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CaN抑制剂对H2O2诱导的大鼠H9c2心肌细胞凋亡的影响

冯 星1,金明华1,刘晓梅1,孙 磊1,杜海英1,孙志伟1,2   

  1. （1.吉林大学公共卫生学院卫生毒理学教研室，吉林 长春 130021；2.首都医科大学公共
  • 收稿日期:2008-12-08 修回日期:1900-01-01 出版日期:2009-03-28 发布日期:2009-08-14
  • 通讯作者: 孙志伟

Effect of inhibitor of calcineurin on myocardial H9c2 cell apoptosis induced by H2O2

FENG Xing1,JIN Ming-hua1,LIU Xiao-mei1,SUN lei1,DU Hai-ying1,SUN Zhi-wei1,2   

  1. （1.Department of Health Toxicology,School of Public Health,Jilin University,Changchun 130021，China;2.Department of Health Toxicology, School of Public Health,Capital University of Medical Sciences,Beijing 100054,China)
  • Received:2008-12-08 Revised:1900-01-01 Online:2009-03-28 Published:2009-08-14
  • Contact: SUN Zhi-wei

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摘要： 目的：探讨过氧化氢（H2O2）诱导的大鼠H9c2心肌细胞凋亡以及钙调神经磷酸酶（CaN）抑制剂他罗利姆（FK506）对细胞的保护作用，为心肌缺血/再灌注所致的心肌损伤及拮抗机制提供实验依据。方法：将大鼠H9c2心肌细胞株体外培养进行实验，实验分为正常对照组（n=6）和H2O2各处理组（n=6）。正常对照组采用生理盐水，实验各组分别用50、100、200和400 μmol•L-1 H2O2处理1、 6和24 h后，用罗丹明（Rhodamme-123）荧光探针检测心肌细胞线粒体膜电位(Δψmt)变化，用Annexin-Ⅴ/PI双染流式细胞术检测心肌细胞凋亡率；并采用100 μmol•L-1 H2O2处理6 h建立大鼠心肌细胞凋亡的损伤模型，给予高、低剂量FK506（0.60和0.15 μmol•L-1）干预，流式细胞术检测心肌细胞凋亡率。结果：①100、200和400 μmol•L-1 H2O2处理组1 h 时线粒体膜电位均较对照组显著升高（P<0.01）， 6和24 h时，200和400 μmol•L-1组Δψmt值均低于100 μmol•L-1 组（P<0.05），且400 μmol•L-1组低于200 μmol•L-1组（P<0.05）；②200和400 μmol•L-1 H2O2处理组1　h 时心肌细胞凋亡率和坏死率均较对照组显著增加（P<0.05），24　h达高峰。③ 高、低剂量FK506干预组与单纯100 μmol•L-1H2O2处理组比较，心肌细胞凋亡率均明显降低（P<0.01），高、低剂量组比较差异无显著性（P>0.05）。结论：H2O2可损伤心肌细胞线粒体，引起线粒体膜电位下降，导致细胞凋亡。CaN抑制剂对H2O2诱导的大鼠H9c2心肌细胞凋亡具有保护作用。

关键词: 钙调神经磷酸酶, 细胞凋亡, 膜电位, 线粒体, 他罗利姆

Abstract: Abstract:Objective To observe H2O2-induced myocardial H9c2 cell apoptosis and protective effect of inhibitor of calcineurin (CaN) and provide experimental basis for study on cardiocytes damage caused by ischemia/reperfusion and related mechanism. Methods Myocardial H9c2 cells cultivated in vitro were treated with saline and various concentrations H2O2 (50,100,200,400 μmol•L-1) at different time (1,6,24 h) in control and experimental groups respectively.Rhodamine 123 fluorescent probe was used to determine membrane potential (Δψmt).Annexin-Ⅴ/PI was used to detect myocardial H9c2 cell apoptosis.The myocardial cell apoptosis models of rats were established,myocardial H9c2 cells was treated with 100 μmol•L-1 H2O2 for 6 h,FK506 were administerted to the model group at low dose (0.15 μmol•L-1)and high dose (0.60 μmol•L-1 )levels,and the apoptotic and necrotic rates were detected by flow cytometry. Results ① Compared with control group,H2O2 caused the significant increase of Δψmt in 100,200,400 μmol•L-1 groups at 1 h（P<0.01）; Δψmt showed the significant decrease in 200 and 400 μmol•L-1 H2O2 treated groups compared with 100 μmol•L-1 group and it was significantly lower in 400 μmol•L-1 group than in 200 μmol•L-1 group at 6 and 24 h (P<0.05). ② Compared with control group,the apoptotic and necrotic rates were significantly increased in 200 and 400 μmol•L-1 H2O2 treated groups at 1 h,and the highest in 400 μmol•L-1 group at 24 h.③Compared with 100 μmol•L-1 H2O2 treated group,the H2O2-induced apoptotic rate was significantly decreased in FK506 treated groups (P<0.01);there was no significant difference between low dose and high dose FK506 groups (P>0.05). Conclusion H2O2 could damage mitochondria,decrease mitochondrial membrane potential and induce the apoptosis and necrosis in myocardial H9c2 cells.The inhibitor of CaN shows protective effect on myocardial H9c2 cell apoptosis induced by H2O2.

Key words: calcineurin, apoptosis, membrane potential, mitochondria, tacrolimus

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