciprocal of the fraction of cells affected minus yielded a linear plot. the slopes and yintercepts of the plots were used to calculate the combination index (ci) with calcusyn version . software (biosoft, cambridge, uk). the ci values were interpreted as follows: < . = synergism; = additive; and > . = antagonism. sirna transfection for bip sirna treatment, cells were transiently transfected with sirna-scramble and sirna-bip (guannan biotech, hangzhou, china). the sirna sequences are as follows: sirna- : ′-gaaaucg aaaggaugguuaau- ′; sirna- : ′-ggagcgcauugauacuaga- ′; sirna- : ′-cagaugaagcuguagcgua- ′; and scramble sirna: ′-uucuccgaacgugucacgu- ′. forty-eight hours after sirna transfection, the cells were treated with cmr for survival analysis and calreticulin staining. immunofluorescent imaging confocal microscopy was used to visualize the formation of cytoplasmic vacuoles in h cells. cells were treated with µm cmr for h, followed by irradiation or no ir as a control. after treatment, the cells were washed twice with pbs and then fixed with % paraformaldehyde for min. triton x- ( . %) was used to permeabilize the cells for - min at room temperature, and the cells were blocked with % bsa in pbs for min. after washing with pbs, the cells were stained with the er marker calreticulin (cell signaling technology, danvers, ma, usa) and the nuclear dye dapi (dako, denmark). immunofluorescent images were acquired using a leica dmi confocal microscope (leica microsystems, german). at least cells from each experiment were counted at random to calculate the percentage of cells as "positive" if they displayed vacuoles. the results from at least three different experiments were averaged. transmission electron microscopy after cmr treatment, cells were fixed in . % glutaraldehyde for h and washed in . m phosphate buffer three times. the samples were immersed in % osmium tetroxide for h, and % uranyl acetate was used for sample dyeing for min. then, the cells were dehydrated by a graded ethanol series for min and % acetone twice. the processed cells were embedded in embedding medium for h and heated at °c for polymerization. the ultrathin sections were obtained by cutting in a microtome. after staining with uranyl acetate and lead citrate, the sections were examined under a transmission electron microscope (hitachi, ibaraki, japan) at kv of accelerating voltage. annexin v-fitc/pi assay apoptosis was examined using an annexin v-fitc apoptosis detection kit (bd biosciences, nj, usa). cells were prepared according to the manufacturer's instructions. briefly,~ × cells were harvested, washed twice with cold pbs and stained with annexin v-fitc and pi in × binding buffer before flow cytomety (fcm) analysis. the populations of apoptotic cells were determined using a becton dickinson facscanto ii. both early apoptotic (annexin v-positive and pi-negative) and late apoptotic (annexin v-positive and pi-positive) cells were included in determining total apoptosis. western blot analysis cells were collected and washed twice with precooled pbs. cells were then lysed in ripa buffer containing protease and phosphatase inhibitors (thermo scientific, ma, usa). twenty microliters of lysates were used for western blot analyses using antibodies against phospho-p , total-p , phospho-erk, total-erk, phospho-jnk, total-jnk, chop, bip, parp, caspase and β-actin (cell signaling technology, danvers, ma, usa). the western blot signals were detected by biorad chemidoc xrs+ (biorad, ca, usa) using a chemiluminescence kit. in vivo study h cells ( × in . ml of × hbss + % hsa) were inoculated subcutaneously into the right thighs of female nu/nu mice ( - weeks of age, charles river, beijing, china). the mice were randomized into four groups when the average tumor volume reached mm . the following treatments were given: (a) methylcellulose/tween as a vehicle; (b) cmr ( mg/kg every day) on days - by intraperitoneal injection; (c) ir ( gy) on day ; and (d) cmr + ir ( gy). tumors were measured twice a week, and tumor volumes were determined from caliper measurements of the tumor length (l) and width (w) and calculated according to the formula (l × w )/ . tumor growth inhibition (tgi) and antitumor efficacy were determined by using the following equation: [%tgi = ( − change in tumor volume in treatment group/change in tumor volume in control group) × ]. immunohistochemistry analysis h xenograft tumors were collected days after the treatment began. the tissue samples were fixed in % formaldehyde solution, embedded in paraffin and sliced into -μm sections. antigen retrieval was conducted on formalin-fixed paraffinembedded (ffpe) tissue sections incubated in retrieval buffer for min (dako, denmark). tissue samples were then incubated with an endogenous peroxidase blocker for min and then incubated with primary antibodies (cc , cell signaling technology, danvers, ma, usa) for min at room temperature. dako envision™ + system-hrp was used as a secondary antibody (dako, denmark). tumor sections were incubated with biotinylated primary antibody (dako, denmark) for ki immunohistochemical analysis. quantification of ki -and cc -positive signals was conducted using the ariol system (genetix, san jose, ca, usa). data are presented as the means ± sd. student's t test was used to determine the significance between groups. significance was defined at the level of p < . . the anticancer effect of cmr (molecular weight: . ) was evaluated in h , a and pc- cells. these cells were treated with cmr ( , , , , and μm) for , and h, and cell viability was then determined. we found that exposure to cmr obviously decreased the viability of h , a and pc- cells in a dose-dependent manner, suggesting that cmr has cytotoxic activity against lung cancer cells. the ic values of cmr in h , a and pc- cells were . , . and . μm at h; . , . and . μm at h; and . , . and . μm at h, respectively (fig. a) . microscopic observation showed that cmr treatment induced extensive cytoplasmic vacuolation in h , a and pc- cells. a significantly higher percentage of cytoplasmic vacuolated cells ( . % ± . % in h cells, . % ± . % in a cells and . % ± . % in pc- cells) was observed when cells were treated with μm cmr compared to that of control cells or cells that were treated with lower doses of cmr (fig. b) . low and medium doses of cmr caused paraptosis but not apoptosis it has been reported that cytoplasmic vacuolation induced by paraptosis is due to er stress [ , ] . as shown in fig. a , immunofluorescent staining showed that the edges of all cytoplasmic vacuoles in cmr-treated cells were stained with the er marker calreticulin (red color), indicating that the formation of vacuoles originated from er dilation. we also noticed that the percentage of cytoplasmic vacuoles formed in these cmr-treated cells increased in a dose-dependent manner. we used transmission electron microscopy (tem) to determine the potential ultrastructural changes in h and a cells in response to cmr treatment. we found that after h of treatment with μm cmr, empty vacuoles with no visible materials observed inside the vacuoles had formed. swollen er cisternae also occurred. however, we did not observe dna fragmentation or chromatin condensation in the nuclei of lung cancer cells after cmr treatment (fig. b ). in addition, annexin v-fitc analysis showed that cmr treatment did not induce significant apoptosis in h and a cells, although slight increases in apoptotic cell death were detected in both cell lines when the cells were treated with μm of cmr for h (fig. c) . cmr caused er stress-dependent paraptosis of lung cancer cells we next analyzed the protein expression levels of the er stress markers bip and chop in lung cancer cells following treatment with cmr. we found that the expression of bip and chop proteins significantly increased in parallel with increasing doses of cmr in h and a cells. however, we did not detect any obvious changes in the protein expression of alg- -interacting protein x (alix), which is an inhibitor of paraptosis [ ] , or the autophagyspecific marker lc b. we also determined the changes in protein expression of three other er transmembrane sensors, inositol requiring enzyme (ire ), protein kinase rna (pkr)-like er kinase (perk) and activating transcription factor (atf ), and our results showed that the expression of ire , p-perk and atf increased in h cells when the cells were treated with μm cmr. in a cells, however, we detected increased protein expression of ire but not p-perk or atf in cmr-treated cells. on the other hand, we did not detect any changes in apoptosis-related caspase- (neither the full-length protein nor cleaved fragments) or its substrate parp in h and a cells after cmr treatment (fig. a) . taken together, these results suggest that exposure to low and medium concentrations of cmr induces paraptosis but not apoptosis in nsclc cells. these observed changes in er stress proteins indicate a potential involvement of er stress in the paraptosis process of cells treated with cmr. we thus evaluated the role of an er stress protein on cmr-induced paraptosis in h cells. we found that knocking down bip by sirna transfection (fig. b) not only decreased the cell-killing effect of cmr treatment (fig. c) but also reduced the percentage of cytoplasmic vacuoles (fig. d) in h cells. therefore, we suggest that cmr exposure induces paraptosis through modulation of er stress proteins. we further evaluated the potential radiosensitization effects of cmr on h and a cells. for this, we performed a clone formation assay to determine the ser. the cells were pretreated with , , or μm cmr and then irradiated with different doses of ir. we found that pretreatment with cmr enhanced the sensitivity of cells to radiotherapy, and the ser values reached . for h cells and . for a cells at a cmr dose of μm, . for h cells and . for a cells at a dose of μm, and . for h cells and . for a cells at a cmr dose of μm (fig. a) . we also determined the ci for cmr combined with ir in h and a cells by treating the cells with constant ratios of corresponding ld of cmr and ir. our results showed that combination treatment with ir and cmr led to an overall ci value of < . , indicating a synergistic radiosensitization effect of cmr on h cells (fig. b) . apoptosis analysis further showed that, while the percentage of apoptotic cells increased in a dose-dependent manner in cells in response to ir treatment, pretreatment with cmr had no effect on the apoptotic response to ir in h or a cells (p > . , fig. c ). however, we detected dramatically increased cell populations with vacuole formation in irradiated h cells (from . % to . %) and a cells (from . % to . %) when cells were pretreated with cmr. notably, no such change was observed in irradiated control cells that were not cotreated with cmr (fig. d) . consistent with the above observations, western blot analysis showed that the expression of bip and chop proteins increased only in the cells treated with the combination of cmr and ir. in addition, we also noticed that when cells were pretreated with μm cmr, the protein levels of ire and atf and the phosphorylation level of perk (p-perk) increased in h cells in response to ir exposure. however, in a cells, only ire protein levels obviously increased in cells that were cotreated with cmr and ir (fig. e) . to understand the detailed mechanism by which cmr treatment induces paraptosis in nsclc cells, we analyzed the signal activation of the p /erk/jnk mapk pathway in cells treated with ir cmr, individually or in combination. western blot analysis revealed that treatment with ir or cmr alone upregulated the phosphorylation levels of erk, jnk and p in h cells compared to those of the control cells. of note, the most significant change in these phosphorylation events was observed in cells treated with the combination of ir and cmr (fig. a) . adding the mapk pathway inhibitor u , which markedly blocked the basal level of the phosphorylation of erk proteins, to the culture medium of h cells before treatment with ir, cmr or the combination, blocked the basal protein expression and increase in bip and chop proteins in response to the cotreatment with ir and cmr (fig. b) , and it also decreased the percentage of vacuolated cells in response to cmr alone (from . % ± . % to . % ± . %) or cmr combined with ir (from . % ± . % to . % ± . %, fig. c ). the combination of cmr and radiation therapy inhibits tumor growth in h xenografts we further extended our study to an in vivo model. in this experiment, h cells were inoculated into nu/nu mice to establish xenografts, and the effects of cmr, ir or cmr combined with ir on tumor growth were assessed. our results showed that treatment with a single dose of ir ( gy) or daily oral treatment with cmr ( mg/kg for days) inhibited in vivo h tumor growth with tgi of . % and . %, respectively. interestingly, we noticed that the combination treatment of ir and cmr caused significantly enhanced tumor growth inhibition up to . % (fig. a) . the tolerability analysis measured mouse body weight and showed that treatments with cmr or cmr combined with ir did not cause obvious body weight changes during the experiment, suggesting that treatment with ir combined with cmr was well tolerated (fig. s ) . western blot analysis showed that cmr treatment or the combination treatment led to an upregulation of bip and chop and activation of p-erk in xenograft tumor tissues (fig. b) . we also noticed that, although treatment with ir or cmr alone reduced ki staining in h xenograft tumors, combination treatment further decreased the level of ki staining in tumor cells. exposure to cmr, however, did not increase the ir-induced positive staining of cleaved caspase (cc ) in the tumor tissues ( fig. c and table s ). radiation therapy is an important component of cancer treatment, and more than % of cancer patients will receive radiotherapy during clinical management of the disease. radiotherapy also contributes to % of the curative treatments for cancer patients. for nsclc patients, concurrent chemotherapy and radiation therapy is the standard care for local advanced patients; however, the clinical outcomes remain unsatisfactory, with a median progression-free survival of - months. recently, a multicenter, open-labeled, randomized phase ii trial showed that targeted therapy with an egfr inhibitor combined with radiation provides a statistically significant pfs improvement ( . vs. . months) compared to that of chemotherapy plus radiotherapy in unresectable stage iii nsclc with an activating egfr mutation, indicating that lung cancer patients with an egfr mutation can benefit from this new therapeutic strategy [ ] , although nearly % of nsclc patients cannot benefit from this treatment because patients have tumors that do not harbor an egfr mutation. however, the success of the combination of ir with egfr-targeting chemotherapy suggests the clinical potential of developing a novel radiotherapeutic strategy for nsclc patients. studies have revealed that natural products can sensitize cancer cells to radiation therapy [ , ] . the mechanisms by which the natural components synergize with ir to facilitate cancer cell killing are usually by enhancing apoptosis, affecting the cell cycle, and/or attenuating angiogenesis [ ] [ ] [ ] [ ] [ ] . in this study, we demonstrated that cmr, a type of diels-alder adduct from mulberry leaves, enhances the radiosensitivity of nsclc cells by inducing paraptosis. previous studies have shown the anticancer activity of cmr in various human cancer cell lines, but little is known about its effects on lung cancer. our current data showed that cmr significantly induced cell killing. of interest, we found that cmr treatment induced er dilation and cytoplasmic vacuolation in nsclc h cells. immunofluorescence analysis and tem imaging further showed that in cmr-treated cells, the er underwent swelling and fusion. in addition, cmr treatment enhanced the expression of er stress markers, including the transcription factors bip and chop. this evidence indicates that cmr kills cancer cells through paraptosis. however, we noticed that cmr treatment did not cause activation of caspase- and parp, which indicates that apoptosis is not a cause of cmrinduced cell death in these cancer cells. in the combination treatment experiment, we also demonstrated that the synergistic effect of cmr on ir-induced cell killing in nsclc cells involved paraptosis but not apoptosis. thus, cmr treatment triggers paraptosis in nsclc cells, which results in significant cancer cell death and a synergistic cell-killing effect of cells in response to ir. of note, however, we noticed that the expression of all three er transmembrane sensors, ire , perk and atf , was enhanced in cmr-treated h cells, while only ire protein expression was increased in a cells, indicating the potential of different er sensors to be induced by er stress in distinct lung cancer cells [ ] . paraptosis is a nonapoptotic pathway whose main features are extensive cytoplasmic vacuolation and the absence of significant cell membrane blebbing and nuclear shrinkage. paraptosis is a molecular pathway that is independent of caspases, and although studies have not fully explored the detailed molecular mechanism of paraptosis, it is proposed that the mapk pathway may also participate in the development and the process of paraptosis [ , , ] . we also found in our study that cmr treatment activated the mapk pathway. however, our results showed no changes in alix protein expression in cmr-treated cells, which is contrary to the results reported in han's study [ ] . the discrepancy in alix involvement in cmr-triggered paraptosis suggests the potential involvement of other molecular signaling pathways in paraptosis in lung cancer cells. regarding the activation of mapk signaling, previous studies have shown that exposure of cells to ir and to a variety of other toxic stresses induces simultaneous compensatory activation of multiple mapk pathways [ ] . interestingly, we observed that radiation treatment combined with cmr enhanced the mapk pathway and increased the percentage of cytoplasmic vacuolation, which could explain the synergistic effect of cmr combined with irradiation. most importantly, we found that paraptosis in cmr-treated cancer cells or in cancer cells treated with the combination of cmr and ir was inhibited when the cells were exposed to the specific phospho-erk inhibitor u , indicating the role of mapk activation in cmr-induced cell killing and paraptosis in nsclc cells. mapk activation mediates cmr-induced paraptosis. a total cell lysates were collected from h cells after the indicated treatments and analyzed for phosphorylation and total erk, jnk and p proteins. anti-β-actin antibody was included as a loading control. b h cells were treated with cmr and radiation, with or without u . cell lysates were collected and analyzed for phosphorylation and total erk, jnk and p protein, as well as chop and bip. c cytoplasmic vacuolation induced in h cells with the indicated treatments was observed by immunocytochemistry; * indicates statistical significance. the scale bar represents μm, and all images are at the same magnification. in conclusion, we report for the first time that the anticancer activity and radiosensitization effect of cmr induces paraptosis. our data suggest that cmr could be considered a potential anticancer agent and radiation sensitizer in future cancer therapeutics. fig. h xenograft tumors were treated with cmr, radiation or the combination. a tumor growth was measured as described in the materials and methods. the growth curves represent the average values of six mice in each group. error bars indicate the standard deviation. b western blot. xenograft tumor tissues were collected after days of the indicated treatments. western blot analysis was performed to test the changes in p-erk, chop and bip. c immunohistochemistry analysis of the expression of ki and the apoptotic marker cc . positive staining was determined for each group (n = animals/group). the scale bar represents μm, and all images are at the same magnification. epidemiology, incidence and mortality of lung cancer and their relationship with the development index in the world targeting immune checkpoints in non small cell lung cancer acquired resistance to egfr targeted therapy in non-small cell lung cancer: mechanisms and therapeutic strategies before or after: evolving neoadjuvant approaches to locally advanced non-small cell lung cancer h inhibits tumor growth and induces apoptosis via intrinsic and extrinsic signaling pathway in human non-small cell lung cancer xenografts cucurbitacin b inhibits cell proliferation and induces cell apoptosis in colorectal cancer by modulating methylation status of btg daucosterol disturbs redox homeostasis and elicits oxidative-stress mediated apoptosis in a cells via targeting thioredoxin reductase by a p dependent mechanism upregulation of bcl in nsclc with acquired resistance to egfr-tki actein inhibits cell proliferation and migration and promotes cell apoptosis in human non-small cell lung cancer cells non-autophagic roles of autophagy-related proteins caspases: a molecular switch node in the crosstalk between autophagy and apoptosis chalcomoracin and moracin c, new inhibitors of staphylococcus aureus enoyl-acyl carrier protein reductase from morus alba constituents of cultivated mulberry tree mulberry diels-alder adducts: synthesis of chalcomoracin and mulberrofuran c methyl ethers paraptosis: mediation by map kinases and inhibition by aip- /alix xanthohumol induces paraptosis of leukemia cells through p mitogen activated protein kinase signaling pathway cell death independent of caspases: a review a novel role for map lc in nonautophagic cytoplasmic vacuolation death of cancer cells uv-b induced changes in the secondary metabolites of morus alba l. leaves mean inactivation dose: a useful concept for intercomparison of human cell survival curves endoplasmic reticulum vacuolation and unfolded protein response leading to paraptosis like cell death in cyclosporine a treated cancer cervix cells is mediated by cyclophilin b inhibition small-cell carcinoma transformation of pulmonary adenocarcinoma after osimertinib treatment: a case report natural product betathujaplicin inhibits homologous recombination repair and sensitizes cancer cells to radiation therapy triolimus: a multi-drug loaded polymeric micelle containing paclitaxel, -aag, and rapamycin as a novel radiosensitizer epigenetic interventions increase the radiation sensitivity of cancer cells promotion of autophagy as a mechanism for radiation sensitization of breast tumor cells icotinib hydrochloride enhances chemo-and radiosensitivity by inhibiting egfr signaling and attenuating rad expression and function in hela s cells study of the radiotherapy sensitization effects and mechanism of capecitabine (xeloda) against non-small-cell lung cancer cell line a danshensu, a major watersoluble component of salvia miltiorrhiza, enhances the radioresponse for lewis lung carcinoma xenografts in mice role of endoplasmic reticulum stress in the anticancer activity of natural compounds taxol induces paraptosis independent of both protein synthesis and mapk pathway chalcomoracin is a potent anticancer agent acting through triggering oxidative stress via a mitophagy-and paraptosis-dependent mechanism mapk pathways in radiation responses this study was supported by grants from the zhejiang provincial natural science foundation (ly h ), the national natural science foundation of china ( , ) and major project of the hangzhou science and technology bureau ( a ). srz, slm and xqc designed the research; srz, xcz, jfl and yyz performed the research study; srz, hxh and hmf made the substantial contributions to analysis and interpretation of data; srz, hxh and xcz wrote the paper. all the authors approved the final version and agreed for publication. the online version of this article (https://doi.org/ . /s - - - ) contains supplementary material, which is available to authorized users.competing interests: the authors declare no competing interests. key: cord- - m m v authors: yu, hai-jun; de geest, bruno g title: nanomedicine and cancer immunotherapy date: - - journal: acta pharmacol sin doi: . /s - - - sha: doc_id: cord_uid: m m v nan this special issue "nanomedicine and cancer immunotherapy" was organized for the celebration of the th anniversary of acta pharmacologica sinica (aps). the prime aim for the special issue is to deliver both high-impact review and research articles to the wide international readership regarding the most recent progress in nano-immunotherapy as well as the approaches for enhancing the efficacy of cancer immunotherapy. primary tumors are conventionally treated by surgery, chemotherapy and radiotherapy. however, tumor relapse and ultimate therapeutic failure remained a formidable challenge in the clinic. the innate and adaptive immune system can make a substantial contribution to the therapeutic effects of conventional cancer treatments. circumstantial evidence in pre-clinical studies suggests that the long-term success of cancer therapies lies in immunotherapy. therefore, cancer immunotherapy is considered as an effective treatment modality to eliminate primary and metastatic tumors as well as to establish immunological memory. nanomedicines can simultaneously deliver various immunological agents to the intended target site (tumor or lymph node). the ultimate application of nanomedicine is to reprogram or modulate the immune response by precisely targeting biological pathways. to increase cancer immunotherapies' impact, we organized this special issue to deepen our understanding about the mechanisms underlying the tumor and immune cells to evade immunity, how nanomedicines can be used to reprogram the tumor microenvironment and how nanomedicines interact with the immune system, and the potential challenges and the critical limitations of immunotherapy approaches, which impede their clinical applications. this issue includes one research article and ten review articles, and brief introductions to each topic are illustrated as below. the advent of immunotherapy is a game changer in cancer therapy with monoclonal antibody and t-cell-based therapeutics being the current flagships. however, small molecule immunotherapeutics might offer advantages over biologicals in terms of complexity, tissue penetration, manufacturing cost, stability and shelf-life. however, small molecule drugs are prone to rapid systemic distribution which might induce severe off-target side effects. nanotechnology could aid in the formulation of these drug molecules to improve their delivery to specific immune cell subsets. the review article by de geest et al. summarizes the current efforts in changing the pharmacokinetic profile of small molecule immunotherapeutics with a strong focus on toll like receptor agonists [ ] . the review article by li and nie et al. introduces the strategies being employed using nanoscale intelligent drug delivery systems to enhance the effects of cancer immunotherapy by promoting drug delivery to the tumor. the review article also provides a perspective on the further possible application of nanoparticles in more effective anti-tumor immunotherapy [ ] . clinical approval of immune checkpoint inhibitors (e.g., ipilimumab, pembrolizumab) has been accelerated by a dramatic long-term survival improvement in a small subset of patients over conventional chemotherapy. however, it should be noted that the majority of treated cancer patients cannot benefit from checkpoint blockade therapies by releasing the t-cell function. poor t-cell response, low immunogenicity and the immunosuppressive environment of tumors, create great challenges for present cancer immunotherapy. nanotechnology can integrate multiple functions within controlled size and shape, and has been explored as a unique avenue for the development of cancer immunotherapy. the review article by luo et al. mainly addresses how nanoengineered vaccine can induce robust t cells response against tumor, as well as how nanomedicine can remodel immunosuppressive tumor microenvironment (itm) to boost anti-tumor immune responses [ ] . advanced nanobiomaterials, including liposomes, polymers, and silica, play a vital role in the codelivery of drugs and immunomodulators. these nanobiomaterial-based delivery systems could effectively promote antitumor immune responses and simultaneously reduce toxic adverse effects. furthermore, nanobiomaterials may also combine with each other or with traditional drugs via different mechanisms, thus giving rise to more accurate and efficient tumor treatments. the review article by qian et al. provides an overview of the latest advancement of nanobiomaterials used for cancer immunotherapy including lipid-or micelle-based nanoparticles, polymer-based scaffolds, inorganic nanosystems, etc… [ ] . a diversity of immunomodulatory agents, including tumorassociated antigens, adjuvants, cytokines and immunomodulators, have been explored for their ability to induce a cascading adaptive immune response. nanoscale metal-organic frameworks (nmofs) are attractive for cancer immunotherapy because they feature tunable pore size, high surface area and loading capacity, and intrinsic biodegradability. the review article by sun et al. overviews the recent progresses in the development of nmofs for cancer immunotherapy, including cancer vaccine delivery and combination of in situ vaccination with immunomodulators to reverse immune suppression. current challenges and future perspectives for rational design of nmof-based cancer immunotherapy are also discussed [ ] . central nervous system (cns) disorders represent a broad spectrum of brain ailments with short and long-term disabilities. despite the various nanomedicine-based platforms, translating therapeutic agents to the clinic is costly and often hampered by regulatory hurdles. a variety of potential drugs have been discovered to treat several neuronal disorders; however, therapeutic success is still limited by the presence of the blood-brain barrier (bbb). furthermore, the unique immune mechanism of the cns demands more consideration of immune-based therapeutic approaches for cns diseases. the review article by shi et al. discusses the efficacy of nanomedicines that utilize immunotherapy to combat cns disorders for enhanced safety, efficacy, and drug delivery specificity [ ] . combining nanomedicines with immunotherapy aims to reinforce the cancer-immunity cycle, via potentiating key steps in the immune reaction cascade, namely antigen release, antigen processing, antigen presentation and immune cell-mediated tumor killing. combination nano-immunotherapy can be realized via three targeting strategies, i.e. by targeting cancer cells, targeting the tumor immune microenvironment and targeting the peripheral immune system. the clinical potential of nano-immunotherapy has recently been demonstrated in a phase iii trial in which nano-albumin paclitaxel (abraxane®) was combined with atezolizumab (tecentriq®) for the treatment of patients suffering from advanced triple-negative breast cancer. the review article by shi and lammers et al. summarizes the bedsides strategies and initial (pre)clinical success stories, and discusses several key challenges in nano-immunotherapy [ ] . as the most powerful antigen-presenting cell type, dendritic cells (dcs) can induce potent antigen-specific immune responses in vivo, hence becoming the prime cell population for vaccination purposes. dcs can be derived ex vivo in large quantity and manipulated extensively to be endowed with adequate immunestimulating capacity. after pulsing with cancer antigens in various ways, the matured dcs are administered back into the patient. dcs home to lymphoid organs to present antigens to and activate specific lymphocytes that react to a given cancer. ex vivo pulsed dc vaccines have been vigorously investigated for decades, registering encouraging results in relevant immunotherapeutic clinical trials, while facing some solid challenges as well. the review article by song et al. reports on the advances of dc vaccine-based cell immunotherapy, in particular the application of mrna technology for the fabrication of dc vaccines [ ] . cancer immunotherapy has attracted extensive attention due to its ability to activate the patients' innate or adaptive immune systems to combat tumors. despite a few clinical successes, further endeavors are still needed to tackle unresolved issues including a limited response rate, development of acquired immune resistance and immune-related toxicities. accumulating evidence has pinpointed tumor microenvironment (tme) as one of the major obstacles against cancer immunotherapy due to its detrimental impacts on tumor-infiltrating immune cells. nanomedicine has been battling with the tme in the past decades and experience obtained could be exploited to improve the current paradigms of immunotherapy. the review article by wang et al. discusses the metabolic features of tme and its influence on different types of immune cells, and summarizes the recent progress of nano-enabled cancer immunotherapy by modulation of immune cells, tumor stroma, cytokines and enzymes to revert immunosuppressive tme [ ] . over the past years, several treatment modalities including radiotherapy, chemotherapy, photothermal and photodynamic therapy, have been exploited to elicit immunogenicity by inducing immunogenic cell death (icd). however, icd-based immunotherapy is restricted by the itm limiting its efficacy in eliciting a long-term anti-tumor immune response as well as severe systemic toxicity. to address these challenges, nanomedicine-based drug delivery strategies have been exploited to improve cancer immunotherapy by boosting icd of tumor cells. nanosized drug delivery systems are promising for increasing drug accumulation at the tumor site and co-delivering icd inducers and immune inhibitors to simultaneously elicit the immune response and relieve the itm. the review article by yu et al. highlights the recent advances in nanomedicine-based immunotherapy utilizing icd-based approaches [ ] . the research article by rock et al. reports enzyme-directed immunostimulant (edi) prodrugs for cancer immunotherapy. the tested edis feature an imidazoquinoline immunostimulant (resiquimod-toll-like receptor / agonist) covalently modified with glycosidase enzyme-directing groups selected from substrates for β-glucuronidase, α-mannosidase, or β-galactosidase. the elicited immunogenicity was compared for different cell types and found to be independent of the glycosidase substrate in the edi or differences in functional glycosidase expression across each cell line. this study highlights the broad applicability of edis to the cancers that exhibit drug efflux, which suggest that edis optimized for drug efflux could be particularly beneficial for improving cancer immunotherapy [ ] . we would like to express our sincere gratitude to all the eminent contributors, who have made such significant contributions to this special issue in acta pharmacologica sinica, and would also like to extend our gratitude to all the peer-reviewers for their insights and thoughtful comments, which significantly improved the quality of this issue. meanwhile, we want to express our deep gratitude to professor jian ding, the editor-in-chief, and mrs. ming-shu wu, manager-in-chief of acta pharmacologica sinica, for their invitation and kindly help throughout the whole process. without their initiative and encouragement, this special issue would not have been possible. last but not least, we must greatly acknowledge ms. juan huang and mr. jia xu, the production editors of acta pharmacologica sinica, for their remarkable work done for this special issue. the last but not the least, it is our hope that all of the articles in this special issue will be found helpful and useful to both established and new investigators in the field of nanomedicine and cancer immunotherapy. nanomedicine-mediated altering of the pharmacokinetic profile of small molecule cancer immunotherapeutics reversal of the immunosuppressive tumor microenvironment by nanoparticle-based activation of immune-associated cells nanoengineered targeting strategy for cancer immunotherapy advanced biomaterials for cancer immunotherapy nanomedicines based on nanoscale metal-organic frameworks for cancer immunotherapy nanomedicine-based immunotherapy for central nervous system disorders cancer nanomedicine meets immunotherapy: opportunities and challenges ex vivo pulsed dendritic cell vaccination against cancer harnessing nanomedicine to overcome immunosuppressive tumor microenvironment engineering nanomedicines for improved cancer immunotherapy by boosting immunogenetic cell death comparing the immunogenicity of glycosidase-directed resiquimod prodrugs mediated by cancer cell metabolism the authors declare no competing interests. key: cord- -y pc qfc authors: zhou, bo-ya; wang, wen-bo; wu, xiao-li; zhang, wen-jie; zhou, guang-dong; gao, zhen; liu, wei title: nintedanib inhibits keloid fibroblast functions by blocking the phosphorylation of multiple kinases and enhancing receptor internalization date: - - journal: acta pharmacol sin doi: . /s - - -y sha: doc_id: cord_uid: y pc qfc keloid is a benign skin tumor characterized by its cell hyperproliferative activity, invasion into normal skin, uncontrolled growth, overproduction and deposition of extracellular matrices and high recurrence rate after various therapies. nintedanib is a receptor tyrosine kinase inhibitor targeting vegf, pdgf, fgf, and tgf-β receptors with proved efficacy in anti-angiogenesis and in treating various types of cancers. in this study, we investigated the effects of nintedanib on keloid fibroblasts in both in vitro and ex vivo models. keloid fibroblasts were prepared from keloid scar samples in active stages collected from patients. we found that nintedanib ( − μm) dose-dependently suppressed cell proliferation, induced g( )/g( ) cell cycle arrest, and inhibited migration and invasion of keloid fibroblasts. the drug also significantly inhibited the gene and protein expression of collagen i (col- ) and iii (col- ), fibronectin (fn), and connective growth factor (ctgf), as well as the gene expression of other pathological factors, such as alpha smooth muscle actin (α-sma), plasminogen activator inhibitor- (pai- ), fk -binding protein (fkbp ), and heat shock protein (hsp ) in keloid fibroblasts. furthermore, nintedanib treatment significantly suppressed the phosphorylation of p , jnk, erk, stat , and smad, enhanced endocytosis of various growth factor receptors. using an ex vivo tissue explant model, we showed that nintedanib significantly suppressed cell proliferation, migration, and collagen production. the drug also significantly disrupted microvessel structure ex vivo. in summary, our results demonstrate that nintedanib is likely to become a potential targeted drug for keloid systemic therapy. keloids are characterized by uncontrolled growth beyond the original lesion, hyperproliferative fibroblasts and overproduction and deposition of extracellular matrices (ecm). thus, keloids are considered as benign skin tumors [ , ] , and are suitable for anticancer strategies [ , ] . in addition, chemotherapy agents targeting signaling pathways are good drug candidates for cancer therapy. previous studies showed the involvement of abnormal signaling pathways, including the transforming growth factor-β (tgf-β)/smad, phosphatidylinositol -kinase (pi k)/mammalian target of rapamycin (mtor) and mitogen-activated protein kinase (mapk)-signaling pathways, in keloid development [ , ] . additionally, enhanced cell proliferative and migratory abilities, abnormal angiogenesis, and vascular hyperpermeability have also been found to be enhanced by vascular endothelial growth factor (vegf), platelet-derived growth factor (pdgf), and fibroblast growth factor (fgf) in keloid development [ , ] . recent progress in this field also demonstrated that inhibitors of multiple signaling pathways are the preferred targeted drugs due to their synergistic mechanisms of antagonizing different signaling pathways [ ] . nintedanib (bibf- ) is a receptor tyrosine kinase inhibitor (tki) targeting vegf receptors (vegfrs), fgf receptors (fgfrs), and pdgf receptors (pdgfrs) [ ] with proven efficacy in antiangiogenesis and in treating various types of cancers, including hepatocellular carcinoma (hcc), renal cell carcinoma (rcc), colorectal cancer (crc), prostate cancer, and gynecologic malignancies [ ] . for example, nintedanib reduced vessel density, vessel permeability, and tumor growth by inhibiting the pi k-akt and mapk-signaling pathways [ ] . nintedanib was also reported to reduce fibrotic progression by inhibiting early events in tgf-β signaling, involving phosphorylation of the type ii tgf-β receptor, activation of smad , and p mapk phosphorylation [ ] . in particular, nintedanib is the first targeted chemotherapy drug for idiopathic pulmonary fibrosis (ipf) therapy in patients, indicating the therapeutic potential of tkis in the treatment of nonmalignant diseases. previous studies have demonstrated that pdgfr, vegfr, and fgfr are usually localized at low-density, detergent-resistant membrane domains called lipid rafts/caveolae [ , ] . lipid rafts/ caveolae are small cholesterol-enriched domains present on the cell membrane. when cholesterol is depleted, receptors are retained in the residual elements of the caveolae, and remain able to bind the ligand, but the downstream cascades are blocked due to steric inhibition and the inability to form a signaling complex [ ] . it has been shown that the keloid mechanism involves multiple signaling pathways, such as vegf, pdgf, fgf, and tgf-β, but there has been no report on a drug that could target more than three growth factors among the studies on drugs targeting keloid cell behavior [ , , ] . this study aimed to explore the targeted drug effect of nintedanib on antagonizing multiple signaling pathways related to vegf-r, fgf-r, pdgf-r, and tgf-β-r and examine its therapeutic effect on keloid fibroblast behaviors, including cell proliferation, cell cycle progression, overproduction of extracellular matrix, cell migration, and cell invasion, using in vitro and ex vivo models. in addition, we also investigated whether disruption of the lipid raft/caveolae structure is involved in the drug effects. patients and keloid samples fifty-four keloid scar samples in active stages were collected from patients without any previous treatment before surgery (see details listed in table ). keloid specimens derived from surgical excision were donated by the patients for research purposes only with written informed consent. the procedures for processing human tissues and cells were approved by the ethics committee of the shanghai jiao tong university school of medicine. chemical reagents nintedanib (selleck chemicals, houston, tx, usa) was dissolved in % dimethyl sulfoxide (dmso) to a stock concentration of mm and stored at − °c under light-protected conditions. the reagent was diluted directly to the desired dose upon use, with an identical final concentration of dmso in both the experimental and control groups. the final concentration of dmso was maintained below . % (v/v) in all of the following experiments to ensure no cytotoxic effect on the cultured kfs. isolation and culture of keloid fibroblasts isolation and culture of keloid fibroblasts were performed according to a previous protocol [ ] . keloid tissues were treated with collagenase nb (serva electrophoresis, heidelberg, germany) dissolved in dulbecco's modified eagle's medium (dmem, hyclone, logan, ut, usa, . % v/v) for h at °c with rotation. after digestion, cells were collected and resuspended in dmem supplemented with % fetal bovine serum (fbs, gibco-invitrogen corp., grand island, ny, usa) and penicillin/streptomycin (gibco) and seeded onto -cm culture dishes. the primary, first-passage cells were utilized in the subsequent experiments. to better represent the keloid cases, kfs derived from three patient tissue samples were mixed as a pooled cell sample. cell counting kit- (cck- ) assay the drug effect on kf proliferation was analyzed by cck- (dojindo laboratories, mashiki, japan). briefly, primary cultured kfs were seeded in -well plates per well and starved for h. then, the medium was replaced with culture medium with or without nintedanib, and the cells were tested with cck- reagent at days , , , , and . the medium optical density value of each well was measured at nm using a microplate reader (thermo fisher scientific, waltham, ma, usa). the assay was carried out in quadruplicate and repeated with three independent pooled cell samples. cell cycle analysis as previously described [ ] , kfs treated with nintedanib at different concentrations ( , . , , , μm) for days were collected. the cells were then fixed in % ethanol at °c overnight followed by staining with a cell cycle analysis kit ( sea biotech, shanghai, china). afterwards, a flow cytometer (beckman coulter, fullerton, ca, usa) equipped with modifit lt v . software was used for flow cytometric analyses, and the experiment was repeated in four independent pooled cell samples. female asian chest female asian arm male asian chest female asian shoulder female asian shoulder male asian chest female asian chest male asian chest female hoechst-positive cells (blue) were imaged under a fluorescence microscope (olympus, tokyo, japan) and counted using image-pro plus (ipp) . software (media cybernetics, silver spring, md, usa). the incorporation ratio of edu-positive cells is shown as the percentage of edu-positive cells of the total hoechst-positive cells. the results were derived from the average cell counts in five randomly selected fields in a well and repeated with three independent pooled cell samples. cell migration assay for the scratch assay, as previously described [ ] , the cell monolayer was scratched using a -μl pipette tip when the cells reached more than % confluence, and the cells were then cultured in serum-free medium with or without nintedanib for h. images were acquired at , , and h after scratching. the results were derived from measured areas in six randomly selected fields in a well and repeated with six independent pooled cell samples. a transwell system ( -μm pore size) was also utilized for the migration assay as reported [ ] . in brief, × serum-starved kfs were seeded in the upper compartment of the boyden chamber in a -well plate in serum-free medium containing nintedanib or vehicle control. after incubation for h, the migrated cells were fixed with % paraformaldehyde and stained with ′, -diamidino- -phenylindole (dapi, sigma, chemical co., st. louis, mo, usa). five high-power fields were randomly selected, and the migrated cells were counted by image-pro plus. this assay was performed in three independent pooled cell samples. furthermore, an oris tm cell migration assay kit (platypus technologies, madison, wi, usa) was used as reported [ ] . briefly, . × kfs were seeded in each well, and after h, the medium was replaced with μl serum-free medium containing different concentrations of nintedanib after removing the stoppers. after another -h incubation, cells were stained with calcein-am (maiyuer bio, shanghai, china). the cell number in the migration zone was counted by image-pro plus. this assay was performed with three independent pooled cell samples. cell invasion assay for the transwell assay, × serum-starved kfs were placed in the upper well of a boyden chamber coated with matrigel (corning). after a -h incubation, invaded cells were fixed and stained with dapi. cell numbers in five randomly selected highpower fields were counted by image-pro plus. the assay was repeated with three independent pooled cell samples. furthermore, an oris tm d embedded invasion assay (platypus technologies) was performed to confirm the inhibitory effect of the drug on kf invasive ability as previously described [ ] . briefly, . × kfs per well were seeded, and after h, the medium was replaced by μl rat-tail collagen i (col- ) solution. after incubation for min, serum-free medium containing nintedanib or vehicle control was added to each well. images of kfs stained with calcein-am were obtained at h post treatment. cells in the detection zone were counted by image-pro plus. this assay was performed with three independent pooled cell samples. ex vivo explant culture of human keloid tissues keloid tissue after epidermal removal was minced into mm × mm × mm fragments. the dermal fragments were equally treated, seeded onto three -cm culture dishes and incubated in dmem containing % fbs until attachment. then, the medium was replaced with fresh medium containing nintedanib or vehicle control. representative images were acquired at the same location on days , , , and after the kfs migrated from the edge of the tissue. on day , the tissue fragments were collected for rt-qpcr and western blotting assays, and the kfs growing in the dishes were collected and counted using a hematocytometer. rna isolation and real-time quantitative pcr after treatment with nintedanib or vehicle control for h in dmem plus % fbs, the cultured kfs were harvested for total rna extraction using trizol reagent (invitrogen life technologies inc., grand island, ny, usa) as previously reported [ ] . for the ex vivo explants, the tissues were minced with scissors and ground by a pro- tissue homogenizer (pro scientific, monroe, ct, usa) to a homogeneous lysate and prepared for rna isolation using trizol reagent. complementary dna (cdna) was synthesized from µg total rna per sample using amv reverse transcriptase (promega, madison, wi, usa). cdna was amplified using rt-qpcr in a realtime thermal cycler (stratagene, la jolla, ca, usa) with power sybr green pcr master mix ( ×) (applied biosystems, foster city, ca, usa) as previously described [ ] . the housekeeping gene β-actin was used as an internal control. each assay was performed in triplicate and repeated with three independent pooled cell samples. the human primers for real-time qpcr analysis are displayed in table . immunofluorescence assay briefly, × kfs per well (for alpha smooth muscle actin, α-sma detection) or × kfs (for col- detection) were seeded in sixwell plates and preincubated for h in regular culture medium. afterwards, the medium was replaced with dmem containing % fbs with or without nintedanib for h (for α-sma detection) or days (for col- detection) followed by fixation in % paraformaldehyde overnight. the primary and secondary antibodies (table ) were diluted according to the instructions. dapi was used for nuclear counterstaining. images of α-sma-positive or col- -positive (green) and dapi nuclear-stained (blue) cells were obtained under a fluorescence microscope (olympus). this assay was performed with three independent pooled cell samples. keloid specimens with a size of mm × mm × mm were fixed in % paraformaldehyde at °c overnight after being cultured in dmem plus % fbs with nintedanib or vehicle control for days. afterwards, the specimens were embedded in paraffin and sectioned to μm thickness. following incubation of the sections with antibodies against col- , fibronectin (fn), cd , and cd (see details in table ), , ′-diaminobenzidine (dab, g , servicebio, wuhan, china) was used as a chromogen to visualize the bound antibodies, and the slides were counterstained with hematoxylin. cd + and cd + vessels were counted in five randomly selected fields under a microscope for semiquantification. this assay was performed with four independent samples. western blotting analysis for ex vivo explants, mg/each of treated or nontreated tissues were washed and cut into small pieces in pbs. the tissues were then homogenized with a pro- tissue homogenizer (pro scientific) in lysis buffer supplemented with a protease inhibitor cocktail (thermo fisher scientific, rockford, il, usa). the lysate was centrifuged at , × g for min at °c. for protein production in cells, kfs were treated with or without nintedanib ( , , μm) for or h to examine cellular signaling molecules or other antigens. protein extraction and western blotting analysis were performed as previously described [ ] . detailed information on the antibodies is listed in table . this assay was performed with three independent samples. to determine whether lipid rafts/caveolae were related to the inhibitory effect of nintedanib on keloid fibroblasts, disruption of lipid rafts/caveolae was carried out by incubating cells in the presence of mm mβcd (sigma) dissolved in double-distilled water (ddh o). cck- and scratch assays were used to measure cell proliferation and migration, respectively. western blotting was also performed to examine the expression of receptors. cells were treated with vehicle control, mβcd ( mm) or nintedanib ( μm) alone or in combination in growth medium. for the combination treatment, the cells were pretreated with mm mβcd for min at °c before the experiment. for the cck- assay, cells were tested on days , , and , and for the scratch assay, images were obtained at and h after scratching. for western blotting assays, cells were collected days after treatment. detailed methods are described above. all the data are presented as the mean ± standard deviation (sd) and were statistically analyzed with one-way anova followed by student-newman-keuls (s-n-k) post hoc test after confirming the normal distribution of the data. for ratio data of the edu assay, nonparametric kruskal-wallis test plus dunn's post hoc test with bonferroni correction was employed. all statistical analyses were performed with the statistical software spss (version . ; spss, inc., chicago, il, usa). a p-value < . was considered statistically significant. nintedanib suppressed kf proliferation and induced g /g cell cycle arrest as shown in fig. a , the cck- assay demonstrated a significant inhibitory effect of nintedanib on kf proliferation in a dosedependent manner during the -day time period compared to the vehicle control group. anova plus the s-n-k test demonstrated significant differences among the groups ( supplementary fig. s , p < . ). next, cell cycle analysis revealed a dose-dependent increase in the cell percentage in g /g phase, whereas a dose-dependent decrease in the cell percentage was found in s phase with significant differences among the groups treated with , and μm nintedanib (p < . , fig. b, c) , indicating that nintedanib induced cell cycle arrest at g /g phase. additionally, an eduincorporation assay also showed significant inhibition of dna synthesis in kfs by nintedanib treatment because much less edu labeling was observed in the treated cells than in the control cells (fig. d) . semiquantitative analysis showed a reduction in edu labeling among the groups (p < . , fig. e ). nintedanib attenuated the migratory capacity of cultured kfs after and h of nintedanib treatment, the scratch assay demonstrated a significant inhibitory effect on kf migration compared to that in nontreated cells when observed under a microscope (fig. a) . at both time points, quantitative analysis further confirmed a significant dose-dependent reduction in cell migration in the treated groups (p < . , fig. b) . similarly, the transwell assay also revealed the inhibitory effect of nintedanib on cell migration by reducing the number of cells that migrated to the bottom surface of the upper chamber in the treated group at h post treatment (fig. c) . semiquantitative analysis showed a dose-dependent reduction in the migrated cell numbers with significant differences among the groups, as revealed by anova plus the s-n-k test (p < . , fig. d) . furthermore, the oris migration assay also confirmed the inhibitory effect of nintedanib on cell migration by both microscopic observation (fig. e ) and quantitative analysis (fig. f) , which revealed a dose-dependent reduction in migrated cell numbers with significant differences among the groups after h of nintedanib treatment (p < . ). nintedanib inhibited the invasive capacity of kfs both the transwell assay and oris invasion assay combined with matrix coating were also performed to investigate the drug effect. as shown in fig. a , b, much fewer cells were able to successfully traverse the matrigel-coated chamber after nintedanib treatment compared to the control group at h post treatment, and the effect was dose-dependent. a similar trend was also confirmed in the oris invasion assay, which used col- coating as the matrix material. as shown in fig. c (microscopic view) and fig. d (quantitative analysis) , a dose-dependent decrease in cell numbers in the detection zone was found with increasing drug concentrations (p < . ). nintedanib suppressed the migration and proliferation of cultured keloid explants to further verify the inhibitory effect of nintedanib on cell migration and proliferation, an ex vivo keloid explant culture model was also established. as shown in fig. e , f, spindle-shaped kfs of the vehicle group migrated out of the edges of the cultured keloid explants and gradually spread over the petri dish within a week with a total cell number of . × after days of culture. however, treatment with increasing concentrations of nintedanib for days significantly inhibited kfs from migrating out of the tissue explants in a dose-dependent manner with significant differences from that of the control group (p < . ). interestingly, the inhibitory effect of the drug was also supported by decreased gene expression of matrix metalloproteinases (mmps). as shown in fig. g , h, mmp and mmp gene levels were significantly reduced in the drug-treated groups, as demonstrated by rt-qpcr analysis (p < . ). nintedanib suppressed the expression of fibrotic factors related to keloid pathogenesis using an in vitro cell culture model, the drug effects on the expression of fibrotic factors at both the gene and protein levels were investigated. as presented in fig. a , at the transcriptional level, treatment with the drug at concentrations of , , and μm for h significantly suppressed the gene expression of col- , fn, connective tissue growth factor (ctgf), plasminogen activator inhibitor- (pai- ), and fk -binding protein (fkbp ) in a dose-dependent manner, with significant differences among the groups (p < . ). the suppression of α-sma and heat shock protein (hsp ) occurred at higher concentrations, and no significant effect was found on col- . consistent with the gene expression results, western blotting also showed significant suppression of the protein production of col- , fn, and ctgf, as shown in fig. b , and semiquantitative analysis revealed significant differences among the different groups (fig. c) . the suppressed protein production of α-sma and col- was also confirmed by immunofluorescence staining of kfs treated with different concentrations of nintedanib (fig. d, e) , which showed decreasing protein expression with increasing drug concentrations. in addition, the drug treatment also disrupted the intracellular collagen pattern (fig. e) . nintedanib inhibited collagen production and disrupted angiogenesis in keloid tissue explants to better mimic the clinical scenario, an ex vivo model of tissue explants was generated for the investigation. immunohistochemical staining of the keloid tissue explants exposed to nintedanib showed a marked decrease in the intensity of col- staining and a reduced fn staining area, which indicated significantly decreased production of col- and fn (fig. a) . consistent with these findings, qpcr and western blotting analyses of tissue explants also showed a reduction in col- and fn expression at both the transcriptional and translational levels (fig. b-d) . furthermore, a reduced number of cd -positive and cd positive microvessels as well as disrupted capillary structure were also observed with nintedanib treatment (fig. a , e, f). to further define the potential mechanism of the above findings, the effect of nintedanib on various signaling pathways was investigated using an in vitro cell culture model. as shown in fig. , treatment of kfs with nintedanib significantly inhibited the phosphorylation of smad and smad at higher concentrations. nintedanib as a multiple signaling blockade for keloids by zhou et al. it also significantly inhibited the phosphorylation of mapk pathway components including p , jnk, and erk, as well as signal transducer and activator of transcription (stat ), in a dosedependent manner (fig. a) . cholesterol is the major structural component of lipid rafts/caveolae; thus, cholesterol depletion was performed to determine whether lipid rafts/caveolae mediate the cellular response to nintedanib. among various approaches, mβcd is the most common method for removing cellular cholesterol [ ] . cck- and scratch assays were used to measure proliferation and migration. the results showed that mβcd treatment alone could have a minor inhibitory effect on cell proliferation and migration, which was significantly different from the control treatment. nevertheless, mβcd was able to rescue to a certain degree the inhibitory effect of nintedanib on kf proliferation and migration when compared to treatment with nintedanib alone (fig. b-d) . interestingly, western blotting assays demonstrated fig. nintedanib inhibits the proliferation of treated keloid fibroblasts. a the cck- assay was used to analyze the proliferation of kfs treated with different concentrations of nintedanib at days , , , , and . b flow cytometry analysis of the cell cycle of treated keloid fibroblasts. c representative cell cycle data were plotted for each group. d after treatment with nintedanib ( , , μm) or vehicle control for h, kfs were incubated with edu for an additional h. the samples were imaged under a fluorescence microscope at × (bar = μm). e the ratio of edu-positive cells to dapi-labeled cells in each group was determined. *p < . vs control, # p < . vs . μm, $ p < . vs μm. abbreviations: g /g , period between the end of m phase and the start of s phase; s, dna duplication phase; g , period between the end of s phase and the start of m phase; m, mitosis. increased protein expression of growth factor receptors in the mβcd and nintedanib combined treatment group relative to the expression levels of nintedanib only treatment group (fig. e) , providing supporting evidence that nintedanib may affect the functions of growth factor receptors by enhancing their endocytosis, which can be disrupted by removing cellular cholesterol in the lipid raft domain. keloids are benign skin tumors in which keloid fibroblasts display tumor cell-like biological behaviors [ , ] . keloids inevitably recur after various treatments, and thus, new therapeutic approaches are needed. targeted therapy is considered an effective strategy for cancer treatment and includes various targets for drug intervention, such as rhodopsin-like g protein-coupled receptors (gpcrs), ion channels, protein kinases, and nuclear hormone receptors, with kinase inhibitors being an important part of targeted drugs for cancer therapy [ , ] . keloid formation is driven by multiple growth factors, including tgf-β, pdgf, and vegf. tgf-β promotes keloid fibroblast proliferation, collagen production, inhibition of mmps [ , ] , and the smad-signaling pathway [ ] . pdgf and vegf also contribute to keloid pathogenesis by stimulating collagen production and angiogenesis [ ] . additionally, pdgf contributes to the chemotaxis of fibroblasts, cell proliferation, migration, and collagen synthesis during wound healing [ ] . studies also demonstrated elevated pdgf receptor expression in keloid fibroblasts, which responded to pdgf more potently than normal dermal fibroblasts [ ] . elevated vegf in keloid tissue and circulation of keloid patients [ ] could lead to imbalanced angiogenesis and contribute to keloid formation [ , ] . moreover, bfgf is also indicated to be involved in keloid angiogenesis [ ] . preclinical data also demonstrated that the inhibition of fgfr signaling could suppress the proliferation of cells derived from many tissue types [ ] [ ] [ ] . this collected evidence provides an excellent strategy for targeted drug therapy of keloids. there have been several studies on the targeted therapy of keloids. palomid (p ), which inhibits the pi k/akt/mtor pathway by targeting both mtor complex (mtorc- ) and mtorc- signaling, caused keloid tissue shrinkage, growth arrest, and apoptosis [ ] . sorafenib, a potent tki targeting vegfr and pdgfrβ, has been shown to effectively inhibit keloid cell proliferation, migration, invasion, and collagen production [ ] . antisense therapy specific to tgf-β mrna transcripts demonstrated downregulation of mmp- in keloid fibroblasts [ ] . however, these studies focused on only one or two targets. we thus hypothesized that the use of a chemical compound that targets multiple signaling pathways would be a more efficient approach to keloid-targeted therapy. nintedanib was reported as an angiogenesis inhibitor of pdgfr-α and β, vegfr- , , and and fgfr- , , and and was proven effective in the therapy of many types of cancers [ ] . moreover, the antifibrotic effects of nintedanib have also been utilized in treating several fibrosis-related disorders, such as ipf and systemic sclerosis [ , ] . recent studies also showed that nintedanib likely targets the tgf-β-signaling pathway [ ] . as tgf-β, vegf, pdgf, and bfgf are involved in keloid mechanisms [ , ] , and nintedanib is a good candidate for targeted drug therapy for keloids, it would be interesting to explore the roles of nintedanib in inhibiting various pathological activities of keloid fibroblasts as preclinical experimental evidence for future clinical studies. this study demonstrated that nintedanib could inhibit various pathological phenotypes, including inhibition of cell proliferation, cell cycle arrest at g /g phase in vitro (fig. ) and inhibition of kf migration and invasion in vitro and ex vivo (figs. and ). in addition, it also significantly inhibited the gene expression of col- , fn, α-sma, and their protein production in vitro (fig. ) . the reduced protein production was also confirmed in an ex vivo model (fig. ). in addition, matrix degradation is likely to be enhanced by drug treatment, as the gene expression of pai- , a serine protease inhibitor that is often highly expressed in keloid fibroblasts [ ] , was significantly inhibited (fig. ) . in addition, nintedanib also significantly inhibited the gene expression of fkbp and hsp , both of which are highly expressed in fibrotic tissues [ , ] . moreover, this study showed that mmp- and mmp- gene expression was inhibited by the drug, which may also partially account for the inhibited cell migration in cultured tissue explants (fig. ) , in which matrix degradation was also required for cell migration out of the tissue explants. as these pathological aspects of keloids are mediated by growth factors, including tgf-β, pdgf, vegf, and bfgf, drug treatments are likely to inhibit the functions of these factors. for example, ctgf, a growth factor downstream of tgf-β [ , ] , was found to be downregulated at both the gene and protein levels, which may account for the inhibition of cell proliferation and matrix production. as further evidence of the inhibition of multiple signaling pathways, the results of fig. demonstrated that nintedanib noticeably inhibited the phosphorylation of smad and smad at higher concentrations and the mapk pathway dose-dependently, including the pathway components p , erk, and jnk. a previous study showed that the mapk pathway is involved in tgf-β transcription, in which p participates in the phosphorylation of smad / in kfs, while erk and jnk promote smad / / complex translocation and then regulate the expression of related genes, such as pai- [ , ] . consistent with the elevated smad and smad phosphorylation levels in keloid fibroblasts which were related to cell proliferation, matrix synthesis, and mmp regulation in keloids [ , ] , downregulation of smad and smad by rna interference resulted in a significant decrease in procollagen expression in keloids [ , ] . thus, the observed inhibition of cell migration and invasion as well as reduced matrix production is likely mediated by drug inhibition of smad-related pathways. moreover, increase in stat activation was also found in kfs and may contribute to keloid formation by promoting cell proliferation, migration, and collagen production [ ] , while stat phosphorylation was inhibited by the drug (fig. a) . in addition, vegf, pdgf, and bfgf are also involved in keloid pathogenesis by enhancing angiogenesis, cell proliferation, and migration through the p , pi k/akt, and mapk pathways. the ex vivo model study demonstrated that nintedanib significantly inhibited the expression of cd and cd and disrupted microvessel formation (fig. ) , as previously reported [ ] . overall, this study demonstrated that nintedanib could exert its therapeutic effects on various aspects of keloid pathogenesis likely by attenuating various signaling pathways and thus could serve as a blocker of multiple signaling pathways. according to the literature, most protein kinase antagonists are competitive inhibitors that interact with the atp-binding pocket to interfere with receptor dimerization and block the phosphorylation of kinases and downstream signaling cascades [ , ] . however, it has also been reported recently that certain drugs indirectly block signaling pathways by promoting receptor internalization and degradation [ ] . most of the protein kinases are localized in regions of cholesterol-rich lipid rafts/caveolae on cell membranes [ ] . studies have shown that mβcd can block receptor internalization by removing cholesterol [ ] . our results also demonstrated that mβcd was able to rescue nintedanibmediated receptor internalization to a certain degree, suggesting a dual mechanism for nintedanib-mediated inhibition of signaling pathways, i.e., it blocked the kinase atp-binding sites as the main mechanism, while it also promoted kinase internalization. the drawback of this drug is that it also inhibited the proliferation of normal dermal fibroblasts (data not shown) and likely affected other physiological activities. a previous study on pulmonary fibrosis also showed that nintedanib had similar inhibitory effects on both normal pulmonary fibroblasts and fibroblasts in ipf [ ] in terms of cell proliferation, α-sma expression, and myofibroblast appearance. this evidence suggests that nintedanib is less likely to be keloid cell specific and that certain toxic effects on normal cells are possible. therefore, localized use of this drug for keloid treatment would be an ideal method. in summary, this study demonstrates that inhibition of the tgf-β/smad and mapk-signaling pathways by nintedanib can effectively inhibit the proliferation, migration, invasion, and ecm deposition of kfs by blocking kinase atp binding and by enhancing kinase internalization. although further exploration of the detailed mechanism is still needed, the results revealed by the current study support the conclusion that nintedanib is likely to become a promising drug candidate for keloid-targeted therapy, which deserves clinical study in the future. human skin keloid fibroblasts display bioenergetics of cancer cells overexpression of insulin-like growth factor- (igf-i) receptor and the invasiveness of cultured keloid fibroblasts keloids and scars: a review of keloids and scars, their pathogenesis, risk factors, and management keloids: the paradigm of skin fibrosis-pathomechanisms and treatment sorafenib exerts an anti-keloid activity by antagonizing tgf-β/smad and mapk/erk signaling pathways keloid disease can be inhibited by antagonizing excessive mtor signaling with a novel dual torc / inhibitor elevated levels of pdgf alpha receptors in keloid fibroblasts contribute to an enhanced response to pdgf the efficiency of multi-target drugs: the network approach might help drug design nintedanib: from discovery to the clinic profile of nintedanib in the treatment of solid tumors: the evidence to date novel mechanisms for the antifibrotic action of nintedanib growth factor receptors, lipid rafts and caveolae: an evolving story vesicular trafficking of tyrosine kinase receptors and associated proteins in the regulation of signaling and vascular function potent dual inhibitors of torc and torc complexes (ku- and ku- ) demonstrate in vitro and ex vivo antikeloid scar activity tgf-beta antisense therapy modulates expression of matrix metalloproteinases in keloidderived fibroblasts compound astragalus and salvia miltiorrhiza extract inhibits cell proliferation, invasion and collagen synthesis in keloid fibroblasts by mediating transforming growth factor-β / smad pathway in vivo tendon engineering with skeletal muscle derived cells in a mouse model enhanced tenogenic differentiation and tendon-like tissue formation by tenomodulin overexpression in murine mesenchymal stem cells sorafenib ameliorates bleomycin-induced pulmonary fibrosis: potential roles in the inhibition of epithelial-mesenchymal transition and fibroblast activation a comprehensive map of molecular drug targets classification of small molecule protein kinase inhibitors based upon the structures of their drug-enzyme complexes the effect of tgf-beta on keloid fibroblast proliferation and collagen synthesis critical role of transforming growth factor beta in different phases of wound healing tgf-beta-induced fibrosis and smad signaling: oligo decoys as natural therapeutics for inhibition of tissue fibrosis and scarring on the nature of hypertrophic scars and keloids: a review altered angiogenic balance in keloids: a key to therapeutic intervention upregulation of transforming growth factor-β and vascular endothelial growth factor in cultured keloid fibroblasts: relevance to angiogenic activity molecular pathways: fibroblast growth factor signaling: a new therapeutic opportunity in cancer fibroblast growth factors (fgfs) in cancer: fgf traps as a new therapeutic approach inhibition of pdgf, vegf and fgf signalling attenuates fibrosis nintedanib inhibits fibroblast activation and ameliorates fibrosis in preclinical models of systemic sclerosis pai- in tissue fibrosis fk -binding protein , a potential novel drug target for idiopathic pulmonary fibrosis effect of heat shock protein on collagen accumulation in keloid fibroblast cells pathogenesis of fibrosis: role of tgf-beta and ctgf adiponectin is involved in connective tissue growth factor-induced proliferation, migration and overproduction of the extracellular matrix in keloid fibroblasts p map kinase mediates transforming growth factor-β transcription in human keloid fibroblasts mechanisms of transforming growth factor β /smad signaling mediated by mitogen-activated protein kinase pathways in keloid fibroblasts aspidin pb, a novel natural anti-fibrotic compound, inhibited fibrogenesis in tgf-β -stimulated keloid fibroblasts via pi- k/akt and smad signaling pathways inhibition of smad expression decreases collagen synthesis in keloid disease fibroblasts modulation of collagen synthesis in keloid fibroblasts by silencing smad with sirna stat contributes to keloid pathogenesis via promoting collagen production, cell proliferation and migration mode of action of nintedanib in the treatment of idiopathic pulmonary fibrosis sorafenib suppresses tgf-β responses by inducing caveolae/lipid raft-mediated internalization/ degradation of cell-surface type ii tgf-β receptors_ implications in development of effective adjunctive therapy for hepatocellular carcinoma pirfenidone and nintedanib modulate properties of fibroblasts and myofibroblasts in idiopathic pulmonary fibrosis this study was financially supported by the national natural science foundation of china (no. ). the authors also thank prof. jung huang from the biochemistry department of st. louis university school of medicine (usa) for his valuable suggestion on part of the experimental design and valuable discussion on related scientific issues. the online version of this article (https://doi.org/ . /s - - -y) contains supplementary material, which is available to authorized users.competing interests: the authors declare no competing interests. key: cord- -pjehkeh authors: zhou, zhong-yan; zhao, wai-rong; xiao, ying; zhou, xiang-ming; huang, chen; shi, wen-ting; zhang, jing; ye, qing; chen, xin-lin; tang, jing-yi title: antiangiogenesis effect of timosaponin aiii on huvecs in vitro and zebrafish embryos in vivo date: - - journal: acta pharmacol sin doi: . /s - - -z sha: doc_id: cord_uid: pjehkeh timosaponin aiii (timo aiii) is a natural steroidal saponin isolated from the traditional chinese herb anemarrhena asphodeloides bge with proved effectiveness in the treatment of numerous cancers. however, whether timo aiii suppresses tumor angiogenesis remains unclear. in the present study, we investigated the antiangiogenesis effects of timo aiii and the underlying mechanisms in human umbilical vein endothelial cells (huvecs) in vitro and zebrafish embryos in vivo. we showed that treatment with timo aiii ( . – µm) partially disrupted the intersegmental vessels (isvs) and subintestinal vessels (sivs) growth in transgenic zebrafish tg(fli- a: egfp)(y ). timo aiii ( . – µm) dose-dependently inhibited vegf-induced proliferation, migration, invasion, and tube formation of huvecs, but these inhibitory effects were not due to its cytotoxicity. we further demonstrated that timo aiii treatment significantly suppressed the expression of vegf receptor (vegfr) and the phosphorylation of akt, mek / , and erk / in huvecs. timo aiii treatment also significantly inhibited vegf-triggered phosphorylation of vegfr , akt, and erk / in huvecs. moreover, we conducted rna-seq and analyzed the transcriptome changes in both huvecs and zebrafish embryos following timo aiii treatment. the coexpression network analysis results showed that various biological processes and signaling pathways were enriched including angiogenesis, cell motility, cell adhesion, protein serine/threonine kinase activity, transmembrane signaling receptor activity, growth factor activity, etc., which was consistent with the antiangiogenesis effects of timo aiii in huvecs and zebrafish embryos. we conclude that the antiangiogenesis effect of timo aiii is mediated through vegf/pi k/akt/mapk signaling cascade; timo aiii potentially exerts antiangiogenesis effect in cancer treatment. cancer, which poses a major threat to public health worldwide, is the second leading cause of death in the usa, and the prevalence and burden of cancer continue to increase in china as well [ , ] . tumor angiogenesis leads to the formation of new blood vessels that transport oxygen and nutrients; this process controls tumor growth and metastasis and plays a key role in the progression of tumors [ , ] . tumor cells secreting angiogenic factors, such as bfgf, vascular endothelial growth factor (vegf), tnf-α, and hif-α, promote the proliferation, migration, and invasion of endothelial cells and initiate the growth of new blood vessels from the preexisting vasculature in tumors [ , ] . thus, targeting inhibition of tumor angiogenesis is considered an important therapeutic strategy in cancer patients [ ] . timosaponin aiii (timo aiii, fig. ) is a natural steroidal saponin isolated from chinese materia medica anemarrhena asphodeloides bge and is commonly used to treat depression, hematochezia, diabetes, cough, hemoptysis, and cardiovascular disease in traditional chinese medicine [ , ] . timo aiii has multiple pharmacological activities, including antiinflammatory [ ] and antiplatelet aggregation activities [ ] , improves learning and memory [ ] and inhibits the passive cutaneous anaphylaxis reaction and pruritus [ ] . interestingly, timo aiii induces hepatotoxicity, and this effect is protected by mangiferin, which is another component in a. asphodeloides bge [ ] . moreover, timo aiii selectively induces cell death in tumor cells but not in normal cells and has been suggested to be a novel potential anticancer compound in recent years [ , ] . timo aiii presents antitumor activity in various cancers, such as non-small cell lung cancer [ ] , pancreatic cancer [ ] , melanoma [ , ] , breast cancer [ ] , hepatocellular carcinoma [ ] , colon cancer [ ] , promyelocytic leukemia [ ] and so on. in addition, chen et al. found that timo aiii also reverses multidrug resistance in human chronic myelogenous leukemia [ ] . various molecular mechanisms, including proapoptosis, antiproliferation, antimigration, antiinvasion, induction of ros and er stress and autophagy, are involved in the anticancer activity of timo aiii [ ] . however, whether timo aiii inhibits the tumor angiogenesis process is not clear. in the present study, we explored the antiangiogenesis effect of timo aiii and its mechanism of action using endothelial cell and zebrafish models. chemicals -( , -dimethyl-thiazol- -yl)− , -diphenyl tetrazolium bromide (mtt), endothelial cell growth supplement (ecgs) and heparin were purchased from sigma aldrich (st louis, mo, usa). vegf was purchased from peprotech (rocky hill, nj, usa). su was purchased from cayman chemical (ann arbor, mi, usa). cell culture reagents were supplied by thermo fisher (waltham, ma, usa). antibodies were purchased from cell signaling technology (danvers, massachusetts, usa). timo aiii (purity by hplc ≥ . %) was purchased from chengdu must bio-technology co., ltd (chengdu, sichuan, china). all experiments related to zebrafish described in this study were in accordance with the animal experimentation ethics committee, shanghai university of traditional chinese medicine. maintenance and embryo handling of zebrafish the transgenic zebrafish line tg(fli- a: egfp) y , which expresses enhanced green fluorescent protein (egfp) in endothelial cells, was kindly provided by prof. simon lee from the university of macau. fish maintenance was performed according to the zebrafish handbook (westerfield, ) . in brief, zebrafishes were kept separately under a h light and h dark cycle and fed brine shrimp twice a day. zebrafish embryos were generated by natural pair-wise mating and incubated at . °c in embryo medium. after h of incubation, chorions were removed by a sharp forceps, and health development embryos were selected for later use. cell viability assay of human umbilical vein endothelial cells human umbilical vein endothelial cells (huvecs) were purchased from american type cell culture (atcc, catalog: atcc® crl- ™) and cultured in f- k medium supplemented with . mg/ml heparin, . mg/ml ecgs, % (v/v) fbs and % penicillin/streptomycin at °c in a % humidified incubator with % co in air. huvecs were seeded at a density of × cells/well in a -well plate and preincubated until they reached - % confluence. after h of starvation in basal f- k medium, huvecs were treated with various concentrations of timo aiii ( . , , , , and μm) with or without vegf ( ng/ml) for h. huvecs were treated with dmso ( . %) as a vehicle control and su ( µm) as a positive control. the cell viability of huvecs was tested by the mtt assay according to the manufacturer's instructions. cell viability was expressed according to the percentage of the vehicle control. real-time endothelial cell proliferation analysis by an rtca huvec cell growth was detected in real time using a real-time cell analysis (rtca) system named xcelligence (aceabio s , hangzhou, china). huvecs were seeded at a density of . × cells/well in a specified -well plate. huvecs were settled for min before being placed in an xcelligence instrument at °c in a % humidified incubator with % co in air. after incubation for h, huvecs were treated with vegf ( ng/ml), timo aiii ( µm), or timo aiii ( µm) + vegf ( ng/ml) for another h. huvecs treated with . % dmso served as a vehicle control. the cell independence, expressed as the cell index, was recorded every hour. the effects of timo aiii on the migration and invasion of huvecs were evaluated using a matrigel (bd biosciences, san jose, ca, usa) transwell system with -well companion plates ( µm pores). in the migration assay, huvecs were directly seeded onto the upper side of the transwell. in each upper well, huvecs were suspended in µl basal f -k medium at a cell density of . × cells/ml that also containing vegf ( ng/ml) with or without various concentrations ( . , , , and µm) of timo aiii. huvecs were treated with . % dmso as a vehicle control and su as a positive control. after incubation for h, huvecs were removed using cotton swabs from the inner surface of the transwell insert. then, the huvecs on the outside of the transwell insert were fixed with % paraformaldehyde for min at room temperature (rt) and stained with hoechst ( μg/ml) for min. the membranes were washed with pbs and mounted on microscope slides. huvecs were excited with uv light and captured at × magnification under a fluorescent inverted microscope. in the invasion assay, the upper and lower sides of the membrane were precoated with matrigel. the drug treatment and observation methods for the following invasion assay were the same as those for the migration assay. the cell migration and invasion ability of huvecs were evaluated by imagej to quantify the cell number in each field. endothelial cell tube formation assay the tube formation assay was performed using a µ-slide angiogenesis system coated with matrigel. huvecs were suspended in µl basal f- k medium at a density of . × cells/ml that also contained vegf ( ng/ml) with or without various concentrations of timo aiii ( . , , , and µm). huvecs were treated with . % dmso as a vehicle control and su as a positive control. after h of incubation, the cells were imaged at × magnification under an inverted microscope. the number of loop formations was counted in each field. western blotting analysis huvecs were incubated with various concentrations ( . , , and µm) of timo aiii for h. huvecs were treated with . % dmso as a vehicle control. after drug treatment, huvecs were washed with ice-cold pbs three times and lysed in lysis buffer containing . m nacl, mm tris, mm edta, . % sds, . % triton x- , and mm pmsf. cell lysates were centrifuged at × g for min at °c. the protein concentration was measured using a bca assay kit (pierce, rockford, il, usa). then, protein was denatured with loading buffer, and µg of total protein from each sample was separated by sds-page and transferred to a polyvinylidene fluoride ( . µm) membrane, which was blocked with % bsa. immunoblots were incubated with primary antibodies, including antibodies against vegfr ( : , cst), phospho-vegfr ( : , cst), vegfr ( : , cst), phospho-akt ( : , cst), akt ( : , , at °c overnight. after washing with tbst three times, the immunoblots were incubated with horseradish peroxidase-conjugated goat anti-rabbit igg ( : , cst) for h at rt. after the final wash, the immunoblots were visualized using an enhanced ecl system (beyotime beyoecl moon, shanghai, china). the membranes were then imaged on an imaging system (ge am , fairfield, ct, usa), and the intensity of each protein band was analyzed by imagej. rna-seq and data analysis rna-seq was performed with the sequencing platform bgiseq- (bgi, shenzhen, china). briefly, huvec cells or tübingen (tu) strain zebrafish embryos ( dpf) were treated with various concentrations of timo aiii for h. then, the huvec cells and zebrafish embryos were harvested, with three and four repetitive samples per group, respectively. the total rna of each sample was extracted, and a sequencing library was constructed. the soapnuke analysis tool (v . . , -l -q . -n . ) was used to analyze the sequence quality and filter low-quality reads with quality scores lower than . hisat (v . . ) was used to map the clean reads to the reference genome (homo sapiens: ucsc_hg ; danio rerio: ncbi_grcz ). then, the clean reads were mapped to the reference transcript by bowtie (v . . ), and the expression values were calculated by rsem (v . . ). degseq (parameter: fold change ≥ and adjusted p value < . ) was used to identify differentially expressed genes (degs) between the control and treatment groups. the transcript level was calculated according to the fragments per kilobase of transcript per million mapped reads (fpkm). for gene ontology (go) analysis of the degs, the go annotation results were classified into official classifications, including molecular function, cellular component, and biological process. the r package function phyper was used to perform go enrichment analysis of degs. the false discovery rate (fdr) was calculated for each p value, and fdr < . was defined as significantly enriched. the same method was used to annotate and enrich the results of the kegg metabolic pathway analysis of the degs. coexpression network analysis based on the gene expression profile was conducted using the r package wgcdna. statistical analysis data are presented as the mean ± s.e.m. from at least three independent experiments. student's t-test and analysis of variance were used to perform statistical evaluations of the differences between the two groups, and p < . was considered significant. the transgenic zebrafish line tg(fli- a: egfp) y , which expresses egfp in endothelial cells [ ] , was employed to test the effect of timo aiii on antiangiogenesis. twenty-four hpf (hours post fertilization) zebrafish embryos were treated with various concentrations of timo aiii for h, and we found that timo aiii inhibited intersegmental vessels (isvs) growth in the tail of zebrafish (fig. a) . statistical graphs indicated that timo aiii dosedependently decreased the intact vessel number and increased the defective vessel number of isvs (fig. b) . consistently, the growth of subintestinal vessels (sivs) was also inhibited after treatment with timo aiii for h (fig. a) . timo aiii also decreased the total area of sivs (fig. b) . these data indicated that timo aiii had an antiangiogenic effect in zebrafish. timo aiii suppressed endothelial cell proliferation in huvecs endothelial cell proliferation initiates angiogenesis [ ] . to detect the effect of timo aiii on endothelial proliferation, we employed huvecs as our experimental model. we found that timo aiii concentration-dependently decreased cell viability and was cytotoxic in huvecs (figs. a and s ). cell viability was significantly decreased at concentrations of and µm, while there was no cytotoxicity over the concentration range of ≤ µm. in addition, timo aiii inhibited vegf-induced cell proliferation at concentrations of , , and µm (fig. b) . consistently, we also found that timo aiii suppressed vegf-induced endothelial cell proliferation using an rtca system (fig. c) . huvecs were treated with the selective vegf receptor (vegfr) inhibitor su as a positive control. these results revealed that timo aiii significantly inhibited endothelial cell proliferation. timo aiii inhibited vegf-induced endothelial cell migration in huvecs the migration of huvecs was evaluated by a classical transwell assay [ ] . huvecs treated with vegf significantly increased the number of migrated cells, while cotreatment with timo aiii and vegf reduced the number of migrated cells in a concentrationdependent manner (fig. a, b) . huvecs were treated with su , a vegfr inhibitor that served as a positive control. huvecs were stained with the nuclear indicator hoechst for easy observation. as shown in figs. a and s , timo aiii was not toxic to huvecs over the concentration range of . - µm. these results indicated that timo aiii suppressed vegf-induced endothelial cell migration and that the cell migration inhibitory effect of timo aiii was not associated with its cytotoxicity. invasion plays vital roles in both tumor angiogenesis and metastasis [ , ] . the invasion ability of huvecs was determined according to the amount of cells that passed through the matrigel and membrane barriers in a transwell system. as shown in fig. a , b, vegf significantly enhanced the invasion cell number of huvecs and timo aiii obviously suppressed the invasion cell number under nontoxic concentrations in a dose-dependent manner. huvecs were incubated with su as a positive control. these results suggested that timo aiii attenuated vegfinduced invasion in huvecs. timo aiii reduced vegf-induced tube formation in huvecs an endothelial cell tube formation assay was used to mimic the process of neovascularization. in the present study, we found that vegf increased the loop number of the network tube structure of huvecs, while timo aiii dramatically inhibited the loop number in each field (fig. a, b) . huvecs were incubated with su as a positive control. in this experiment, huvecs were also treated with timo aiii at nontoxic concentrations from . to µm (figs. a and s ), and timo aiii reduced vegf-induced tube formation in a concentration-dependent manner. we also tested the effects of fig. timo aiii inhibits endothelial cell proliferation in huvecs. a huvecs were treated with various concentrations of timo aiii ( . , , , , and µm) for h, and cell viability was tested by the mtt assay. b huvec cells were cotreated with vegf ( ng/ml) and various concentrations of timo aiii ( . , , , and µm) or su ( µm), which served as a positive control, for h, and cell viability was determined by the mtt assay. c cell impedance, which was expressed as the cell index and measured by the xcelligence real-time cell analysis (rtca) system, was used to evaluate the proliferation in huvecs. huvecs were treated with vegf ( ng/ml), timo aiii ( µm), or vegf ( ng/ml) + timo aiii ( µm) for h, and cell impedance was detected at -h intervals. data are presented as the percentage of the control group. the results are presented as the means ± s.e.m. ## p < . and ### p < . versus the control group. *p < . , ***p < . versus the vegftreated group vegf and timo aiii on cell proliferation during the tube formation process and found that vegf treatment with or without timo aiii did not significantly affect the cell proliferation of huvecs after incubation for h (fig. c) . therefore, we concluded that the tube formation inhibitory effect of timo aiii was not associated with its inhibition of vegf-induced endothelial cell proliferation. timo aiii affected vegf/pi k/akt/mapk signaling in huvecs endothelial cell proliferation, migration, and invasion are dramatically regulated by the vegf/vegfr/mapk signaling pathway [ , ] . as shown from the western blotting results presented in fig. a , timo aiii downregulated the protein levels of vegfr and . the phosphorylation levels of akt, mek / , and erk / were attenuated by treatment with timo aiii, while the phosphorylation levels of p and jnk were not significantly changed (fig. b-f ). timo aiii also decreased the expression of jnk (fig. d ). in addition, timo aiii suppressed the phosphorylation of vegfr , akt, and erk / triggered by vegf (fig. a-c) . these results suggested that the underlying mechanism of the antiangiogenic effect of timo aiii might be related to the inhibition of the vegf/ pi k/akt/mapk signaling pathway. transcriptome analysis of timo aiii-regulated gene expression in huvecs and zebrafish embryos to further clarify the underlying mechanism of the antiangiogenesis effect of timo aiii, we conducted rna-seq to examine the genome-wide transcriptional changes between the timo aiii-treated group and the control group in both huvecs and tu strain zebrafish embryos. the degs between the control group and the timo aiii-treatment group were identified in both huvec cells and zebrafish. in addition, we performed coexpression network analysis based on the expression profile normalized by the fpkm using the r package wgcdna. network analysis indicated that and modules were constructed in timo aiii-treated huvec cells (fig. s a) and zebrafish (fig. s b) , respectively. among them, we detected many significant traitrelated modules (fig. s a, b) . go and kegg enrichment analyses of the highly connected genes in these trait-related modules demonstrated that many processes and pathways were significantly enriched, including angiogenesis, cell motility, cell adhesion, protein serine/threonine kinase activity, transmembrane signaling receptor activity, growth factor activity, etc. (fig. s c-e) . these findings were consistent with our experimental results that showed timo aiii inhibited angiogenesis in endothelial cells and zebrafish. the present study explored the antiangiogenesis effect of timo aiii (fig. ) on huvecs in vitro and zebrafish in vivo. in zebrafish, we found that timo aiii inhibited the growth of isvs and sivs in a concentration-dependent manner. in huvecs, we demonstrated that timo aiii attenuated cell proliferation, migration, invasion, and tube formation. the underlying mechanism might involve the vegf/pi k/akt/mapk signaling pathway. this study was the first to investigate the potential antiangiogenic properties of timo aiii. angiogenesis plays vital roles in tumor growth and metastasis. timo aiii was proposed to be a potential natural compound for [ ] . although the anticancer effect of timo aiii has been demonstrated in various types of cancers, the antiangiogenesis effect of timo aiii has not been elucidated. in our previous studies, our group established the transgenic zebrafish line tg(fli- a: egfp) y and huvecs as effective models for antiangiogenesis compound screening and studying its underlying mechanism [ , ] . in the present study, we found that timo aiii inhibited isvs and sivs growth in zebrafish embryos in a dose-dependent manner (figs. and ) . these results provide evidence for the antiangiogenesis effect of timo aiii in vivo. in previous studies, timo aiii inhibited cell proliferation, migration, and invasion at concentrations of , , , and µm in the lung cancer cell line a [ ] . timo aiii suppressed the metastasis of renal carcinoma cells at concentrations of , , and µm [ ] . timo aiii attenuated the oncogenic phenotype of breast cancer cells at concentrations of and µm [ ] . thus, the concentrations of timo aiii used in the anticancer studies varied depending on the cancer types and experimental conditions. endothelial cells play critical roles in vascular functions, including angiogenesis [ , , ] . endothelial cell proliferation initiated the growth of new blood vessels, and timo aiii inhibited the proliferation of huvecs (fig. a) . vegf is an angiogenesis driver and plays a vital role in the process of tumor angiogenesis [ ] . timo aiii reduced vegf-induced endothelial cell proliferation in huvecs regardless of its cytotoxicity (fig. b) . consistently, an rtca system, which can detect the impendence of attached cells, also indicated real-time inhibition of endothelial cell proliferation by timo aiii (fig. c ). in addition, endothelial cell migration and invasion were detected by a classic transwell system. we found that timo aiii suppressed vegf-induced endothelial cell migration and invasion in huvecs over the range of nontoxic concentrations of timo aiii (figs. and ). these results were consistent with the antiproliferation, antimigration, and antiinvasion effects of timo aiii found in various types of cancer cells [ , ] . the tube formation of endothelial cells, which mimicked -d network vascular growth, was measured by an easily operated µ-slide coated with matrigel. timo aiii disrupted the tube formation of huvecs (fig. a, b) , while timo aiii and vegf did not affect cell proliferation in huvecs (fig. c ). huvecs treated with the specific vegfr inhibitor su served as positive controls in these experiments. thus, we concluded that timo aiii reduced endothelial cell proliferation, migration, invasion, and tube formation and had an antiangiogenic effect in huvecs in vitro. however, timo aiii was not cytotoxic and did not affect cell apoptosis or the cell cycle in huvecs (fig. s and table s ). we also tested the cytotoxicity of timo aiii on the rat aortic smooth muscle cell line a r and found that timo aiii did not significantly inhibit cell proliferation at concentrations of . , , and µm (fig. s ) . previous studies revealed that timo aiii induced apoptosis though the activation of the caspase cascade via the jnk / pathway [ ] . timo aiii reduced hepatocyte growth factor-induced invasion by erk inhibition in a breast cancer cell line [ ] . thus, the mapk signaling pathway might also be recruited in mechanism of the antiangiogenesis effect of timo aiii in endothelial cells. furthermore, activation of mapk signaling mediated by vegf and vegfr is an essential mechanism in angiogenesis [ ] . in this study, timo aiii downregulated the protein levels of vegfr and (fig. a) . timo aiii suppressed the phosphorylation of akt, mek / , and erk / but not p and jnk in huvecs (fig. b-f ). timo aiii also decreased the expression of jnk (fig. d) . moreover, vegf stimulated the phosphorylation of vegfr , akt, and erk / , and timo aiii also attenuated these processes (fig. a-c) . cotreatment with timo aiii fig. timo aiii attenuates the vegf/pi k/akt/mapk signaling pathway in huvecs. a-f huvecs were treated with various concentrations of timo aiii ( . , , and µm) for h. huvecs incubated with . % dmso served as a vehicle control. phosphorylated and total proteins were detected by western blotting using the specific antibodies. the ratio of phosphorylated and total proteins was calculated. data are presented as the fold of the control group. the results are presented as the means ± s.e.m. *p < . , **p < . , and ***p < . versus the control group and vegf did not decrease the expression of vegfr (fig. a) , while treatment with timo aiii alone decreased vegfr expression (fig. a) in huvecs. these results might be due to the complicated regulation of vegf in endothelial cells. these results revealed that timo aiii inhibited the vegf/pi k/akt/mapk signaling pathway in endothelial cells, which might be the underlying mechanism of the antiangiogenesis effect of timo aiii. in addition, the transcriptome data indicated that several cellular processes and pathways, including angiogenesis, cell motility, cell adhesion, protein serine/ threonine kinase activity, transmembrane signaling receptor activity, and growth factor activity, were related to the treatment of huvecs and zebrafish embryos with timo aiii (fig. s ) . these results further confirmed the antiangiogenesis effect of timo aiii. in summary, timo aiii partially disrupted sivs and isvs growth in zebrafish embryos. timo aiii inhibited endothelial cell proliferation, migration, invasion, and tube formation in huvecs. the underlying mechanism of the antiangiogenesis effect of timo aiii might be involved in the inhibition of the vegf/pi k/akt/mapk signaling pathway. for the first time, we demonstrated the antiangiogenesis effect of timo aiii in huvecs in vitro and zebrafish in vivo. cancer statistics estimates of cancer incidence and mortality in china angiogenesis biomarkers and their targeting ligands as potential targets for tumor angiogenesis tumor angiogenesis: therapeutic implications angiogenesis inhibitors: current strategies and future prospects targeting vasculature in urologic tumors: mechanistic and therapeutic significance role of angiogenesis in the pathogenesis of cancer timosaponin aiii, a saponin isolated from anemarrhena asphodeloides, ameliorates learning and memory deficits in mice the genus anemarrhena bunge: a review on ethnopharmacology, phytochemistry and pharmacology timosaponin aiii and its metabolite sarsasapogenin ameliorate colitis in mice by inhibiting nf-κb and mapk activation and restoring th /treg cell balance timosaponin aiii induces antiplatelet and antithrombotic activity via gq-mediated signaling by the thromboxane a receptor inhibitory effects of steroidal timosaponins isolated from the rhizomes of anemarrhena asphodeloides against passive cutaneous anaphylaxis reaction and pruritus timosaponin a induces hepatotoxicity in rats through inducing oxidative stress and down-regulating bile acid transporters timosaponin aiii: a novel potential antitumor compound from anemarrhena asphodeloides timosaponin aiii is preferentially cytotoxic to tumor cells through inhibition of mtor and induction of er stress timosaponin aiii inhibits migration and invasion of a human non-small-cell lung cancer cells via attenuations of mmp- and mmp- by inhibitions of erk / , src/fak and βcatenin signaling pathways anemarrhena asphodeloides bunge and its constituent timosaponin-aiii induce cell cycle arrest and apoptosis in pancreatic cancer cells timosaponin aiii induces apoptosis and autophagy in human melanoma a -s cells timosaponin aiii inhibits melanoma cell migration by suppressing cox- and in vivo tumor metastasis timosaponin aiii suppresses hepatocyte growth factor-induced invasive activity through sustained erk activation in breast cancer mda-mb- cells a novel mechanism of xiap degradation induced by timosaponin aiii in hepatocellular carcinoma cytotoxic and antineoplastic activity of timosaponin a-iii for human colon cancer cells timosaponin aiii mediates caspase activation and induces apoptosis through jnk / pathway in human promyelocytic leukemia cells timosaponin a-iii reverses multidrug resistance in human chronic myelogenous leukemia k /adm cells via downregulation of mdr and mrp expression by inhibiting pi k/akt signaling pathway an andrographolide derivative agp- b exhibiting anti-angiogenic activity in huvecs and zebrafish via blocking the vegfa/vegfr signaling pathway spatholobi caulis extracts promote angiogenesis in huvecs in vitro and in zebrafish embryos in vivo via upregulation of vegfrs microrna- inhibits the proliferation and migration of endothelial cells in patients with atherosclerosis by targeting importin-α and regulating inflammatory responses invadosomes are coming: new insights into function and disease relevance inhibin is a novel paracrine factor for tumor angiogenesis and metastasis anti-angiogenic effect of furanodiene on huvecs in vitro and on zebrafish in vivo timosaponin aiii inhibits metastasis of renal carcinoma cells through suppressing cathepsin c expression by akt/mir- - p axis timosaponin a-iii inhibits oncogenic phenotype via regulation of pcg protein bmi in breast cancer cells ferulic acid relaxed rat aortic, small mesenteric and coronary arteries by blocking voltage-gated calcium channel and calcium desensitization via dephosphorylation of erk / and mypt endothelial-dependent and independent vascular relaxation effect of tetrahydropalmatine on rat aorta zyz conducted the experiments and wrote the manuscript. wrz and yx conducted the experiments and analyzed the data. ch analyzed the transcriptome data. xmz, wts, and jz conducted the experiments. qy and xlc designed the study. jyt designed the study and revised the manuscript. the online version of this article (https://doi.org/ . /s - - -z) contains supplementary material, which is available to authorized users.competing interests: the authors declare no competing interests. fig. timo aiii suppresses the vegf-activated vegf/pi k/akt/mapk signaling pathway in huvecs. a-c huvecs were treated with vegf ( ng/ml) with or without timo aiii ( µm) for h. huvecs incubated with . % dmso served as a vehicle control. phosphorylated and total proteins were detected by western blotting using the specific antibodies. the ratios of phosphorylated and total proteins were calculated. data are presented as the fold of the control group. the results are presented as the means ± s.e.m. *p < . and **p < . versus the control group. # p < . , ## p < . , and ### p < . versus the vegf-treated group key: cord- - t qg fp authors: papoutsis, konstantinos; kapelouzou, alkistis; georgiopoulos, georgios; kontogiannis, christos; kourek, christos; mylonas, konstantinos s; patelis, nikolaos; cokkinos, dennis v; karavokyros, ioannis; georgopoulos, sotirios title: tissue-specific relaxin- is differentially associated with the presence/size of an arterial aneurysm and the severity of atherosclerotic disease in humans date: - - journal: acta pharmacol sin doi: . /s - - - sha: doc_id: cord_uid: t qg fp circulating or tissue-related biomarkers are of clinical value for risk stratification in patients with abdominal aortic aneurysms. relaxin- (rl ) has been linked to the presence and size of arterial aneurysms, and to the extent of atherosclerosis in human subjects. here, we assessed the expression levels of rl in aneurysmal (aa, n = ) and atherosclerotic (ath, n = ) arteries, and established the correlation between rl levels and the presence/size of aa and the clinical severity of atherosclerosis. the expression levels of metalloproteinases (mmps) and endothelial nitric oxide synthetase (enos) were also detected for correlations with different phenotypes of atherosclerosis and aa. temporal artery biopsy specimens (n = ) and abdominal aortic tissues harvested from accident victims during autopsy (n = ) were used as controls. quantitative tissue biomarker analysis revealed that tissue-specific rl was increased in patients with larger or symptomatic aa compared to subjects with atherosclerotic disease and healthy controls. in situ rl levels were proportional to the size and the severity of aneurysmatic disease, and were substantially elevated in patients with symptomatic aneurysm of any diameter or asymptomatic aneurysm of a diameter > % of that of the normal artery. in contrast, tissue rl was inversely associated with the clinical severity of atherosclerotic lesions. correlation between rl and mmp was different between ath and ath , depending on atherosclerosis grade. overall, tissue rl is differentially associated with discrete phenotypes of arterial disease and might exert multipotent biological effects on vascular wall integrity and remodeling in human subjects. abdominal aortic aneurysms (aaas) represent the most widely studied lesions among arterial aneurysms (aas). although the prevalence and incidence rates of aaas have decreased during the past two decades [ ] , early detection is crucial for timely and successful management because the morbidity [ ] and mortality [ ] [ ] [ ] [ ] are high in patients with a ruptured aaa (raaa) who manage to reach the hospital alive in order to undergo emergency open or endovascular repair. biomarkers potentially related to aneurysm formation and expansion have been investigated and reviewed twice [ , ] in order to augment timely diagnoses, but currently, there is no clinical applicability for any of the studied biomarkers due to either no association or a weak association with the natural history of aneurysms. our group has recently discovered a positive correlation between the serum levels of the novel biomarker relaxin- (rl ) and the presence/size of aas in human subjects [ ] . rl , a nonglycosylated peptide with a structural and post-translational processing resemblance to insulin and insulin-like growth factors (ilgf), was initially identified as a reproductive hormone implicated in vasoregulation during pregnancy [ ] . ever since the discovery [ ] of its receptor, known today as relaxin family peptide receptor- (rfxp ), research on rl has expanded on various tissues and systems, including cardiovascular tissues such as arteries, veins, and the atrial and ventricular myocardium [ ] [ ] [ ] . we hypothesized [ ] that serum rl is related to the presence and size of an aa based on the knowledge that rl upregulates the synthesis of matrix metalloproteinase (mmp)- and mmp [ ] [ ] [ ] , which are mmps with a stronger association with aas [ , ] . meanwhile, existing knowledge suggests that rl upregulates the synthesis of nitric oxide (no) [ , [ ] [ ] [ ] . reduction in endothelium-derived no is, along with oxidative stress, the common pathway of action of all major risk factors leading to endothelial dysfunction and atherosclerosis [ ] [ ] [ ] [ ] [ ] , and our group demonstrated recently [ ] that serum rl is inversely correlated with the severity of atherosclerotic disease. in the present study, we aimed to verify and expand the findings of our preliminary study for serum rl [ ] by investigating the levels of tissue-specific rl in aneurysmal (aa) and atherosclerotic (ath) arteries and establishing a correlation of tissue-specific rl levels with the presence/size of aa and the clinical severity of atherosclerosis. comparisons were made between the aa and ath artery groups and the control artery group, including temporal artery biopsy specimens (tab) and abdominal aortic tissues harvested from accident victims during autopsy (ag). moreover, we carried out tissue-specific measurements of mmp , mmp and endothelial nitric oxide synthetase (enos) in the aforementioned groups of arterial specimens to investigate further correlations with different phenotypes of atherosclerosis and aa. in this study, a total of subjects were enrolled. our interventional groups consisted of patients who had open surgery for different forms of atherosclerotic disease and patients who had open surgery for an aa. these patients underwent surgery at laiko hospital in athens, greece and along with patients who underwent tab in the same center were the same subjects originally recruited in our preliminary study [ ] . ten accident victims who underwent a postmortem autopsy (ag) in the department of anatomy, medical school of athens, kapodistrian university, were further enrolled to serve as controls. the study was conducted in accordance with the declaration of helsinki. apart from the accident victims, all subjects provided informed consent for the use of their clinical and laboratory results for scientific purposes. data collection and processing remained anonymous. phenotypes per group study groups and subcategorization along with baseline characteristics have been previously described [ ] . in brief, our study population was allocated to the following groups: aa group. the aa group included patients ( . ± . , mean ± sd; age; male sex) with aaas (n = ), thoracic aortic aneurysms (taa) (n = ), internal iliac aneurysms (n = ), or popliteal aneurysms (n = ). further subcategorization with respect to the size and clinical presentation of aneurysmatic disease was implemented as described previously [ ] . ( ) aneurysm group (aa ) (n = ; male sex): patients with an asymptomatic aneurysm of a diameter %- % of that of the normal artery. ( ) aneurysm group (aa ) (n = , male sex): patients with an asymptomatic aneurysm of a diameter %- % of that of normal artery. details about diagnostic modalities and diagnostic approaches in classifying the severity of atherosclerosis can be found elsewhere [ ] . tab and ag control groups. the tab group (n = ) ( . ± . , means ± sd; age; male/female sex) had a normal pathology report of temporal arteritis and had no history or clinical signs of aa, an extracranial carotid artery, or pad. the ag group (n = ) ( . ± . , means ± sd; age; male/female sex) included accident victims who were autopsied within h postmortem without aa found during autopsy and without a history of atherosclerotic disease. as stated in our previous report [ ] , the aa, ath, and tab study groups were well matched for age, gender, medications, medical comorbidities, smoking status, and renal and liver biochemistry. ag group subjects were substantially younger and did not have any medical comorbidities. tissue collection and preservation arterial specimens from all subjects were collected with an aseptic technique at room temperature. in the aa group, part of the aneurysmatic sac, consisting of all arterial layers, was collected during open repair of the aneurysm. in the ath group, only the atherosclerotic plaque was collected during carotid endarterectomy or the lower extremity revascularization procedure. in the ta group, part of the temporal artery, consisting of all arterial layers, was collected during tab. in the ag group, part of the infrarenal abdominal aorta, consisting of all arterial layers, was collected during autopsy. the samples were flushed with normal saline and immediately stabilized with a commercially available tissue reagent (allprotect tissue reagent, qiagen gmbh, d- hilden, germany). stabilized tissues were transported within the time and temperature limits set by the manufacturer and stored at − °c until analysis was performed. analysis of atherosclerotic and aneurysm tissues immunohistochemical staining. ten serial paraffin sections of μm thickness along the arterial specimens were mounted on polylysine-coated microscope slides and allowed to dry overnight, pending immunohistological staining. sections were deparaffinized in xylene and a series of graded ethanol and finally stained with the appropriate antibodies (a) rabbit anti-human relaxin- (meridian, life science, inc., uk) (working concentration: µg/ml), (b) rabbit anti-human mmp (mbl, usa) (working concentration: µg/ml), (c) rabbit anti-human mmp (thermo fisher scientific, usa) (working concentration: µg/ml), (d) enos (thermo fisher scientific, usa) (working concentration: µg/ml). the zytochem plus detection kit (germany) was used for the development as described by the manufacturer. qrt-pcr. total rna was isolated from acquired samples using the tri reagent (sigma, saint louis, mo, usa), according to the manufacturer's protocol [ ] . cdna was synthesized by rt (m-mlv, reverse transcriptase; sigma), and real-time quantitative polymerase chain reaction was performed by using sybr green the thermal cycling protocol was as follows: min at °c, followed by cycles of °c for s and then °c for min. negative pcr controls were run to verify the absence of genomic dna contamination (no reverse transcription control). fluorescence was recorded at regular intervals following the °c annealing/extension segment of the pcr, and real-time data showing the relative fluorescence versus cycle number were analyzed. because of the paucity of good internal pcr controls for tissue specimens, rl , mmp , mmp , and enos expression (for consistency of measurement) was determined from a Δct value [expression = (−ΔΔct)] where Δct was derived for each individual specimen and calculated by subtracting the mean ct value for all targets measured from the individual ct value of a given pcr target, as previously described. the results were then reported as the mean ± sem for each peptide (rl , mmp , mmp , and enos) measured in tissue. statistical analysis data are presented as the mean ± standard deviation (mean ± sd). statistical significance between groups was calculated using tukey's multiple comparison test. correlation between measured parameters was assessed by pearson analysis. differences were considered significant when a two-tailed p < . was calculated. all statistical calculations were performed using graphpad prism, version . (graphpad, inc., ca, usa). immunohistochemical tissue biomarkers-qualitative analysis tissue biomarkers are presented in fig. . our qualitative analysis showed that mmps and enos were colocalized to residual elastic fiber fragments in aneurysmal tissue and in atherosclerotic plaques. interestingly, we found that relaxin- lined endothelial cells (ecs) in all groups; the tunica media was composed of alternate layers of elastic lamellae in both aa and ath. metalloproteinase staining was also found in endothelial cells of both aa and ath. we also found thickening of the tunica media marked with disarrangement of smooth muscle cells (in all groups). in both the aa and ath groups, we observed an accumulation of amorphous material and plasma membrane rupture, while in the tab group, mmp staining showed a diffusion of the endothelial lining bound by tight junctions. finally, enos staining showed features of endothelial cell vacuolation (all groups) and plasma membrane rupture in the aa group. expression tissue mrna levels of rl , mmp , mmp , and enos in all groups in all three ath subgroups, the rl level was significantly higher than those of mmp , mmp , and enos (p < . ) (fig. ) . similarly, rl tissue levels were increased in comparison to mmp , mmp , and enos levels (p < . ). in tab patients, rl tissue levels were increased compared to those of mmps and enos (p < . for all). there was no significant difference between rl and mmps and enos in the ag group. all data of the mrna tissue levels are presented in supplementary table for the aa and ath groups and supplementary table for the tab and ag groups. differences in tissue rl , mmps, and enos mrna levels across increasing severities of aneurysmatic and atherosclerotic disease across increasing severity of the ath group, mmp , mmp , and enos expression decreased in patients with a more severe presentation of atherosclerotic disease (p < . ) (fig. a) . in contrast, within the aneurysmatic disease (subgroup aa to aa ), rl , mmp , and mmp as well as enos tissue levels increased (p < . ) (fig. b) . comparison of rl , mmps, and enos mrna levels between all study groups rl , mmps, and enos mrna levels were substantially higher in the aa group than in the ath, tab, and ag groups (supplemental graphs a, b, c and d). there were no significant differences between rl , mmp , and enos levels in the ath and tab groups. only mmp was significantly higher in the ath group than in the tab group. the ag group had significantly lower levels of rl , mmps, and enos mrna than the other groups had. comparison of rl , mmps, and enos mrna levels between subcategorized study groups in the ath subgroups, further statistical analysis (fig. a-d) revealed that rl , mmps, and enos mrna levels were significantly higher in the ath and ath subgroups than in the tab group, with the exception of rl mrna levels in the ath subgroup, which were not significantly different from tab. mrna levels were lower or not significantly different in patients with clinically severe atherosclerotic disease (ath subgroup) compared to the tab group. finally, the ag group had significantly lower mrna levels than all subgroups had. further statistical analysis revealed that only in arterial specimens from larger or symptomatic aneurysms (aa and aa groups) were the rl , mmps, and enos mrna levels consistently higher than those in the ath subgroups or in the tab and ag groups. specimens from smaller aneurysms (aa group) had significantly higher rl , mmps, and enos mrna levels only compared to the levels in patients with clinically severe atherosclerotic disease (ath subgroup) and to the levels in the ag group and generally had significantly lower or not significantly different levels compared to those in less clinically advanced atherosclerotic disease (ath and ath subgroups) and in the tab group. correlation between rl and mmps and enos in the aa and ath groups in patients with mild atherosclerosis (group ath ), rl was positively correlated with enos and inversely correlated with mmp , whereas it was positively associated with mmp and enos in moderate atherosclerotic disease (ath ) ( table , upper right). in severe atherosclerosis (ath ), no correlations were found between rl and mmp , mmp , and enos (table , upper right). with respect to aas, rl was positively associated with enos and inversely correlated with both mmp and mmp only in patients with mild dilatation (aa group). in contrast, no significant correlations of rl were found in patients with moderate and severe arterial aneurysmatic disease (aa and aa ) ( table , lower left). furthermore, rl was positively correlated with mmp , mmp , and enos in the tab group. in the ag group, rl did not correlate with either mmps or enos (p > . ). the principal finding of this study is that tissue-specific rl is increased in patients with a larger or symptomatic aa in comparison to subjects with atherosclerotic disease and to healthy controls. notably, in situ rl levels were proportionally increased according to the size and severity of aneurysmatic disease and were substantially elevated in patients with a symptomatic aneurysm of any diameter or an asymptomatic aneurysm of a diameter > % of that of the normal artery in comparison to (a) patients with asymptomatic or smaller aas, (b) subjects with atherosclerosis, and (c) control subjects. on the other hand, an inverse association of tissue rl with the clinical severity of atherosclerotic lesions was observed. these findings are generally consistent with our previously published data on serum rl [ ] , and to the best of our knowledge, this is the first report on the correlation of tissue-specific rl mrna levels in human subjects with aneurysms and atherosclerosis. further to our previous work, our current study indicated that tissue mmps and enos fluctuate with exactly the same pattern as rl in various phenotypes of both aneurysmatic and atherosclerotic disease, and importantly, rl was not related to mmps in moderate and severe aneurysmatic and atherosclerotic disease, suggesting an independent role of this multipotent molecule on vascular wall integrity and remodeling. aas are characterized by structural alterations of the vascular wall resulting, in part, from the degradation of collagen and elastin fibers. these changes are associated with inflammatory infiltrates [ ] and excessive production of mmps [ , ] , which are assumed to orchestrate the widespread matrix destruction. an association between aa and mmps has been described in both aaa [ , ] and pad [ , ] . mmp transcripts expressed physiologically at low levels increase rapidly in the presence of inflammation-associated vascular remodeling. notably, increased expression of mmps has been observed by other authors in human aneurysm tissue [ ] and has been linked to extracellular matrix degradation and increased risk of rupture in response to distending intra-arterial pressure [ , ] . previous studies have shown a complementary role of mmp and mmp in aneurysmal dilatation. in brief, mmp primarily acts as a collagenase that initiates cleavage of the collagen triple helix, and subsequently, single chains are subject to degradation by mmp [ , ] . in our study, we found that increased expression of both mmps is related to a more severe phenotype of aa disease, even though previous studies have reported contradicting conclusions on the clinical utility of circulating plasma [ ] and serum [ ] levels of mmp and mmp . in addition, we observed an inverse correlation of rl with mmps in the early stages of aa. however, the association of mmps with rl in advanced stages was not noted; this suggests that further mechanisms are involved. endothelial cells express enos to serve as an important source of no, a potent vasodilator and inhibitor of inflammation, platelet aggregation, and smooth muscle cell proliferation [ ] . interestingly, enos gene polymorphisms have been associated with vascular diseases [ ] ; two recent meta-analyses have identified the t c polymorphism of enos as a predictor for the development of intracranial aneurysms in the cerebral vascular system [ , ] , and various polymorphisms of enos have been linked to aaa [ ] [ ] [ ] . our findings of increased enos levels in larger aneurysms are in accordance with a recent animal study [ ] , in which increased enos activity reduced smooth muscle α-actin and upregulated mmps during flow-induced intracranial aneurysm formation. however, experiments in mice have shown that enos deficiency results in an increased incidence of aaa formation [ ] , and aoki et al. [ ] showed that enos suppresses cerebral aneurysm formation by reducing hemodynamic stress to arterial walls. currently, there is no clinical utility of enos as a biomarker for predicting the natural history of human aneurysms, which are a multifactorial disease, and the contradicting evidence suggests that enos may play a lesser role in aneurysm formation and progression than other factors. destabilization of plaques is implicated as a clinical manifestations of atherosclerotic disease [ , ] . among various proteinases, mmp and mmp have been shown to be the predominant ones secreted by t lymphocytes and macrophages [ , ] across atherosclerotic plaque development [ ] . mmp is constitutively expressed in all human vascular cells [ ] , but plaques express increased mmp compared to that in normal vessels, especially unstable ones [ ] . overall, mmp levels are increased in patients with atherosclerotic [ , ] pad, especially in combination with type ii diabetes [ ] or acute coronary syndromes [ , ] . additionally, circulating mmp levels are increased in patients with atherosclerosis [ ] , acute coronary syndromes [ , ] , and type ii diabetes [ ] . there is also substantial evidence for a link between heightened mmp and plaque vulnerability [ , ] through facilitation of basement membrane breakdown, smooth muscle cell migration, plaque neovascularization, and inflammatory cell infiltration [ , ] . indeed, in our current study, increased levels of mmp and mmp were related to the severity of atherosclerotic disease, while the rl level was inversely associated with atherosclerotic burden. our group has recently shown serum rl to be increased in the early clinical stages of atherosclerosis and decreased in more advanced stages of atherosclerotic disease [ ] , and it was hypothesized that rl upregulation represents a form of protective mechanism; however, it was unclear whether this early increase was actually beneficial. the inverse correlation of rl with mmp in mild atherosclerosis and the positive association with enos in moderate atherosclerosis is suggestive of a beneficial effect. enhanced no bioavailability through enos upregulation and decreased expression of h s generating enzymes might increase plaque stability and integrity [ , ] . it remains unclear why this protective mechanism is ameliorated in advanced stages of atherosclerosis, but our findings provide further evidence of potential therapeutic use of rl in atherosclerotic disease, as other investigators have recently demonstrated [ ] . there is a good size of evidence that rl increases no synthesis in both an acute and a delayed fashion, although the actual mechanism and whether this is related directly or indirectly to rfxp is not clear [ , ] . the activity and expression of not only enos [ ] [ ] [ ] [ ] but also inducible-nos (inos) [ , , [ ] [ ] [ ] [ ] [ ] [ ] [ ] and neuronal-nos (nnos) is increased by rl . due to the production of no and activation of guanyl cyclase, nos activation by rl leads to an increase in cyclic guanyl monophosphate (cgmp) [ , , , ] . based on activation of nos and cgmp, no production by rl can be further enhanced by an increase in enos activity by akt (protein kinase b) phosphorylation [ ] , upregulation of inos activity following stimulation of nuclear factor kappa-light-chain-enhancer of activated b cells (nf-κΒ)- table . correlation matrix of assessed biomarkers tissue mrna levels in each aneurysm and atherosclerosis group separately, depending on the severity of atherosclerosis. down-left illustrated the aa groups. upright illustrated the ath groups. − represents the aa or ath group; − represent the aa or ath group; − represent aa or ath group. r values indicate pearson's correlation coefficients. significant association between two tissue biomarkers is presented with green color; no statistical significance presented with red color; while the minus symbol (−) in green boxes indicates an inverse association. aa aneurysm group, ath atherosclerosis group, rl relaxin- , mmp matrix metalloproteinase, enos endothelial nitric oxide synthetase relaxin- in aneurysmatic and atherosclerotic disease k papoutsis et al. controlled transcription [ , ] , and finally by increased activity of enos, following activation of the endothelin b (et b ) receptor by increased activity of mmp and mmp by rl [ ] . interestingly, inhibition of nf-κΒ-mediated transcription reduces the rl -related increase in mmp expression and activity [ ] , suggesting a relationship between the effects of rl upon mmps and no [ ] . furthermore, rl can decrease the expression of tissue inhibitors of mmps (timps) [ ] [ ] [ ] , and recently, it was shown that the known pathways of rl /rfxp signaling [including activation of phosphoinositide -kinase (pi k), akt, protein kinase c (pkc)-ζ, and extracellular signal-regulated protein kinases and (erk / )] are connected with the increased expression of mrna for mmp [ ] . these insights could be useful for investigating the utility of rl in the clinical setting [ ] . certain limitations should be acknowledged in our study. we enrolled four relatively small groups of patients, which might have hampered the identification of associations. furthermore, our study is limited by the fact that the ag group was not matched with other groups for age and comorbidities, while in the ath group, subjects presented with heterogeneous arterial pathologies. these limitations warrant caution when interpreting the results and drawing conclusions. in our study, we included data from autopsy subjects, as gupta et al. [ ] have shown that under conditions similar to ours, autopsy specimens are reliable for conduction molecular estimations. importantly, no definite conclusions can be made regarding the underlying mechanisms of rl in vascular diseases. contemporary literature does not provide mechanistic data on possible rl effects on aas. consequently, the potential applicability of rl as a novel therapeutic target merits further investigation. a postulation may be advanced that rl represents a feedback protective mechanism to counteract an ongoing detrimental process. in conclusion, tissue-specific rl is higher in patients with an aa, showing a positive relationship with the size of the aneurysmatic dilatation. levels of rl are also inversely correlated with the severity of atherosclerotic disease. future studies with larger cohorts are warranted to verify and expand our results with the ultimate aim to reveal possible etiopathogenetic backgrounds of atherosclerosis and aneurysm formation. kp wrote the paper and designed the study; ak performed the research; gg and np analyzed the data; c. kontogiannis, c. kourek, ksm and dvc wrote the paper; and ik and sg designed the study. estimation of global and regional incidence and prevalence of abdominal aortic aneurysms contemporary open repair of ruptured abdominal aortic aneurysms a french randomized controlled trial of endovascular versus open surgical repair of ruptured aorto-iliac aneurysms a randomised trial of endovascular and open surgery for ruptured abdominal aortic aneurysm-results of a pilot study and lessons learned for future studies endovascular or open repair strategy for ruptured abdominal aortic aneurysm: day outcomes from improve randomized trial amsterdam acute aneurysm trial collaborators. endovascular repair versus open repair of ruptured abdominal aortic aneurysms: a multicenter randomized controlled trial novel biomarkers of abdominal aortic aneurysm disease: identifying gaps and dispelling misperceptions potential circulating biomarkers for abdominal aortic aneurysm expansion and rupture-a systematic review associations between serum relaxin , aneurysm formation/size and severity of atherosclerosis: a preliminary prospective analysis relaxin: a pleiotropic hormone activation of orphan receptors by the hormone relaxin relaxin family peptides and their receptors evidence for local relaxin ligand-receptor expression and function in arteries localization of relaxin receptors in arteries and veins, and region-specific increases in compliance and bradykinin-mediated relaxation after in vivo serelaxin treatment vascular matrix metalloproteinase- mediates the inhibition of myogenic reactivity in small arteries isolated from rats after short term administration of relaxin matrix metalloproteinase induction by relaxin causes cartilage matrix degradation in target synovial joints effects of recombinant h relaxin on the expression of matrix metalloproteinases and tissue inhibitor metalloproteinase in cultured early placental extravillous trophoblasts size matters: the relationship between mmp- expression and aortic diameter matrix metalloproteinases and work in concert to produce aortic aneurysms relaxin- in cardiometabolic diseases: mechanisms of action and future perspectives evolution of the relaxin-like peptide family: from neuropeptide to reproduction signalling by insulin and igf receptors: supporting acts and new players angiotensin ii-mediated hypertension in the rat increases vascular superoxide production via membrane nadh/nadph oxidase activation: contribution to alterations of vasomotor tone hypercholesterolemia increases endothelial superoxide anion production free radicals mediate endothelial cell dysfunction caused by elevated glucose effects of diabetes on monocyte-endothelial interactions and endothelial superoxide production in fructose-induced insulinresistant and hypertensive rats does adma cause endothelial dysfunction? single-step method of rna isolation by acid guanidinium thiocyanate-phenol-chloroform extraction immunophenotypic analysis of the aortic aneurysm wall suggests that vascular dendritic cells are involved in immune responses expression and localization of macrophage elastase (matrix metalloproteinase- ) in abdominal aortic aneurysms matrix metalloproteinase- production and its binding to the matrix are increased in abdominal aortic aneurysms systemic dilation diathesis in patients with abdominal aortic aneurysms: a role for matrix metalloproteinase- expression of matrix metalloproteinases and timps in human abdominal aortic aneurysms distribution of inflammatory mediators in carotid and femoral plaques the pathophysiology of abdominal aortic aneurysm growth: corresponding and discordant inflammatory and proteolytic processes in abdominal aortic and popliteal artery aneurysms prevention of abdominal aortic aneurysm progression by targeted inhibition of matrix metalloproteinase activity with batimastat-loaded nanoparticles proteolysis of the abdominal aortic aneurysm wall and the association with rupture matrix metalloproteinase- is an interstitial collagenase inhibition of matrix metalloproteinases blocks lethal hepatitis and apoptosis induced by tumor necrosis factor and allows safe antitumor therapy the plasma level of matrix metalloproteinase may predict the natural history of small abdominal aortic aneurysms. a preliminary study aminoterminal propeptide of type iii procollagen and matrix metalloproteinases- and - failed to serve as serum markers for abdominal aortic aneurysm accelerated atherosclerosis, aortic aneurysm formation, and ischemic heart disease in apolipoprotein e/endothelial nitric oxide synthase double-knockout mice endothelial nitric oxide synthase gene sequence variations and vascular disease relationship between endothelial nitric oxide synthase (enos) and natural history of intracranial aneurysms: meta-analysis association between three enos polymorphisms and intracranial aneurysms risk: a meta-analysis. medicine (baltimore) allele frequency of human endothelial nitric oxide synthase gene polymorphism in abdominal aortic aneurysm enos g t polymorphism as a mild predisposing factor for abdominal aortic aneurysm enos g t polymorphism and abdominal aortic aneurysms endothelial nitric oxide synthase and superoxide mediate hemodynamic initiation of intracranial aneurysms complementary inhibition of cerebral aneurysm formation by enos and nnos coronary plaque disruption coronary risk factors and plaque morphology in men with coronary disease who died suddenly comparison of mmp- and mmp- secretion from t helper , and lymphocytes alone and in coculture with macrophages inflammation and plaque vulnerability the immune response in atherosclerosis: a double-edged sword matrix metalloproteinases in vascular remodeling and atherogenesis: the good, the bad, and the ugly matrix metalloproteinase is associated with stable and matrix metalloproteinases and with vulnerable carotid atherosclerotic lesions: a study in human endarterectomy specimen pointing to a role for different extracellular matrix metalloproteinase inducer glycosylation forms gene expression levels of matrix metalloproteinases in human atherosclerotic plaques and evaluation of radiolabeled inhibitors as imaging agents for plaque vulnerability increased expression of matrix metalloproteinases and matrix degrading activity in vulnerable regions of human atherosclerotic plaques plasma levels and zymographic activities of matrix metalloproteinases and in type ii diabetics with peripheral arterial disease genetic polymorphisms and plasma levels of matrix metalloproteinases and their relationships with developing acute myocardial infarction matrix metalloproteinase: investigation from gene to protein as effective factor in myocardial infarction peripheral blood levels of matrix metalloproteases- and - are elevated in patients with acute coronary syndromes plasma brain natriuretic peptide, endothelin- , and matrix metalloproteinase expression and significance in type diabetes mellitus patients with ischemic heart disease prospective study of matrix metalloproteinase- and risk of myocardial infarction and stroke in older men and women associations of matrix metalloproteinase- and monocyte chemoattractant protein- concentrations with carotid atherosclerosis, based on measurements of plaque and intima-media thickness matrix metalloproteinase- and - differentially regulate smooth muscle cell migration and cell-mediated collagen organization vascular endothelial growth factor and matrix metalloproteinase expression in human carotid atherosclerotic plaques: relationship with plaque destabilization via neovascularization amelioration of atherosclerotic inflammation and plaques via endothelial adrenoceptor-targeted enos gene delivery using redox-sensitive polymer bearing l-arginine reciprocal regulation of enos, h s and co-synthesizing enzymes in human atheroma: correlation with plaque stability and effects of simvastatin efficacy and safety of oral porcine relaxin (prlx) in adjunct to physical exercise in the treatment of peripheral arterial disease (pad) emerging role of relaxin in renal and cardiovascular function relaxin receptors and nitric oxide synthases: search for the missing link influence of relaxin on the neurally induced relaxant responses of the mouse gastric fundus reversal by relaxin of altered ileal spontaneous contractions in dystrophic (mdx) mice through a nitric oxide-mediated mechanism relaxin up-regulates the nitric oxide biosynthetic pathway in the mouse uterus: involvement in the inhibition ofmyometrial contractility relaxin- in aneurysmatic and atherosclerotic disease k papoutsis et al relaxin depresses small bowel motility through a nitric oxide-mediated mechanism: studies in mice relaxin depresses platelet aggregation: in vitro studies on isolated human and rabbit platelets relaxin activates the l-arginine-nitric oxide pathway in vascular smooth muscle cells in culture locally produced relaxin may affect testis and vas deferens function in rats relaxin upregulates inducible nitric oxide synthase expression and nitric oxide generation in rat coronary endothelial cells relaxin inhibits the activation of human neutrophils: involvement of the nitric oxide pathway protective effect of relaxin in cardiac anaphylaxis: involvement of the nitric oxide pathway relaxin potentiates the expression of inducible nitric oxide synthase by endothelial cells from human umbilical vein in in vitro culture relaxin activates the l-arginine-nitric oxide pathway in human breast cancer cells relaxin-induced matrix metalloproteinase- expression is associated with activation of the nf-b pathway in human thp- cells relaxin modulates synthesis and secretion of procollagenase and collagen by human dermal fibroblasts inhibition of markers of hepatic stellate cell activation by the hormone relaxin relaxin inhibits effective collagen deposition by cultured hepatic stellate cells and decreases rat liver fibrosis in vivo postmortem cardiac tissue maintains gene expression profile even after late harvesting relaxin- in aneurysmatic and atherosclerotic disease k papoutsis et al the online version of this article (https://doi.org/ . /s - - - ) contains supplementary material, which is available to authorized users.competing interests: the authors declare no competing interests.