内质网应激介导过氧化氢诱导的心肌细胞凋亡

摘要

Objective To investigate relationship between the apoptosis of cardiomyocyte induced by oxidative stress and the endoplasmic reticulum stress(ERS). Methods The cultured H9C2 cells were divided into 2 groups:low concentrationC 100 jLimol/L H2O2)group and high concentrationC500 jumol/L H2O2)group at different time points. The apoptosis rate was detected by flow cytometry and the typical cardiomyocyte apoptosis was observed by hematoxylin-eosinC HE) staining. The expression of ERS marker proteins p-PERK and CHOP was detected by using Western blot. The chemical chaperone 4-phenylbutyric acid (PBA)was used to inhibit ERS in myocardial cells and the expression of p-JNK, p-PERK and CHOP was detected by using Western blot. The activities of Caspase-12 and Caspase-3 were measured by using flow cytometry. Results ? The highest rate of apoptosis was observed in the myocardial cells treated with H2O2 (100 jumol/Dfor 8 h. (2) The expression of ERS marker proteins p-PERK and CHOP was significantly higher in the H2O2 groups. (3)PBA could effectively inhibit H2O2-induced apoptosis of cardiomyocytes,significantly down-regulate the expression of ERS marker proteins p-PERK and CHOP(P<0. 01) ,and significantly reduce the activities of Caspase-3 and Caspase-12 (P<0. 01). Conclusion The low concentration of H2O2 (100 jLtmol/L)significantly induced the apoptosis of myocardial cells via triggering ERS regulation mechanism and high concentration of H2 O2 (500 jLimol/Dresulted in myocardial necrosis. Our results could provide new clues for prevention and treatment of cardiovascular diseases.%目的 探讨氧化应激诱导心肌细胞凋亡是否与内质网应激(endoplasmic reticulum stress,ERS)的调控机制有关.方法 给予乳鼠心肌细胞高低两种浓度(500、100 μmol/L)和不同时间(0、4、8、12、24 h)过氧化氢(H2O2)刺激.采用流式细胞仪检测各组心肌细胞的凋亡率;苏木精-伊红染色法(HE)观察心肌细胞凋亡的典型形态;通过Western blot检测ERS标志蛋白p-PERK 和CHOP的表达变化.进一步采用ERS抑制剂化学伴侣PBA(4-phenylbutyric acid)抑制心肌细胞ERS,Western blot检测p-JNK、p-PERK 和CHOP蛋白的表达变化;采用流式细胞仪检测Caspase-12 和Caspase-3的活性.结果 ①低浓度H2O2可显著诱导心肌细胞凋亡,高浓度H2O2则导致心肌细胞发生坏死;100 μmol/L H2O2刺激心肌细胞8 h时其凋亡率最高.②心肌细胞发生凋亡时ERS标志蛋白p-PERK 和CHOP表达显著增高.③ERS抑制剂PBA预处理心肌细胞,可有效抑制H2O2诱导的p-PERK、CHOP表达及Caspase-3、Caspase-12活性的上调,与单纯H2O2处理组相比差异有统计学意义(P＜0.01).结论 低浓度H2O2可通过促发ERS整合调控机制介导心肌细胞凋亡.这一结果将从ERS整合调控细胞应激的角度为心血管疾病的防治提供新思路.