Effect And Mechanism Of Subchronic Vinyl Chloride Exposure Combined With High-Fat Diet On Hepatic Steatosis In Mice

Objective:In this study,mice was treated with a 13-week exposure:sub-chronic vinyl chloride monomer inhalation exposure,high-fat diet,and a combination of the two,by detected oxidative stress,ERS related indicators and intestinal flora changes,to explore the effects of high-fat diet,VCM subchronic exposure and the combination of the two on hepatic steatosis in mice and its mechanism.Methods:Forty-eight healthy SPF male C57BL/6J mice were randomly divided into six groups:control group and high-fat diet group,low dose vinyl chloride monomer exposure group(160mg/m~3),high-dose vinyl chloride monomer exposure group(800 mg/m~3)and low-dose combined with high-fat diet group(160mg/m~3),high-dose combined with high-fat diet group(800mg/m~3).The inhalation was carried out by means of static inhalation,5 times/week,2h/time.After 13 weeks,the rats were sacrificed after anesthesia,and various biological materials such as serum,liver tissue,stool,and the like were collected.Some livers were subjected to HE staining and oil red O staining sections.The serum levels of ALT and AST were measured to evaluate liver damage.The serum low-density lipoprotein VLDL content was measured to evaluate the changes of liver lipid transport.The serum and liver TG and TC levels were used to reflect the metabolism of liver fat.The contents of metabolic enzymes CYP2E1 and oxidative stress related indicators MDA,GSH,SOD and GSH-Px in the liver were measured.Real-time quantitative PCR(RT-PCR)and Western-Blot were used to detect endoplasmic reticulum stress marker GRP-78,SREBP-1,and fatty acids in liver tissue,respectively.Synthesis of related enzyme genes acetyl CoA carboxylase(ACC)and fatty acid synthase(FAS)mRNA expression levels and protein expression levels The composition and the diversity of the intestinal flora of mice in each group were analyzed by 16S rDNA amplicon sequencing.SPSS 24.0 software for statistical analysis.Analysis of the interaction between two factors by using factorial design analysis.Results:1.High-fat diet can induce pathological changes such as hepatic steatosis and hepatocyte injury in mice.HE staining showed scattered vacuolar degeneration,oil red O staining showed fatty red staining of liver tissue,and liver pathological changes caused by vinyl chloride monomer and high-fat diet combined were more serious.2.Serum ALT,AST,TG,TC levels in the high-fat group,VCM low-dose combined group and VCM high-dose combined group were higher compared with control group(P<0.05).The serum ALT level in the high-dose VCM exposure group was higher compared with control(P<0.05).The serum TG and TC levels in the VCM low-dose combined group were higher than those in the low-dose exposure group.The VCM high-dose combined group had higher serum TG,TC and liver TC levels than the vinyl chloride high-dose exposure group(P<0.05).There was no difference in serum VLDL levels between the groups(P>0.05).3.Changes in CYP2E1:the levels of CYP2E1 in the liver of each group were higher than those in the control group,the VCM low-dose combined group was higher than the high-fat group,and the VCM high-dose combined group was higher than the high-fat group and high-dose VCM group(P<0.05).Compared with the control group,the MDA content of the liver and the SOD level of the two combined mice were decreased(P<0.05),and the MDA content of the high-dose VCM group was higher than that of the high-fat group and the high-dose VCM group.In the high-fat group and high-dose VCM exposed group(P<0.05).The GSH content in the liver of the high-dose VCM exposed group and the two combined groups was lower than control(P<0.05).There was no difference in GSH-Px levels(P>0.05).4.Endoplasmic reticulum stress and its related indicators in the downstream:GRP-78 gene mRNA expression in high-fat group,high-dose VCM exposed group and VCM high-dose combination group were higher than the control group(P<0.05),but only the relative expression of protein in the VCM high-dose combined group was higher than that in the control group(P<0.05).The mRNA expression level of SREBP-1 gene in VCM high-dose combined group was consistent with the relative expression of protein,which was higher than that in the control group(P<0.05),and the expression of SREBP-1 protein in VCM high-dose combined group was also higher than that in the high-dose combined group(P<0.05).The mRNA expression level of FAS gene in the VCM high-dose combination group was consistent with the expression of protein,which was higher than that in the control group and single exposure group(high-fat group,low-dose VCM exposure group and high-dose VCM exposure group),with statistical difference(P<0.05),the relative expression of FAS gene protein in VCM high-dose combined group was also higher than that in the control group and single exposure group(high-fat group,low-dose VCM exposure group and high-dose VCM exposure group),with statistical difference(P<0.05).The relative expression of ACC gene and the relative expression of protein were not different(P>0.05).5.Compared with the control group,the composition and diversity of intestinal microflora in each treatment group changed to different degrees.The changes of the main dominant bacteria,Firmicutes and Bacteroidetes,were obvious.The high-fat diet group was similar to the VCM low-dose combination group,the content of thick-walled bacteria was increased,and the level of Bacteroides was decreased.The intestinal microbial types detected by the high-fat group were lower than the VCM high-dose combined group and the VCM low-dose exposure group(P<0.05).The VCM low-dose combined group was lower than the control group,the VCM high-dose combined group and the low-dose VCM exposure group(P<0.05).Compared with the control,the intestinal microbial composition and diversity of all treatment groups were different,but the species composition of the high-fat group and the VCM low-dose combined group were very similar,and the differences within the group were small.Conclusion:1.High-fat diet can induce elevated blood lipids and hepatic lipids,cause the hepatocyte damage,hepatic steatosis,and a low concentration of vinyl chloride monomer exposure has not detected detectable liver damage,but the level of hepatic steatosis and hepatocyte injury in the high-fat diet group was higher than that in the single-factor exposure group(high-fat group and VCM low-and high-dose exposure groups),and vinyl chloride monomer exposure can enhance liver damage caused by high-fat diet.2.Both vinyl chloride and high-fat diets can cause the increase of cytochrome P450 enzyme CYP2E1 content.VCM low-dose combined group and VCM high-dose combined group can cause statistically significant changes in oxidative stress index,VCM high-dose combined high-fat diet can cause oxidative stress and ERS,then the SREBP-1 were activated.The expression of ACC and FAS mRNA and protein in lipid de novo synthesis is increased,which may lead to increased fat synthesis in the liver,leading to hepatic steatosis has occurred.3.High-fat diet and inhalation of vinyl chloride can induce intestinal flora imbalance in mice.In the distribution of dominant flora,compared with the control group,the mice have different degrees of the proportion of Firmicutes increased,and Bacteroidetes had different proportions of reduction.There was no significant difference in the intestinal microbial composition between the low-dose VCM combined group and the high-fat group,but the high-dose VCM combined group and the low-dose VCM combined group produced different intestinal microbial compositional changes.Exposure to high-fat diet,vinyl chloride monomer and the combination of both can lead to varying degrees of intestinal flora imbalance.