黄芪总皂苷通过调控LncRNA MIR22HG和Caspase⁃3通路促进类风湿关节炎滑膜成纤维细胞的凋亡

Total astragalus saponins promote apoptosis of fibroblast⁃like synoviocytes in rheumatoid arthritis by regulating lncRNA mir22HG and caspase⁃3 pathway

目的:通过数据挖掘和细胞实验探究黄芪总皂苷(AST)通过调控lncRNA MIR22HG(MIR22HG)/Caspase⁃3对类风湿关节炎(RA)凋亡指标Bax、Caspase⁃3、Caspase⁃8和高凝指标PAF、PGI2的作用机制。方法:收集RA患者30例、正常人30例,提取外周血单个核细胞(PBMCs),定量逆转录PCR法(RT⁃qPCR)检测MIR22HG的表达,使用数据挖掘探索及其与凋亡、凝血指标的相关性。建立RA⁃FLS细胞系,构建MIR22HG过表达质粒,转染至RA⁃FLS中,用cell counting kit8(CCK⁃8)、RT⁃qPCR、酶联免疫吸附法(ELISA)、蛋白质免疫印迹法(WB)、免疫荧光法(IF)、流式细胞术检测黄芪总皂苷(AST)对RA⁃FLS的凋亡和凝血指标的影响。结果:与正常组相比,RA组MIR22HG高表达,PAF、Bcl⁃2mRNA升高,PGI2、Bax mRNA降低(P<0.05)。Spearman相关性分析结果显示,MIR22HG与PAF、Bcl⁃2呈正相关,MIR22HG与PGI2、Bax呈负相关(P<0.05)。关联规则结果显示,MIR22HG表达升高与Bax、PGI2降低和PAF、Bcl⁃2升高的支持度>45%、置信度均>95%、提升度均>1。CCK⁃8结果显示,AST对RA⁃FLS最佳干预浓度和时间分别为2 mg·mL^(-1)和48 h。经AST处理后,RA⁃FLS中MIR22HG、Bcl⁃2蛋白表达降低,Bax、Caspase⁃3和Caspase⁃8蛋白表达升高,细胞凋亡增多,PAF降低、PGI2升高(均P<0.05)。过表达MIR22HG,以上结果可逆。使用Caspase⁃3信号通路激动剂PETCM处理RA⁃FLS,效果和AST一致。结论:AST可以通过抑制MIR22HG、协同Caspase⁃3信号通路起到促进RA⁃FLS凋亡、抑制RA⁃FLS高凝状态的作用。

Objective:To investigate the effect of total astragalus saponins(AST)on apoptosis indicators Bax,Caspase⁃3 and Caspase⁃8,and hypercoagulability indicators PAF and PGI2 in rheumatoid arthritis(RA)by regulating the lncRNA MIR22HG and Caspase⁃3 pathway via data mining and cell experiments.Methods:Included were 30 RA patients and 30 normal subjects.From all subjects,peripheral blood mononuclear cells(PBMCs)were collected and subjected to detection of MIR22HG expression by quantitative reverse transcription PCR(RT⁃qPCR).Data mining was used to explore the relationship of MIR22HG expression with apoptosis and blood coagulation.A cell line of fibroblast⁃like synoviocytes in RA(RA⁃FLS)was established,and transfected with MIR22HG⁃overexpressing plasmid.The effects of AST on RA⁃FLS apoptosis and blood coagulation were examined by using cell counting kit8(CCK⁃8),RT⁃qPCR,ELISA,Western blotting(WB),immunofluorescence(IF)assay and flow cytometry.Results:Compared with the normal group,the RA group showed higher expression levels of MIR22HG,PAF and Bcl⁃2 mRNA,and lower expression levels of PGI2 and Bax mRNA(P<0.05).Spearman correlation analysis showed that MIR22HG was positively correlated with PAF and Bcl⁃2,and was negatively correlated with PGI2 and Bax(P<0.05).Computation of association rules between increased MIR22HG expression,increased PAF and Bcl⁃2,and decreased Bax and PGI2 yielded a support value>45%,a confidence value>95%and a lift value>1.CCK⁃8 assay showed that the optimal concentration and duration of AST intervention on RA⁃FLS were 2 mg·mL-1 and 48 h,respectively.After AST treatment,RA⁃FLS showed decreased expression of MIR22HG and Bcl⁃2 proteins,increased expression of Bax,Caspase⁃3 and Caspase⁃8 proteins,with more cell apoptosis.There was a reduction in PAF and an increase in PGI2(all P<0.05).Overexpression of MIR22HG could reverse these effects.Treatment of RA⁃FLS with PETCM,a Caspase signaling pathway agonist,led to identical effects as by AST.Conclusion:AST can promote apoptosis of RA⁃FLS and inhibit its hypercoagulability by inhibiting MIR22HG and cooperating with the Caspase⁃3 signaling pathway.