黄芪甲苷对miRNA-1过表达诱导大鼠心力衰竭的保护作用及其机制

Protective effect of astragaloside Ⅳ on heart failure induced by over-expression of miRNA-1 and its mechanism

目的:研究黄芪甲苷(ASⅣ)对微小RNA-1(miRNA-1)过表达诱导大鼠心力衰竭(HF)的保护作用,探讨其可能的作用机制。方法:将32只SD大鼠随机分为正常对照组、miRNA-1mimics阴性病毒对照(miRNA-1 mimics NC)组、miRNA-1 mimics组和ASⅣ+miRNA-1组,每组8只。通过左心室壁注射miRNA-1慢病毒建立大鼠HF模型。超声心动图检测各组大鼠左心室射血分数(EF)和左心室短轴缩短率(FS),实时荧光定量PCR(RT-qPCR)法检测各组大鼠心肌组织中miRNA-1表达水平。将H9C2心肌细胞分为正常对照组、miRNA-1 mimics阴性病毒对照(miRNA-1mimics NC)组、miRNA-1 mimics组和ASⅣ+miRNA-1 mimics组。采用miRNA-1慢病毒感染H9C2心肌细胞,RT-qPCR法检测H9C2心肌细胞中miRNA-1表达水平,MTT法测定各组大鼠H9C2心肌细胞存活率并筛选ASⅣ的最适浓度,钙测试盒检测各组大鼠心肌H9C2细胞中钙离子(Ca^(2+))水平,RT-qPCR法检测各组大鼠心肌H9C2细胞中钙离子/钙调蛋白依赖性蛋白激酶Ⅱ(CaMKⅡ)和兰尼碱受体2(RyR2)mRNA表达水平,Western blotting法检测各组大鼠心肌H9C2细胞中CaMKⅡ和RyR2蛋白表达水平。结果:与正常对照组和miRNA-1 mimics NC组比较,miRNA-1 mimics组大鼠心肌组织中miRNA-1表达水平明显升高(P<0.01),EF和FS明显降低(P<0.01);与miRNA-1mimics组比较,ASⅣ+miRNA-1 mimics组大鼠心肌组织中miRNA-1表达水平明显降低(P<0.01),EF和FS明显升高(P<0.01)。与正常对照组和miRNA-1 mimics NC组比较,miRNA-1 mimics组大鼠H9C2心肌细胞中miRNA-1表达水平和Ca^(2+)水平明显升高(P<0.01),CaMKⅡmRNA和蛋白表达水平明显升高(P<0.01),RyR2 mRNA和蛋白表达水平明显降低(P<0.01);与miRNA-1mimics组比较,ASⅣ+miRNA-1 mimics组大鼠H9C2心肌细胞中miRNA-1表达水平、Ca^(2+)水平和CaMKⅡmRNA和蛋白表达水平明显降低(P<0.01),RyR2 mRNA和蛋白表达水平均明显升高(P<0.01)。结论:ASⅣ可能是通过下调miRNA-1的表达,参与心肌细胞中Ca2+水平的调控,进而改善HF,起到心脏保护作用。

Objective:To study the protective effect of astragalosideⅣ(ASⅣ)on the heart failure(HF)induced by over-expression of microRNA-1(miRNA-1)in the rats,and to clarify the possible mechanism.Methods:A total of 32 SD rats were randomly divided into normal control group,miRNA-1 mimics negative virus control(miRNA-1 mimics NC)group,miRNA-1 mimics group,and ASⅣ+miRNA-1 mimics group,and there were 8 rats in each group.The HF models of rats were established by injecting miRNA-1 lentivirus in left ventricular wall.Real-time fluorescence quantitative PCR(RT-qPCR)method was used to detect the expression level of miRNA-1 in myocardium tissue of the rats in various groups,and echocardiography was used to examine the left ventricular injection fraction(EF)and left ventricular fraction shortening(FS)of the rats in various groups.The H9C2 cardiomyocytes were divided into normal control group,miRNA-1 mimics negative virus control(miRNA-1 mimics NC)group,miRNA-1 mimics group and ASⅣ+miRNA-1 mimics group.The H9C2 cardiomyocytes were infected with miRNA-1 lentivirus.RT-qPCR method was used to detect the expression levels of miRNA-1 in the H9C2 cardiomyocytes in various groups,and the survival rates of the H9C2 cardiomyocytes in various groups were detected by MTT method to screen the optimal concentration of ASⅣ.The calcium test box was used to detect the expression levels of calcium ions(Ca2+)in the H9C2 cardiomyocytes in various groups,RT-qPCR method was used to detect the expression levels of calcium/calmodulin dependent protein kinaseⅡ(CaMKⅡ)and ryanodine receptor type 2(RyR2)mRNA in the H9C2 cardiomyocytes in various groups,and Western blotting method was used to detect the expression levels of CamKⅡand RyR2 proteins in the H9C2 cardiomyocytes in various groups.Results:Compared with normal control group and miRNA-1 mimics NC group,the expression level of miRNA-1 in myocardium tissue of the rats in miRNA-1 mimics group was increased significantly(P<0.01),and the EF and FS were decreased significantly(P<0.01);compared with miRNA-1 mimics group,the expression level of miRNA-1 in myocardium tissue of the rats in ASⅣ+miRNA-1 mimics group was decreased significantly(P<0.01),and the EF and FS were increased significantly(P<0.01).Compared with normal control group and miRNA-1 mimics NC group,the expression level of miRNA-1 and Ca^(2+)level in the H9C2 cardiomyocytes of the rats in miRNA-1 mimics group were increased significantly(P<0.01),the expression levels of CaMKⅡmRNA and protein were increased significantly(P<0.01),and the expression levels of RyR2 mRNA and protein were decreased significantly(P<0.01);compared with miRNA-1 mimics group,the expression levels of miRNA-1,Ca^(2+)level and the expression levels of CamKⅡmRNA and protein in the H9C2 cardiomyocytes of the rats in ASⅣ+miRNA-1 mimics group were decreased significantly(P<0.01),and the expression levels of RyR2 mRNA and protein were increased significantly(P<0.01).Conclusion:ASⅣmay be involved in the regulation of Ca^(2+)level in the cardiomyocytes by down-regulation of the expression of miRNA-1,thus improves the HF and plays a protective role in the heart.