ACh对COCl2化学缺氧诱导H9C2心肌细胞损伤的保护作用及其分子机制  

Acetylcholine Protect H9C2 Cardiac Cells from CoCl_2-induced Hypoxic Injury and Its Molecular Mechanism

作　　者：吴涛[1] 耿登峰[1] 符宇[1] 高佳佳[1] 谈智[2,3] WU Tao GENG Deng-feng FU Yu GAO Jia-jia TAN Zhi(Department of Cardiology, Sun Yat-sen Memorial Hospital, Sun Yat-sen University, Guangzhou 510120, China Department of Physiology, Zhongshan School of Medicine, Guangzhou 510080, China Institute of Hypertension, Zhongshan School of Medicine, Guangzhou 510080, China)

机构地区：[1]中山大学孙逸仙纪念医院心内科,广东广州510120 [2]中山大学中山医学院生理教研室,广东广州510080 [3]中山大学中山医学院高血压研究所,广东广州510080

出　　处：《中山大学学报（医学科学版）》2016年第5期641-648,共8页Journal of Sun Yat-Sen University:Medical Sciences

基　　金：国家自然科学基金(81270377);广东省自然科学基金(2014A030313092,2014A030313062)

摘　　要：【目的】探讨乙酰胆碱(Ach)对抗氯化钴(CoCl_2)诱导H9C2心肌细胞缺氧损伤的作用及其分子机制。【方法】应用600μmol/L CoCl_2处理H9C2心肌细胞12 h以建立缺氧损伤细胞模型,应用ACh 10^(-3) mol/L预处理8 h建立心肌细胞保护模型,实验分为1空白对照组(control);2损伤模型组(CoCl_2600μmol/L);3 ACh预处理组(ACh 10^(-3) mol/L+CoCl_2600μmol/L);4ɑ7胆碱能受体(ɑ7n ACh R)拮抗剂组:ACh+甲基牛扁碱柠檬酸盐(MLA)+CoCl_2组(ACh 10^(-3) mol/L+CoCl_2600μmol/L+MLA 10-6mol/L)。应用CCK-8试剂盒检测细胞存活率。双氯荧光素(DCFH-DA)染色荧光显微镜照相法检测胞内活性氧(ROS)水平。JC-1荧光显微镜照相法检测细胞线粒体膜电位水平改变。Hoechst33258核染色荧光显微镜照相法测定凋亡细胞的形态及数量的变化。Fluo4-AM荧光显微镜照相法测定细胞胞质内钙离子水平的改变。Western Blot检测Drp1、caspase-3蛋白水平。【结果】600μmol/L CoCl_2处理显著损伤H9C2心肌细胞,线粒体膜电位丢失,ROS生成、细胞凋亡率、胞内钙离子显著增加(P<0.001),明显上调Drp1、caspase-3表达水平(P<0.001);应用ACh预处理可明显提高H9C2心肌细胞存活率(P<0.001),ROS水平下降,线粒体膜电位丢失减少(P<0.001),细胞凋亡率、胞内钙离子明显下降(P<0.001),显著抑制CoCl_2对Drp1、caspase-3表达的上调作用(P<0.01);ACh+MLA预处理可对抗ACh上述作用过程。【结论】ACh具有保护缺氧H9C2心肌细胞的作用,其机制可能与通过激动ɑ7nAChR抑制钙离子-Drp1通路有关。[ Objective ] To investigate the cytoprotection of acetylcholine （ACh） against cobalt chloride （CoCl2）-induced hypoxic injury in H9C2 cells and its molecular mechanism. [ Methods] H9C2 cardiac cells were treated with 600 μmol/L COC12 for 12 h to establish H9C2 cell injury model; H9C2 cardiac cells were pretreated with 10^-3 mol/L Ach for 8 h to establish H9C2 cytopmtection model. The cells were divided into the following four groups： ①control group; ②cell injury model group （COC12 600 p, mol/L ）; ③ ACh preconditioning group： ACh ＋COC12 group （ACh 10^-3 mol/L＋ COC12 600 μmol/L）; ④ a7 nicotinic acetylcholine receptor （u7nAChR） antagonist group： ACh＋Methyllycaconitine citrate （MLA） ＋COC12 group （ACh 10^-3 mol/L＋CoC12 600 p, mol/L＋MLA 10^-6 mol/L）. The cell viability was tested by cell counter kit 8 （CCK-8）. The intracellular levels of Calcium and reactive oxygen species （ROS） were measured by Fluo4-AM stainning and DCFH-DA staining respectively. Mitochondrial membrane potential （MMP） was detected by JC-1 staining and photofluorography. The changes of the morphology and the number of apoptotic cells were tested by Hoechst 33258 nuclear staining. The expression levels of Drpl and caspase-3 proteins were measured by Western blot assay. [Results] Exposure of H9C2 cardiac cells to 600 μmol/L COC12 markedly induced cell injury and dramatically upregulated the expression level of Drpl and caspase-3 （P 〈 0.001 ） ,while the accumulation of intracellular calcium and ROS, loss of MMP and the apoptosis rate were increased significantly （P 〈 0.001 ）. Pretreated cells with ACh before exposuring to COC12 significantly increased cell viability （P 〈 0.001 ）, markedly inhibited upregulating the levels of Drpl and caspase-3 induced by CoC12（P〈0.01 ）, sigificantly attennuated the accumulation of intracellular calcium and ROS （P 〈 0.001 ）, while the dissipation of MMP and the apoptosis rate were decreased （P 〈 0.001）. Methyllycaconit

关 键 词：乙酰胆碱 H9C2心肌细胞 心肌缺氧 细胞凋亡 

分 类 号：R54[医药卫生—心血管疾病]