论文部分内容阅读
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。
Objective To investigate whether oxidative stress-induced cardiomyocyte apoptosis is associated with the regulation of endoplasmic reticulum stress (ERS). Methods Cardiomyocytes were exposed to H2O2 at different concentrations (500, 100μmol / L) and different time (0, 4, 8, 12, 24h). The apoptotic rate of cardiomyocytes in each group was detected by flow cytometry. The typical morphology of cardiomyocyte apoptosis was observed by hematoxylin-eosin staining (HE staining). The expressions of p-PERK and CHOP in ERS were detected by Western blot . The ERS inhibitor PBA (4-phenylbutyric acid) was further used to inhibit the ERS expression in cardiomyocytes, and the expressions of p-JNK, p-PERK and CHOP were detected by Western blot. The expressions of Caspase-12 and caspase-3 were detected by flow cytometry active. Results ① Low concentration of H2O2 significantly induced cardiomyocyte apoptosis, and high concentration of H2O2 resulted in necrosis of cardiomyocytes. The apoptosis rate of cardiomyocytes was the highest when stimulated with 100μmol / L H2O2 for 8h. ② The apoptosis of cardiomyocytes ERS marker protein p-PERK and CHOP expression was significantly increased. ③ Pretreatment with cardioprotective agents PBA could effectively inhibit the H2O2-induced p-PERK and CHOP expression and the up-regulation of Caspase-3 and Caspase-12 activity, as compared with H2O2 alone group (P <0.01) . Conclusions Low concentration H2O2 can induce cardiomyocyte apoptosis by inducing ERS integration and regulation. This result will provide new ideas for the prevention and treatment of cardiovascular diseases from the perspective of ERS integration and regulation of cell stress.