丹参酮Ⅱ_A抑制同型半胱氨酸诱导的大鼠主动脉血管平滑肌细胞增殖和迁移及其机制研究

Study on Tanshinone Ⅱ_A Inhibiting the Proliferation and Migration Induced by Homocysteine of VSMCs and Its Mechanism

目的:研究丹参酮Ⅱ_A抑制同型半胱氨酸(Hcy)诱导的大鼠主动脉血管平滑肌细胞(VSMCs)增殖和迁移及其机制。方法:取VSMCs用于试验。在验证丹参酮Ⅱ_A抑制Hcy诱导的VSMCs增殖和迁移试验(验证试验)中,将细胞分为对照组、Hcy组(1 000μmol/L)及丹参酮ⅡA低、中、高剂量组(5、10、20μg/L)。在丹参酮作用机制试验(细胞通路试验)中,将细胞分为对照组、丹参酮Ⅱ_A组(20μg/L)、雷帕霉素组(20 nmol/L)、MHY1485组(10μmol/L)、丹参酮Ⅱ_A+雷帕霉素组(丹参酮ⅡA,20μg/L+雷帕霉素,20 nmol/L)、丹参酮Ⅱ_A+MHY1485组(丹参酮ⅡA,20μg/L+MHY1485,10μmol/L)。验证细胞通路P70S6K和p-P70S6K的表达时,雷帕霉素(抑制试验)与MHY1485(激动试验)分别进行试验。酶标仪测定VSMCs的增殖,细胞划痕试验、Transwell法测定VSMCs的迁移,增强化学发光法测定VSMCs中P21、P27、基质金属蛋白酶(MMP)-2、MMP-9、P70S6K和p-P70S6K的表达。结果:验证试验中,与对照组比较,Hcy组细胞24、48 h吸光度值,迁移面积和VSMCs穿透数量,MMP-2、MMP-9和p-P70S6K表达水平显著升高,P21、P27表达水平显著降低(P<0.01);与Hcy组比较,丹参酮Ⅱ_A低剂量组细胞48 h,中、高剂量组24、48 h吸光度值,MMP-2水平,低、中、高剂量组细胞迁移面积和VSMCs穿透数量,MMP-9和p-P70S6K表达水平显著降低,丹参酮Ⅱ_A中、高剂量组细胞中P21、P27表达水平显著升高(P<0.01);各组细胞P70S6K水平比较,差异无统计学意义(P>0.05)。细胞通路试验中,与对照组比较,丹参酮Ⅱ_A、雷帕霉素组细胞24、48 h吸光度值,迁移面积和VSMCs穿透数量显著降低,MHY1485组24、48 h吸光度值显著升高(P<0.01);与丹参酮ⅡA组比较,丹参酮Ⅱ_A+雷帕霉素组细胞24、48 h吸光度值显著降低,丹参酮Ⅱ_A+MHY1485组细胞12、24、48 h吸光度值,迁移面积和VSMCs穿透数量显著升高(P<0.01)。抑制试验中,与对照组比较,丹参酮Ⅱ_A组细胞p-P70S6K表达水平显著降低(P<0.01);与丹参酮ⅡA组比较、雷帕霉素、丹参酮ⅡA+雷帕霉素组细胞p-P70S6K表达水平显著降低(P<0.01)。激动试验中,丹参酮Ⅱ_A组细胞p-P70S6K水平显著降低(P<0.01);与丹参酮Ⅱ_A组比较,MHY1485、丹参酮Ⅱ_A+MYH1485组细胞p-P70S6K水平显著升高(P<0.01)。结论:丹参酮Ⅱ_A可以抑制VSMCs的增殖和迁移,并通过抑制mTOR/P70S6K信号通路而发挥作用。

OBJECTIVE：To study tanshinone ⅡA inhibiting the proliferation and migration induced by homocysteine（Hcy） of rat aortic vascular smooth muscle cells（VSMCs） and its signal pathway.METHODS：VSMCs were selected for the following experiments.In order to validate Tanshinone ⅡA inhibiting the proliferation and migration induced by Hcy of VSMCs,VSMCs were divided into control group,Hcy group（1 000（xmol/L）,Tanshinone ⅡA low-dose,medium-dose and high-dose groups（5,10,20 μg/L）.In mechanism study test,VSMCs were divided into control group,Tanshinone ⅡA group（20 μg/L）,rapamycin group（20 nmol/L）,MHY1485 group（10 μmol/L）,Tanshinone ⅡA＋rapamycin group（Tanshinone ⅡA,20 μg/L＋rapamycin,20 nmol/L）,Tanshinone ⅡA＋MHY1485 group（Tanshinone ⅡA,20 μg/L＋MHY1485,10 nmol/L）.In validation test of P70S6 K and p-P70S6 K pathway expression,rapamycin and MHY1485 were used for inhibitory test and activation test,respectively.The proliferation of VSMCs was determined by ELIASA,and Transwell chambers and wound healing test were employed to test the migratory ability of VSMCs.Western blotting were used to investigate the expressions of P21,P27,MMP-2,MMP-9,P70S6 K and p-P70S6 K in VSMCs.RESULTS：In validation test,compared with control group,24,48 h absorbance,migration area,the number of VSMCs penetration and the expression of MMP-2,MMP-9 and p-P70S6 K increased significantly,while the expression of P21 and P27 decreased significantly（P〈0.01）.Compared with Hcy group,48 h absorbance of Tanshinone ⅡA low-dose group,24,48 h absorbance and the expression of MMP-2 of Tanshinone ⅡA medium-dose and high-dose groups,migration area,the number of VSMCs penetration,the expression of MMP-9 and p-P70S6 K in low-dose,medium-dose and high-dose groups all decreased significantly;the expression of P21 and P27 increased significantly in Tanshinone ⅡA medium-dose and high-dose groups（P〈0.01）;there was no statistical significance in P70S6 K level among those groups（P〈0.05）.In cell pathway test,compared with control group,24,48 h absorbance,migration area and the number of VSMCs penetration decreased significantly in Tanshinone ⅡA group and rapamycin group,while 24,48 h absorbance increased significantly in MHY1485 group（P〈0.01）.Compared with Tanshinone ⅡA group,24,48 h absorbance decreased significantly in Tanshinone ⅡA＋rapamycin group,while 12,24,48 h absorbance,migration area and the number of VSMCs penetration increased significantly in Tanshinone ⅡA＋MHY1485 group（P〈0.01）.In inhibitory test,compared with control group,the expression of p-P70S6 K decreased significantly in Tanshinone ⅡA group（P〈0.01）;compared with Tanshinone ⅡA group,the expression of p-P70S6 K decreased significantly in rapamycin group and Tanshinone ⅡA＋rapamycin group（P〈0.01）;in activation test,the expression of p-P70S6 K decreased significantly in Tanshinone ⅡA group（P〈0.01）,compared with Tanshinone ⅡA group,the expression of p-P70S6 K increased significantly in MHY1485,Tanshinone ⅡA＋MHY1485group（P〈0.01）.CONCLUSIONS：Tanshinone ⅡA can inhibit the proliferation and migration of VSMCs by suppressing mTOR/P70S6 K signal pathway.