附子汤对类风湿性关节炎滑膜成纤维细胞MH7A增殖和miR-155表达的影响

Effect of Fuzitang on Proliferation of Human Rheumatoid Arthritis Synovial Fibroblast Cell Line MH7A and Expression of miR-155

目的:观察附子汤对类风湿性关节炎滑膜成纤维细胞MH7A增殖的影响,研究附子汤对miR-155表达的影响并进一步探讨其抗类风湿性关节炎的作用机制。方法:体外培养MH7A细胞,设空白组、附子汤高剂量组(25 g·L^(-1))、低剂量组(12.5 g·L^(-1))和硫酸羟氯喹阳性药组(0.00625 g·L^(-1)),采用细胞增殖与活性检测(CCK-8)试剂盒法检测细胞增殖,流式细胞术检测MH7A细胞周期的改变;应用实时荧光定量聚合酶链式反应(Real-time PCR)法检测药物处理后miR-155及其下游基因含SH2结构域的肌醇5-磷酸酶-1(SHIP-1)、蛋白激酶B3(Akt3)和哺乳动物雷帕霉素靶蛋白(mTOR)mRNA表达,蛋白免疫印迹法(Western blot)检测磷脂酰肌醇3-激酶(PI3K)、Akt3和mTOR的蛋白表达。结果:附子汤体外在质量浓度6.25 g·L^(-1)以上时,能明显抑制MH7A增殖,且呈明显的量-效关系和时-效关系。与空白组比较,附子汤高、低剂量组G2/M期细胞比例均明显增加(P<0.05),高剂量组G0/G1期细胞比例明显降低(P<0.05),其细胞周期的阻滞作用呈明显的量效关系。与空白组比较,附子汤高、低剂量组miR-155的表达均明显下调(P<0.05);附子汤高剂量组SHIP1 mRNA表达明显上调、Akt3 mRNA表达明显下调(P<0.05),附子汤高、低剂量组mTOR mRNA表达均明显下调(P<0.05)。与空白组比较,附子汤高、低剂量组的PI3K、Akt3和mTOR的蛋白表达均明显下调(P<0.05)。结论:附子汤对MH7A细胞增殖具有显著的抑制作用,作用机制与下调miR-155的表达,从而促进SHIP-1的表达,抑制其下游PI3K/Akt3/mTOR信号通路的基因表达有关。

Objective: To observe the effects of Fuzitang(FZT)on the proliferation of MH7A cells,the human rheumatoid arthritis synovial fibroblasts, and the expression of miR-155 and explore its antirheumatoid arthritis mechanism. Method: MH7A cells were cultured in vitro and divided into a blank group,high-(25 g·L^(-1))and low-dose(12.5 g·L^(-1))FZT groups,and a positive drug group(hydroxychloroquine,0.006 25 g·L^(-1)). The cell proliferation was detected by cell counting kit-8(CCK-8)method,and the change in the MH7A cell cycle was detected by flow cytometry. The mRNA expression of miR-155 and its downstream genes,including SH2 domain-containing inositol 5-phosphatase-1(SHIP-1),protein kinase B 3(Akt3),and mammalian target of rapamycin(mTOR),was detected by Real-time fluorescence quantitative polymerase chain reaction(Real-time PCR),and the protein expression of phosphatidylinositol 3-kinase(PI3K),Akt3,and mTOR was detected by Western blot. Result: FZT in vitro in a concentration of 6.25 g·L^(-1)above could inhibit the proliferation of MH7A cells in the significant dose-and time-effect manner. Compared with the blank group,the FZT groups showed increased proportions of cells in the G2/M phase(P<0.05),and the high-dose FZT group showed a decreased proportion of cells in the G0/G1phase(P<0.05). The arresting effect of FZT on the cell cycle was in a significant dose-effect manner. Compared with the blank group,the FZT groups showed down-regulated miR-155 and mTOR mRNA expression(P<0.05),and the high-dose FZT group showed up-regulated SHIP1mRNA expression and down-regulated Akt3 mRNA expression(P<0.05). Compared with the blank group,the FZT groups showed reduced protein expression of PI3K,Akt3,and mTOR(P<0.05). Conclusion: FZT can significantly inhibit the proliferation of MH7A cells,and the mechanism is related to the promotion of the expression of SHIP-1 and down-regulation of the gene expression of the PI3K/Akt3/mTOR signaling pathway by down-regulating the expression of miR-155.