内质网应激介导过氧化氢诱导的心肌细胞凋亡

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、100μmol/L)和不同时间(0、4、8、12、24h)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;100μmol/L H2O2刺激心肌细胞8h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。

Hydrogen Peroxide-induced Cardiomyocyte Apoptosis via Triggering Endoplasmic Reticulum Stress

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 concentration(100 μmol/L H2O2)group and high concentration(500 μmol/L H2O2)group at different time points.The apoptosis rate was detected by flow cytometry and the typical cardiomyocyte apoptosis was observed by hematoxylin-eosin(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 ① The highest rate of apoptosis was observed in the myocardial cells treated with H2O2(100 μmol/L)for 8 h.② The expression of ERS marker proteins p-PERK and CHOP was significantly higher in the H2O2 groups.③PBA 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 μmol/L)significantly induced the apoptosis of myocardial cells via triggering ERS regulation mechanism and high concentration of H2O2(500 μmol/L)resulted in myocardial necrosis.Our results could provide new clues for prevention and treatment of cardiovascular diseases.