促红细胞生成素对缺氧复氧心肌细胞的保护作用及其机制

目的探讨促红细胞生成素(EPO)对缺氧复氧(H/R)心肌细胞是否具有保护作用及其机制。方法以乳鼠心肌细胞株(H9C2细胞)为研究对象,将其分为对照组、H/R组及EPO干预组。其中对照组常规培养;H/R组缺氧2 h后复氧24 h;EPO干预组在缺氧2 h后加入不同浓度的EPO(5、10、20 IU/ml)处理,然后复氧24 h。培养结束后分别检测各组细胞上清液中乳酸脱氢酶(LDH)浓度,使用MTS法检测细胞存活率,使用Western blot检测细胞内Cleaved Caspase-3表达变化,使用线粒体和胞浆蛋白制备试剂盒分离线粒体和胞浆蛋白,使用Western blot分别检测线粒体和胞浆内Omi/HtrA2蛋白表达变化。结果与对照组比较,H/R组细胞存活率明显下降,细胞上清液LDH释放明显增加,细胞内Cleaved Caspase-3表达增强,胞浆内Omi/HtrA2蛋白表达增强,差异均有统计学意义(P0.05);而与H/R组相比,EPO干预组细胞存活率上升,细胞上清液LDH释放降低,Cleaved Caspase-3表达减弱,胞浆内Omi/HtrA2蛋白表达减少,差异均有统计学意义(P0.05),但各指标均未恢复至对照组水平。结论 H/R可诱发心肌细胞凋亡,EPO可减轻H/R诱导的心肌细胞凋亡,其机制可能为抑制线粒体内Omi/HtrA2蛋白的转位,从而抑制Caspases通路激活有关。

Protective effect of erythropoietin on myocardial cells apoptosis induced by hypoxia-reoxygenation and its mechanism

Objective To investigate the protective effect of erythropoietin （EPO） on hypoxia-reoxygenation （H/R） myocardial cells and its mechanism. Methods Neonatal rat myocardial cells （H9C2 cells） were divided into 3 groups：control group, H/R group and EPO group. The cells of control group were routine cultured; the cells of H/R group were anoxia for 2 h and re-oxygen for 24 h; EPO intervention group was treated with different concentrations of EPO （5 IU/ml, 10 IU/ml, 20 IU/ml） after hypoxia. The concentration of LDH in cell supernatant was assessed by ELISA; the cell viability was measured by MTS assay; the intracellular expression change of cleaved Caspase3 was detected by Western blot. Mitochondria/cytosol Isolation Kit was used to isolate Mitochondria from cytosol, Western blot was used to detect the expression of Omi/HtrA2 protein in the mitochondria and cytosol. Results Compared with the control group, H/R group cell survival rate decreased significantly, cell supernatant LDH concentration increased significantly, the expression of cleaved caspase-3 was increased, the expression of Omi/HtrA2 protein in the cytoplasm was increased,the differences were statistically significant （P〈0.05）; compared with H/R group, EPO intervention group cell survival rate increased, cell supernatant concentrations of LDH, cleaved caspase-3 and the Omi/HtrA2 protein in the cytoplasm expression decreased, the difference was statistically significant （P〈0.05）, but the indexes were not restored to the control group level. Conclusion H/R can induce apoptosis of myocardial cell, which can be reduced by EPO, and its mechanism may be related to the inhibition of Omi/HtrA2 protein in mitochondrial translocation, thus inhibiting the caspases pathway activation.