Mechanism Of Steatosis Induced By Vinyl Chloride Metabolites Combined With Fatty Acids In HepG2 Cells

Objective:In this study,HepG2 cells were treated with chloroacetaldehyde(the main metabolite of vinyl chloride)and palmitic acid(one of the important components of saturated fatty acids).To investigate the role of oxidative stress and endoplasmic reticulum stress in low-concentration vinyl chloride combined with a high-fat diet-induced hepatic steatosis,which provides a scientific basis for the formulation of occupational health and safety measures for workers exposed to vinyl chloride.Methods:HepG2 cells were treated with different concentrations of chloroacetaldehyde(CAA)or palmitic acid(PA)or N-acetyl-L-cysteine(NAC,antioxidants)or 4-phenylbutyric acid(4-PBA,endoplasmic reticulum stress inhibitors)for 24 h or 48 h.The CCK-8 method was used to detect the effects of different toxicants on cell survival.According to this,they were divided into control group,4.5μM CAA group,9μM CAA group,PA(100 μM)group,4.5μM CAA + PA group,9μM CAA + PA group,NAC(2 m M)group,NAC +4.5μM CAA + PA group,NAC + 9μM CAA + PA group,4-PBA(0.5 m M)group,4-PBA + 4.5μM + PA group and 4-PBA + 9 μM CAA + PA group.After exposure 48 h,Oil Red O staining,quantification and determination of TG and TC content were performed to evaluate lipid accumulation.The kits were used to detect the levels of MDA,SOD,GSH-Px and GSH in cells to assess the oxidative stress.The relative expression levels of endoplasmic reticulum stress marker(GRP-78)and fatty acid synthesis-related proteins(SREBP-1,FAS,ACC)in cells were detected by Western-blot assay.SPSS24.0software was used to conduct one-way ANOVA and factorial design ANOVA for the data,and the test level was 0.05.Results:1.Cell survival rate: The survival rate of HepG2 cells gradually decreases as the dose of CAA or PA or NAC or 4-PBA increases.2.Intracellular lipid content: The number of red-stained lipid droplets,TG and TC content in HepG2 cells in the 9μM CAA group,PA group and combination groups were higher than those in the control group(P < 0.05).The number of red-stained lipid droplets,TG and TC content in the 9μM CAA combined with PA group were higher than the single-exposure groups(that is,the PA group,4.5μM CAA group and 9μM CAA group)(P < 0.05).3.The content of intracellular oxidative stress indicators: The MDA content of HepG2 cells in the 9μM CAA group,PA group and CAA combined with PA groups increased compared with the control group and 4.5μM CAA group,and the SOD level was lower than that of the control group and 4.5μM CAA group(P < 0.05).The MDA content in HepG2 cells of the 9μM CAA combined with PA group was higher than that of the single-exposure groups,and the SOD content was lower than that of the single-exposure groups(P < 0.05).The levels of GSH-Px and GSH in HepG2 cells in the PA group and CAA combined with PA groups were lower than those in the control group(P < 0.05),and the levels of GSH-Px and GSH in HepG2 cells in the CAA combined PA groups were lower than those in the single-exposure groups(P < 0.05).4.The relative expression of endoplasmic reticulum stress and de novo lipogenesis-related proteins: The expression of GRP-78 and ACC in HepG2 cells of the PA group and CAA combined with PA groups was higher than that of the control group(P < 0.05).The expression of GRP-78 and ACC in the cells of different doses of CAA combined with PA groups was higher than that of corresponding doses of CAA group(P< 0.05).The expression of SREBP-1 in HepG2 cells in the CAA combined with PA group was higher than that in the control group(P < 0.05),and the expression of SREBP-1 in HepG2 cells in the 9μM CAA combined with PA group was higher than that in the CAA-exposed groups(P < 0.05).The expression of FAS in the HepG2 cells of the9μM CAA group,PA group and CAA combined with PA groups was higher than that of the control group(P < 0.05),and the expression of FAS in the cells of different doses of CAA combined with PA group was higher than that of the corresponding dose of CAA group(P < 0.05).5.After the inhibitor NAC or 4-PBA intervention in the combined exposure group,the expression of lipid content,oxidative stress,endoplasmic reticulum stress and de novo lipogenesis-related proteins:(1)The number of red-stained lipid droplets,TG and TC content in CAA combined with PA groups pretreated with NAC or 4-PBA were significantly less than those in the corresponding dose of CAA combined with PA group(P < 0.05).(2)NAC or 4-PBA pretreatment alleviated the oxidative stress induced by CAA combined with PA(P < 0.05).(3)The relative expression of GRP-78 in the cells of the 4-PBA pretreatment with9μM CAA combined with PA group was lower than that in the 9μM CAA combined with PA group(P < 0.05).The relative expression of SREBP-1,FAS and ACC in the cells of NAC or 4-PBA pretreatment with 9μM CAA combined with PA group was significantly lower than that of 9μM CAA combined with PA group(P < 0.05).6.All the results of the factorial analysis found no interaction between CAA and PA,and the joint action between the two may be additive.Conclusion:1.CAA aggravated the steatosis of HepG2 cells induced by PA.2.Low-dose vinyl chloride activated oxidative stress and endoplasmic reticulum stress,and then up-regulated the expression of de novo lipogenesis-related proteins,thereby promoting hepatic steatosis caused by a high-fat diet.3.Vinyl chloride combined with a high-fat diet has an additive effect in inducing hepatic steatosis.