羟基红花黄色素A调控miR-20a-5p/TLR4轴抑制人类风湿关节炎成纤维样滑膜细胞凋亡和炎性因子的表达

Effects of HSYA on the apoptosis and expression of inflammatory factors in human rheumatoid arthritis fibroblastlike synovial cells by regulating the miR-20a-5p/TLR4 axis

目的探究羟基红花黄色素A(HSYA)对人类风湿关节炎成纤维样滑膜细胞凋亡和炎性因子表达的影响及可能机制。方法体外培养成纤维样滑膜细胞MH7A,分为对照组、TNF-α组、不同剂量(0.1、1.0、10μmol/L)HSYA+TNF-α组、miR-NC+TNF-α组、miR-20a-5p+TNF-α组、anti-miR-NC+10μmol/L HSYA+TNF-α组、anti-miR-20a-5p+10μmol/L HSYA+TNF-α组,CCK-8法检测细胞增殖活性,流式细胞术检测细胞凋亡,酶联免疫吸附法检测细胞培养上清液中IL-1β和IL-6表达,RT-qPCR法检测细胞中miR-20a-5p表达,蛋白质印迹法检测细胞中TLR4蛋白表达。双荧光素酶报告基因实验验证miR-20a-5p与TLR4调控关系。结果与对照组比较,TNF-α组MH7A细胞增殖活性降低(P<0.05),细胞凋亡率及细胞培养上清液中IL-1β和IL-6表达水平升高(P<0.05),细胞中miR-20a-5p表达降低(P<0.05),而TLR4蛋白表达升高(P<0.05)。与TNF-α组比较,不同剂量(0.1、1.0、10μmol/L)HSYA+TNF-α组MH7A细胞增殖活性升高(P<0.05),细胞凋亡率及细胞培养上清液中IL-1β和IL-6表达水平降低(P<0.05),细胞中miR-20a-5p表达升高(P<0.05),而TLR4蛋白表达降低(P<0.05)。与miR-NC+TNF-α组比较,miR-20a-5p+TNF-α组MH7A细胞增殖活性升高(P<0.05),细胞凋亡率及细胞培养上清液中IL-1β和IL-6表达水平降低(P<0.05),细胞中TLR4蛋白表达降低(P<0.05)。与anti-miR-NC+H-HSYA+TNF-α组比较,anti-miR-20a-5p+HHSYA+TNF-α组MH7A细胞增殖活性降低(P<0.05),细胞凋亡率及细胞培养上清液中IL-1β和IL-6表达水平升高(P<0.05),细胞中TLR4蛋白表达升高(P<0.05)。TLR4是miR-20a-5p的靶基因,且miR-20a-5p抑制MH7A细胞中TLR4蛋白表达(P<0.05)。结论HSYA对TNF-α诱导的成纤维样滑膜细胞MH7A凋亡和炎性因子表达具有抑制作用,其作用机制可能与调控miR-20a-5p/TLR4轴有关。

Objective To investigate the effects of hydroxysafflor yellow A(HSYA)on the apoptosis and expression of inflammatory factors in human rheumatoid arthritis fibroblast-like synovial cells,and to explore its possible action mechanism.Methods Fibroblast-like synovial cells MH7A were cultured in vitro and divided into control group,TNF-αgroup,different doses(0.1,1.0,10μmol/L)HSYA+TNF-αgroup,miR-NC+TNF-αgroup,miR-20a-5p+TNF-αgroup,anti-miR-NC+H-HSYA+TNF-αgroup,and anti-miR-20a-5p+H-HSYA+TNF-αgroup.The CCK-8 method was used to detect cell proliferation activity;cell apoptosis was detected by flow cytometry;ELISA was used to detect the expression levels of IL-1βand IL-6 in cell culture supernatants;RT-qPCR method was used to detect the expressions of miR-20a-5p in cells.Moreover Western Blot was used to detect the expression of TLR4 protein,and the dual luciferase reporter gene experiment was used to verify the regulatory relationship between miR-20a-5p and TLR4.Results Compared with that in control group,the proliferation activity of MH7A cells in TNF-αgroup was significantly decreased(P<0.05),but the apoptosis rate and the expression levels of IL-1βand IL-6 in cell culture supernatant were significantly increased(P<0.05),and the expression levels of miR-20a-5p in cells were decreased,however,the expression levels of TLR4 protein were significantly increased(P<0.05).Compared with those in TNF-αgroup,the proliferation activities of MH7A cells in the different doses HSYA+TNF-αgroup was increased,but the apoptosis rate and the expression levels of IL-1βand IL-6 in cell culture supernatants were significantly decreased(P<0.05),and the expression levels of miR-20a-5p in cells were significantly increased,however,the expression levels of TLR4 protein were significantly decreased(P<0.05).Compared with that in miR-NC+TNF-αgroup,the proliferation activity of MH7A cells in miR-20a-5p+TNF-αgroup was increased,but the apoptosis rate and the expression levels of IL-1βand IL-6 as well as TLR4 protein d in cell culture supernatants were significantly decreased(P<0.05).Compared with those in anti-miR-NC+10μmol/L HSYA+TNF-αgroup,the proliferation activity of MH7A cells in anti-miR-20a-5p+10μmol/L HSYA+TNF-αgroup was significantly decreased,but the apoptosis rate and the expression levels of IL-1βand IL-6 as well as TLR4 protein in cell culture supernatant were significantly increased(P<0.05).Moreover TLR4 was the target gene of miR-20a-5p,and miR-20a-5p inhibited the expression of TLR4 protein in MH7A cells(P<0.05).Conclusion HSYA has inhibitory effects on the apoptosis and inflammatory factor expression of fibroblast-like synovial cells MH7A induced by TNF-α,and its action mechanism may be related to the regulation of miR-20a-5p/TLR4 axis.