不同时间慢性间歇低氧下CB1R/AMPK/PGC-1α信号通路对H9C2大鼠心肌细胞的调控作用

Regulatory effect of CB1R/AMPK/PGC-1α signaling pathway on H9C2 rat cardiomyocytes under chronic intermittent hypoxia at different time

目的:探讨慢性间歇低氧(chronic intermittent hypoxia,CIH)对H9C2大鼠心肌细胞的影响,以及大麻素1型受体(cannabinoid type 1 receptor,CB1R)/AMP活化蛋白激酶(AMP-activated protein kinase,AMPK)/过氧化物酶体增殖物激活受体γ辅激活因子1α(peroxisome proliferator-activated receptor-γcoactivator-1α,PGC-1α)信号通路在其中发挥的作用。方法:将H9C2大鼠心肌细胞分为正常对照(normal control,NC)组、6 h CIH组、18 h CIH组和18 h CIH+AM251(CB1R拮抗剂)组,间歇低氧仓中制备CIH细胞模型。流式细胞术检测细胞凋亡及活性氧水平,JC-1检测线粒体膜电位,MitoTracker染色后激光共聚焦显微镜观察线粒体的形态。Western blot和细胞免疫荧光分析CB1R、AMPK、磷酸化AMPK(phosphorylated AMPK,p-AMPK)和PGC-1α的表达情况。结果:与NC组相比,6 h CIH组活性氧轻度增高,线粒体膜电位显著升高,线粒体明显增多,CB1R、AMPK、p-AMPK和PGC-1α蛋白水平显著升高(P<0.05或P<0.01);18h CIH组细胞凋亡增加,活性氧显著增高,线粒体膜电位显著降低,线粒体碎片化显著增多,CB1R表达显著增多,AMPK、p-AMPK和PGC-1α蛋白水平显著降低(P<0.05或P<0.01);与18 h CIH组相比,18 h CIH+AM251组细胞凋亡和活性氧减少,线粒体膜电位显著升高,线粒体碎片化显著减少,CB1R表达显著降低,AMPK、p-AMPK和PGC-1α蛋白水平显著升高(P<0.05或P<0.01)。结论:AMPK/PGC-1α信号通路参与了CIH对H9C2大鼠心肌细胞的影响,CB1R可作为CIH介导的H9C2大鼠心肌细胞损伤的治疗靶点。

AIM:To investigate the effects of chronic intermittent hypoxia(CIH)on H9C2 rat cardiomyocytes and the role of cannabinoid type 1 receptor(CB1R)/AMP-activated protein kinase(AMPK)/peroxisome proliferator-activated receptor-γcoactivator-1α(PGC-1α)signaling pathway in the development of CIH-induced cardiomyocyte injury.METHODS:The H9C2 rat cardiomyocytes were divided into normal control(NC)group,6 h CIH group,18 h CIH group and 18 h CIH+AM251(CB1R antagonist)group.The CIH cell models were prepared in intermittent hypoxia chamber.Apoptosis and reactive oxygen species(ROS)were detected by flow cytometry.The mitochondrial membrane potential was measured by JC-1.The mitochondria in the cells were observed by confocal laser scanning microscopy after MitoTracker staining.Western blot and cellular immunofluorescence were used to analyze the protein levels of CB1R,AMPK,phosphorylated AMPK(p-AMPK)and PGC-1α.RESULTS:Compared with NC group,the ROS elevated slightly,and the mitochondrial membrane potential,number of mitochondria,and the protein levels of CB1R,AMPK,p-AMPK and PGC-1αwere significantly increased in 6 h CIH group(P<0.05 or P<0.01).Apoptosis,ROS,mitochondrial fragmentation and CB1R expression were significantly increased,while the mitochondrial membrane potential and the protein levels of AMPK,p-AMPK and PGC-1αwere significantly decreased in 18 h CIH group(P<0.05 or P<0.01).AM251 prevented the increase in CIH-induced CB1R expression.Compared with 18 h CIH group,apoptosis,ROS and mitochondrial fragmentation were significantly decreased,while the mitochondrial membrane potential and the protein levels of AMPK,pAMPK and PGC-1αwere significantly increased in 18 h CIH+AM251 group(P<0.05 or P<0.01).CONCLUSION:The AMPK/PGC-1αsignaling pathway is involved in the effect of CIH on H9C2 rat cardiomyocytes,and CB1R can be used as a therapeutic target for CIH-induced H9C2 rat cardiomyocyte injury.