miR-19b通过调节线粒体功能抑制H_(2)O_(2)诱导H9c2心肌细胞凋亡

miR⁃19b inhibits H_(2)O_(2)induced apoptosis of H9c2 cardiomyocytes by regulating mitochondrial function

目的:研究microRNA(miR)-19b抑制H_(2)O_(2)诱导的心肌细胞凋亡并探讨其机制。方法:使用过氧化氢(H_(2)O_(2))诱导H9c2心肌细胞损伤模型,将H_(2)O_(2)诱导的H9c2细胞分别转染miR-19b模拟物、抑制剂及其阴性对照。大鼠H9c2心肌细胞株分为6组,对照(Control)组,H_(2)O_(2)组,H_(2)O_(2)+miR-19b mimic阴性对照(NC)组,H_(2)O_(2)+miR-19b mimic组,H_(2)O_(2)+miR-19b inhibitor组,H_(2)O_(2)+miR-19b inhibitor NC组。采用活性氧(ROS)试剂盒检测细胞氧化应激水平,JC-1法检测细胞线粒体膜电位(MMP)变化,试剂盒检测细胞色素c与线粒体共定位,Western Blot法检测Bax、Bcl-2和Cleaved caspase3和线粒体动力相关蛋白1(Drp1)、线粒体融合蛋白2(Mfn2)及视神经萎缩蛋白1(OPA1)表达水平。结果:与Control组相比,H_(2)O_(2)组心肌细胞ROS(红色荧光强度)水平明显升高,MMP(JC-1绿红荧光强度比值)显著降低,细胞色素c(绿色荧光强度)释放明显增加,细胞促凋亡蛋白Cleaved caspase-3、Bax、Drp1蛋白的表达上调,抗凋亡蛋白Bcl-2、Mfn2、OPA1蛋白的表达均下调(P<0.05)。与H_(2)O_(2)+miR-19b mimic NC组相比,miR-19b转染可显著减少ROS的生成,稳定MMP,抑制细胞色素c释放,降低Cleaved caspase-3、Bax、Drp1表达水平,Bcl-2、Mfn2、OPA1的表达显著上升(P<0.05)。与H_(2)O_(2)+miR-19b inhibitor NC组相比,转染miR-19b抑制剂可阻断miR-19b对H_(2)O_(2)诱导H9c2心肌细胞的保护作用,表现为ROS生成增加,MMP降低,细胞色素c释放增加,Cleaved caspase-3、Bax、Drp1的表达增加,Bcl-2、Mfn2、OPA1的表达下降(P<0.05)。结论:miR-19b高表达可显著抑制H9c2细胞的氧化应激损伤,其可能与改善线粒体动力学、维持心肌细胞线粒体的动态稳定性以及调节凋亡相关蛋白表达从而抑制细胞凋亡有关。

Objective:To investigate the inhibition of microRNA(miR)‐19b on hydrogen peroxide(H_(2)O_(2))‐induced cardiomyocyte apoptosis and its mechanism.Methods:H9c2 cardiomyocytes were injured by H_(2)O_(2).H_(2)O_(2)‐induced H9c2 cells were transfected with miR‐19b mimics,inhibitors,and negative con‐trols.Rat H9c2 myocardial cell strain was divided into 6 groups:control group,H_(2)O_(2)group,H_(2)O_(2)+miR‐19b mimic NC group,H_(2)O_(2)+miR‐19b mimic group,H_(2)O_(2)+miR‐19b inhibitor group,and H_(2)O_(2)+miR‐19b inhibitor NC group.The reactive oxygen species(ROS)kit was used to detect the ox‐idative stress level of cells,the JC‐1 method was used to detect the changes in mitochondrial mem‐brane potential(MMP),and the kit was used to detect the co‐localization of cytochrome c and mito‐chondria.The expression levels of Bax,Bcl‐2,cleaved caspase3,mitochondrial dynamin‐related pro‐tein 1(Drp1),mitochondrial fusion protein(Mfn2),and optic atrophy 1(OPA1)were detected by Western blot.Results:Compared with that respectively in the control group,the level of ROS(red fluorescence intensity)in cardiomyocytes of the H_(2)O_(2)group was significantly increased,MMP(JC‐1 green red fluorescence intensity ratio)was significantly reduced,the release of cytochrome c(green flu‐orescence intensity)was significantly increased,the expression of apoptosis promoting proteins cleaved caspase‐3,Bax,and Drp1 were up‐regulated,and the expression of anti‐apoptotic proteins Bcl‐2,Mfn2,and OPA1 was down‐regulated(all P<0.05).Compared with H_(2)O_(2)+miR‐19b mimic NC group,miR‐19b transfection can significantly reduce the production of ROS,stabilize MMP,inhibit the release of cytochrome c,reduce the expression level of Cleaved caspase‐3,Bax,and Drp1,and significantly increase the expression of Bcl‐2,Mfn2,and OPA1(all P<0.05).Compared with the H_(2)O_(2)+miR‐19b inhibitor NC group,transfection of miR‐19b inhibitor could block the protective ef‐fect of miR‐19b on H_(2)O_(2)induced H9c2 cardiomyocytes,which showed that ROS production in‐creased,MMP decreased,cytochrome c release increased,the expression of Cleaved caspase‐3,Bax,and Drp1 increased,and the expression of Bcl‐2,Mfn2,and OPA1 decreased,with statistical significance(all P<0.05).Conclusion:The overexpression of miR‐19b can significantly inhibit the oxidative stress injury of H9c2 cells,which may be related to improving mitochondrial dynamics,maintaining the dynamic stability of myocardial mitochondria,and regulating the expression of apopto‐sis‐related proteins to inhibit apoptosis.