氧化应激在镉致大鼠肝细胞凋亡中的作用

摘要

[Objective] The role of oxidative stress on the apoptosis of rat hepatocytes induced by cadmium was studied. [Method] Rat hepatocytes were isolated by a two-step perfusion technique. After 24 h planting, hepatocytes were treated with cadmium acetate, or treated with Cd in the presence or absence of Z-VAD-fink or NAC. Cell viability was measured with MTT assay, the apoptosis, reactive oxygen species (ROS) generation, mitochondrial membrane potential (△Ψm) collapse were measured by flow cytometry. The hepatocytes hotnogenate was prepared to detect the levels of caspase-3, malondialdehyde (MDA) and reduced glutathione hormone (GSH) in spectro photometric assay. [Result] The results showed that cellular viability decreased and apoptosis rate increased significantly or very significantly (P＜0.05 or P＜0.01) in a dose-dependent manner after exposure to 2.5, 5 and 10 μmol·L-1 Cd. Cd (2.5 and 5 μmol·L-1) induced ROS generation significently in 1.5 hours (P＜0.05 or P＜0.01). Cd induced △Ψm collapse. NAC effectively protected hepatocytes against apoptosis induced by Cd. Cd induced ROS generation and △Ψm collapse were blocked by NAC (P＜0.05). The GSH content decreased with the increase of the content of Cd. There was very significant difference in partial groups than the control group at 12 h (P＜0.01), but GSH content increased with the increase of the dose of Cd at 24 h. The activities of MDA level increased. The caspase-3 activity didn't increase. No effect of Z-VAD-fink on apoptosis was observed. [Conclusion] These results showed that ROS generation and oxidative stress by Cd triggers apoptosis via caspase-independent pathway.%[目的]探讨氧化应激在镉诱导大鼠肝细胞凋亡中的作用.[方法]采用两步灌流法获得大鼠肝细胞,经过24 h培养,用醋酸镉处理细胞,或者Z-VAD-fmk、NAC和镉共同处理细胞.应用MTT法检测细胞存活率,流式细胞仪检测细胞凋亡率、细胞内ROS水平和线粒体膜电位,分光光度法检测caspase-3活性、GSH和MDA含量.[结果]结果表明,肝细胞暴露于浓度为2.5、5、10μmol·L-1的镉后,细胞相对存活率显著下降(P＜0.01),凋亡率显著或极显著升高(P＜0.05或P＜0.01),呈剂量-效应关系；2.5和5μmol·L-1镉组在1.5h之前导致细胞内ROS水平显著或极显著升高(P＜0.05或P＜0.01)；镉暴露可使肝细胞△φm显著或极显著降低(P＜0.05或P＜0.01); NAC能够显著减少锅引起的凋亡细胞数量和极显著降低凋亡率(P＜0.01),可以显著降低单独镉暴露组ROS水平升高和阻止△ψm降低(P＜0.05)；细胞内GSH含量12h时随镉剂量增高而降低,部分剂量组极显著低于对照组(P＜0.01),24 h时随剂量增高而升高,部分剂量组显著或极显著高于对照组(P＜0.05或P＜ 0.01)；细胞内MDA含量随着镉浓度的增大而升高,10 μmol·L-1剂量组MDA的含量显著高于对照组(P＜0.05)；未见caspase-3活性升高,caspase抑制剂Z-VAD-fmk对镉致细胞凋亡无影响.[结论]醋酸镉致肝细胞凋亡与其引起ROS产生并导致氧化损伤的非caspase途径有关.