二氮嗪预处理对培养H9C2心肌细胞缺氧/复氧损伤的保护作用  

Protective effect of diazoxide preconditioning on hypoxia/reoxygen injury of cultured H9C2 myocytes

作　　者：李洪[1] 肖颖彬[1] 王明军[2] 杨天德[1] 陶军[1] 

机构地区：[1]第三军医大学新桥医院麻醉科,重庆400037 [2]解放军总医院,北京100853

出　　处：《重庆医学》2007年第16期1587-1589,共3页Chongqing medicine

基　　金：国家自然科学基金资助项目(30200089)

摘　　要：目的探讨线粒体KATP(Mito-KATP)通道特异性开放剂二氮嗪预处理在H9C2心肌细胞缺氧、复氧损伤中的作用及其机制。方法将培养的H9C2心肌细胞随机分为5组,即(Ⅰ)对照组;(Ⅱ)缺氧/复氧组;(Ⅲ)二氮嗪(DZ)预处理组;(Ⅳ)二氮嗪加ROS清除剂2-巯基丙酰氨基乙酸(MPG)预处理加缺氧/复氧组;(Ⅴ)二氮嗪加PKC的特异性抑制剂氯化白屈菜赤碱(CH)预处理加缺氧/复氧组。对照组常规培养;缺氧/复氧组缺氧培养100min,复氧30min;其余各组则加入相应的药物预处理10min,DZ、MPG和CH的终浓度分别为200、400mmol/L和2mmol/L,更换无血清培养基培养20min,然后再缺氧100min,复氧30min。采用Hoechst33258染色、JC-1荧光标记和Westernblotting等方法分别检测H9C2心肌细胞的凋亡率、线粒体膜电位变化和细胞色素C的释放量。结果二氮嗪预处理能减少缺氧/复氧损伤后H9C2心肌细胞的凋亡率(P<0.01);阻止线粒体膜电位的下降(P<0.01);减少线粒体释放细胞色素C(P<0.01);预处理过程中加入MPG、CH在一定程度上可抑制二氮嗪预处理的心肌保护效应。结论缺氧/复氧损伤可通过降低H9C2心肌细胞线粒体膜电位,引发线粒体释放细胞色素C,从而诱导心肌细胞的凋亡。开放Mito-KATP通道能减轻H9C2心肌细胞的缺氧/复氧损伤,其机制可能与开放Mito-KATP通道、释放信号分子ROS和激活PKC有关。Objective To explore the protective effect and mechanisms of mitochondrial ATP sensitive potassium channel opener diazoxide（DZ） preconditioning on hypoxia/ reoxygen（H/R） injury of cultured H9C2 myocytes. Methods Cultured H9C2 myocytes 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）group Ⅴ （DZ＋CH preconditioning＋ H/R）. N-2-mercaptopropionylglycine（MPG） is a free radical scavengers, and CH（Chelerythrine） is a selective PKC inhibitor. Except for group Ⅰ and group Ⅱ , the H9C2 myocytes in other groups were subjected to 100 min of hypoxia and 30 min of reoxygen after being pretreated simultaneouly with DZ or both DZ and MPG or both DZ and CH for 10 min and then being washed-out for 20min. The final concentration of DZ, MPG,CH was 200mmol/L,400mmol/L and 2mmol/L respectively. The rate of apoptotic cells, mitochondrial membrane potential and cytochrome C released from mitochondria were respectively measured by Hoechst33258 staining, JC-1 staining and western blotting. Results Preconditioning with DZ significantly decreased the apoptotic rate of H9C2 myocytes（P〈0.01）, suppressed the loss of mitochondrial membrane potential（P〈0.01 ） and reduced the release of cytochrome C released from mitochondria（P〈0.01）. Both MPG and CH could eliminate the protective effects of DZ preconditioning. Conclusion Hypoxia/ reoxygen injury could induce apoptosis of H9C2 myocytes by decreasing the mitochondrial membrane potential and reducing the release of cytochrome C from mitochondria to cytoplasm. Pre-opening the Mito-KATP channels could induce the protective effects of H9C2 myocytes against hypoxia/ reoxygen injury. The mechanisms of DZ preconditioning might be opening Mito-KATP channels, releasing ROS and PKC activation.

关 键 词：二氮嗪 H9C2心肌细胞 预处理 缺氧 线粒体KATP通道 

分 类 号：R365.542[医药卫生—病理学]