促红细胞生成素对缺氧诱导大鼠心肌细胞凋亡的影响及机制

Effect of erythropoietin on hypoxia-induced apoptosis of cardiomyocytes in rats

目的探讨促红细胞生成素(EPO)对缺氧诱导大鼠心肌细胞凋亡的影响及机制。方法建立体外培养的缺氧新生大鼠心肌细胞模型,设立正常对照组、缺氧组及低、中、高EPO干预组、LY294002干预组、IGF-1干预组共7组,正常对照组于CO2培养箱中培养,不经缺氧处理或药物干预。缺氧组细胞经缺氧处理,不予任何药物干预。低EPO干预组:以6.25 IU/mL的EPO预处理细胞24 h,再予缺氧处理。中EPO干预组:以25 IU/mL的EPO预处理细胞24 h,再予缺氧处理。高EPO干预组:以100 IU/mL的EPO预处理细胞24 h,再予缺氧处理。LY294002干预组:以LY294002(终浓度50μmol/L)预处理1 h,后予缺氧处理。IGF-1干预组:以IGF-1(200 ng/mL)预处理细胞1 h,后予缺氧处理。采用比色法检测肌酸激酶(CK)、乳酸脱氢酶(LDH)活力,采用流式细胞术检测细胞Caspase-3阳性表达率和细胞凋亡率。结果经EPO预处理,缺氧诱导的心肌细胞CK、LDH活力降低,呈剂量依赖性,低EPO干预组、中EPO干预组、高EPO干预组组间CK、LDH活力比较,P均<0.01;LY294002干预组与缺氧组CK、LDH活力增加;IGF-1干预组CK、LDH活力降低(P均<0.01)。缺氧组Caspase-3蛋白阳性表达率高于正常对照组(P<0.01)。低、中、高EPO干预组Caspase-3蛋白阳性表达率均低于缺氧组(P均<0.01),低、中、高EPO干预组间比较差异有统计学意义(P均<0.01)。正常对照组、缺氧组、低、中、高EPO干预组、LY294002干预组及IGF-1干预组心肌细胞凋亡率分别为0.63%±0.1%、35.6%±1.3%、22.4%±2.1%、12.9%±2.3%、5.39%±1.1%、83.5%±2.1%、5.01%±1.1%,各组间比较,P均<0.01。结论 EPO可抑制缺氧诱导大鼠心肌细胞凋亡,其机制可能与PI3K/Akt信号通路有关。

Objective To investigate the effect of erythropoietin (EPO) on cardiomyocyte apoptosis induced by hypoxia in rats and its molecular mechanism. Methods Primarily cultured rat cardiomyocytes were divided into 7 groups: the normal control group, hypoxia group, different concentrations EPO (6.25, 25, and 100 IU/mL) intervention groups (taken as the low, medium, and high concentration groups), LY294002 intervention group, and IGF-1 intervention group. The cells in the normal control group was cultured in CO 2 incubator without hypoxia treatment or drug intervention. Cells in the hypoxic group were treated with hypoxia without any drug intervention. Cells in the low concentration EPO intervention group were treated with 6.25 IU/mL EPO for 24 h, and then treated with hypoxia. Cells in the medium concentration EPO intervention group were pretreated with 25 IU/mL EPO for 24 h and then treated with hypoxia. Cells in the high concentration EPO intervention group were treated with 100 IU/mL EPO for 24 h and then were treated with hypoxia. Cells in the LY294002 intervention group were treated with LY294002 (final concentration 50 mol/L) for 1 h and then treated with hypoxia. Cells in the IGF-1 intervention group were pretreated with IGF-1 (200 ng/mL) for 1 h, and then treated with hypoxia. The activities of creatin kinase (CK) and lactate dehydrogenase (LDH) were measured by colorimetric method, the positive expression rate of Caspase-3 and apoptotic rate were measured by flow cytometry. Results The pretreatment of EPO decreased the activities of CK and LDH in a dose-dependent manner (both P <0.01), whereas pretreatment of LY294002 increased the activities of CK and LDH. Pretreatment of IGF-1 decreased the activities of CK and LDH. The hypoxia group had a higher positive expression rate of Caspase-3 than the normal control group ( P <0.01). The positive expression rates of Caspase-3 in the low, medium, and high concentration EPO intervention groups were lower than that in the hypoxia group, with statistically significant differences (all P <0.01). The apoptotic rates of cardiomyocytes were 0.63%±0.1%, 35.6%±1.3%, 22.4%±2.1%, 12.9%±2.3%, 5.39%±1.1%, 83.5%±2.1%, and 5.01%±1.1%, respectively, in the normal control group, hypoxia group, low concentration EPO intervention group, medium concentration EPO intervention group, high concentration EPO intervention group, LY294002 intervention group, and IGF-1 intervention group (all P <0.01). Conclusion EPO significantly inhibits hypoxia-induced cardiomyocyte apoptosis, and its mechanism may be related to the PI3K/Akt signal pathway.