The Effects Of Resveratrol On Proliferation,Apoptosis And Autophagy Levels Of CD4~+T Cells In Peripheral Blood Of Patients With Sj?gren’s Syndrome

Objective:Using network pharmacology to predict the possible mechanisms of resveratrol(Res)for Sj?gren’s syndrome(SS),And in vitro experiments were conducted to verify whether the therapeutic effect of Res on SS was realized by regulating the proliferation,apoptosis,differentiation and autophagy levels of CD4~+T cells,exerting immune regulation and maintaining the homeostasis and function of T cells.Methods:Possible therapeutic targets for Res treatment of SS were screened in TCMSP database and Swiss Target Predictio database,In Gene Cards database(https://www.genecards.org/),OMIM database screening of SS related disease targets.Venn was used to draw the Venn map of gene intersection of drug disease action targets.The"component-target"network diagram was constructed by Cytoscape 3.7.1software to find out the core target.The nodes in the network diagram represent the relationship between Res,SS and potential target.The screened targets were imported into the STRING database,and the species"Homo sapiens"was selected to obtain the protein-protein interaction network.Go program was used to analyze the possible mechanisms of Res treatment of SS,including biological processes,molecular functions and cell composition.KEGG program was used to perform pathway enrichment analysis on potential targets of Res in the treatment of SS to predict the possible mechanism of Res in the treatment of SS.After being approved by the ethics committee of the hospital and signing the informed consent form with the patients and healthy controls,the peripheral blood of SS patients and healthy subjects were collected,and CD4~+T cells were obtained by magnetic beadseparation,and they were randomly divided into healthy control group,blank control group and Res intervention group.Flow cytometry detection of CD4~+T cell proliferation,apoptosis,proportion of Th17/Treg,RT-q PCR detection of CD4~+T cell autophagy genes Atg5 and LC3Ⅱexpression level.Western blot method to detect each group of CD4~+T cell autophagy protein Atg5 and LC3Ⅱexpression level.Results:1.The possible mechanism of network pharmacology to predict the therapeutic effect of Res on SS.A total of 177 Res treatment targets were screened in TCMSP database and Swiss Target Prediction database,and 1200 SS disease targets were screened in Gene Cards database and OMIM database,and 68 interset of Res and SS disease targets were screened.The core genes were IL1B,MAPK1,IL6,AKT1,NOS3,STAT3,CCL2,IL10,MAPK8,MMP9,JN,Casp3 and CXCL8.Core targets of biological process mainly involves cell factor receptor activity,phosphatase activity,activity of cytokines,DNA-binding transcription factor binding activity,protease activity,protein phosphatase activity,cracking enzyme activity,protein tyrosine kinase activity,die structure domain binding activity,can affect cell proliferation,apoptosis,differentiation and other biological processes.The core target enrichment pathways were apoptosis,Th17 cell differentiation,and autophagy-related pathways PI3K-Akt signaling pathway and MAPK signaling pathway.2.The therapeutic effect of Res on SS was verified by in vitro experiment(1)Compared with healthy control group,the proliferation index of CD4~+T cells in blank control group was significantly increased(P<0.001);Compared with the blank control group,the 100μM Res,200μM Res,and 500u M Res intervention groups could inhibit the excessive proliferation of CD4~+T cells(P<0.001),and there was no significant difference in the proliferation index of CD4~+T cells between the 200μM Res intervention group and the 500μM Res intervention group.(2)Compared with healthy control group,the apoptosis rate of CD4~+T cells in blank control group was significantly decreased(P<0.001).Compared with the blank control group,the percentage of CD4~+T cell apoptosis in the 100u M Res intervention group was not significantly different,but the 200μM Res intervention group could promote the apoptosis of CD4~+T cells(P<0.05),and there was no significant difference compared with the healthy control group(P>0.05).500μM Res could promote CD4~+T cell apoptosis(P<0.001).(3)Compared with healthy control group,the proportion of Th17 cells in blank control group and 100μM Res intervention group was significantly increased(P<0.001).Compared with the blank control group,the proportion of Th17 cells in the 100μM Res intervention group was not significantly changed,while the proportion of Th17 cells in the 200μM Res and 500μM Res intervention groups was significantly decreased(P<0.001),and there was no significant difference compared with the healthy control group.(4)Compared with healthy control group,the proportion of Treg cells in blank control group,100μM and 200μM Res intervention groups was significantly decreased(P<0.001),while there was no significant difference in the proportion of Treg cells in500μM Res intervention group(P>0.05).Compared with the blank control group,the proportion of Treg cells in the 100u M Res intervention group was not significantly changed(P>0.05),but the proportion of Treg cells in the 200μM Res and 500μM Res intervention groups was significantly increased(P<0.01).(5)Compared with the healthy control group,blank control group CD4~+T cells Atg5 m RNA expression levels(P<0.01),LC3Ⅱm RNA expression levels(P<0.001);200μM Res intervention group of CD4~+T cell Atg5 and LC3Ⅱm RNA expression levels were elevated(P<0.001);Compared with blank control group,the Res intervention group of CD4~+T cell Atg5 and LC3Ⅱm RNA expression levels were elevated,statistically significant difference(P<0.001).(6)Compared with the healthy control group,blank control group Atg5 protein expression levels of CD4~+T cells(P<0.05),LC3Ⅱprotein expression levels(P<0.01);Res intervention group of CD4~+T cells Atg5 and LC3Ⅱprotein expression levels(P<0.001);Compared with the blank control group,Res intervention group of CD4~+T cell Atg5 and LC3Ⅱprotein expression levels(P<0.01).Conclusion:1.Network pharmacological methods suggested that Res might regulate apoptosis,Th17 cell differentiation,and autophagy-related PI3K-Akt and MAPK signaling pathways in the treatment of SS.2.In vitro experiments confirmed that Res can inhibit the proliferation and apoptosis of peripheral blood CD4~+T cells in SS patients,inhibit the differentiation of peripheral blood CD4~+T cells to Th17 cells,and promote the differentiation of peripheral blood CD4~+T cells to Treg cells,The best concentration was 200μM.3.Res may inhibit the excessive proliferation of CD4~+T cells to treat SS by promoting CD4~+T autophagy in peripheral blood of SS patients,thus providing theoretical basis and basis for clinical treatment of SS treated by Res.