亚砷酸钠对人正常肝细胞脂质代谢及因子调控的作用

Effects of sodium arsenite on lipid metabolism in human hepatocytes and regulatory factors

背景:肝脏作为砷毒作用的主要靶器官,成为砷毒性作用机制相关研究的焦点。目的:探讨亚砷酸钠对人正常肝细胞脂质代谢、细胞增殖、细胞凋亡及相关调控因子表达的影响。方法:MIHA正常人肝细胞株暴露于0,10,20,30μmol/L亚砷酸钠48 h,光学显微镜观察细胞形态变化,CCK-8法检测细胞活性,单试剂COD-PAP法、单试剂GPO-PAP法及酶微板法检测细胞上清总胆固醇、三酰甘油及总胆汁酸水平,油红O染色法检测细胞内脂质含量,Edu-488渗入法检测细胞增殖,PI染色法及Annexin V-FITC/PI双标记法结合流式细胞术分别检测细胞周期和细胞凋亡,实时荧光定量PCR和Western blot检测肝细胞核因子4α、胆固醇7α-羟化酶及法尼醇X受体的m RNA及蛋白表达水平。结果与结论:(1)与对照组(0μmol/L亚砷酸钠)相比,随着亚砷酸钠浓度的增加:MIHA细胞活性逐渐降低;细胞上清中总胆固醇、三酰甘油水平逐渐增高,总胆汁酸水平逐渐降低;细胞内脂质含量逐渐增多;细胞停滞于S期及G_2/M期细胞比例逐渐增多;细胞凋亡率逐渐升高;肝细胞核因子4α mRNA表达水平未见明显变化,胆固醇7α-羟化酶与法尼醇X受体mRNA表达水平下降;肝细胞核因子4α、胆固醇7α-羟化酶、法尼醇X受体蛋白表达水平逐渐下降;(2)亚砷酸钠具有细胞毒性,显著降低MIHA细胞活性,诱导细胞脂肪变性,阻滞细胞增殖,诱发细胞凋亡等损伤;亚砷酸钠可下调肝细胞核因子4α蛋白表达以及胆固醇7α-羟化酶、法尼醇X受体的转录和蛋白表达,进一步引起肝细胞的脂质代谢紊乱。

BACKGROUND:The liver,as the main target organ for arsenic toxicity,has become the focus of studies related to the mechanism of action of arsenic toxicity.OBJECTIVE:To investigate the effects of sodium arsenite(NaAsO2)on lipid metabolism,cell proliferation,apoptosis,and expression of related regulatory factors in human normal hepatocytes.4956|中国组织工程研究|第29卷|第23期|2025年8月METHODS:MIHA normal human hepatocyte cell lines were exposed to 0,10,20,and 30µmol/L NaAsO2 for 48 hours.Cell morphology changes were observed by light microscopy.Cell viability was detected by CCK-8 assay.The cell serum total cholesterol,triacylglycerol,and total bile acids were detected by single-agent COD-PAP assay,single-agent GPO-PAP assay,and enzyme microplate assay.The intracellular lipid content was detected by oil red O staining.Cell proliferation was detected by Edu-488 infiltration.Cell cycle and apoptosis were detected by PI staining and Annexin V-FITC/PI dual-labeling combined with flow cytometry.The mRNA and protein expression levels of hepatocyte nuclear factor 4 alpha,cholesterol 7α-hydroxylase,and farnesoid X receptor were detected by real-time fluorescence quantitative PCR and western blot assay,respectively.RESULTS AND CONCLUSION:(1)Compared with the control group(0µmol/L NaAsO2),with the increase of NaAsO2 concentration:MIHA cell viability decreased gradually.The content of total cholesterol and triacylglycerol in cell supernatant increased gradually,while the contents of total bile acids decreased gradually.The content of intracellular lipid increased gradually.The proportion of cells stagnating in S phase and G2/M phase gradually increased,and the apoptosis rate gradually increased.The expression level of hepatocyte nuclear factor 4 alpha mRNA did not show significant changes,while cholesterol 7α-hydroxylase and farnesoid X receptor mRNA expression levels decreased.The protein expression levels of hepatocyte nuclear factor 4 alpha,cholesterol 7α-hydroxylase,and farnesoid X receptor decreased gradually.(2)NaAsO2 has cytotoxicity,significantly reduces MIHA cell viability,induces cell steatosis,inhibits cell proliferation,and induces cell apoptosis.NaAsO2 down-regulates hepatocyte nuclear factor 4 alpha protein expression and the transcription and expression of cholesterol 7α-hydroxylase and farnesoid X receptor,which further induces lipid metabolism disorders in hepatocytes.