曲美他嗪对人脑血管平滑肌细胞增殖、迁移及活性氧表达的影响及其机制

Effect of trimetazidine on proliferation,migration and reactive oxygen expression on human cerebral vascular smooth muscle cells and its mechanism

目的探讨曲美他嗪(TMZ)对人脑血管平滑肌细胞(HBVSMCs)增殖、迁移及活性氧(ROS)表达的影响及其机制。方法选用生长状况良好的对数生长期HBVSMCs分两部分进行实验,A实验:将HBVSMCs分为5组,对照组:不处理;血小板衍生生长因子(PDGF)组:加入10 ng/ml PDGF;0.2μmol/L、1.0μmol/L和5.0μmol/L TMZ组:在给予PDGF基础上,分别加入终浓度为0.2μmol/L、1.0μmol/L和5.0μmol/L TMZ。B实验:将HBVSMCs分为5组,对照组:不处理;PDGF组:给予10 ng/ml PDGF;TMZ组:在给予PDGF基础上,加入浓度为5.0μmol/L TMZ;XAV939组:在给予PDGF基础上,加入浓度为2.0μmol/L XAV939;TMZ+XAV939组:在给予PDGF基础上,加入5.0μmol/L TMZ和2.0μmol/L XAV939共同处理。采用噻唑蓝法、划痕实验、二氯荧光素法分别检测各组HBVSMCs的增殖活力、迁移率及ROS表达水平,采用蛋白质免疫印迹法检测HBVSMCs中Wnt/β-连环蛋白(catenin)信号通路相关蛋白β-catenin、细胞周期蛋白D1(CyclinD1)、基质金属蛋白酶(MMP)-9及原癌基因蛋白质(c-Myc)的表达水平。结果A实验中,对照组、PDGF组、0.2μmol/L TMZ组、1.0μmol/L TMZ组、5.0μmol/L TMZ组HBVSMCs的OD值、迁移率、ROS、β-catenin、Cyclin D1、MMP-9及c-Myc表达水平比较,差异均有统计学意义(P<0.05);其中PDGF组上述指标均高于对照组,各浓度TMZ组上述指标均低于PDGF组,且呈浓度依赖性降低(P<0.05)。B实验中,对照组、PDGF组、TMZ组、XAV939组、TMZ+XAV939组HBVSMCs的OD值、迁移率及ROS表达水平比较差异均有统计学意义(P<0.05),其中PDGF组上述指标均高于对照组;TMZ组和XAV939组上述指标均低于PDGF组;TMZ+XAV939组上述指标均低于TMZ组(P<0.05)。结论TMZ可能通过抑制Wnt/β-catenin信号通路活化抑制HBVSMCs增殖、迁移及ROS表达。

Objective To explore the effect of trimetazidine(TMZ)on proliferation,migration and reactive oxygen(ROS)expression on human cerebral vascular smooth muscle cells(HBVSMCs)and its mechanism.Methods HBVSMCs in logarithmic growth phase with good growth conditions were selected for two parts experiment.Experiment A:HBVSMCs were divided into 5 groups.Control group:no treatment;PDGF group:10 ng/ml PDGF was added;0.2μmol/L,1.0μmol/L and 5.0μmol/L TMZ groups:on basis of PDGF,0.2μmol/L,1.0μmol/L and 5.0μmol/L TMZ were added respectively.Experiment B:HBVSMCs were divide into 5 groups.Control group:no treatment;PDGF group:10 ng/ml PDGF was added;TMZ group:on basis of PDGF,5.0μmol/L TMZ was added;XAV939 group:on basis of PDGF,2.0μmol/L XAV939 was added;TMZ+XAV939 group:on basis of PDGF,5.0μmol/L TMZ and 2.0μmol/L XAV939 were added.Thiazole blue method,scratch test and dichlorofluorescein method were used to detect the proliferation activity,mobility and ROS expression level of HBVSMCs in each group.Western blotting was used to detect Wnt/β-catenin signaling pathway related proteins includingβ-catenin,Cyclin D1,matrix metalloproteinase(MMP)-9 and proto-oncogene protein(c-Myc)expression levels in HBVSMCs.Results In experiment A,differences of OD value,migration rate,ROS,β-catenin,Cyclin D1,MMP-9 and c-Myc expression levels of HBVSMCs among control group,PDGF group,0.2μmol/L,1.0μmol/L and 5.0μmol/L TMZ group were statistically significant(P<0.05),and above indicators in PDGF group were higher than those in the control group,above indicators in TMZ group of various concentrations were lower than those in PDGF group with concentration-dependent decrease(P<0.05).In experiment B,differences of OD value,migration rate,ROS expression level of HBVSMCs among control group,PDGF group,TMZ group,XAV939 group,TMZ+XAV939 group were statistically significant(P<0.05),and above indicators in PDGF group were higher than those in control group,above indicators in TMZ group and XAV939 group were lower than those in PDGF group,above indicators in TMZ+XAV939 group were lower than those in TMZ group(P<0.05).Conclusion TMZ can inhibit the proliferation,migration and ROS expression of HBVSMCs by inhibiting Wnt/β-catenin signaling pathway activation.