同型半胱氨酸经PERK磷酸化激活CHOP-ERO1α通路介导心肌细胞凋亡的研究

Homocysteine mediates cardiomyocyte apoptosis by phosphorylating PERK and activating CHOP-ERO1α pathway

目的探讨同型半胱氨酸(Hcy)对心肌的损伤作用及其可能机制。方法选用H9C2心肌细胞,分别用不同浓度Hcy、4-苯基丁酸(4-PBA)干预细胞。将H9C2细胞分为对照组、H400组、H400P2组,对照组使用普通培养基,H400组加入400μmol/L的Hcy,H400P2组在H400组基础上加入2mmol/L的4-PBA。CCK-8检测细胞存活率,TUNEL染色评估细胞凋亡,免疫细胞化学检测内质网氧化还原酶1α(ERO1α)表达,Western blot检测蛋白表达差异。结果 Hcy对H9C2心肌细胞损伤呈浓度依赖性(F=2 039.958,P<0.01)。与对照组比较,H400组细胞凋亡分数及胰腺内质网激酶(PERK)、磷酸化PEPK(p-PERK)、CCAAT增强子结合蛋白同源蛋白(CHOP)、ERO1α表达均增加(P<0.01);H400P2组细胞凋亡分数及PERK、p-PERK、CHOP、ERO1α表达均有所下降,与H400组比较均差异有统计学意义(P<0.05)。结论 Hcy通过内质网应激机制介导心肌细胞凋亡。

Objective To investigate the effects of homocysteine（Hcy） on myocardial injury and its possible mechanisms. Methods The selected H9C2 cardiomyoeytes were intervened with various concentrations of Hcy and 4-phenyl butyric acid（4- PBA）. The H9C2 cells were divided into the control group, H400 group and H400P2 group. The control group used the common medium,the H400 group was added with 400 vmol/L Hcy,the H400P2 group was added with 2 mmol/L 4-PBBA on the basis of H400 group. The cell livability was detected by using cell counting kit-8（CCK-8）. Apoptosis was evaluated by using the terminal deoxynucleotidyl transferase mediated nick-end labelling（TUNEL） staining. The EROla expression was determined by using immu- nocytochemistry,and the protein expression difference was determined by using Western blot. Results The injury of Hcy on H9C2 cardiomyocytes showed a concentration-dependent manner（F= 2 039. 958, P〈0.01）. Compared with the control group, the apopto- sis percentages and expression levels of PERK,p-PERK, CHOP and EROla in the H400 group were increased（P〈0.01） while which in the H400P2 group were decreased,the difference was statistically significant（P〈0.05）. Conclusion Hcy mediates myo- cardial apoptosis through endoplasmic reticulum stress mechanism.