艾塞那肽改善缺氧复氧条件下心肌H9c2细胞线粒体功能的机制研究

摘要

目的:研究艾塞那肽改善缺氧复氧(H/R)条件下心肌H9c2细胞线粒体功能的机制.方法:体外培养H9c2细胞,分为空白对照组、药物对照(艾塞那肽200 nmol/L)组、模型(H/R)组、预处理(艾塞那肽200 nmol/L+H/R)组、胰高血糖素样肽1受体(GLP-1R)抑制剂[Exendin-(9-39)100 nmol/L+艾塞那肽200 nmol/L+H/R]组、环磷腺苷(cAMP)抑制剂(Rp-cAMPS 1μmol/L+艾塞那肽200 nmol/L+H/R)组和蛋白激酶A(PKA)抑制剂(H-895μmol/L+艾塞那肽200 nmol/L+H/R)组.除前2组外其余各组建立H/R模型,建模前30 min加入艾塞那肽,加入艾塞那肽前10 min加入相应的抑制剂.采用透射电镜观察线粒体形态结构,流式细胞术检测线粒体钙离子(Ca2+m)水平及线粒体膜电位(ΔΨm),酶标仪检测细胞三磷酸腺苷(ATP)水平.结果:与空白对照组比较,模型组细胞线粒体嵴肿胀增加,密度降低,呈空泡化;Ca2+m水平升高,ΔΨm和ATP水平降低(P<0.05).与模型组比较,预处理组细胞线粒体嵴肿胀减轻,密度增加,空泡化程度减轻;Ca2+m水平降低,ΔΨm和ATP水平升高(P<0.05).与预处理组比较,3种抑制剂组细胞Ca2+m水平升高,ΔΨm和ATP水平降低(P<0.05).结论:艾塞那肽可减轻H/R条件下H9c2细胞线粒体钙超载,升高ΔΨm,增加ATP生成.其机制可能与激活GLP-1R/cAMP/PKA信号通路有关.%OBJECTIVE:To study the mechanism of improvement effects of exenatide on mitochondrial function of H9c2 cells under hypoxia/reoxygenation (H/R) condition. METHODS:H9c2 cells were cultured in vitro and were divided into blank control group,drug control group(exenatide 200 nmol/L),model group(H/R),pretreatment group(exenatide 200 nmol/L+H/R),gluca-gon-like peptide-1 receptor (GLP-1R) inhibitor [Exendin-(9-39) 100 nmol/L+exenatide 200 nmol/L+H/R], cAMP inhibitor (Rp-cAMPS 1 μmol/L+exenatide 200 nmol/L+H/R)group and PKA inhibitor(H-895 μmol/L+exenatide 200 nmol/L+H/R)group. Except for first 2 groups,H/R model was established in other groups,and they were given exenatide 30 min before modeling and relevant inhibitor 10 min before giving exenatide. Morphology of mitochondria was observed by TEM,and mitochondrial calcium (Ca2+m)and the mitochondrial membrane potential(ΔΨm)were determined by flow cytometry. Cellular ATP content was measured by microplate reader. RESULTS:Compared with blank control group, mitochondrial cristae swelling was enhanced in model group,while density decreased,showing vacuolization;Ca2+m level increased while ΔΨm and ATP decreased (P<0.05). Com-pared with model group,mitochondrial cristae swelling relieved in pretreatment group,while density increased,showing vacuoliza-tion relieved;Ca2+m level decreased,while ΔΨm and ATP increased(P<0.05). Compared with pretreatment group,the levels of Ca2+m increased in 3 kinds of inhibitors group,while ΔΨm and ATP decreased(P<0.05). CONCLUSIONS:Exenatide attenuates H9c2 cell mitochondria Ca2+m accumulation,increases ΔΨm and ATP production. Which indicate its mechanism may be associated with activating GLP-1R/cAMP/PKA pathway.