线粒体钙超载激活线粒体凋亡途径在甲基汞致神经元凋亡中的作用

目的 探讨线粒体钙超载并诱导线粒体凋亡途径活化在甲基汞致神经元凋亡过程中的作用及其机制。 方法 取出生24 h内的新生昆明小鼠,解剖取脑皮质,进行原代神经元培养。细胞发育成熟后,应用含不同浓度甲基汞(methyl-mercury,MeHg,0、0.25、0.5、1 μmol/L)的培养液分别处理神经元1、3、6 h,测定神经元细胞活力,确定适宜暴露时间后,测定神经元线粒体内钙离子水平,线粒体膜电位,细胞色素c(cytochrome c,Cyt c)、凋亡诱导因子(apoptosis induce factor,AIF)、天冬氨酸蛋白水解酶3(cysteinyl aspartate specific proteinase 3, caspase 3)蛋白表达水平及细胞凋亡率。 结果 神经元经不同浓度MeHg处理后,细胞活力降低。在0.25、0.5、1 μmol/L MeHg处理组,神经元线粒体内钙离子水平升高,线粒体膜电位下降,Cyt c、AIF、caspase3蛋白表达水平升高,细胞早期凋亡率升高,且都呈现剂量效应关系。在1 μmol/L MeHg处理组,上述指标与对照组比较差异均有统计学意义(P<0.01)。 结论 甲基汞暴露会诱发神经元线粒体钙超载并激活线粒体凋亡途径进而导致细胞凋亡。

Effect of mitochondrial calcium overload mediating mitochondrial apoptosis pathway activation on methylmercury-induced neuronal apoptosis

Objective To explore the role and mechanism of mitochondrial calcium overload and mitochondrial apoptosis pathway activation in the process of neuron apoptosis induced by methylmercury (MeHg) poisoning. Methods The brains of newborn mice were anatomized within 24 hours after birth for primary culture of mouse cortical neurons. The cells were incubated with different concentration of MeHg (0 μmol/L, 0.25 μmol/L, 0.5 μmol/L and 1 μmol/L) in the culture medium for 1, 3 and 6 h respectively, and then cell viability was measured. After suitable exposure time was determined, mitochondrial calcium level, mitochondrial membrane potential, cytochrome c (Cyt c), apoptosis induce factor (AIF), cysteinyl aspartate specific proteinase 3 (caspase 3) protein expression level and apoptosis rate were detected. Results Cell viability was decreased after exposure to different concentration of MeHg. For the 0.25 μmol/L, 0.5 μmol/L and 1 μmol/L MeHg treatment groups, mitochondrial calcium levels, Cyt c, AIF and caspase 3 protein expression levels and cell early apoptosis rates were elevated, while mitochondrial membrane potential was declined, all in a dose-dependent manner. There were statistically significant differences in the above-mentioned indicators between 1 μmol/L MeHg treatment group and the control group (all P<0.01). Conclusions MeHg exposure can induce mitochondrial calcium overload, activate mitochondrial apoptosis pathway, and then lead to neuronal apoptosis.