依那普利上调miR-133a减轻缺氧/复氧诱导的心肌细胞损伤

Enalapril Reduces Hypoxia/Reoxygenation-induced Cardiomyocyte Injury by Up-regulating miR-133a

目的探讨依那普利对缺氧/复氧诱导的心肌细胞损伤的影响和分子机制.方法建立H9 c2细胞缺氧/复氧损伤模型.设置对照(Con)组、模型(Model)组、实验1组、实验2组、实验3组、实验3+anti-miR-NC组和实验3+anti-miR-133a组.采用细胞计数试剂盒(CCK-8)、集落形成实验检测细胞活力、克隆形成能力.流式细胞术分析细胞凋亡和周期分布.实时荧光定量PCR(RT-qPCR)分析miR-133a表达.免疫印记法(Western blot)检测半胱氨酸蛋白酶3前体(pro-caspase3)和裂解的caspase3(Cleaved-caspase3)蛋白表达.结果与对照组比较,模型组H9c2细胞活力、克隆形成数、S期细胞比例、miR-133a表达、pro-caspase3蛋白表达显著降低,凋亡率、Cleaved-caspase3蛋白表达显著升高(P<0.01).与模型组比较,实验2组、实验3组H9c2细胞活力、克隆形成数、S期细胞比例、miR-133a表达、pro-caspase3蛋白表达显著升高,凋亡率、Cleaved-caspase3蛋白表达显著降低(P<0.01).与实验3+anti-miR-NC组比较,实验3+anti-miR-133a组H9c2细胞活力、克隆形成数、S期细胞比例、pro-caspase3蛋白表达显著降低,凋亡率、Cleaved-caspase3蛋白表达显著升高(P<0.01).结论一定浓度的依那普利能够减轻缺氧/复氧诱导的心肌细胞损伤,其机制可能与上调miR-133a表达有关.

Objective To investigate the effect of enalapril on hypoxia/reoxygenation-induced cardiomyocyte injury and-its molecular mechanism.Methods Hypoxia/reoxygenation injury H9c2 cell model in vitro was established.H9c2 cells were divided into control(Con)group,-model(Model)group,experiment 1 group,experiment 2 group,experiment 3 group,experiment 3+anti-miR-NC group,and experiment 3+anti-miR-133a group.Cell counting kit(CCK-8)and colony formation experiment were used to detect cell viability and clone formation ability.Flow cytometry was applied to analyze apoptosis and cycle distribution.Real-time fluorescent quantitative PCR(RT-qPCR)was used to analyze miR-133a expression.Western blotting was used to detect the expression of cysteine protease 3 precursor(pro-caspase3)and cleaved-caspase3 proteins.Results Compared with the control group,the cell viability,clone formation number,S phase cell ratio,miR-133a expression and pro-caspase3 protein expression of H9c2 cells in model group were significantly reduced,and the apoptosis rate and cleaved-caspase3 protein expression were significantly increased(P<0.01).Compared with Model group,the cell viability,clone formation number,S phase cell ratio,miR-133a expression and pro-caspase3 protein expression of H9c2 cells in experimental 2 and experimental 3 groups were significantly increased,and apoptosis rate and cleaved-caspase3 protein expression were significantly decreased(all P<0.01).Compared with the experi mental 3+anti-miR-NC group,the cell viability,the number of clones,the proportion of S-phase cells and the expression of pro-caspase3 protein of H9c2 cells in experimental 3+anti-miR-133a group were significantly reduced,the apoptosis rate and the cleaved-caspase3 protein expression were increased significantly(all P<0.01).Conclusion A certain concentration of enalapril can reduce hypoxia/reoxygenation-induced cardiomyocyte injury,and the mechanism may be related to the up-regulation of miR-133a expression.