Study On The Mechanism Of Matrine In Rheumatoid Arthritis Based On PI3K/AKT Signaling Pathway

Part 1 Network pharmacology study on the mechanism of matrine intervention in RAObjective:Prediction of potential targets of matrine on RA by network pharmacology,to explore the mechanism of matrine intervention in RA.Methods:(1)The targets of matrine action were collected from five databases including Herb,Superpred,Swiss Target,TCMSP and CTD.Five databases including CTD,DrugBank,GeneCard,OMIM and TTD were used to collect RA-related targets.(2)Unified Gene Name of Matrine Target and RA Target in Uniport and GeneCard Databases.(3)Obtain the intersection of bitter ginseng targets and RA targets online using Venny platform,and import the results into STRING platform for protein interaction network analysis,results imported into Cytoscape software and analyzed the intersection targets with its CentiScaPe2.2 plug-in,the key target of matrine intervention in RA was selected according to the thresholds of betweenness,closeness and degree,and it was enriched analyzed by R language.Result:Number of matrine targets collected from 5 databases:11,161,60,10,27 respectively,a total of 239 targets were obtained by combining and removing duplication.The five disease-related databases collected 176,176,1085,163,126 RA targets respectively,a total of 1340 targets were obtained after merging and removing duplicate targets.83 matrine intervention targets in RA were obtained by venny platform,16 key targets were identified after CentiScaPe 2.2 plug-in analysis,AKT1 is the most vital because of its maximum Degree.GO analysis showed that the biological processes related to RA were mainly cell proliferation and endogenous apoptosis signal regulation.KEGG analysis showed that TNF signaling pathway,HIF-1 signaling pathway,PI3K/AKT signaling pathway,JAK/STAT signaling pathway and IL-17 signaling pathway were closely related to matrine intervention in RA.Nine of the 16 key targets,including AKT1、TNF、IL6、TP53、MYC、MTOR、TLR4、JAK2、AR、PIK3R1 were enriched in the PI3K/AKT signaling pathway.Conclusion:Matrine may interfere with the development of RA by regulating PI3K/AKT signaling pathway.Part 2 Effect of matrine on PI3K/AKT signaling pathway activation in CIA rat FLSObjective:To study the effect of matrine on PI3K/AKT signaling pathway in FLS of TNF-α-induced collagen CIA rats.Methods:The CIA rat model was established by bovine type Ⅱ collagen in all groups except the normal control group,the synovial tissue of each group of rats was stripped,FLS was extracted,and cell culture was performed.Inverted microscope observation of FLS growth,Immunofluorescence staining for identification of FLS in CIA rats.The inflammatory cell group was only given TNF-α intervene,matrine group was given TNF-α+0.75 mg/ml matrine intervention,the inhibitor group was given TNF-α+20μmol/L LY294002 intervention.Determination of p-PI3K,p-AKT and AKT protein expression in each group of rats FLS by Western Blot,determination of PI3K mRNA,AKT mRNA expression by RT-PCR.Result:Under ordinary microscope,it was shown that in addition to the normal control group,the arrangement of cells was dense and the number of cells was significantly increased.Fluorescence immunoassay showed positive expression of Vimentin.The results of Western Blot:compared with normal control group,the expression of p-PI3K and p-AKT protein in inflammatory cell group increased(P<0.01),compared with the inflammatory cell group,the expression of p-PI3K and p-AKT protein in matrine group and inhibitor group decreased(P<0.01,P<0.05),there was no difference in the expression of unactivated AKT protein among groups(P>0.05),RT-PCR:compared with normal control group,the expression of PI3K mRNA and AKT mRNA in inflammatory cell group increased(P<0.01),compared with the inflammatory cell group,the expression of PI3K mRNA and AKT mRNA in matrine group and inhibitor group were significantly inhibited(P<0.01),compared with the inhibitor group,the expression of PI3K mRNA and AKT mRNA in matrine group decreased(P<0.01).Conclusion:Matrine inhibited the activation of PI3K/AKT signaling pathway in FLS of CIA rats.