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【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。
【Aim】 To investigate the effect of acetylcholine (Ach) on the hypoxic injury of H9C2 cardiomyocytes induced by cobalt chloride (CoCl2) and its molecular mechanism. 【Method】 H9C2 cardiomyocytes were treated with 600 μmol / L CoCl 2 for 12 h to establish hypoxic injury model. Cardiomyocyte protection model was established by pretreatment with ACh 10 -3 mol / L for 8 h. The experimental groups were divided into 1 blank control group (control group); 2 injury model group (CoCl_2600μmol / L); 3 ACh pretreatment group (ACh 10 ~ (-3) mol / L + CoCl_2600μmol / L); 4ɑ7 cholinergic receptor (ɑ7n ACh R) : ACh + methylbitaline citrate (MLA) + CoCl_2 group (ACh 10 ~ (-3) mol / L + CoCl_2600μmol / L + MLA 10 ~ 6mol / L). Cell viability was measured using CCK-8 kit. Intracellular reactive oxygen species (ROS) levels were examined by dichlofluorescein (DCFH-DA) staining and fluorescence microscopy. JC-1 fluorescence microscopy to detect changes in mitochondrial membrane potential levels. Hoechst33258 nuclear staining fluorescence microscopy for the determination of apoptotic cells morphology and quantity changes. Fluo4-AM Fluorescence Microscopy Determination of Cytosolic Ca2 + in Cells. Western Blot detection Drp1, caspase-3 protein levels. 【Result】 The results showed that the injury induced by 600 μmol / L CoCl 2 could obviously damage the H9C2 myocardial cells, the loss of mitochondrial membrane potential, the generation of ROS, the rate of apoptosis and the increase of intracellular calcium (P <0.001), significantly upregulated the expressions of Drp1 and caspase-3 <0.001). Pretreatment with ACh significantly increased the survival rate of H9C2 cardiomyocytes (P <0.001), decreased the level of ROS, decreased the loss of mitochondrial membrane potential (P <0.001), and decreased the rate of apoptosis <0.001), and significantly inhibited the up-regulation of Drp1 and caspase-3 expression by CoCl_2 (P <0.01). ACh + MLA pretreatment could counteract the above-mentioned action of ACh. 【Conclusion】 ACh can protect H9C2 cells from hypoxia, and its mechanism may be related to the inhibition of calcium-Drp1 pathway by activating ɑ7nAChR.