The Effect Of FSN On Autophagy Of Synovial Cells In Rheumatoid Arthritis Rats Via JAK3/STAT3 Signaling Pathway

Objective:Fengshining（FSN） had a very good clinical effect in the treatment of rheumatoid arthritis（RA）.Based on network pharmacological analysis and prediction,this study understanded the mechanism of FSN’s intervention in treating rheumatoid arthritis model rats from the perspective of autophagy,which can provide experimental evidence and new clinical ideas for FSN treatment of RA.Methods:1.The method was to analyzed the targets and pathways of FSN in the treatment of rheumatoid arthritis with the help of network pharmacology related platforms and software.First,TCMSP database,chemical source network database,Pubchem and Swiss Target Prediction platform were used to screen the active ingredients and targets of Fengshining.Uniprot database was used to obtain the corresponding gene names of the targets.Gene Cards,OMIN and drugbank database were used to collect the disease targets of RA.Then,Cytoscape software was used to construct the network of Fengshining drug-active ingredient-common target gene network and target interaction network.Finally,KEGG pathway enrichment analysis and GO analysis of biological processes were performed on the common target genes through the DAVID database.2.The effects of collagen induction,wind-cold-damp induction and collagen induction combined with wind-cold-damp induction on the general state of rats,ankle joint swelling,knee joint pathology and synovial cell autophagy protein LC3Ⅰ,LC3Ⅱand Beclin-1levels were compared.Determined the appropriate model preparation method by comparing the results.3.The selected model preparation method was used to prepare rheumatoid arthritis rat models.The rats were divided into model control group,tofacitinib group(15 mg·kg^-1),FSN low,medium and high dose groups(7,14,28 g·kg^-1)according to the random number table method.Normal control group was set up with 12 rats like other groups.The rats were given intragastric administration with distilled water or corresponding drugs,once a day for 28 consecutive days.After the gavage,rat serum,knee joint synovium and ankle joint tissue were taken.We recorded and observed the body weight and rectal temperature of each group of rats.The degree of swelling in the ankle joints of rats was measured after treatment.The contents of IL-2,IL-7,IL-9 and TNF-αin rat serum were detected by ELISA.The levels of JAK3 and STAT3 in the synovium of each group were detected by RT-PCR.Western-blot method was used to detect the levels of Beclin-1 and p62 in synovium of rats.The ultrastructure of rat synovial cells was observed by transmission electron microscope.Results:1.241 common target genes were screened through network pharmacology.Protein interaction network analysis found that AKT1,IL6,MAPK3,VEGFA,TP53,TNF,MAPK1,EGFR,CASP3,EGF,STAT3,PTGS2,MAPK8,JUN,SRC were key core proteins.These proteins were related to autophagy.The target protein of FSN was mainly involved in the positive regulation of RNA polymerase Ⅱ promoter transcription,drug response,positive regulation of cell proliferation,positive regulation of transcription,DNA template,inflammatory responser functional regulation.The biological process of target gene enrichment was related to autophagy.KEGG pathway enrichment analysis showed that FSN’s treatment of rheumatoid arthritis was related to JAK/STAT pathway.2.Compared with the normal control group,the weight of rats in each model group decreased（P<0.05）;the swelling degree of joints in the CIA model group and the wind-cold-damp CIA model group increased（P<0.05）,and the pathological changes of synovium and cartilage were obviously accompanied Synovial hyperplasia and inflammatory cell infiltration;LC3Ⅱ,LC3Ⅱ/LC3Ⅰand Beclin-1 levels in the wind-cold-damp CIA model group increased,among which Beclin-1 levels increased（P<0.05）.By comparing the three model preparation methods,it was determined that collagen induction combined with wind-cold-damp induction can be used as an animal model preparation method for studying RA from the perspective of autophagy.3.From the experimental results of the effect of FSN on the autophagy level of synovial cells in rheumatoid arthritis model rats:Compared with the normal control group,the swelling degree of the joints in the model control group increased significantly（P<0.05）.The expressions of IL-2,IL-7,IL-9,TNF-α,JAK3 mRNA,STAT3 mRNA and Beclin-1 protein increased and p62 protein decreased in each treatment group（P<0.05）.Compared with the model control group,beclin-1 protein expression increased and p62protein expression decreased in the synovium of the knee joint of the rats in each treatment group（P<0.05）.The expressions of IL-7,TNF-αand JAK3 mRNA in the low-dose group and high-dose group were reduced（P<0.05）.The expressions of IL-2,IL-7,IL-9,TNF-α,JAK3 m RNA,and STAT3 m RNA were decreased in the middle-dose group（P<0.05）.Compared with the normal control group,the expressions of joint swelling degree,IL-2,IL-7,JAK3 m RNA,Beclin-1 increased and p62 protein decreased in each treatment group（P<0.05）.Conclusion:Rheumatoid arthritis can be treated with FSN,which be related to the fact that it reduces the expression of inflammatory factors and thus regulates JAK3/STAT3signaling pathway.The regulated JAK3/STAT3 signaling pathway regulates autophagy related proteins and enhances the autophagy of synovial cells,thus inhibiting the continuous proliferation of RA synovium.