key: cord-0776873-q6mfg79p
authors: Jia, Shanshan; Luo, Hua; Liu, Xinkui; Fan, Xiaotian; Huang, Zhihong; Lu, Shan; Shen, Liangliang; Guo, Siyu; Liu, Yingying; Wang, Zhenzhong; Cao, Liang; Cao, Zeyu; Zhang, Xinzhuang; Zhou, Wei; Zhang, Jingyuan; Li, Jialin; Wu, Jiarui; Xiao, Wei
title: Dissecting the novel mechanism of Reduning injection in treating Coronavirus Disease 2019 (COVID-19) based on network pharmacology and experimental verification
date: 2021-01-22
journal: J Ethnopharmacol
DOI: 10.1016/j.jep.2021.113871
sha: 68febb5506ae9a5e0b764398afbc0d34c713bf25
doc_id: 776873
cord_uid: q6mfg79p

ETHNOPHARMACOLOGICAL RELEVANCE: Reduning injection (RDNI) is a patented Traditional Chinese medicine that contains three Chinese herbal medicines, respectively are the dry aboveground part of Artemisia annua L., the flower of Lonicera japonica Thunb., and the fruit Gardenia jasminoides J.Ellis. RDNI has been recommended for treating Coronavirus Disease 2019 (COVID-19) in the "New Coronavirus Pneumonia Diagnosis and Treatment Plan". AIM OF THE STUDY: To elucidate and verify the underlying mechanisms of RDNI for the treatment of COVID-19. METHODS: This study firstly performed anti-SARS-CoV-2 experiments in Vero E6 cells. Then, network pharmacology combined with molecular docking was adopted to explore the potential mechanisms of RDNI in the treatment for COVID-19. After that, western blot and a cytokine chip were used to validate the predictive results. RESULTS: We concluded that half toxic concentration of drug CC50 (dilution ratio) =1:1280, CC50=2.031 mg crude drugs/mL (0.047 mg solid content/mL) and half effective concentration of drug (EC50) (diluted multiples) =1:25140.3, EC50=103.420μg crude drugs/mL (2.405μg solid content/mL). We found that RDNI can mainly regulate targets like carbonic anhydrases (CAs), matrix metallopeptidases (MMPs) and pathways like PI3K/AKT, MAPK, Forkhead box O s and T cell receptor signaling pathways to reduce lung damage. We verified that RDNI could effectively inhibit the overexpression of MAPKs, PKC and p65 nuclear factor-κB. The injection could also affect cytokine levels, reduce inflammation and display antipyretic activity. CONCLUSION: RDNI can regulate ACE2, Mpro and PLP in COVID-19. The underlying mechanisms of RDNI in the treatment for COVID-19 may be related to the modulation of the cytokine levels and inflammation and its antipyretic activity by regulating the expression of MAPKs, PKC and p65 nuclear factor NF-κB.

In December 2019, a coronavirus that is capable of human-to-human transmission was discovered 76 and 1:2560 total 8 dilutions. The cell culture medium in the 96-well culture plate was discarded, 158 diluted drugs were added (100µL/well), replicates of wells were used, then each well was added 159 with 100 µL blank DMEM medium (containing 2% double-antibody and 16 µg /mL trypsin). The 160

control group was treated with 200 µL DMEM medium (containing 2% double-antibody and 16μg 161 /mL trypsin) The cells were incubated at 37℃ in a 5% CO 2 incubator for 4-5 days. The cytopathic 162 effect (CPE) was observed under the light microscope. The change of CPE in the cells was 163 recorded as "+", and no change of CPE was recorded as "-". 164

Using a sterile 96-well culture plate, 100µL of Vero E6 cells at a concentration of 5 × 10 4 cells/mL 166 was seeded onto each empty well and cultured for 24h at 37℃ with 5% CO 2 . The test drugs were 167 diluted for 8 concentrations (100 µL/well), 4 replicates of wells were used for each concentration, 168 and then an equal volume (100 µL/well) of 100 TCID50 virus was added to each well, followed 169 by incubation in a 5% CO 2 incubator at 37℃ for 1 h. Meanwhile, the control group was treated 170 with 200 µL of DMEM medium (containing 2% antibody and 16 µL /mL trypsin) per well, and the 171 model group was treated with 100 µL virus solution containing 100 TCID50 per well at 37 °C for 172 1 h. The supernatant was aspirated after 1 h. 200 µL/well DMEM medium containing 2% antibody 173 and 16 µL /mL trypsin was added to the cell wells. The cells were then incubated at 37℃ in a 5% 174 CO 2 incubator for 4-5 days. The CPE was observed under the light microscope. The change of 175 CPE in the cells was recorded as "+", and no change of CPE was recorded as "-". compounds. To ensure the accuracy and comprehensiveness of target screening, we set the 190 probability filter of SwissTargetPrediction to above zero, while "minimum required interaction 191 score" was set at higher confidency (0.700) and "max number of interactors to show" was set at no 192 more than 50 interactors. In the attachment of the literature report (Wang, et al., 2020) , single-cell 193 sequencing of colonic epithelial cells identified 5,556 genes that are co-expressed with ACE2. 194 Cytoscape software (Shannon, et al., 2003) (version 3.7.1, http://cytoscape.org/) was used to take 195 the intersection to acquire the genes that are co-expressed with ACE2 for the targets of RDNI. 196

The collected targets were sorted and imported into Cytoscape to create a network to visualize the 198 pharmacological mechanisms. We downloaded all the human high-quality binary protein-protein 199 interactions from the high-quality proteomics database HINT (High-quality INTeractomes, last 200 updated in April, 2019) (Das, J., 2012), which was applied to construct the human genome-wide 201 protein-protein interaction (PPI) network. All the targets of RDNI were mapped onto the HINT 202 network. Compound targets and the edges between them were extracted, and only the largest 203 connected branch was retained to build the target network. 204

The DAVID (Kanehisa, 2020) platform (https://david.ncifcrf.gov/, Version 6.8) was employed for 206 disease cluster analysis (functional annotation clustering), the species and background were set to 207 " homo sapiens ". Subsequently, gene ontology (GO) and Kyoto Encyclopedia of Genes and 208

Genomes (KEGG) enrichment analyse of important targets were conducted by the clusterProfiler 209 package (Burley, et al., 2017) was also used to draw the heatmap of molecular docking. anti-p-NF-κB-p65 (ser276) (IPW0781), anti-p-NF-κB-p65 (ser536) (IPW1630) were purchased 237 from Nanjing Pathway Biological Technology Co., Ltd (Nanjing, China). Anti-p38 (KGYT3514) 238 and anti-p-p38 (KG11253) were obtained from Nanjing KeyGen Biotech Co., Ltd (Nanjing, 239

China). While the secondary antibodies were purchased from Jackson Co., Ltd (Lancaster, 240 Pennsylvania). 241 BEAS-2B cells were purchased from Shanghai Bioleaf Biotech Co., Ltd (Shanghai, China). 242

PolyI:C was purchased from Sigma. The cells were cultured in tissue culture flasks, at 37℃ with 5% 243 CO 2 . The culture medium was discarded when 90% of them were fused. These cells were washed 244 twice with PBS and were digested with 0.25% trypsin-EDTA solution. After centrifugation at 800 245 rpm for 4 minutes, the supernatant was discarded. 3 mL of complete medium was added to 246 resuspend the cell pellet, and 1 mL was inoculated in a tissue culture flask at a density of 1×10 6 247 cells/mL. 5 mL complete culture medium was added, and the cells were cultured at 37°C with 5% 248 CO 2 . After 60%~70% of them were fused, the culture medium was discarded and the cells were 249 washed twice with PBS. 250

The cells were divided into 3 groups (blank, model and sample groups). The original medium in 251 the culture flask was discarded. 2 mL serum-free medium was added to the cells for the blank 252 group, and the cells were then incubated for 24 h. 50 μg/mL PolyI:C serum-free medium was 253 added to the model and sample groups. After incubating for 60 mins, the samples were collected 254 for western blot analysis. 

The QAH-INF-3 chip purchased from Raybiotech (Georgia, America) was used to detect the 273 effect of RDNI on PolyI:C-induced cytokine secretion in BEAS-2B cells. In this study, blank, 274 model and sample groups were used, each group had 3 replicate wells. Before the formal 275 experiment, the medium was discarded and serum-free medium was added to starve the cells for 1 276 h. According to the needs of the experiment, 200 μL of the test drug solution was prepared and 277 administered with serum-free medium (the monomer compound concentration was 60 μM and the 278 compound concentration was 50 μg/mL). At the same time, 200 μL serum-free blank medium was 279 added to the blank and model groups. After 1 h of drug treatment, a certain amount of PolyI:C 280 mother liquor was added to the model and sample groups to make the final concentration of 281

PolyI:C to be 50 μg/mL, and the incubation time was 24 h. After treatment, the supernatant was 282 collected and centrifuged at 4 ℃ to take 100 μL for chip detection. The Raybiotech kit containing 283

Blocking Buffer, Cytokine Standard Mix, Wash Buffer, Detection Antibody (Biotin-conjugated 284

Anti-Cytokines), Sample Diluent and Cy3 equivalent dye-conjugated streptavidin was used for 285 experimental samples, which was followed by Raybiotech's standard operating procedures 286 including sample preparation, chip hybridization, washing and detection. The Axon Genepix chip 287 scanner (GenePix 4000B, Axon Instruments, USA) was used to scan the cytokine chip. 

The drug was diluted with eight different concentrations, and the results were shown in Table 1 . 292

The results showed that dilution of 1:640 (4.063 crude drugs mg/mL, 0.094 mg solid content/mL) 293 and above dilutions were toxic to the cells. When the dilution was 1:1280 (2.031 mg crude drugs 294 /mL, 0.047 mg solid content /mL), 2 of the 4 compound pores were toxic to the cells. According to 295 the calculation formula of CC 50 , CC 50 (dilution ratio) =1:1280, CC 50 =2.031 mg crude drugs /mL 296 (0.047 mg solid content /mL). 297 "+" means that the cells have CPE changes, "-" means that the cells have no CPE changes or was 299 in normal cell morphology. 300

The drug was diluted with eight different concentrations, and the results showed that the drugs that 302 were in 1:10000 dilution (260.000 μg crude drugs/mL, 6.045 μg solid content/mL) or higher 303 concentration had inhibition effects on the virus. When the dilution ratio was 1:20 000 (130.000μg 304 crude drugs/mL, 3.023 μg solid content/mL), 3 of the 4 compound pores had inhibition effects on 305 the virus, as shown in Table 2 . According to the formula of drug toxicity EC 50 , we concluded that 306 EC 50 (diluted multiples) = 1:25140.3, EC 50 =103.420μg crude drugs /mL (2.405μg solid content 307 /mL). 308 "+" means that the cells have CPE changes, "-" means that the cells have no CPE changes or was 310 in normal cell morphology, and "/" refers to cytotoxicity. The purple nodes represent the compounds, the yellow nodes represent the targets co-expressed 331 with ACE2, the red nodes represent the targets that are related to fever, and the green nodes 332 represent the other targets. All the nodes were sorted by degree. The color round represents the 

The PPI network consisted of a total of 617 nodes that were linked through 2480 edges (Figure  342 2B). The average degree value of the nodes in the network was 8.03, and the target with the 343 highest degree was HSP90AB1 (degree = 109). The degree values of a total of 167 targets were 344 higher than the average degree value of these targets. 345

We conducted GO and KEGG enrichment analysis and disease functional clustering analysis on 347 167 targets was conducted with the degree value above average. A total of 1491 items with 348 epithelial and endothelial cell apoptosis, which is acute lung injury (ALI) and ARDS 447 (Channappanavar, et al., 2017) . Therefore, the use of cytokine storm and immunoregulation 448 therapy are necessary for the treatment of COVID-19. Several cytokines were involved in the 449 target of RDNI treatment, which initially proved that RDNI had certain regulatory effects on 450 cytokine storm (Tisoncik, et al., 2012) . In addition, cytokines also participate in several pathways 451 that are related to fever induction, hence they are categorized as a group called pyrogenic 452 cytokines. The major pyrogenic cytokines include interleukin-1 (IL-1α and IL-1β), TNF-α, IL-6, 453 and interferon gamma, IL-8 and IFN-α, which possess pyrogenic activity all can be directly or 454

indirectly and can be regulated by RDNI (Zampronio, et al., 2015) . This suggested that RDNI 455 might be anti-pyretic. 456

From the analysis of PPI network, heat shock protein 90 alpha family class B member 1 457 (HSP90AB1) and heat shock protein 90 alpha family class A member 1 (HSP90AA1) have the 458 highest degree values. TP53 and EGFR also showed high degree values. For heat shock protein 90 459 (HSP90), a 90-kDa and ATP-dependent molecular chaperone, can regulate inflammatory signaling 460 networks. Its intracellular concentrations can be increased by stressors, e.g., increased temperature 461 (fever), oxidative stress, ethanol, and infection that induce protein unfolding, misfolding, or 462 aggregation (Tukaj, 2016; Lilja, et al., 2015) . Importantly, an existing study proved that HSP90 463 inhibition could also prevent LPS-mediated inflammatory in human lung cells (Lilja, et al., 2015) . The disease clustering and functional enrichment analyses showed that RDNI is highly associated 478 with pulmonary diseases. GO entries are closely related to the process of ALI and ARDS. It is 479 mostly related to cell death and phosphorylation. Pathway enrichment analysis showed that 18 480 signal transduction pathways, 12 immune system pathways, 6 cell growth and death related 481 pathways and 10 viral infectious disease pathways are related to RDNI treatment. MAPK/NF-κB 482 signaling is associated with the pathogenesis and progression of ALI and inflammation, and is 483 considered as major molecular pathways for ALI and ARDS (Wu, et From the results of molecular docking, the average binding energies between compounds and 504 ACE2, PLP and Mpro were less than -5kJ / mol. This indicated that the compounds have potential 505 to inhibit the virus from entering into the human body and replicating. Some components with 506 lower binding free energies are considered as potential antiviral active ingredients. 507

In experiment session, western blot experiment verified the regulation of key pathways by RDNI 508 and its monomer compounds. ERK, p38 MAPK, and JNK are belonged to MAPK superfamily, It's inhibition significantly reduced inflammation in LPS-stimulated cells (Bin, et al., 2019) . 514

Phosphorylated p65 (Ser536) migrates from the cytoplasm to the nucleus, activates multiple 515 NF-kappa B target genes, and is one of the main mediators of ALI development. It is involved in 516 the transcriptional regulation of proinflammatory cytokines, and the continuous elevation of 517 proinflammatory cytokines in the lungs is associated with an increase in ALI mortality (Krupa, et 518 al., 2009 ). In addition, PKC plays an important role in lung permeability, contraction, migration, 519 hypertrophy, proliferation, apoptosis and secretion (Dempsey, et al., 2007) . Our experimental 520 results showed that RDNI could effectively inhibit the abnormal increase of these factors. This 521 might be the mechanism by which RDNI protects the patients with COVID-19 from lung injury. 522

The results of protein microarray analysis preliminarily verified the regulation of cytokines by 523 RDNI. It could effectively inhibit the expression of cytokines like IL-1α, IL-1β, IL-4, IL-6, IL-8 524 and TNF-α, thereby regulating cytokine storm. Therefore, RDNI could protect COVID-19 patients 525 from lung injury and help to relieve fever and COVID-19 symptoms. 

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Supplementary materials contain seven tables. 607