目的 研究亚砷酸钠(NaAsO2)对人肝星状细胞(LX-2)铁死亡的影响.方法 体外培养LX-2细胞,采用成组设计将不同浓度NaAsO2(5 μmol/L、10μmol/L、15 μmol/L)染毒LX-2细胞24h,构建体外NaAsO2致LX-2活化模型,同时设立对照组;透射电子显微镜(transmission electron microscope,TEM)观察NaAsO2处理LX-2细胞线粒体结构变化情况;荧光显微镜和荧光酶标仪检测细胞Fe2+水平;比色法检测丙二醛(malondialdehyde,MDA)含量;蛋白免疫印迹法检测溶质载体家族7号成员11(solute carrier family number 7member 11,SLC7A11)、谷胱甘肽过氧化物酶 4(glutathione peroxidase 4,GPX4)、α-平滑肌肌动蛋白(α-SMA)蛋白表达水平.结果 TEM结果显示,NaAsO2组细胞线粒体膜完整性破损,线粒体嵴减少和消失;此外,与对照组比较,NaAsO2处理组Fe2+水平均升高(P＜0.05);不同剂量NaAsO2组MDA含量差异均有统计学意义(F=7.18,P均＜0.05).其中,与对照组比较,5、15μmol/L NaAsO2组LX-2细胞MDA含量均高于对照组(P＜0.05);在翻译水平上,纤维化指标α-SMA蛋白表达水平随着NaAsO2剂量的升高而表达上调(P＜0.05),铁死亡指标SLC7A11和GPX4蛋白表达水平随着NaAsO2剂量的升高而减少(P＜0.05).结论 铁死亡参与NaAsO2致LX-2细胞活化过程.
The Effect of Ferroptosis on the Activation of Human Hepatic Stellate Cells Induced by Inorganic Arsenic
Objective To investigate the effect of NaAsO2 on ferroptosis in human hepatic stellate cells(LX-2).Methods LX-2 cells were cultured in vitro,and different concentrations of NaAsO2(5μmol/L,10μmol/L,15μmol/L)were infected with LX-2 cells for 24h in a group design to construct the activation model of LX-2 induced by NaAsO2 in vitro,and a control group was set up.Mitochon-drial structure of LX-2 cells treated with NaAsO2 was observed by transmission electron microscopy(TEM).Fe2+levels were detected by fluorescence microscope and fluorescent enzyme label.The content of malondialdehyde(MDA)was determined by the colorimetric meth-od.The protein expression levels of SLC7A11,GPX4,and α-smooth muscle actin(α-SMA)were detected by Western blot.Results TEM showed that mitochondrial membrane integrity was damaged and mitochondrial ridges were reduced and disappeared in the NaAsO2group.In addition,compared with the control group,Fe2+levels in NaAsO2 treatment groups were increased(P＜0.05).There were statistically significant differences in MDA content among different doses of NaAsO2groups(F=7.18,P＜0.05).Compared with the control group,MDA content of LX-2 cells in 5 and 15μmol/L NaAsO2groups was higher than that in the control group(P＜0.05).At the translation level,the expression of fibrosis index α-SMA protein level was up-regulated with the increase of NaAsO2dose(P＜0.05),the protein expression levels of ferroptosis index SLC7A11 and GPX4decreased in a dose-dependent manner with the increase of the dose of NaAsO2(P＜0.05).Conclusion Ferroptosis is involved in the activation of LX-2 cells induced by NaAsO2.
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