摘要
目的:探讨低温刺激诱导心肌细胞损伤及凋亡,以及细胞外信号调节蛋白激酶5(ERK5)/凋亡调节蛋白Bim途径的调控作用。方法:基于乳鼠原代心肌细胞,低温培养箱施加低温刺激;使用特异性小干扰RNA(siRNA)下调ERK5或Bim的表达;细胞凋亡使用流式细胞仪检测;各蛋白表达水平通过Western blot检测;细胞内钙离子、活性氧簇(ROS)水平、线粒体膜电位(ΔΨm)通过荧光标记及流式细胞仪检测。结果:在低温刺激下的心肌细胞中,ERK5 siRNA可加剧Bim蛋白表达;Bim siRNA不影响ERK5,但减轻p-ERK5表达;ERK5 siRNA导致更高的细胞凋亡率,Bim siRNA则可减弱ERK5 siRNA的促凋亡作用;ERK5 siRNA加重了细胞内钙离子超载、ROS激活、线粒体膜电位破坏,Bim siRNA则可对抗上述损伤性作用。结论:在低温干预下的心肌细胞中,下调ERK5可释放对Bim的表达抑制,加重凋亡、细胞内钙离子超载、ROS爆发,造成更严重的线粒体膜电位破坏,而下调Bim则产生反向的保护作用。结果显示ERK5/Bim途径在低温诱导心肌细胞损伤中的重要调控作用。
Objective： To explore the regulative role of extracellular regulated protein kinase-5 （ERK5）/Bcl-2 interacting mediator of cell death （Bim） pathway in hypothermal stimulation induced neonatal rat’s cardiomyocytes （CMs） damage and apoptosis. Methods： CMs were cultured for hypothermal stimulation and the speciifc siRNA was used to down-regulate the ERK5 or Bim in CMs. The cell apoptosis was detected by lfow cytometry, protein expression was examined by Western blot analysis, the intracellular Ca2＋, reactive oxygen species （ROS） and mitochondrial membrane potential （ΔΨm） were evaluated by lfuorescent labeling and lfow cytometry. Results： In hypothermal stimulated CMs, ERK5 siRNA could promote Bim protein expression, but Bim siRNA could not inlfuence ERK5, while attenuated p-ERK5 expression. ERK5 siRNA induced higher apoptosis rate, while Bim siRNA could decrease such effect. ERK5 siRNA increased the intracellular Ca2＋overloading, ROS activation andΔΨm damage, while Bim siRNA played the role to against those effects in hypothermal stimulated CMs. Conclusion： Our study revealed that ERK5/Bim pathway played the important regulative roll in hypothermal stimulation induced neonatal rat’s CMs damage and apoptosis.
出处
《中国循环杂志》
CSCD
北大核心
2014年第7期540-544,共5页
Chinese Circulation Journal
关键词
低温刺激
心肌细胞
细胞外信号调节蛋白激酶5
凋亡调节蛋白Bim
Hypothermal stimulation
Cardiomyocytes
Extracellular regulated protein kinase-5
Bcl-2 interacting mediator of cell death
