key: cord-0028769-qorxl1r8
authors: Fang, Hua; Li, Hua-Feng; Yang, Miao; Liao, Ren; Wang, Ru-Rong; Wang, Quan-Yun; Zheng, Peng-Cheng; Zhang, Fang-Xiang; Zhang, Jian-Ping
title: NF-κB signaling pathway inhibition suppresses hippocampal neuronal apoptosis and cognitive impairment via RCAN1 in neonatal rats with hypoxic-ischemic brain damage
date: 2019-05-03
journal: Cell Cycle
DOI: 10.1080/15384101.2019.1608128
sha: b44dd88a63fec3795c86b30c82b69043a0bfef20
doc_id: 28769
cord_uid: qorxl1r8

NF-κB is a core transcription factor, the activation of which can lead to hypoxic-ischemic brain damage (HIBD), while RCAN1 plays a protective role in HIBD. However, the relationship between NF-κB and RCAN1 in HIBD remains unclear. This study aimed to explore the mechanism of NF-κB signaling pathway in hippocampal neuron apoptosis and cognitive impairment of neonatal rats with HIBD in relation to RCAN1. Initially, microarray analysis was used to determine the differentially expressed genes related to HIBD. After the establishment of HIBD rat models, gain- or loss-of-function assay was performed to explore the functional role of NF-κB signaling pathway in HIBD. Then, the learning and memory ability of rats was evaluated. Expression of RCAN1, NF-κB signaling pathway-related genes and glial fibrillary acidic protein (GFAP), S-100β and acetylcholine (Ach) level, and acetylcholinesterase (AchE) activity were determined with neuron apoptosis detected to further explore the function of NF-κB signaling pathway. RCAN1 could influence the development of HIBD. In the HIBD model, the expression of RCAN1 and NF-κB-related genes increased, and NF-κB p65 showed a significant nuclear shift. By activation of NF-κB or overexpression of RCAN1, the number of neuronal apoptosis, S-100β protein level, and AchE level increased significantly, Ach activity decreased significantly, and GFAP positive cells increased. In addition, after the activation of NF-κB or overexpression of RCAN1, the learning and memory ability of HIBD rats was inhibited. All the results show that activation of NF-κB signaling pathway promotes RCAN1 expression, thus increasing neuronal apoptosis and aggravating cognitive impairment in HIBD rats.

Hypoxic-ischemic brain damage (HIBD) is a disease which can induce inflammatory lung injury [1] . It is also one of the major causes of death and long-term neurological impairment in infants and children [2] . Hypoxic-ischemic encephalopathy induced by HIBD is a severe brain disorder for children but without effective treatment [3] . It has been proved that the pathological mechanisms of HIBD are involved in factors like oxidative stress, inflammation and nerve cell apoptosis [4, 5] . It requires extensive resources and wealth for the treatment and care of the sequelae of HIBD, and there is frequently little improvement in the overall ability of children with the disease even with the best care [6] . Therefore, it is necessary to elucidate the molecular mechanism underlying HIBD to explore a treatment regimen for HIBD.

Nuclear factor κB (NF-κB) is involved in many types of tumors in which it plays a confounding role [7] . Moreover, as a core transcription factor of many signaling pathways, NF-κB also plays an important role in cell survival and apoptotic celldeath via regulating the relative expression of selected genes [8] . A previous study has proved that when NF-κB signaling pathway is inhibited, the apoptosis of damaged nerve cells can be reduced resulting in an improvement of learningmemory function in HIBD rats [9] . Moreover, the regulator of calcineurin 1 (RCAN1) has been proved to be regulated by NF-κB [10] . RCAN1 can regulate the activity of calcineurin phosphatase and suppress inflammation [11] . Recently, evidence suggests that RCAN1 is highly expressed around the infarct area after experimental stroke and are associated with brain ischemia/reperfusion injury [12] . Overexpression of RCAN1 has been proved to induce caspase-9 and caspase-3 and subsequently promotes neuronal apoptosis in primary neurons in Alzheimer disease [13] . Based on these findings, it is of great importance to study the exact roles of NF-κB signaling pathway and RCAN1 in the treatment of HIBD. Thus, in this study, we aim to explore the hypothesis that RCAN1 down-regulation by the inhibition of the NF-κB signaling pathway can suppress the apoptosis of hippocampal neurons and improve cognitive impairment of rats with HIBD.

This study was approved by the ethics committee of Guizhou Provincial People's Hospital and was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals. Best efforts were made to minimize the sufferings of animals.

The HIBD microarrays, GSE2161 and GSE11686, were retrieved from the National Center for Biotechnology information (NCBI). The data of the microarrays were downloaded from the Gene Expression Omnibus (GEO) database (http://www. ncbi.nlm.nih.gov/geo). GSE2161 (https://www. ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE2161) included four HIBD samples and four normal samples with annotation probe [Mouse430_2] Affymetrix Mouse Genome 430 2.0 Array (GPL1261). GSE11686 (https://www.ncbi.nlm.nih. gov/geo/query/acc.cgi?acc = GSE11686) included 6 HIBD samples and 10 normal samples with annotation probe [HG-U133A] Affymetrix Human Genome U133A Array (GPL96). The "limma" package (http://www.bioconductor.org/packages/ release/bioc/html/limma.html) in the R Language Programming based on Bioconductor was used to select important differentially expressed genes through Empirical Bayes [14] . At last, the differentially expressed genes were annotated using the "annotate" package (http://www.bioconductor.org/ packages/release/bioc/html/annotate.html). Differentially expressed mRNAs were judged by the threshold of p< 0.05 and | Log2FoldChange| >1.5.

Sixty healthy Sprague-Dawley (SD) rats (no limitation with gender; aged 7 days; weighing 12-20 g), provided by Experimental Animal Center of Guizhou University, were selected and randomly divided into two groups: the sham group (n = 10) and the HIBD group (n = 50). Artery ligation improved by Nakajima et al. [15] was conducted to establish hypoxia-ischemia animal models at room temperature of 20°C ± 5°C after all animals were weighed preoperatively. The neonatal rats were anesthetized with ether and placed in a supine position with limbs fixed on a small operation board. After the skin was sterilized with 75% alcohol, an incision was made in the midline of the neck, and the left common carotid artery was aseptically isolated and ligated twice with a 7-0 sterile surgical silk. The wound was sutured and the skin was disinfected again. The animals were allowed to recover in an incubator at 32.5°C for 2 h. The surgical rat was then placed in a 2 L airtight jar, and the bottom layer was covered with sodium lime (to absorb CO 2 and moisture). The jar was submerged in a 37°C water bath to maintain a constant thermal environment. A mixture of 8% oxygen and 92% nitrogen were introduced at a rate of 1 to 2 L/min, and the internal oxygen concentration was measured with an oxygen meter to maintain the oxygen concentration in the hypoxic chamber at about 8% (7%9 %) with continuous hypoxia kept for 2 h. After successful establishment of HIBD model, the rats were placed in air at room temperature for about 1 2 h, and finally put back into the dams for breastfeeding. The entire procedure was completed within 40 min and the anesthetic time should not be too long. Intraoperative procedures should be refined to minimize bleeding and to avoid stimulating the surrounding tissue. After surgery, the bloodstains of neonatal rats should be wiped off to prevent the dams from refusing to breastfeed because of strange smells. Sham-operated rats underwent the same operative procedures except that the exposed carotid artery was not ligated. In addition, after the skin was sutured, no hypoxic treatment was performed, and the rats were returned to the dams at the same time as the model group.

The state of consciousness, general behavior, and physical activity of the rats were observed before the experiment, after the anesthesia, after the surgery, during the hypoxia, and at the end of the hypoxia. At the 7 th d after establishment of HIBD model, three rats in the sham group or the HIBD group were randomly selected for anesthesia. The rats were fixed on a surgical plate to open the chest cavity. A 20 mL sterile medical syringe attached to a puncture needle was carefully inserted to the left ventricle of the neonatal rat, and the right atrial appendage of the rat was cut, which was injected with sterile saline continuously until the effluent fluid became clear and then injected with about 10 to 20 mL of 4% paraformaldehyde. After the limbs and liver of rats became white and hardened, the brain was extracted, observed with naked eyes and externally fixed in paraformaldehyde. The section was prepared vibrantly. The structure, neuronal morphology and the arrangement of the rat cerebral cortex and hippocampus were observed by the Nissl staining under a normal optical microscope.

The paraffin-embedded sections were routinely dewaxed and immersed in a 1% toluidine blue solution for 10 min at 37°C. The excess staining solution was washed away with distilled water. The sections were color-saturated by 80-95% gradient alcohol to an appropriate rate and observed under a microscope. After the cytoplasmic staining could be clearly compared, the sections were dehydrated with anhydrous alcohol, cleared with transparent xylene, and sealed with neutral balsam. Then the morphological changes of neurons in the hippocampus were observed under the microscope.

Besides the sham group (n = 10) and the HIBD group (n = 10), the remaining successfully modeled rats were randomly divided into the following groups:

Phorbol-12-myristate-13-acetate (PMA) group: PMA (ab120297, Abcam Inc., Cambridge, MA, USA) was dissolved in dimethyl sulfoxide (DMSO) with the final concentration 100 mM and intraperitoneally injected into the HIBD rats (n = 10); BAY-11-7805 group: The NF-κB inhibitor BAY- (ab141574, Abcam Inc., Cambridge, MA, USA) was dissolved in DMSO with the final concentration 10 μM and injected intraperitoneally into the HIBD rats (n = 10); si-RCAN1 group: RCAN1 AAV siRNA (iAAV05614009, ABM Inc., Richmond, BC, Canada) packaged viruses were injected into the left hippocampus of successfully modeled HIBD rats by stereo injection (n = 10); PMA + si-RCAN1 group: PMA and RCAN1 AAV siRNA packaged viruses were injected stereoscopically into the successfully modeled HIBD rats (n = 10).

The Morris water maze (MWM) test and swimming training were performed when rats were 1-month-old to evaluate the spatial learning and memory of rats. Before the day of training, the platform was removed, and a fixed point was selected on the wall of the pool. The rats swam into the water in the direction of facing the pool wall, and swimming trajectory were recorded. The purpose was to allow the rats to adapt to the environment, to perform preliminary tests on the swimming ability of the rats and to eliminate those who have obvious abnormalities. The navigation test was adopted to test the ability of rats to learn and memorize the water maze. The rats were continuously trained. Each day, the rats were placed into the water from different fixed points facing the pool wall. In each experiment, the time that the rat sought and boarded the platform (escape latency) was recorded as the learning and memory performance. If the rat failed to find the platform within the set time, the computer would stop tracking and record the time. Rats failing to find the platform would be brought to the platform by the experimenter. The training interval (after a training session, the rats were allowed to rest on the platform for a while) was very important for rats that were highly stressed in water. It can prevent rats from jumping back into the water and guarantee the success of the experiment. After the day's training, the rats would be washed, dried and placed next to the heater to prevent them from hypothermia. Then, the average escape latency for each group of rats was calculated. And the spatial probe trail was used to detect rats' exact memory of the spatial location of the platform after the rats learn to search for the platform, namely the memory retention ability. After the navigation test (the 5 th d of MWM test), the platform was withdrawn, and then a fixed quadrant was selected randomly as the entry and the rat was placed here to swim into water in the direction of facing the pool wall. The swimming trajectory within 120 s and the times of crossing the original platform position was recorded. At the end of the spatial probe trail, after rats rested for 1 d (the 6 th d of MWM test), a water maze test was performed again on all rats. The method was the same as the spatial probe trail to exam the ability of long-term memory retention.

The total RNA was extracted by the Trizol onestep method, and the microplate reader (DNM-9606, Beijing Perlong New Technology Co., Ltd., Beijing, China) was used to determine the purity and concentration of the sample RNA. The RNA was reversely transcribed into cDNA Using the PrimeScript TM RT-PCR Kit (Perfect Real-Time) Kit (RR047A, Takara Bio Inc., Otsu, Shiga, Japan), which was used as a template. The expression of RCAN1 was determined using β-actin as an internal control. The reverse transcription reaction conditions were: at 37°C for 60 min, and at 85°C for 5 min. The relative expression of mRNA was measured by using the SYBR Green Real-time fluorescence quantitative PCR kit (1725270, Bio-Rad, Hercules, CA, USA) on the Bio-Rad 9700 instrument (10021337, Bio-Rad, Hercules, CA, USA), and the experiment was repeated 3 times. The RT-qPCR system was 25 µL, including 21 μL of 1 × SYBR premix Ex Taq mix (Takara Bio Inc., Otsu, Shiga, Japan), 2 μL of cDNA and 1 μL of (10 nM) forward and reverse primers, respectively. The reaction conditions were as follows: predenaturation at 95°C for 3 min, 45 cycles of denaturation at 95°C for 15 s, annealing at 60°C for 20 s and extension at 72°C for 30 s. the mRNA level in each group was calculated using the 2 −ΔΔCt method [16] . The used primers are shown in Table 1 .

The total protein was extracted from the tissue homogenate. The lysate was collected after centrifugation at 4°C and 45 × g for 15 min. The protein concentration was determined by the Bradford method. Then, 30 μg of the sample protein was obtained and mixed well with immobilized pH gradient (IPG) loading buffer and underwent electrophoresis with 10% sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE) gel for 2 h. The protein on the gel was transferred onto a polyvinylidene fluoride (PVDF) membrane. After being blocked with 5% skimmed milk for 2 h at room temperature, the membrane was washed 3 times with tris-buffered saline with tween (TBST) buffer, and incubated with rabbit anti-rat NF-κB p65 polyclonal antibody (ab16502, 1: 500), NF-κB phosphorylated (p)-p65 polyclonal antibody (ab86299, 1: 2000), RCAN1 monoclonal antibody (ab185931, 1: 500) and β-actin monoclonal antibody (ab8227, 1: 2000) overnight at 4°C. βactin was used as an internal reference. Then, the membrane was washed three times with TBST buffer and then incubated with goat anti-rabbit secondary antibody (ab6721, 1: 2000) at room temperature for 2 h. Subsequently, the membrane was washed 3 times with TBST buffer. The primary and secondary antibodies were all obtained from Abcam Inc. (Cambridge, UK). The color was developed by Electro-Chemi-Luminescence (ECL) method. After tableting and computer scanning, the gray value of the target bands was measured by the image analysis software Image J (National Institutes of Health, Bethesda, Maryland, USA). The relative expression of the target proteins was expressed by the ratio of the gray value of the target protein bands to that of the reference protein band. Statistical mapping was performed and the experiment was repeated 3 times.

The apoptotic cells in hippocampus tissues of rats in each group were detected by TUNEL staining and counted. The hippocampus tissues were dehydrated by gradient alcohol, cleared by xylene, immersed in wax, embedded in paraffin, and cut into sections. The continuous coronal sections were obtained by the paraffin machine from the chiasma opticum to the posterior horn of the lateral ventricle and loaded to the slides treated by polylysine. The sections were dewaxed with xylene for 10 min, immersed in 100-75% gradient alcohol for 1 min, respectively, and soaked in distilled water for 1 min. 

The soluble protein-100β (S-100β) content in the brain tissues was determined by the rat S-100β protein ELISA kit (R&D Systems, Minneapolis, MN, USA). The tissue homogenate (10%, 1000 μL) was centrifuged by a high-speed cryogenic centrifuge at 2863 × g at 4°C for 10 min, and the supernatant was obtained. The quantitative standard substance was added with 2 mL of distilled water to prepare a 20 ng/mL standard solution. Eight standard tubes were set-up. The first standard tube was added with 900 μL of the sample dilution, and the second to the seventh tubes were added with 500 μL sample dilution. Standard solution (100 μL, 20 ng/mL) was added to the first tube and mixed, and 500 μL of the mixture was aspirated with a pipette and transferred into the second tube. Dilutions were repeated until 500 μL of the mixture was removed from the 7 th tube. The 8 th tube was a blank control. The 10 × sample dilution was diluted with distilled water at the ratio of 1: 10, and the 20 × concentrated washing buffer were diluted with double distilled water at the ratio of 1: 20. Each tube was added with 100 μL of standard or test sample. After being mixed thoroughly, the plate was incubated at 37°C for 120 min. The subsequent operations were carried out following the kit instructions. A standard curve was plotted with standards of 2000, 1000, 500, 250, 62.5, 31.2 and 0 pg/mL as the abscissa and optical density (OD) as the ordinate. According to the OD value of the sample on the graph, the corresponding S-100β content was calculated.

Colorimetry was applied to determine the levels of acetylcholine (Ach) and acetylcholinesterase (AchE) activity in the rat hippocampus. The tissue homogenate (10%) was centrifuged at 2863 × g for 10 min at 4°C and the supernatant was obtained.

According to the instructions of the kit (Nanjing Jiancheng Bioengineering Institute, Nanjing, Jiangsu, China), the sample tubes, standard tubes, blank tubes and control tubes were prepared. The mixture in the tube was centrifuged were mixed and centrifuged at 2863 × g for 5 min at 4°C to obtain the supernatant. The OD value of Ach was measured with the spectrophotometer, and λ was set to 550 nm. The supernatant was placed in a slit cuvette and zeroed with distilled water, and the OD value of each tube was measured. The Ach level of the sample was calculated. The OD value of AchE was measured with the same procedure except for λ was set to 412 nm.

The samples were sectioned and put in a 60°C incubator for 20 min. The sections were dewaxed, added into 200 mL of ethylenediaminetetraacetic acid (EDTA) solution, steamed in a high-pressure cook for 10 min, and immersed in 3% H 2 O 2methanol for 10 min. Afterwards, the sections were incubated with rabbit anti-rat glial fibrillary acidic protein (GFAP, 1: 400, Beijing Bioss Biotechnology Co., Ltd., Beijing, China) and rabbit anti-rat NF-κB p65 polyclonal antibody (ab16502, 1: 500, Abcam Inc., Cambridge, UK) at 4°C overnight. After washing with PBS, the sections were added with CY-3-conjugated goat anti-rabbit Immunoglobulin G (IgG) and incubated in a wet box at 37°C for 30 min under the environment void of light. After PBS washing under the environment void of light, 4',6-diamidino-2-phenylindole (DAPI) solution was added to counterstain the nucleus for 30 s under the environment void of light at 37°C. And after PBS washing under the environment void of light and distilled water washing, the sections were sealed with 50% buffer glycerol. Except for sealing, after each step was completed, the samples were washed with PBS three times, 10 min each time. Sealing, antibody incubation, and rinsing steps were all performed on a shaker. Finally, the brain sections were spread on the slides. The hippocampal CA1 region was observed, and the number of GFAP immunepositive cells in three adjacent fields per section was counted, and the results were averaged from five fields. After the calculation of the number of GFAP positive cells, the nuclear shift of NF-κB p65 was observed using the NIS-Elements Viewer 4.0 software under a laser confocal microscope, and the emission wavelengths of CY-3 and DAPI were 565 nm and 460 nm, respectively.

The statistical analyses were conducted by the SPSS17.0 statistical software (SPSS Inc., Chicago, IL, USA). The experimental data were expressed as mean ± standard deviation. The escape latency among multiple groups was analyzed by repeated measures analysis of variance (ANOVA). The times of crossing platforms and the long-term memory latency in the spatial probe trial were analyzed by one-way ANOVA. Pairwise comparison within group was analyzed by means of Least Significant Difference (LSD) test. Data between two groups were compared by independent sample t test, with Bonferroni method for correction analysis. p < 0.05 was considered statistical significance.

GSE2161 and GSE11686 microarrays were downloaded from the GEO database. From the GSE2161 microarray, 2094 differentially expressed genes were screened out, among which 985 differentially expressed genes were upregulated and 1109 were downregulated. From the GSE11686 microarray, 241 differentially expressed genes were screened out with 106 genes were upregulated and 135 were downregulated. Previous studies suggest that RCAN1-encoded protein interacts with calcineurin A, inhibits phosphatase-dependent signaling pathway, and influences the central nervous system development, such as HIBD [17, 18] . RCAN1 has been reported to serve as an oncogene in many cancers [19, 20] . However, the specific effect of RCAN1 on HIBD is elusive. With RCAN1 as a candidate gene, we aimed to explore the relevance of RCAN1 to drug in HIBD and its clinical significance. Among the differentially expressed genes, RCAN1 has a high rank. Through the expression heatmaps of the first 50 differentially expressed genes from GSE2161 microarray (Figure 1(a) ) and GSE11686 microarray (Figure 1(b) ), we found that RCAN1 existed in the first 50 differentially expressed genes of both GSE2161 and GSE11686 microarrays and that RCAN1 was highly expressed in these two microarrays, especially in GSE2161 microarray. Moreover, the intersection of the first 50 differentially expressed genes of the microarrays were obtained, suggesting that only RCAN1 existed in the intersection (Figure 1(c) ). The NF-κB signaling pathway could cure neuroglioma by regulating RCAN1 gene [18, 21] . Thus, we hypothesized that the NF-κB signaling pathway could influence HIBD by regulating RCAN1 gene.

The Nissl-stained tissue sections were observed under the microscope to detect the pathological changes in the sham group and the HIBD group. The study found that there was no obvious neuronal damage on both sides of the section in the sham group. The morphology and structure of the cells were clear and complete, the nucleolus was clear, and the Nissl bodies without vacuoles were evenly distributed around the nucleus in order. It was demonstrated that in the HIBD group obvious pathological changes on the left cerebral hemisphere modeling side were observed, showing a large number of nuclear pyknosis, nuclear debris. Nissl body shape blurred or disappeared with vacuolar formation, and Nissl body was disorderly arranged forming a net. Compared with the sham group, the morphology of pyramidal cells in the HIBD group was changed significantly with disordered cell arrangement, significantly reduced volume, concentrated cytoplasm, slightly stained GSM296826  GSM296827  GSM296828  GSM296829  GSM296830  GSM296831  GSM296832  GSM296833  GSM296834  GSM296835  GSM296836  GSM296837  GSM296838  GSM296839  GSM296840  GSM296841   PVALB  CDC42  GOLGA8N  NOTCH2NL  HSPA2  TRDN  CEBPD  COL4A2  MT1E  CXCL14  LPL  FABP3  UGT2B4  PCDHGB6  RBM17  ADH6  KCNC3  CTNNA2  CST5  C10orf95  CD69  GPR85  OBSCN  NUSAP1  TSPYL2  GOLGA8A  BDH1  LIPE  TGM2  CBR1  KCNJ8  HMGCS2  RCAN1  SLC12A8  GPRC5D  AMOT  LRRN3  CACNA2D3  KIR3DL1  CHAD  LCAT  DPY19L2P2  CYP26B1  HSF2BP  MST1  KRT1  CLTC−IT1  NANOG  TRAIP  HCG18 type type HIBD normal Nissl bodies, and deformed or disappeared nucleus. There were apparent cell death and cell loss with normal pyramidal cells distributing among the dead cells ( Figure 2) . Therefore, the HIBD model was successfully established.

The mRNA and protein levels of RCAN1 and the NF-κB signaling pathway-related factors in hippocampal tissues were determined by RT-qPCR and Western blot analysis. The results showed that compared with the sham group, the HIBD group had significantly increased mRNA and protein levels of RCAN1 and the protein level of NF-κB p65 (all p< 0.05) (Figure 3 ), suggesting that RCAN1 and NF-κB p65 were highly expressed in the HIBD rats.

Activation of Nf-κB signaling pathway upregulates RCAN1 expression RT-qPCR and Western blot analysis were used to detect the expression of RCAN1 with activation of NF-κB signaling pathway. Compared with the sham group, the mRNA and protein levels of RCAN1 in the HIBD group, the PMA group, and the PMA + si-RCAN1 group were significantly increased (all p < 0.05), whereas in the BAY-11-7805 and si-RCAN1 groups, the expression of RCAN1 was not statistically different (all p > 0.05). Compared with the HIBD group, the mRNA and protein levels of RCAN1 in the PMA group were significantly increased (p < 0.05), whereas in the BAY-11-7805 group, si-RCAN1, and PMA + si-RCAN1 groups, the mRNA and protein levels of RCAN1 were significantly decreased (all p < 0.05) (Figure 4) . The results demonstrated that the activation of NF-κB signaling pathway up-regulated the expression of RCAN1.

Silencing RCAN1 has no significant effect on the NF-κB signaling pathway

The subcellular localization of NF-κB p65 was detected by immunofluorescence confocal laser scanning ( Figure 5(a) ). Blue was a DAPI marker indicating the nuclear region; red was a CY-3 marker indicating NF-κB p65; two fluorescent confocal overlapped into pink, indicating the translocation of NF-κB p65 into the nucleus. The observation of hippocampal tissues in the sham group revealed that a small amount of NF-κB p65 always presented in the cytoplasm, with no apparent nuclear translocation, and was mainly located in the cytoplasm. Compared with the sham group, in the HIBD, PMA, si-RCAN1, and PMA + si-RCAN1 groups, significant amount of NF-κB p65 was observed moving from cytoplasm to the nucleus, and the protein levels also increased significantly (all p < 0.05), while there was no significant difference in the BAY-11-7805 group (p > 0.05). Compared with the HIBD group, the PMA group, and the PMA + si-RCAN1 group appeared a large amount of NF-κB p65 moving from cytoplasm to the nucleus, and the relative protein expression HIBD Sham 25µm 25µm Figure 2 . The HIBD model is established which was identified by Nissl staining (400 ×). HIBD, hypoxic-ischemic brain damage.

was also significantly increased (all p < 0.05). However, in the si-RCAN1 group, NF-κB p65 had no significant sign of nuclear shift, and there was no statistical difference in the number of positive cells and the relative protein expression (p > 0.05). In the BAY-11-7805 group, NF-κB p65 showed no significant nuclear shift (p > 0.05), but the relative protein expression and the number of positive cells decreased (p < 0.05) ( Figure 5(b-d) ). It was suggested that the activation of NF-κB signaling pathway up-regulated the expression of NF-κB p65, but RCAN1 silencing had no significant effect on the NF-κB signaling pathway.

The number of GFAP-positive cells with activation of the NF-κB signaling pathway was measured by immunohistochemical staining. After immunohistochemical staining of rat hippocampal tissue, it was found that GFAP-positive cells were stained with green. Compared with the sham group, the number of positive cells in the HIBD, PMA, si-RCAN1, and PMA + si-RCAN1 groups increased significantly (all p < 0.05), whereas in the BAY-11-7805 group, GFAP positive cells showed no statistical difference between the two groups (p > 0.05). Compared with the HIBD group, the number of positive cells in the BAY-11-7805 group and the si-RCAN1 group decreased significantly, while it increased significantly in the PMA group ( Figure 6 ). All these results showed that with the activation of the NF-κB signaling pathway, the GFAP expression in glial cells of the hippocampus was elevated.

The changes of neuronal apoptosis with inhibition of NF-κB and RCAN1 silencing were observed by TUNEL staining. Under a light microscope, there was no obvious apoptotic cell was observed in the hippocampal tissues of the sham group. Compared with the sham group, the HIBD, PMA, si-RCAN1 and PMA + si-RCAN1 groups showed different degrees of apoptosis. As shown in Figure 7 , visible bright red fluorescence increased, and the number of apoptosis was also significantly increased (p < 0.05). Compared with the HIBD group, the apoptosis of the PMA group was significantly increased (p < 0.05), while the number of apoptosis cells in the BAY-11-7805 group and the si-RCAN1 group was significantly decreased (p < 0.05), and there was no statistical difference in the PMA + si-RCAN1 group (p > 0.05). All these results suggested that inhibition of NF-κB and silencing of RCAN1 reduced the neuronal apoptosis.

Inhibition of NF-κB and RCAN1 silencing decreases the levels of S-100β and AchE but increases Ach activity ELISA and colorimetry were applied to determine the levels of S-100β, Ach and AchE in the rat hippocampus. Compared with the sham group, S-100β protein content and AchE activity were significantly increased but the Ach activity was significantly decreased in the HIBD group, the PMA group, the si-RCAN1 group, and the PMA + si-RCAN1 group (all p < 0.05), and there was no statistical difference in BAY-11-7805 group (all p > 0.05). Compared with HIBD group, S-100β protein content and AchE activity significantly decreased in the BAY-11-7805 and si-RCAN1 groups (all p < 0.05) and increased in the PMA group, while the Ach activity increased significantly in the BAY-11-7805 and si-RCAN1 groups, but significantly decreased in the PMA group (all p < 0.05), and there was no statistical difference in the PMA + si-RCAN1 group (all p > 0.05) (Figure 8 ). Based on these results, it was found that inhibition of NF-κB and RCAN1 silencing decreased the levels of S-100β and AchE but increased the Ach activity.

The NF-κB signaling pathway correlates to longterm learning ability of rats

The MWM test was used to test the learning and memory ability of rats in each group (Figure 9 ). In each group, on the first day of swimming training, it was found that all rats had normal swimming ability. In the spatial probe trail, the escape latency of neonatal rats in each group changed with time, and the trend decreased most obviously in the sham group, and the memory remained good on the 6 th d. From the 2 nd d, compared with the sham group, the differences in the HIBD, PMA, si-RCAN1, and PMA + si-RCAN1 groups gradually increased with time. Compared with the HIBD group, PMA, si-RCAN1 and BAY-11-7805 groups differed greatly, and the difference was most obvious at the 6 th d (p < 0.05). It was suggested that hypoxia-ischemia impairs the spatial learning acquisition ability and recent memory ability of rats, which was related to the NF-κB signaling pathway. On the 6 th d of training, rats in the sham group still quickly found a hidden platform with a short swimming trajectory and a clear goal, while the swimming trajectories in the HIBD, PMA, si-RCAN1, and PMA + si-RCAN1 groups were disorganized. Compared with the HIBD group, there were significant differences in the PMA, si-RCAN1, and BAY-11- 7805 groups, suggesting that the rats lost their ability to locate and maintain memory after HIBD, and their long-term memory deficits were associated with the NF-κB signaling pathway. On the 5 th day of the MWM test, rats in the sham group crossed the platform significantly more than those in the HIBD, PMA, si-RCAN1 and PMA + si-RCAN1 groups. Compared with the HIBD group, there were significant differences in the PMA, si-RCAN1 and BAY-11-7805 groups (p < 0.05). It was demonstrated that the shamoperated rats had better spatial memory retention and spatial localization accuracy than the HIBD rats, which was associated with the NF-κB signaling pathway.

HIBD is one of the main causes of infant mortality and neurologic damage including cognitive impairment and mental retardation [22] . More and more evidence shows that the increase of apoptosis plays an important role in HIBD, which has led to an in-depth study of the mechanism of targeted therapy for HIBD [23] . Therefore, how to inhibit cell apoptosis in patients with HIBD is of great importance. Previous findings have shown that when NF-κB is activated in different situations, it can inhibit or promote cell apoptosis [24] . Besides, it has also been proved that RCAN1 is involved in neuronal apoptosis and the abnormal expression of RCAN1 can induce neurological disease [25] . Thus, aiming to determine the role of NF-κB signaling pathway and RCAN1 in apoptosis of hippocampal neurons and cognitive impairment, this study found that RCAN1 down-regulated by inhibition of NF-κB signaling pathway suppressed the apoptosis of hippocampal neurons and improves the cognitive impairment in rats with HIBD. Initially, our results found that RCAN1 was overexpressed and that the NF-κB signaling pathway was activated in HIBD rats. Furthermore, we also observed downregulation of RCAN1 after treatment of the NF-κB signaling pathway inhibitor. A previous study shows that in HIBD, NF-κB was activated [8] . Besides, it has also been proved that in rats with hypoxic-ischemic encephalopathy, the level of NF-κB p65 was significantly higher [26] . Moreover, a study reveals the overexpression of RCAN1-4 in brain ischemia/ reperfusion (I/R) injury [12] . In line with our findings, a previous study has shown that the NF-κB signaling pathway can up-regulate RCAN1 [10] . More specifically, it has been proved that overexpression of RCAN1 can induce neuronal apoptosis, but when the NF-κB signaling pathway is inhibited, the expression of RCAN1 is downregulated [27] . Furthermore, this study showed that the inhibition of NF-κB signaling pathway or silencing of RCAN1 reduced the apoptosis of hippocampal neurons in HIBD rats, which was indicated by the decrease of S-100β and AchE expression and increase of Ach activity. A previous study shows that S-100β protein was closely associated with HIBD, therefore the level of S-100β is of predictive values in patients with HIBD [28] . Another study points out that apoptosis may be associated with increased level of AchE [29] . Ach is a major neurotransmitter which is closely related to the inflammatory response [30] . And it has been found that Ach has the ability to suppress apoptosis induced by Fas [31] . As a transcription factor, NF-κB can control the expression of many genes which play a dominating role in cell programmed death and cell survival [32] . Consistent with our study, a previous research verifies that in neurons with hippocampal damage, the NF-κB signaling pathway is activated and the expression of NF-κB is increased [33] . Furthermore, another study confirms that the inhibition of the NF-κB signaling pathway leads to suppressed apoptosis of hippocampal neurons and promoted memory recovery [34] . A previous study reports that RCAN1 can regulate calcineurin signaling which is able to control apoptosis [35] . Additionally, another finding Hypoxia Apoptosis Figure 10 . Molecular mechanism underlying the NF-κB signaling pathway regulated HIBD progression. The NF-κB signaling pathway is activated in HIBD rats. Activation of the NF-κB signaling pathway promotes RCAN1 expression, which increased the neuronal apoptosis and aggravated the cognitive impairment in HIBD rats.

shows that when RCAN1 expression is decreased, neuronal apoptosis can be inhibited [36] . The present study also proved that the cognitive impairment of rats with HIBD was attenuated via the inhibition of NF-κB signaling pathway or RCAN1 silencing. Consistent with our study, a previous research finds that lycopene can ameliorate cognitive impairment through inactivating NF-κB signaling pathway [37] . Another study proves that naringenin can be used in pretreatment to improve cognitive impairment via inhibiting inflammation regulated by NF-κB [38] . Furthermore, it has also been proved that when RCAN1 isoform 4 is overexpressed, cognitive behavioral impairment can be aggravated [39] . These findings confirm our result that inhibition of NF-κB signaling pathway can improve the cognitive impairment of rats with HIBD.

Based on the previous researches, the present study has confirmed that the inhibition of NF-κB signaling pathway reduce the RCAN1 expression in HIBD rats, which decreases the hippocampal neuronal apoptosis and improve the cognitive impairment in rats with HIBD ( Figure 10 ). These findings identify the NF-κB signaling pathway and RCAN1 as potential therapeutic targets for the treatment of HIBD. Given that all the experiments in the present study were conducted in rats, we will try to perform human clinical trials in future studies.

Different genes and non-coding RNAs were also reported to be differentially expressed in several eye-related neurodegenerative disorders [40] [41] [42] . Therefore, in order to develop the idea that they might be used as molecular targets for future clinical trials, the possibility to realize a transcriptomic experiment should be conducted to analyze which genes and non-coding RNAs could be implicated in biochemical pathway involving NF-κB and RCAN1. For instance, glyoxalase I (GLO1), one of the oxidative stress-related enzymes, was involved both in oxidative stress and apoptotic pathway mediated by NF-κB [43] , that was also found to be associated with other pathologies caused by neuronal death, such as retinitis pigmentosa [44] . Any new discoveries of genes or non-coding RNA or mechanisms in HIBD, may ultimately improve knowledge of pathologies and benefit therapeutically from neurodegenerative diseases. Another point was that the cause of RCAN1 over-expression in HIBD rats should be evaluated, and RCAN1 promoter should be analyzed to evaluate if the presence of variants could alter binding of transcription factors. Therefore, the dual luciferase reporter gene assay, which was already applied to other neurodegenerative pathologies, such as Stargardt disease [45] , should be conducted to validate the promoter integrity in the future study.

Hypoxicischemic brain damage induces distant inflammatory lung injury in newborn piglets

Clinical pathophysiology of hypoxic ischemic brain injury after cardiac arrest: a "two-hit" model

Marine compound xyloketal B reduces neonatal hypoxic-ischemic brain injury

Plasma biomarkers of oxidative stress in neonatal brain injury

Bax inhibiting peptide reduces apoptosis in neonatal rat hypoxic-ischemic brain damage

Endogenous IL-6 of mesenchymal stem cell improves behavioral outcome of hypoxic-ischemic brain damage neonatal rats by supressing apoptosis in astrocyte

Inflammation meets cancer, with NF-kappaB as the matchmaker

A dual role of the NF-kappaB pathway in neonatal hypoxic-ischemic brain damage

Mesenchymal stem cells suppress neuronal apoptosis and decrease IL-10 release via the TLR2/NFkappaB pathway in rats with hypoxic-ischemic brain damage

Regulator of calcineurin 1 gene transcription is regulated by nuclear factor-kappaB

Regulator of calcineurin 1 differentially regulates TLR-dependent MyD88 and TRIF signaling pathways

Regulator of calcineurin 1 (Rcan1) has a protective role in brain ischemia/reperfusion injury

Regulator of calcineurin 1 (RCAN1) facilitates neuronal apoptosis through caspase-3 activation

limma powers differential expression analyses for RNA-sequencing and microarray studies

The influence of immaturity on hypoxic-ischemic brain damage in the rat

Rcan1 deficiency impairs neuronal migration and causes periventricular heterotopia

Regulator of Calcineurin 1 gene isoform 4, down-regulated in hepatocellular carcinoma, prevents proliferation, migration, and invasive activity of cancer cells and metastasis of orthotopic tumors by inhibiting nuclear translocation of NFAT1

The regulator of calcineurin 1 (RCAN1) inhibits nuclear factor kappaB signaling pathway and suppresses human malignant glioma cells growth

The effect of RCAN1 on the biological behaviors of small cell lung cancer

Down syndrome critical region 1 positively correlates with angiogenesis in hypopharyngeal cancer

The RCAN1 inhibits NF-kappaB and suppresses lymphoma growth in mice

Erythropoietin improved neurologic outcomes in newborns with hypoxic-ischemic encephalopathy

Hyperbaric oxygen suppresses hypoxic-ischemic brain damage in newborn rats

MicroRNA-224-5p regulates adipocyte apoptosis induced by TNFalpha via controlling NF-kappaB activation

Regulation of RCAN1 translation and its role in oxidative stress-induced apoptosis

NF-kappaB signaling is involved in the effects of intranasally engrafted human neural stem cells on neurofunctional improvements in neonatal rat hypoxic-ischemic encephalopathy

Lycopene inhibits regulator of calcineurin 1-mediated apoptosis by reducing oxidative stress and down-regulating Nucling in neuronal cells

Functional expression, characterization, and application of human S100B

Study of acetylcholinesterase activity and apoptosis in SH-SY5Y cells and mice exposed to ethanol

Comparative study of the expression of cholinergic system components in the CNS of experimental autoimmune encephalomyelitis mice: acute vs remitting phase

Antiapoptotic effect of acetylcholine in fas-induced apoptosis in human keratocytes

NF-kappaBdependent and -independent pathways in the protective effects of activated protein C in hippocampal and cortical neurons at excitotoxicity

Role of GABAB receptors and p38MAPK/NF-kappaB pathway in paclitaxel-induced apoptosis of hippocampal neurons

Down-regulated long non-coding RNA ANRIL restores the learning and memory abilities and rescues hippocampal pyramidal neurons from apoptosis in streptozotocin-induced diabetic rats via the NF-kappaB signaling pathway

Histone deacetylase 3 promotes RCAN1 stability and nuclear translocation

Amyloid-beta precursor protein facilitates the regulator of calcineurin 1-mediated apoptosis by downregulating proteasome subunit alpha type-5 and proteasome subunit beta type-7

Supplementation of lycopene attenuates oxidative stress induced neuroinflammation and cognitive impairment via Nrf2/ NF-kappaB transcriptional pathway

Naringenin pre-treatment inhibits neuroapoptosis and ameliorates cognitive impairment in rats exposed to isoflurane anesthesia by regulating the PI3/Akt/PTEN signalling pathway and suppressing NF-kappaB-mediated inflammation

Overexpression of RCAN1 isoform 4 in mouse neurons leads to a moderate behavioral impairment

miRNAexpression profile of retinal pigment epithelial cells under oxidative stress conditions

Non-coding RNAome of RPE cells under oxidative stress suggests unknown regulative aspects of Retinitis pigmentosa etiopathogenesis

Role of oxidative stress in Retinitis pigmentosa: new involved pathways by an RNA-Seq analysis

Nicotine induces apoptosis in human osteoblasts via a novel mechanism driven by H2O2 and entailing Glyoxalase 1-dependent MG-H1 accumulation leading to TG2-mediated NF-kB desensitization: implication for smokers-related osteoporosis

GLO1 gene polymorphisms and their association with retinitis pigmentosa: a case-control study in a Sicilian population

Stargardt phenotype associated with two ELOVL4 promoter variants and ELOVL4 downregulation: new possible perspective to etiopathogenesis?

We would like to thank our researchers for their hard work and reviewers for their valuable advice.

No potential conflict of interest was reported by the authors.