阿托伐他汀对同型半胱氨酸诱导的心肌细胞MEK/ERK通路及心肌线粒体损伤的影响

Effects of atorvastatin on homocysteine-induced MEK/ERK pathway and mitochon-drial damage in cardiomyocytes

目的探讨阿托伐他汀对同型半胱氨酸(Hcy)诱导的心肌细胞H9c2丝裂原细胞外信号调节激酶(MEK)/细胞外调节蛋白激酶(ERK)通路及心肌线粒体损伤的影响。方法细胞计数试剂盒8(CCK-8)检测不同浓度Hcy对H9c2细胞存活率的影响,筛选Hcy诱导浓度和时间。将诱导后的H9c2细胞分为模型组、5μmol/L阿托伐他汀组、10μmol/L阿托伐他汀组和15μmol/L阿托伐他汀组,另取正常H9c2细胞为对照组。流式细胞仪检测各组细胞凋亡率;JC-1法检测各组细胞线粒体膜电位的变化;DCFH-DA法检测各组细胞内活性氧(ROS)水平;酶联免疫吸附法(ELISA)检测各组细胞中丙二醛(MDA)、超氧化物歧化酶(SOD)和过氧化氢酶(CAT)含量;Western blot检测各组细胞MEK1/2和ERK1/2磷酸化水平。结果与对照组相比,2μmol/L Hcy可显著降低H9c2细胞存活率(P<0.05),本研究使用2μmol/L Hcy处理24 h诱导H9c2细胞。与对照组相比,模型组H9c2细胞凋亡率、ROS水平、MDA含量显著升高(P<0.05),线粒体膜电位、SOD、CAT含量及MEK1/2、ERK1/2磷酸化水平显著降低(P<0.05);与模型组相比,5μmol/L阿托伐他汀组、10μmol/L阿托伐他汀组和15μmol/L阿托伐他汀组H9c2细胞凋亡率、ROS水平、MDA含量依次降低(P<0.05),线粒体膜电位、SOD、CAT含量及MEK1/2、ERK1/2磷酸化水平依次升高(P<0.05)。结论阿托伐他汀可能通过激活MEK/ERK通路降低Hcy诱导的H9c2细胞氧化应激反应,减轻心肌线粒体损伤。

Aim To investigate the effects of atorvastatin(Ato)on homocysteine(Hcy)-induced mitogen extracellular signal-regulated kinase(MEK)/extracellular regulatory protein kinase(ERK)pathway and myocardial mitochondrial damage in H9c2 cardiomyocytes.Methods Cell counting kit-8(CCK-8)was used to detect the effect of different concentrations of Hcy on the survival rate of H9c2 cells to screen the induction concentration and time of Hcy.H9c2 cells were divided into model group,5μmol/L Ato group,10μmol/L Ato group and 15μmol/L Ato group,and another normal H9c2 cells were taken as control group.The apoptosis rate was detected by flow cytometry;the change of mitochondrial membrane potential was detected by JC-1 method;the level of intracellular reactive oxygen species(ROS)was measured by DCFH-DA method;enzyme linked immunosorbent assay(ELISA)was used to detect the contents of malondialdehyde(MDA),superoxide dismutase(SOD)and catalase(CAT);and the phosphorylation levels of MEK1/2 and ERK1/2 were detected by Western blot.Results Compared with control group,2μmol/L Hcy significantly reduced the survival rate of H9c2 cells(P<0.05),so that,in this study,H9c2 cells were treated with 2μmol/L Hcy for 24 h.Compared with control group,the apoptosis rate,ROS level and MDA content of H9c2 cells were significantly increased in the model group(P<0.05),and the mitochondrial membrane potential,SOD,CAT contents and phosphorylation levels of MEK1/2 and ERK1/2 were significantly decreased(P<0.05).Compared with model group,the apoptosis rate,ROS level and MDA content of H9c2 cells in 5μmol/L Ato group,10μmol/L Ato group and 15μmol/L Ato group decreased in turn(P<0.05),and the mitochondrial membrane potential,SOD,CAT contents and phosphorylation levels of MEK1/2 and ERK1/2 increased in turn(P<0.05).Conclusion Ato may reduce the oxidative stress induced by Hcy in H9c2 cells by activating MEK/ERK pathway and alleviate myocardial mitochondrial damage.