线粒体ATP敏感钾通道介导缺氧预处理期细胞外信号调节蛋白激酶活化的机制

The mechanism of ERK activation in anoxia preconditioning phase mediated by mK_(ATP) channel

目的 探讨线粒体ATP敏感钾通道 (mKATP)与细胞外信号调节蛋白激酶 (ERK1/2 )在缺氧预处理延迟保护机制中的相互关系。 方法 采用SD大鼠心肌细胞培养复制、缺氧 /复氧损伤模型及缺氧预处理模型 ,建立对照组、缺氧 /复氧组、缺氧预处理组、二氮嗪组、5 羟基癸酸 +二氮嗪组、5 羟基癸酸 +缺氧预处理组、二氮嗪 +MGP组、缺氧预处理 +2 巯基丙酰基甘氨酸 (MGP)组、二氮嗪 +PD980 5 9组 ,以细胞存活率等作为反映心肌细胞损伤的指标 ,并于预处理后不同时间点分别测定细胞内活性氧含量及ERK1/2 的活性。 结果 缺氧预处理组及二氮嗪组细胞存活率和细胞内超氧化物歧化酶活性〔(81 9± 11 4 ) %、(13 6± 3 7)U/L ,(79 2± 12 4 ) %、(16 5± 4 6 )U/L〕均显著高于缺氧 /复氧组〔(4 2 2± 7 3) %、(8 8± 2 8)U/L〕 ,缺氧预处理组及二氮嗪组乳酸脱氢酶释放〔(10 1 9± 18 9)U/L、(97 5± 17 7)U/L〕均显著低于缺氧 /复氧组〔(2 5 0 5± 4 3 6 )U/L ,均为P <0 0 1〕。缺氧预处理及二氮嗪均可快速诱导细胞内大量的活性氧生成并激活ERK1/2 ,但这些作用均可被mKATP阻滞剂 5 羟基癸酸及氧自由基清除剂MGP所阻断。二氮嗪的细胞保护作用还可被ERK1/2 阻滞剂PD980 5 9所抵消。 结论 mKATP可通过诱导活性?

Objective To investigate the relationship of mitochondrial ATP-sensitive potassium channel (mK_ ATP ) and extracellular signal-regulated kinase (ERK_ 1/2 ) in the delayed protection of anoxia preconditioning (APC). Methods The amoxia/reoxygention(A/R) and APC models were established in cultured neonatal rat cardiomyocytes. The cellular injury was evaluated by the measurement of cellular superoxide dismutase activity,cell viability and lactate dehydrogenase release. The cellular reactive oxygen species (ROS) generation and ERK_ 1/2 activation were determined at different time point from the beginning of preconditioning with anoxia or diazoxide (a mK_ ATP opener). Results The cells viability and superoxide dismutase activity in APC 〔(81.9±11.4)%,(13.6±3.7)U/L 〕and diazoxide groups 〔(79.2±12.4)%,(16.5±4.6)U/L〕 were significantly higher than those in anoxia/reoxygenation (A/R) group 〔(42.2± 7.3 )%,(8.8 ± 2.8 )U/L,P <0.01〕for all comparisons. The lactate dehydrogenase activity in APC group (101.9±18.9) U/L and diazoxide group (97.5 ±17.7) U/L were significantly lower than those in A/R group (250.5± 43.6) U/L,all P <0.01. Both APC and diazoxide facilitated the cellular abundant ROS generation and quick ERK_ 1/2 activation. But these effects of APC and diazoxide were completely attenuated by 5-hydroxydecanoate (mK_ ATP blocker) or the free radical scavenger,2-mercaptopropionylglycine. In addition,ERK_ 1/2 inhibitor PD98059 also abolished the cellular protective effects induced by diazoxide. Conclusions mK_ ATP might mediate the ERK_ 1/2 activation in anoxia preconditioning phase through generating ROS and then triggers the delayed protection of APC.