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）. Methyllycaconitine citrate （MLA） can reversed the above effects of ACh. [Conclusion] ACh has cardioprotective effect, its mechanism may be associated with the inhibition of calcium-Drpl pathway by activating the α7 cholinergic receptor.
作者
吴涛
耿登峰
符宇
高佳佳
谈智
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)
出处
《中山大学学报（医学科学版）》
CAS
CSCD
北大核心
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)
作者简介
吴涛，硕士研究生，研究方向：心血管疾病的损伤与保护机制，E-mail：1193826744@qq．com
