摘要： 目的探讨亚硫酸钠（Na₂SO₃）暴露对人正常肝细胞自噬的影响及其作用机制。方法人正常肝细胞HL-7702分别暴露于含10%胎牛血清的DMEM培养基（阴性对照组）、0.2% DMSO （溶剂对照组）、Na₂SO₃暴露组（0.1、1、2.5、5 mmol/L）和20 mmol/L CCl₄ （阳性对照组） 2 h、48 h。采用CCK-8法检测不同浓度Na₂SO₃暴露2 h、48 h后各组细胞存活率；免疫荧光法检测Na₂SO₃各浓度暴露组2 h、48 h自噬相关蛋白LC3B表达水平；Western blotting检测Na₂SO₃各浓度暴露组2 h、48 h自噬相关蛋白LC3B、p62和AMPK，细胞凋亡相关蛋白caspase-3表达水平；荧光素酶化学发光法测定HL-7702细胞内Na₂SO₃各浓度暴露组2 h、48 h ATP含量。结果与阴性对照组比较，1、2.5、5 mmol/L Na₂SO₃暴露组2 h、48 h细胞存活率随Na₂SO₃浓度的升高而降低（P<0.05）；阳性对照组细胞存活率也明显下降（P<0.05）。与阴性对照组比较，2.5 mmol/L、5 mmol/L Na₂SO₃暴露组和阳性对照组在48 h时，HL-7702细胞中LC3B免疫荧光强度明显减弱。与阴性对照组比较，HL-7702细胞中LC3B-Ⅱ、p62蛋白在48 h 5 mmol/L Na₂SO₃暴露组表达显著降低（P<0.05）；活化的caspase-3蛋白在48 h 1、2.5、5 mmol/L Na₂SO₃暴露组表达升高（P<0.05）。与阴性对照组比较，暴露2 h、48 h时0.1、1、2.5、5 mmol/L Na₂SO₃暴露组HL-7702细胞中ATP含量显著升高（P<0.05）。结论高浓度Na₂SO₃ （5 mmol/L）可能抑制人正常肝细胞自噬而致细胞损伤，其机制可能是通过降低LC3B和p62蛋白表达来实现的。

亚硫酸钠暴露对人正常肝细胞自噬的影响及其作用机制

李明虹¹, 杨舒筠², 李丹¹, 李星道¹, 刘希冲¹, 高文婷¹, 姬晓彤¹, 白剑英¹

1. 山西医科大学公共卫生学院环境卫生学教研室, 太原 030001;
2. 太原市疾病预防控制中心有害生物控制科, 太原 030001

收稿日期:2021-08-23出版日期:2022-05-30发布日期:2022-05-28
通讯作者:白剑英
作者简介:李明虹(1994-),女,硕士研究生.
基金资助:山西省自然科学基金（201701D121140）


关键词: 亚硫酸钠暴露, 人正常肝细胞, 自噬, 影响, 作用机制
Abstract: Objective To investigate the effects of sodium sulfite (Na₂SO₃) exposure on autophagy of normal human hepatocytes and the underlying mechanism of action. Methods HL-7702 cells were exposed to a sodium medium with final concentrations of 0 (negative control),0.1,1,2.5,or 5 mmol/L Na₂SO₃ or to 20 mmol/L CCl₄ (positive control) for 2 and 48 h,respectively. Cell viability was assayed by a CCK-8 kit in each group of Na₂SO₃ for 2 h and 48 h to measure the cell survival rates. Fluorescence intensity of the autophagy-related protein,LC3B,was detected by immunofluorescence in each group of Na₂SO₃-exposed cells for 2 h and 48 h. Western blotting analysis was used to detect the expression levels of the autophagy-related proteins,LC3B,p62,and AMPK,and the apoptosis-related protein,caspase-3,in each Na₂SO₃-exposed group for 2 h and 48 h. The content of ATP in HL-7702 cells was determined by luciferase chemiluminescence exposure to each concentration of Na₂SO₃ within 2 h and 48 h. Results Compared with the negative control group,the cell survival rates of the 1,2.5,and 5 mmol/L Na₂SO₃ exposure groups decreased for 2 h and 48 h (P< 0.05). With an increase in Na₂SO₃ dosage,the survival rate of HL-7702 cells showed a decreasing trend. The survival rate of cells in the positive control group also decreased significantly (P< 0.05). Compared with the negative control group,the immunofluorescence intensity of LC3B in HL-7702 cells decreased significantly in the 2.5 and 5 mmol/L Na₂SO₃ exposure groups and in the positive control group for 48 h. Compared with the negative control group,the protein expression of LC3B-Ⅱ and p62 in HL-7702 cells was significantly decreased in the 5 mmol/L Na₂SO₃ exposure group for 48 h (P< 0.05) and the protein expression of cleaved caspase-3 was increased in the 1,2.5,and 5 mmol/L Na₂SO₃ exposure groups for 48 h (P< 0.05). Compared with the negative control group,the ATP content in HL-7702 cells increased significantly in the groups exposed to 0.1,1,2.5,and 5 mmol/L Na₂SO₃ for 2 h and 48 h (P< 0.05). Conclusion High concentrations of sodium sulfite (5 mmol/L) may inhibit autophagy of normal human hepatocytes and cause cell damage. The underlying mechanism of action may be associated with a reduction in the expression of LC3B and p62.
Key words: sodium sulfite exposure, human normal hepatocytes, autophagy, effect, mechanism
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