活性氧在二氮嗪预处理减轻大鼠心肌细胞缺氧复氧损伤中的作用  

The role of reactive oxygen species in the protection of cultured adult rat cardiomyocytes by diazoxide preconditioning against hypoxia-reoxygenation injury

作　　者：李洪[1] 肖颖彬[2] 杨天德[1] 黄河[1] 方国兴[1] 

机构地区：[1]第三军医大学新桥医院麻醉科,重庆市400037 [2]第三军医大学新桥医院心血科外科,重庆市400037

出　　处：《中华麻醉学杂志》2007年第1期62-65,共4页Chinese Journal of Anesthesiology

基　　金：国家自然科学基金资助项目（30200089）

摘　　要：目的 探讨活性氧（ROS）在线粒体KATP（Mito-KATP）通道特异性开放剂二氮嗪预处理减轻大鼠心肌细胞缺氧复氧损伤中的作用及其机制。方法 培养的成年大鼠心室肌细胞，随机分为5组：对照组、缺氧复氧组、二氮嗪预处理＋缺氧复氧组、二氮嗪＋ROS清除剂2-硫基丙酰氨基乙酸（MPG）预处理＋缺氧复氧组、二氮嗪＋蛋白激酶C特异性抑制剂氯化白屈菜赤碱预处理＋缺氧复氧组。对照组常规培养；缺氧复氧组缺氧30min复氧40min；其余各组则加入相应的药物预处理10min，二氮嗪、MPG、氯化白屈菜赤碱的终浓度分别为200、400、2μmol·L^-1，更换无血清培养基培养20min，然后缺氧30min复氧40min。采用MTT法、CK检测试剂、Na^＋-K^＋-ATPase活性检测试剂和Western blot等方法分别检测心肌细胞的活力、CK活性、Na^＋-K^＋-ATPase活性、细胞浆和细胞膜PKcε表达情况，计算细胞膜PKCε表达百分比。结果 缺氧复氧可导致心肌细胞存活率及Na^＋-K^＋-ATPase活性降低，CK活性升高；二氮嗪预处理能增加细胞存活率及Na^＋-K^＋-ATPase活性，降低CK活性，增加细胞膜PKCε表达百分比；MPG抑制了二氮嗪预处理的心肌保护作用，并且在一定程度上抑制了PKCε的转位；CH可完全阻滞PKCε的转位，并且可降低二氮嗪预处理的心肌保护作用。结论 Mito-KATP通道开放后释放的ROS参与了二氮嗪预处理减轻大鼠心肌细胞缺氧复氧损伤，其机制与PKCε的转位激活有关。Objective To investigate the role of reactive oxygen species （ROS） produced by mitochondrial ATP sensitive potassium （ Mito-KATP ） channel opening in the protection of cardiomyocytes against hypoxigreoxygenafion （H/R） injury by diazoxide （DZ） preconditioning.Methods Cultured adult rat cardiomyocytes were randomly assigned to 5 groups： group Ⅰ control; group Ⅱ H/R; group Ⅲ DZ preconditioning ＋ H/R; group Ⅳ DZ ＋ MPG preconditioning ＋ H/R and groupV DZ ＋ CH preconditioning ＋ H/R. MPG （N-2- mercaptopropionylglycine） is a free radical scavenger and CH （chelerythrine） is a selective PKC inhibitor. The cardiomyocytes in group Ⅱ - Ⅴ were subjected to 30 min of hypoxia and 40 rain of reoxygenation and were pretreated with DZ in group Ⅲ, DZ＋ MPG in group Ⅳ and DZ ＋ CH in groupV for 10 min. The final concentration of DZ, MPG and CH was 200μmol·L^-1 , 400 μmol·L^- 1 and 2 μmol· L^-1 respectively. The cardiomyocytes were then transferred to a new culture medium before H/R. The cell viability, the release of creatine kinase from the cells, the activity of Na^＋-K^＋-ATPase and expression of PKCε in cardiomyocytes were measured by using MTT, CH test kit, Na^＋-K^＋-ATPase test kit and Western blot technique. Results Preconditioning with DZ significantly increased the number of live cardiomyocytes and activity of Na^＋-K^＋-ATPase, reduced the release of creatine kinase and induced PKC epsilon translocation from cytosolic fraction to particulate fraction. MPG suppressed the protection of cardiomyocytes by DZ preconditioning against H/R injury and depressed the translocation of PKCε（ P 〈 0.01 ）. CH could completely block the translocation of PKCε and attenuate the protection of cardiomyocytes by DZ preconditioning（ P 〈 0.01 ）. Conclusion ROS released by Mito-KATP channel opening is involved in the protection of cultured rat cardiomyocytes by DZ preconditioning through activating the translocation of PKCε.

关 键 词：活性氧 心肌再灌注损伤 二氮嗪 细胞 

分 类 号：R686[医药卫生—骨科学]