亚硫酸钠暴露对人正常肝细胞自噬的影响及其作用机制

Effect of sodium sulfite exposure on autophagy of normal human hepatocytes and its mechanism

目的探讨亚硫酸钠(Na_(2)SO_(3))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)、Na_(2)SO_(3)暴露组(0.1、1、2.5、5 mmol/L)和20 mmol/L CCl_(4)(阳性对照组)2 h、48 h。采用CCK-8法检测不同浓度Na_(2)SO_(3)暴露2 h、48 h后各组细胞存活率;免疫荧光法检测Na_(2)SO_(3)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Na_(2)SO_(3)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Na_(2)SO_(3)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 Na_(2)SO_(3)暴露组2 h、48 h细胞存活率随Na_(2)SO_(3)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 Na_(2)SO_(3)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 Na_(2)SO_(3)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 Na_(2)SO_(3)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 Na_(2)SO_(3)暴露组HL-7702细胞中ATP含量显著升高(P<0.05)。结论高浓度Na_(2)SO_(3)(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。

Objective To investigate the effects of sodium sulfite(Na_(2)SO_(3))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 Na_(2)SO_(3)or to 20 mmol/L CCl_(4)(positive control)for 2 and 48 h,respectively.Cell viability was assayed by a CCK-8 kit in each group of Na_(2)SO_(3)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 Na_(2)SO_(3)-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 Na_(2)SO_(3)-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 Na_(2)SO_(3)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 Na_(2)SO_(3)exposure groups decreased for 2 h and 48 h(P<0.05).With an increase in Na_(2)SO_(3)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 Na_(2)SO_(3)exposure groups and in the positive control group for 48 h.Compared with the negative control group,the protein expression of LC3B-Ⅱand p62 in HL-7702 cells was significantly decreased in the 5 mmol/L Na_(2)SO_(3)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 Na_(2)SO_(3)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 Na_(2)SO_(3)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.