苦参碱对血管平滑肌细胞增殖和凋亡的影响及机制

Effects and mechanism of matrine on proliferation and apoptosis of vascular smooth muscle cells

目的观察苦参碱对血管平滑肌细胞增殖和凋亡的影响,并探讨其作用机制。方法取血管平滑肌细胞MOVAS进行培养,分别加入0.5、1、5、10、100μmol/L苦参碱溶液,作用24、48、72 h后,采用CCK-8法检测细胞增殖情况,根据增殖曲线进行回归计算,苦参碱对MOVAS细胞增殖抑制的EC 50是7.6μmol/L,因此选择5、10μmol/L作用浓度进行后续实验。取对数生长期细胞,分为5μmol/L苦参碱组、10μmol/L苦参碱组及对照组,分别加入5、10μmol/L苦参碱和无菌PBS溶液。采用PI染色法检测各组细胞周期,ELISA法检测细胞凋亡率和凋亡相关蛋白Caspase-3活性,Western blotting法检测AKT/mTOR信号通路相关蛋白AKT、mTOR及其下游底物p70S6K、4EBP1磷酸化蛋白表达。结果与对照组比较,5μmol/L苦参碱组、10μmol/L苦参碱组G 1期细胞比例升高,S期和G 2/M期细胞比例降低(P<0.05或<0.01);细胞凋亡率升高,Caspase-3活性增加(P<0.05或<0.01);p-AKT、p-mTOR及p-p70S6K、p-4EBP1蛋白表达均降低(P均<0.05);与5μmol/L苦参碱组比较,10μmol/L苦参碱组上述指标的改善更明显(P均<0.05)。结论苦参碱能诱导MOVAS细胞发生细胞凋亡和细胞周期阻滞,从而抑制MOVAS细胞增殖;其机制可能是通过抑制AKT/mTOR通路活性发挥作用。

Objective To observe the effects of matrine on the proliferation and apoptosis of vascular smooth muscle cells and to explore its mechanism.Methods MOVAS cells were cultured and 0.5,1,5,10 and 100 mol/L matrine solutions were added for 24,48 and 72 h.Cell proliferation was detected by CCK-8 to select the optimal concentration of matrine on MOVAS cells.According to the regression calculation of proliferation curve,the EC 50 of matrine on MOVAS cell proliferation inhibition was 7.6 mol/L,so 5 and 10 mol/L were selected for the subsequent experiments.Cells in the logarithmic phase were divided into 5 mol/L matrine group,10 mol/L matrine group,and control group,and 5 and 10 mol/L matrine and aseptic PBS solution were added,respectively.Cell cycle was detected by PI staining,apoptosis rate and Caspase-3 activity were detected by ELISA,and AKT/mTOR protein phosphorylation and substrate protein expression(p-AKT,AKT,p-MTOR,mTOR,p70S6K,p-p70s6K,p-4EBP1 and 4EBP1)were detected by Western blotting.Results Compared with the control group,the proportion of cells in the G 1 phase increased in the 5 mol/L matrine group and 10 mol/L matrine group,while the proportion of cells in the S phase and G 2/M phase decreased.The apoptosis rate increased and the activity of Caspase-3 increased.The expression of p-AKT and p-mTOR protein decreased,while the expression of AKT and mTOR protein did not change significantly.Phosphorylation of substrate p70S6K and 4EBP1 by P-P70S6K and P-4EBP1 was reduced.Conclusion Matrine can induce apoptosis and cell cycle arrest of MOVAS cells,and thus inhibit the proliferation of MOVAS cells by inhibiting phosphorylation of the AKT/mTOR pathway.