大黄酸对过氧化氢致心肌细胞线粒体损伤的保护作用机制

Protective effect and mechanism of rhein on hydrogen peroxideinduced mitochondrial injury in cardiomyocytes

目的探讨大黄酸是否通过调节线粒体功能保护过氧化氢(H_(2)O_(2))诱导的心肌细胞损伤。方法应用H_(2)O_(2)建立心肌细胞损伤模型,实验分为3组:对照组(不用H_(2)O_(2)处理)、H_(2)O_(2)组(以H_(2)O_(2)诱导心肌细胞H9c2损伤)、大黄酸组(H_(2)O_(2)刺激心肌细胞H9c2造模,1μg/mL大黄酸干预)。采用噻唑蓝法检测细胞活力,电镜检测线粒体超微结构,JC-1检测线粒体膜电位,Western blot检测蛋白p-Drp1、Drp1、OPA1、Bax、Bcl-2及Caspase 3表达水平。结果与对照组相比,H_(2)O_(2)组能明显抑制细胞活力(P<0.05),大黄酸组中3个浓度均能显著增加细胞活力,大黄酸浓度为1μg/mL细胞活力最显著(P<0.01)。对照组线粒体轮廓完整,棱嵴清晰;H_(2)O_(2)组线粒体肿胀,轮廓模糊,棱嵴消失,可见空泡化改变;大黄酸组线粒体与H_(2)O_(2)组相比,形态得到明显改善。与对照组比较,H_(2)O_(2)组细胞浆中绿色荧光增强、线粒体内红色荧光减弱;与H_(2)O_(2)组相比,大黄酸组线粒体内红色荧光增强、细胞浆中绿色荧光减弱。与对照组比较,H_(2)O_(2)组OPA1和Bcl-2表达降低,而p-Drp1、Bax和Caspase 3显著增加(P<0.05);大黄酸组OPA1、Bcl-2明显增加,而p-Drp1、Bax和Caspase 3显著降低(P<0.05)。结论大黄酸能够减轻H_(2)O_(2)诱导的心肌细胞损伤,其可能是通过保护线粒体形态和功能,调节线粒体动力学蛋白,抑制凋亡而实现。

Objective Mitochondria play important roles in the oxidative stress injury of cardiomyocytes.This article investigated whether rhein protects hydrogen peroxide(H_(2)O_(2))induced cardiomyocyte injury by regulating mitochondrial function.Methods The model of H_(2)O_(2) injury was established and the experiment was divided into 3 groups:control group,H_(2)O_(2) induction group(H_(2)O_(2) stimulation of cardiomyocyte H9c2 modeling),and rhein intervention group(H_(2)O_(2) stimulation of cardiomyocyte H9c2 modeling,1μg/mL rhein intervention).Cell viability was determined by methyl thiazolyl tetrazolium assay.The mitochondrial ultrastructure was measured by electron microscopy,mitochondrial membrane potential was detected by JC-1assay.Protein expression levels of p-Drp1,Drp1,OPA1,Bax,Bcl-2,and Caspase 3 were analyzed by Western blot.Results Compared to the H_(2)O_(2) model,the survival rate of cardiomyocytes was increased to(67.5±2.1)%after rhein treatment(P<0.05).The mitochondrial ultrastructure was significantly improved,and mitochondrial membrane potential was increased.The expression ofpDrp1,Bax,and Caspase 3 were decreased,while OPA1 and Bcl-2 expressions were increased.Conclusion Rhein can alleviate myocardial cell injury induced by H_(2)O_(2),which may be achieved by protecting mitochondrial morphology and function,regulating mitochondrial kinetic protein,and inhibiting apoptosis.