FUNDC1通过调控线粒体分裂影响高糖损伤H9c2心肌细胞凋亡的机制研究  

Mechanism of FUNDC1 affacting apoptosis of H9c2 cardiomyocytes with high glucose injury by regulating mitochondrial fission

作　　者：郑君毅[1] 张莹莹[1] 刘园园[1] 陈孟英[1] 郭绪昆[1] ZHENG Junyi;ZHANG Yingying;LIU Yuanyuan;CHEN Mengying;GUO Xukun(Department of Cardiology,Tianjin Chest Hospital,Tianjin Institute of Cardiovascular Disease,Tianjin 300222,China)

机构地区：[1]天津市胸科医院心内科,天津市心血管病研究所,300222

出　　处：《天津医药》2022年第8期791-795,共5页Tianjin Medical Journal

摘　　要：目的 探讨含 FUN14域蛋白 1(FUNDC1)在高糖损伤的 H9c2心肌细胞中通过调控线粒体分裂影响心肌细 胞凋亡的作用机制。方法 体外培养 H9c2心肌细胞并建立高糖损伤模型,分别进行细胞转染,使用腺苷酸活化蛋白 激酶(AMPK)激活剂 5-氨基-4-咪唑羧基酰胺核苷(AICAR)后,分为正常对照组(CTRL组)、高糖组(HG组)、HG+ shRNA-NC组(转染 shRNA-NC的 HG组细胞)、HG+shRNA-FUNDC1组(转染 shRNA-FUNDC1的 HG组细胞)和 HG+ AICAR组(使用 AICAR的 HG组细胞)。MTT法检测细胞存活率;酶标仪检测细胞乳酸脱氢酶(LDH)释放量;激光共 聚焦显微镜观察线粒体结构;Western blot检测线粒体动力相关蛋白 1(DRP1)、分裂蛋白 1(FIS1)、Bcl-2相关 X蛋白 (Bax)、B淋巴细胞瘤-2(Bcl-2)、活化的胱天蛋白酶 3(Cleaved Caspase-3)、FUNDC1和 GAPDH的蛋白表达水平。结 果 与 CTRL 组比较,HG 组细胞存活率降低,LDH 释放量升高,线粒体 DRP1(DRP1-mito)、FIS1、Bax 和 Cleaved Caspase-3蛋白表达水平升高,胞浆 DRP1(DRP1-cyto)和 Bcl-2蛋白表达水平降低(P<0.05)。与 HG组比较,HG+ shRNA-FUNDC1组细胞存活率升高,LDH释放量明显降低,DRP1-mito、FIS1、Bax和 Cleaved Caspase-3蛋白表达水平 降低,DRP1-cyto和 Bcl-2蛋白表达水平升高(P<0.05)。HG+shRNA-NC组和 HG组比较,各指标差异均无统计学意 义(P>0.05)。CTRL组线粒体主要呈线状结构,HG组和 HG+shRNA-NC组线粒体均主要呈点状结构;与 HG组比较, HG+shRNA-FUNDC1组线粒体线状结构增多,点状结构减少。与 CTRL组比较,HG组 FUNDC1蛋白表达水平升高 (P<0.05)。与 HG组比较,HG+AICAR组 FUNDC1蛋白表达水平降低(P<0.05)。结论 FUNDC1通过调控线粒体 分裂影响高糖引起的 H9c2心肌细胞凋亡,AMPK信号通路参与该过程。Objective To investigate the mechanism of FUNDC1 affecting high glucose injured H9c2 cardiomyocyte apoptosis by regulating mitochondrial fission.Methods H9c2 cardiomyocytes were cultured in vitro,and the high glucose induced injury model of cells was established.After cell transfection or AMPK activation by AICAR,the cells were divided into the control(CTRL)group,the high glucose injury(HG)group,the HG with transfected shRNA-NC(HG+shRNA-NC)group,the HG with transfected FUNDC1 shRNA(HG+shRNA-FUNDC1)group and the HG with AICAR(HG+AICAR)group.MTT method was used to detect the cell survival rate.The level of released LDH was measured with microplate reader.The structure of mitochondria was observed by laser confocal microscope.The proteins expression levels of mitochondrial dynamin-related protein1(DRP1),fission protein 1(FIS1),Bcl-2 associated X protein(Bax),B-lymphocytoma2(Bcl-2),cleaved cysteine-containing aspartate-spicific protease 3(Cleaved Caspase-3),FUNDC1 and GAPDH were detected by Western blot assay.Results Compared with the CTRL group,the cell survival rate decreased,and the protein levels of DRP1-cyto and Bcl-2 were also decreased,the release of LDH and the protein expression levels of DRP1-mito,FIS1,Bax and Cleaved Caspase-3 increased in the HG group(P<0.05).Compared with the HG group,knocking down FUNDC1 increased the cell survival rate and the protein levels of DRP1-cyto and Bcl-2,decreased the release of LDH and the protein expression levels of DRP1-mito,FIS1,Bax and Cleaved Caspase-3(P<0.05).There was nodifference in each index between the HG group and the HG+shRNA-NC group(P>0.05).The mitochondria were mainly linear structure in the CTRL group,and the mitochondria were mainly pucta structure in the HG group and the HG+shRNA NC group.Compared with the HG group,the mitochondrial linear structure increased and the puncta structure decreased in the HG+shRNA-FUNDC1 group.Compared with the CTRL group,the protein expression level of FUNDC1 increased in the HG group(P<0.05).Compared with the HG group,the pr

关 键 词：肌细胞 心脏 线粒体 细胞凋亡 FUNDC1 信号通路 

分 类 号：R541[医药卫生—心血管疾病]