PTD4-Cu/ZnSOD融合蛋白对缺氧复氧损伤模型大鼠心肌细胞H9C2心肌线粒体功能的影响

The Influence of PTD4-Cu/ZnSOD on the Myocardial Mitochondrial Function of H9C2 Myocardial Cells in Rats with Hypoxia/Reoxygenation Injury

目的探讨蛋白质转导结构域4-铜锌超氧化物歧化酶(PTD4-Cu/ZnSOD)融合蛋白对缺氧/复氧损伤心肌线粒体功能的影响。方法 H9C2大鼠心肌细胞采用厌氧培养箱培养(85%N_2,10%H_2,5%CO_2)制作缺氧复氧损伤模型,之后在缺氧复氧损伤细胞培养液中不加任何处理因素作为HRI组,分别加入10μmol/L Cu/ZnSOD作为Cu/ZnSOD组、10μmol/L PTD4-Cu/ZnSOD作为PTD4-Cu/ZnSOD组,且以正常培养心肌细胞作为正常组。采用JC-1试剂盒检测线粒体膜电位,TUNEL法检测心肌细胞凋亡,生化测定心肌细胞线粒体三磷酸腺苷(ATP)酶活性、超氧化物歧化酶活性及丙二醛含量,Western blot检测心肌细胞线粒体B淋巴细胞瘤-2基因(Bcl-2)蛋白、Bax蛋白的表达。结果 HRI组线粒体膜电位显著低于正常组;PTD4-Cu/ZnSOD组线粒体膜电位较正常组较低。各处理组心肌细胞凋亡指数均明显高于正常组(P<0.01);PTD4-Cu/ZnSOD组心肌细胞凋亡指数较HRI组明显降低(P<0.01)。PTD4-Cu/ZnSOD组线粒体ATP酶活性明显高于HRI组,且超氧化物歧化酶活性较高,显著降低丙二醛含量(P<0.01)。HRI组Bcl-2蛋白表达低于正常组,Bax蛋白表达高于正常组(P<0.01);与HRI组比较,PTD4-Cu/ZnSOD融合蛋白能显著增加Bcl-2蛋白表达,降低Bax蛋白表达(P<0.01)。结论 PTD4-Cu/ZnSOD融合蛋白可提高线粒体ATP酶活性,减轻线粒体损伤;提高线粒体SOD活性,减轻自由基对线粒体损伤;提高线粒体膜Bcl-2蛋白表达,降低Bax蛋白表达,从而发挥心肌保护作用。

Objective To investigate the effect of protein transduction domain4（PTD4）-Cu/Zn superoxide dismutases（SOD）on the myocardial mitochondrial function of H9C2 myocardial cells in rats with hypoxia/reoxygenation injury（HRI）.Methods H9C2 myocardial cells were cultured in anaerobic incubator（85% N2,10% H2,5% CO2）to establish the HRI model.H9C2 myocardial cells cultured in vitro were randomized into 4 groups：normal group,HRI group,HRI＋Cu/ZnSOD group,and HRI＋PTD4-Cu/ZnSOD group.HRI was achieved by exposing cardiomyocytes to 4 hours hypoxia followed by 2 hours reoxygenation.The changes of mitochondrial membrane potential was detected by JC-1.The myocardial cell apoptosis was detected by TUNEL method.The mitochondrial ATPase activity,SOD activity,and malondialdehyde（MDA）content were detected by biochemical assay.The expressions of B-cell lymphoma-2（Bcl-2）and Bcl-2 associated X protein（Bax）in myocardial mitochondria were detected by western blot.Results The mitochondrial membrane potential in HRI group was significantly lower than that in normal group.The mitochondrial membrane potential in HRI＋PTD4-Cu/ZnSOD group was lower than that in normal group.The apoptosis index of cardiomyocytes in each treatment group was significantly higher than that in normal group（P 〈0.01）.The myocardial cell apoptosis index in HRI＋PTD4-Cu/ZnSOD group was significantly lower than that in HRI group（P〈 0.01）.The activity of mitochondrial ATPase in HRI＋PTD4-Cu/ZnSOD group was significantly higher than that in HRI group,and the activity of SOD was higher,the content of MDA was significantly reduced（P〈 0.01）.The expression of Bcl-2 protein in HRI group was lower than that in normal group,and the expression of Bax was higher than that in normal group（P 〈0.01）.Compared with HRI group,the expression of Bcl-2 protein significantly increased while the expression of Bax protein decreased in HRI＋PTD4-Cu/ZnSOD group（P 〈0.01）.Conclusion PTD4-Cu/ZnSOD can improve the activity of mitochondrial ATP enzyme,reduce mitochondrial damage,improve the activity of mitochondrial SOD,attenuates the damage of free radicals to mitochondria,improve the expression of Bcl-2 protein in mitochondrial membrane,reduce the expression of Bax protein,and thus play the role of myocardial protection.