白芍总苷对类风湿性关节炎滑膜细胞增殖、凋亡和炎性因子的影响

Effects of total glucosides of paeony on proliferation,apoptosis and inflammatory factors of synovial cells in rheumatoid arthritis

目的探讨白芍总苷(total glucosides of paeony TGP)对类风湿性关节炎成纤维样滑膜细胞(FLS)增殖、凋亡和炎性因子的影响及分子机制。方法分离培养FLS,实验分为对照(NC)组(不作任何处理)、不同浓度白芍总苷组(0.5、1.0、1.5 mg·mL^(-1)白芍总苷)、miR-NC组(转染miR-34a模拟物对照)、miR-34a组(转染miR-34a模拟物)、anti-miR-NC组(转染miR-34a抑制剂对照)、anti-miR-34a组(转染miR-34a抑制剂)、白芍总苷+anti-miR-NC组(1.5 mg·mL^(-1)白芍总苷+转染miR-34a抑制剂对照)、白芍总苷+anti-miR-34a组(1.5 mg·mL^(-1)白芍总苷+转染miR-34a抑制剂)。细胞计数试剂盒8(CCK-8)检测细胞增殖活力;流式细胞术检测细胞凋亡;蛋白质印迹(Western blot)法检测细胞增殖核抗原Ki67(Ki67)、裂解的半胱氨酸天冬氨酸蛋白酶-3(Cleaved caspase-3)、磷酸化p65(p-p65)、磷酸化核因子κB抑制蛋白α(p-IкBα)蛋白表达;酶联免疫吸附试验(ELISA)试剂盒检测白细胞介素-6(IL-6)、肿瘤坏死因子-α(TNF-α)水平;实时荧光定量PCR(RT-qPCR)检测miR-34a表达水平。结果与对照组相比,不同浓度白芍总苷组Ki67表达水平降低,Cleaved-caspase-3表达水平升高,细胞活力降低,细胞凋亡率升高(P<0.05);白芍总苷组IL-6、TNF-α含量显著降低,miR-34a表达水平显著升高,p-p65、p-IкBα蛋白表达水平降低(P<0.05)。过表达miR-34a抑制FLS增殖和炎性因子释放,诱导细胞凋亡;而抑制miR-34a表达作用相反。且抑制miR-34a表达逆转了白芍总苷对FLS细胞增殖、凋亡、炎症因子释放及核因子-κB(NF-κB)信号通路的影响。结论白芍总苷可能通过上调miR-34a表达抑制NF-κB信号通路抑制类风湿性关节炎成纤维样滑膜细胞增殖和炎性因子释放,并诱导细胞凋亡。

Objective To investigate the effect and molecular mechanism of total paeony glycoside on the proliferation,apoptosis and inflammatory factors of rheumatoid arthritis fibroblast-like synoviocytes(FLS).Methods FLS were isolated and cultured;the experiment was divided into control(NC)group(without any treatment),different concentrations of total glycosides of paeony group(0.5,1.0,1.5 mg·mL^(-1)total glycosides of paeony),miR-NC group(transfected with miR-34 a mimics control),miR-34 a group(transfected with miR-34 a mimics),anti-miR-NC group(transfected with miR-34 a inhibitor control),anti-miR-34 a group(transfected with miR-34 a inhibitor),total paeony glycoside+anti-miR-NC group(1.5 mg·mL^(-1)total paeony glycoside+transfected with miR-34 a inhibitor control),total paeony glycoside+anti-miR-34 a group(1.5 mg·mL^(-1)total paeony glycoside+transfected with miR-34 a inhibitor).Cell counting kit 8(CCK-8)was used to detect cell proliferation activity;flow cytometry to detect cell apoptosis;Western blot was used to detect cell proliferation nuclear antigen Ki67(Ki67),cleaved cysteine-containing aspartate-specific proteases-3(Cleaved caspase-3),phosphorylated p65(p-p65),phosphorylated nuclear factor kappa B inhibitor protein alpha(p-IкBα)protein expression;enzyme-linked immunosorbent assay(ELISA)kit was used to detected the leve of interleukin-6(IL-6)and tumor necrosis factor-α(TNF-α);real-time fluorescence quantitative PCR(RT-qPCR)to detect miR-34 a expression.Results Compared with the control group,the expression of was Ki67 decreased,the expression of Cleaved-caspase-3 was increased,the cell viability was decreased,and the apoptosis rate was increased(P<0.05)in different concentrations of total glycosides of paeony group;the leves of IL-6 and TNF-αwere significantly reduced,the expression of miR-34 a was significantly increased,and the expressions of p-p65 and p-IкBαprotein were decreased(P<0.05)in total glycosides of paeony group.Overexpression of miR-34 a inhibits FLS proliferation and release of inflammatory factors,and induces cell apoptosis;whileinhibition of miR-34 a expression has the opposite effect.Inhibition of miR-34 a expression reversed the effects of total paeony glycosides on FLS cell proliferation,apoptosis,inflammatory factor release and nuclear factor-κB(NF-κB)signaling pathway.Conclusion Total glycosides of paeony may inhibit the proliferation of rheumatoid arthritis fibroblast-like synoviocytes and release of inflammatory factors,and induce apoptosis via inhibit the NF-κB signaling pathway by up-regulating miR-34 a expression.