Network Pharmacology-based Identification Of The Key Mechanism Of Qinghuo Rougan Formula Alleviates Uveitis

Objective:1.Using a comprehensive approach including bioinformatics and systems biology methods,the network pharmacology analysis of Qinghuo Rougan Formula,which has a clear clinical curative effect,reveals the target of Qinghuorougan active compound and clarifies the Drug-Gene-target-disease network relationships and enrichment analysis of targets for uveitis treatment.2.The experimental autoimmune uveitis(EAU)rat model was established.The inhibitory effect of Qinghuo Rougan Recipe on the uveitis process was verified by experiments and the possible anti-inflammatory mechanism of Qinghuo Rougan Formula was explored.Methods:1.Qinghuo Rougan Formula is prepared by the combination of Ten herbs.The bioactive compounds from the Ten plants were collected from Traditional Chinese Medicine Systems Pharmacology(TCMSP)、 Herbal Ingredients Targets Database Introduction(HIT)and Traditional Chinese Medicine Integrated Database(TCMID).OB(oral bioavailability)and DL(drug likeness)were used for the candidate active ingredients screening according to in silico ADME(Absorption Distribution Metabolism Excretion)integrative model.We set DL?≥?0.18? and OB ≥ 30% as the threshold of candidate compounds.The structures of the compounds were found in the Pub Chem.Afterward,the target proteins corresponding to compounds screened from Pharmmapper database and Pub Med database were standardized in Uniprot databases.And finally,Cytoscape 3.6.0 software was collected and construct a ‘QHRGF-compoundtarget’ network.Uveitis-related targets were mined out from OMIM database、Dis Ge NET database and Gene Cards databases.All of the disease gene targets were normalized in the R environment using bioconductor package when the redundancy was deleted.Screening for Drug-Disease crossover genes.Based on the previous steps,two set of target list was prepared: Drug related gene and disease targets.The crossover genes were filtrated in the R environment using Venn Diagram package.String 11.0 database was used for analysis the protein-protein interaction(PPI)of intersection and the common targets were counted in the R environment.Finally,Cytoscape3.6.0 was used for determine the network of Drug-compound-target-Disease.2.The crossover expressed proteins were applied to do bioinformatics annotation in the R environment using bioconductor package,including a panther classification system and a gene ontology(GO)annotation database website,KEGG pathway enrichment analysis.3.Healthy 6-8-week-old female Lewis rats(about 160–180 g)were randomly divided into 4 groups(Normal control group 、EAU group、Conventional-dose QHRGF group、High-dose QHRGF group)of 16 rats in each group.EAU group、Conventional-dose QHRGF group and High-dose QHRGF group every rat received 200μL IRBP peptide emulsions,while rats in control group were treated with the same method injected with the identical volume of mixed solutions of TB,CFA and PBS.Conventional-dose QHRGF group and High-dose QHRGF group were respective given QHRGF(2ml/100g)and QHRGF(4ml/100g)administration for 28 days.On day 13 after immunization,rats in both groups were euthanized and eyeballs were extracted.Eyeballs from 3 individuals in each group were randomly selected,fixed in 4% paraformaldehyde and embedded in paraffin,and then sections were cut to perform H&E staining.Samples were cut into 4μm continuous sections and stained with histopathological examination(H&E)(Magnified 200×).4.The inflammation severity was assessed for every eye and was scored on a scale of 0-4 in half-point increments according to a semi-quantitative system.On day 0、7、13、18 after immunization,inguinal liver were obtained from each groups rats.Cells were isolated from liver by passage through a nylon wool column;then,the cells were collected by Ficoll-Hypaque density gradient centrifugation and cultured in 37 °C carbon dioxide incubator for another 12 h.To obtain antigen-specific T lymphocytes.Subsequently,The percentage values of Th1,Th17 and NKT cells in each group of cultured T cells were analyzed by flow cytometry and sorted.(BD;FACS Verse).Finally,the dynamic changes of NKT and Th1 and Th17 after immunization were examined.1.In the present study,according to the screening conditions,we collected a total of 80 kinds of 100 compound removals from 10 herbs of QHRGF.Topological analysis of protein interaction network nodes,screening key nodes to remove duplication,a total of 1932 target protein.For Disease targets Identifying,we collected uveitis-related diseases from OMIM,Dis Ge NET and Gene Cards database,we retrieved 729 target genes for disease targets,and obtained 649 key nodes by screening.We obtain the Venn diagram as shown total of 259 proteins.At the same time,we constructed an interactive network of QHRGF-compound-target-uveitis.2.GO annotation revealed the expressed proteins of Drug-Disease were mainly associated with cytokine receptor binding,chemokine activity,Toll-like receptor binding,acute inflammatory response,MAP kinase activity and enzyme inhibitor activity.Moreover,KEGG enrichment analysis showed many target genes were tightly interact with Cytokine-cytokine receptor interaction,Th17 cell differentiation、Natural killer cell mediated cytotoxicity and MAPK signaling pathway 、 T cell receptor signaling pathway,which belongs to Autoimmune thyroid disease and inflammatory immune signaling pathway respectively,should be crucial pathways.3.We found that NKT cells in EAU group reached a peak on the 7th day after immunization(pre-inflammation)compared with normal control rats(P<0.01).NKT cell levels were significantly increased in the QHRGF group compared with the EAU group on the 13 th day of immunization(P<0.05).Its growth trend is opposite to that of Th1 and Th17 cells.This demonstrates that the inhibition of the differentiation of Th1 /Th17 cells by QHRGF may be achieved by stimulating the action of NKT cells.Meanwhile,on day 13 after immunization,the expressions of IL-10,IL-4 m RNA were significantly increased in liver T cells(P<0.01),the expressions of IL-17 and IFN-γm RNA were significantly depressed in liver T cells after treatment with QHRGF(P<0.01).The protein expression of IFN-γ,IL-17,IL-10 and IL-4 in the liver of rats treated with QHRGF was consistent with the trend of gene expression.Conclusions:1.Based on the network pharmacology analysis,the prediction of the interaction between the traditional Chinese medicine molecule and the target protein of Qinghuo Rougan Formula combined with the biological path-based experimental verification is a more effective method to explain the mechanism of action of compound Chinese medicine.Results:2.Anti-inflammatory stress response is the main biological process of Qinghuo Rougan Recipe.Qinghuo Rougan Formula may regulate the development of uveitis through the regulation of NKT and the inhibition of MAPK signaling pathway.This study laid the foundation for Qinghuo Rougan Formula modulates local immune responses and may be a promising,long-lasting therapeutic strategy for refractory and recurrent uveitis,as well as other inflammatory eye diseases.