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

doi:10.12307/2025.509

Abstract

Abstract: 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.
METHODS: MIHA normal human hepatocyte cell lines were exposed to 0, 10, 20, and 30 µmol/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 (0 µmol/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.

Key words: sodium arsenite, MIHA cell, cholesterol 7α-hydroxylase, farnesol X receptor, cell cycle, cell apoptosis, oil red O staining, lipid metabolism

CLC Number:

Tian Zhenli, Zhang Xiaoxu, Fang Xingyan, Xie Tingting. Effects of sodium arsenite on lipid metabolism in human hepatocytes and regulatory factors[J]. Chinese Journal of Tissue Engineering Research, 2025, 29(23): 4956-4964.

Figures/Tables 2

2.1 NaAsO2抑制MIHA细胞活性且呈浓度依赖性根据CCK-8结果计算IC50值，确定NaAsO2处理时间为48 h，见图1A。与对照组(0 μmol/L NaAsO2)相比，10-30 μmol/L NaAsO2组细胞活性均显著降低(P < 0.05)，且随着NaAsO2浓度的增加，MIHA细胞活性逐渐降低，见图1B，呈剂量-效应关系。 2.2 NaAsO2导致MIHA细胞形态改变倒置显微镜观察显示，对照组(0 μmol/L NaAsO2)细胞状态良好，胞膜光滑完整，胞质丰富饱满，呈多边形以铺路石样排列紧密，见图1C。与对照组相比，实验组随着NaAsO2浓度的增加，胞膜逐渐模糊不清，细胞逐渐失去正常形态，胞质减少，空泡逐渐增多；细胞融合率逐渐降低，间隙增宽，破碎裂解及脱落漂浮的细胞越来越多。 2.3 NaAsO2引起MIHA细胞脂质代谢紊乱与对照组(0 μmol/LNaAsO2)比较，10-30 μmol/L NaAsO2组细胞上清总胆固醇、三酰甘油水平显著增高(P < 0.05)，见图2A，B，且呈剂量-效应关系。与之相反，10-30 μmol/L NaAsO2组细胞上清总胆汁酸水平显著降低(P < 0.05)，见图2C，呈剂量-效应关系。 2.4 NaAsO2引起MIHA细胞脂肪变性油红O染色检测各组MIHA细胞内脂质含量及分布，结果显示，对照组(0 μmol/L NaAsO2)细胞结构正常，胞内无脂滴形成，见图2D。与对照组相比，10-30 μmol/L NaAsO2组细胞肿胀，多数细胞胞浆内脂肪空泡形成，脂滴大量形成；细胞结构明显异常。 2.5 NaAsO2阻滞细胞增殖 0，10，20，30 μmol/L NaAsO2组细胞增殖率分别为(35.71±0.39)%，(30.84±0.57)%，(24.90±0.26)%，(19.73±0.77)%，与对照组(0 μmol/L NaAsO2)相比，10，20，30 μmol/L NaAsO2组细胞增殖率显著降低(P < 0.05)，见图3，且随着NaAsO2浓度的增加，细胞增殖抑制愈加明显，呈剂量-效应关系。 2.6 NaAsO2诱导细胞周期停滞 0，10，20，30 μmol/L NaAsO2组细胞S期细胞比例分别为(15.21±0.36)%，(19.19±0.18)%，(25.21±1.03)%，(28.63±0.87)%，G2/M期细胞比例分别为(6.08±0.30)%，(11.02±0.29)%，(13.21±0.40)%，(16.81±0.72)%，与对照组(0 μmol/L NaAsO2)相比，10，20，30 μmol/L NaAsO2组周期被阻滞在S期及G2/M期的细胞比例显著增加(P < 0.05)，且随着NaAsO2浓度的增加，细胞S期及G2/M期阻滞引起细胞增殖抑制愈加明显，呈剂量-效应关系，见图4。"

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