促红细胞生成素对缺氧／复氧乳鼠心肌细胞的抗凋亡作用及机制研究

The anti-apoptosis effect of erythropoietin on neonatal rat cardiocytes during hypoxia/reoxygenation injury and its possible mechanism

目的观察促红细胞生成素（EPＯ）对缺氧／复氧心肌细胞凋亡的影响，并初步探讨蛋白激酶c（PKc）和线粒体ATP敏感性钾通道（mitoKATP）与EPO在抗凋亡信号通路中的相互关系。方法分离培养SD大鼠乳鼠心肌细胞，并分为对照组、缺氧／复氧组、EPO组和PKC抑制剂白屈菜红碱组，建立缺氧／复氧模型；用流式细胞术检测心肌细胞凋亡率，激光共聚焦显微镜扫描观察细胞黄素蛋白自体荧光强度变化，监测钾通道开放情况。结果缺氧／复氧组心肌细胞凋亡率明显高于对照组[（42．56±8．00）％比（17．88±2．00）％，P〈0．053，黄素蛋白自体荧光强度值与对照组比较差异无统计学意义[（0．278±0．170）×10-2比（0．149±0．050）×10，P〉0．053；EPO组心肌细胞凋亡率明显低于缺氧／复氧组[（22．73±5．00）％比（42．56±8．00）％，P〈0．053，黄素蛋白自体荧光强度值则较缺氧／复氧组明显增强[（2．201±1．090）×10。比（0．278±0．170）×10-2，P〈0．01]；白屈菜红碱对EPO抗凋亡和增强黄素蛋白自体荧光强度有阻断作用[细胞凋亡率：（46．72±17．00）％比（22．73±5．00）％，荧光强度：（0．986±0．320）×10-2比（2．201±1．090）×10-2。，P〈0．01和P〈0．05；。结论通过激活PKC继而开放mitoKATP通道可能是EPO抗缺氧／复氧心肌细胞凋亡的信号通路之一。

Objective To investigate the anti-apoptosis effect of erythropoietin （EPO） on myocardial cells after hypoxia/reoxygenation in vitro, and the relationship among protein kinase C （PKC）, the mitochondrial ATP-sensitive potassium （mitoKATP） channel and EPO in the anti-apoptotic signaling pathways. Methods Cardiocytes were harvested from neonatal rats and cultured. Cultured myocardial cells were divided into the control group, the hypoxia/reoxygenation group, the EPO group and the chelerythrine group, and a hypoxia/reoxygenation model of eardiocytes was reproduced. Apoptosis rate was assayed by flow cytometry. Flavoprotein fluorescence was scanned by confocal laser microscope to assess the mitoKATP channel activity. Results Apoptosis rate was significantly higher in hypoxia/reoxygenation group than that of control group [（42.56＋8.00）% vs. （17.88±2.00）%, P〈0.05]. There was no statistically significant difference in flavoprotein fluorescence between this group and the control group [（0. 278±0. 170） × 10-2 vs. （0. 149±0. 050）× 10-2, P〈0. 053. Myocardial cell apoptosis rate in EPO group was lower than that in hypoxia/reoxygenation group [（22.73±5.00）× vs. （42.56±8.00）%, P〈0.05], and flavoprotein fluorescence intensity was significantly enhanced when compared with hypoxia/reoxygenation group （（2. 201±1. 090）⌒10-2 vs. （0. 278±0. 170）×10 2, P〈0. 013. However, when chelerythrine was added, the anti-apoptosis effect of EPO was blocked, and the intensity of cardiocytes flavoprotein fluorescence was decreased [the apoptosis rate was （46.72 ±17.00）% and the flavoprotein fluorescence intensity was （0. 986±0. 320）× 10 -23] When compared with EPO group there was statistically significant difference （P〈 0.01 and P〈0.05）. Conclusion Myocardial cell apoptosis occurs in hypoxia/reoxygenation injury, and EPO can protect rat cardiomyoeytes from hypoxia/reoxygenation induced apoptosis. The protective effect is partly associated with the PKC/mitoKATP pathway.