镉诱导大鼠肝细胞BRL-3A铁死亡及其作用机制

Cadmium induces ferroptosis in rat hepatocytes BRL-3A and its mechanism

用4μmol/L氯化镉处理大鼠肝脏细胞BRL-3A 12,24,36 h后,通过CCK-8法测定细胞活力,比色法检测细胞上清中乳酸脱氢酶(LDH)的含量,流式细胞术检测细胞中活性氧(ROS)的含量及线粒体膜电位水平,比色法检测细胞中氧化还原相关指标丙二醛(MDA)、还原型谷胱甘肽(GSH)和氧化型谷胱甘肽(GSSG)含量的变化,Western blot检测铁死亡相关蛋白的表达,荧光探针法检测细胞中亚铁离子(Fe^(2+))的含量,电子显微镜观察细胞的超微结构。结果显示,镉(Cd)处理后,BRL-3A细胞活力下降,LDH水平显著升高;ROS、MDA、GSH、GSSG和Fe^(2+)含量极显著增加(P<0.01),FTH1、TfR1、HO-1蛋白表达水平极显著升高(P<0.01),GPX4蛋白表达水平极显著下降(P<0.01),SLC7A11蛋白水平变化不显著,细胞线粒体萎缩变小、嵴减少或消失。结果表明,Cd处理可致BRL-3A细胞发生脂质过氧化物和Fe^(2+)的蓄积,GPX4水平下降,从而介导细胞发生铁死亡。

The purpose of this study was to investigate the effect and mechanism of cadmium(Cd)on ferroptosis in rat hepatocytes(BRL-3A).In this study,BRL-3A cells were treated with 4μmol/L cadmium chloride for 12,24 and 36 h.Cell viability was determined by CCK-8 method,and lactate dehydrogenase(LDH)content in cell supernatant was detected by colorimetric method.Reactive oxygen species(ROS)concentration and the level of mitochondrial membrane potential in cells were detected using flow cytometry.Contents of redox-related indicators including malondialdehyde(MDA),reduced glutathione(GSH)and oxidized glutathione(GSSG)in cells were detected by colorimetric method.The expression levels of ferroptosis-related proteins were detected by Western blot.The content of ferrous ions(Fe^(2+))in cells was detected by fluorescent probe method,and the ultrastructure of cell was observed using transmission electron microscope.The results showed that the viability of BRL-3A cells decreased after cadmium treatment,the level of LDH in the culture supernatant increased significantly,the contents of ROS,MDA,GSH,GSSG and Fe^(2+)increased significantly,and the protein expression levels of FTHl,TfR1 and HO-1 increased significantly.The protein expression levels of GPX4 decreased significantly,whereas no significant changes of SLC7A1l were observed.The mitochondria of the cells shrank and became smaller,the cristae disappeared,and the membrane density increased.Therefore,Cd treatment can induce the accumulation of lipid peroxides and Fe^(2+)in BRL-3A cells,and decrease the protein level of GPX4,thereby mediating cell ferroptosis.This study lays a foundation for elucidating the mechanism of Cd toxicity to hepatocytes.