二参颗粒对氯化钴诱导的H9C2细胞氧化应激介导的细胞凋亡的保护作用

Protective Effect of Ershen Granules on CoCl2-Induced Apoptosis by Suppressing Oxidative Stress in H9C2 Cells

目的:通过观察二参颗粒对氯化钴(CoCl2)诱导的缺氧损伤和大鼠心肌细胞H9C2凋亡的影响,阐明二参颗粒对心脏的保护作用。方法:以500μmol/L的CoCl2与H9C2细胞共同作用24 h,建立心肌细胞缺氧缺血模型。将H9C2细胞分为空白对照组、模型对照组、二参颗粒50μg/mL、100μg/mL、200μg/mL组。酶联免疫法检测胞外培养液中乳酸脱氢酶(LDH)的含量,流式细胞术检测胞内活性氧(ROS)含量、线粒体膜电位(MMP)和凋亡水平,蛋白质印迹分析法检测细胞内Bcl-2/Bax、Cleaved Caspase-3、Cleaved Caspase-9和Cleaved PARP的表达水平。结果:与空白对照组比较,模型对照组细胞活力下降、活性氧(ROS)荧光表达值升高、线粒体膜电位JC-1单体阳性细胞显著升高以及细胞培养液中LDH含量升高,凋亡率显著升高,凋亡相关蛋白Cleaved Caspase-3、Cleaved Caspase-9和Cleaved PARP的蛋白表达上调,Bcl-2/Bax比值降低(P<0.05或P<0.01);与模型对照组比较,二参颗粒能有效改善细胞活力,降低LDH的含量,显著降低胞内ROS荧光表达值,改善线粒体膜电位和凋亡水平,下调凋亡相关蛋白Cleaved Caspase-3、Cleaved Caspase-9和Cleaved PARP的蛋白表达,升高Bcl-2/Bax的比值(P<0.05或P<0.01)。结论:二参颗粒可以抑制CoCl2诱导的心肌细胞ROS水平,减少氧化应激发生,进而改善氧化应激介导的心肌细胞的细胞凋亡。

Objective: To observe the effects of Ershen granules on hypochloric injury induced by cobalt chloride(CoCl2) and H9 C2 apoptosis in rat cardiomyocytes, in order to study its protective effect on the heart.Methods: A model of cardiomyocyte hypoxia and ischemia was established by 500 μmol/L CoCl2 co-incubation with H9 C2 cells for 24 h. H9 C2 cells were divided into the control group,the model group, Ershen granules 50 μg/mL, 100 μg/mL and 200 μg/mL groups. Enzyme-linked immunosorbent assay was used to detect the content of lactate dehydrogenase(LDH) in extracellular culture medium. Flow cytometry was used to detect the levels of intracellular reactive oxygen species(ROS),mitochondrial membrane potential(MMP) and apoptosis. The expression levels of Bcl-2/Bax,Cleaved Caspase-3,Cleaved Caspase-9 and Cleaved PARP were determined by western blot. Results: Compared with the control group, the cell viability was decreased, the expressions of reactive oxygen species(ROS) fluorescence and the mitochondrial membrane potential JC-1 monomer-positive cells were significantly increased in the model group, the LDH content and the percentage of apoptotic cells were increased,the protein expressions of apoptosis-related proteins Cleaved Caspase-3,Cleaved Caspase-9 and Cleaved PARP were up regulated, and the Bcl-2/Bax ratio was decreased(P<0.05 or P<0.01). Compared with the model group, Ershen granules effectively improved the cell viability, reduced LDH content, significantly reduced intracellular ROS fluorescence expression,improved mitochondrial membrane potential and apoptosis level, down regulated the protein expressions of apoptosis-related proteins Cleaved Caspase-3、Cleaved Caspase-9 and Cleaved PARP, also increased the ratio of Bcl-2/Bax.(P<0.05 orP<0.01)Conclusion: Ershen granules can inhibit the ROS level of cardiomyocytes induced by CoCl2, reduce the occurrence of oxidative stress, therefore to improve the cardiomyocyte apoptosis induced by oxidative stress.