Effect Of DIM In Improving Nonalcoholic Fatty Liver And Regulating Gut Microbiota In Mice

Background and ObjectiveNonalcoholic fatty liver disease(NAFLD)is a chronic,non-communicable metabolic disease that can pose a serious threat to human health.Previous studies have shown that 3’3-Diindolylmethane(DIM),which is widely found in cruciferous natural plants,can improve fat accumulation,inflammation,glucose tolerance and insulin sensitivity,and DIM also has a regulatory effect on gut microbiota.It is not clear whether DIM can influence hepatic glucolipid metabolism and inflammatory response through gut microbiota in a high-fat diet-induced NAFLD mouse model,and thus can prevent and improve the development of NAFLD.The aim of this study is to explore the effect of DIM on the progression of NAFLD mice and the regulation of their intestinal flora,so as to find new ways and methods for the prevention and treatment of NAFLD.MethodsIn this study,a mouse model of non-alcoholic fatty liver was constructed using the nutritional disorders method.SPF grade 4-week-old male C57BL/6J mice were divided into standard diet control group(Control):given standard chow+gavage(PBS buffer+0.1%DMSO),high-fat group(HFD)group:high-fat chow+gavage(PBS buffer+0.1%DMSO),DIM low-dose intervention group(HFD+DIM5):high-fat feed+gavage(5 mg/Kg DIM solution+0.1%DMSO),DIM high-dose intervention group(HFD+DIM20):high-fat feed+gavage(20 mg/Kg DIM solution+0.1%DMSO)for 19 weeks 6 times a week.At the 18th week of the experiment,one mouse in each of the Control and HFD groups was dissected and its liver removed.The livers of the mice were observed visually and then examined histopathologically and the hepatocytes showed fatty lesions,indicating that the model was successfully constructed.Mice were monitored daily for food intake replacement,body weight was monitored at a fixed time each week,and feces were collected every two weeks.Oral glucose tolerance test was performed 2 weeks before the end of modeling and insulin resistance test was performed 1 week before.At the end of the experiment,the livers,white fat and brown fat of the mice were weighed,and the livers were stained with H&E and oil red O.The levels of liver triglyceride(TG)and total cholesterol(TC)were measured by enzymatic assay,and the levels of alanine transaminase(ALT)and aspartate transaminase(AST)were measured by mouse serum.RT-qPCR was performed to analyze the expression levels of liver lipid metabolism and inflammatory factors,and intestinal barrier function factors in mice.High-throughput sequencing of 16S r RNA gene was performed in mouse feces,and Alpha diversity,species distribution differences at phylum and genus levels,marker species differences,spearman correlation analysis of intestinal flora and lipid metabolism indicators,predictive analysis of intestinal flora function.Statistical analysisData were statistically processed and analyzed using SPSS 25.0 and Graphpad prism 8.0 statistical software.Data in the graphs are expressed as mean± standard deviation(x±s),and one-way ANOVA was used to assess whether the differences were statistically significant.p-values are expressed as*for P<0.05 or**for P<0.01,and***for P<0.001 to consider the differences statistically significant;ns,not statistically significant.Results1.Effects of DIM on body weight and glucose metabolism in mice with high-fat-induced non-alcoholic fatty liverThe mice in the HFD group showed significant weight gain,liver weight and white fat compared to the Control group,and the mice showed reduced glucose tolerance and insulin sensitivity.After DIM treatment,it was found that the body weight and liver weight of mice in the two dose groups were significantly reduced,and the glucose tolerance and insulin sensitivity were improved toward the Control group;while the weight of white fat only decreased significantly in the HFD+DIM20 group,and no significant difference in brown fat intake of mice in each group was observed.2.Effect of DIM intervention on hepatic lipid metabolism in mice with nonalcoholic fatty liver diseaseThe appearance of the liver of mice in HFD group increased in size,light color and blunt rounded edges,after DIM treatment the liver of mice in HFD+DIM5 group was light red in color,the volume and size did not change significantly compared with the appearance of Control group,the appearance of the liver of mice in HFD+DIM20 group tended to Control group liver.The results of H&E and oil red O staining showed that the fat of hepatocytes of mice in HFD+DIM5 group and HFD+DIM20 group became to significantly improved compared with HFD group,and the hepatocytes of mice in HFD+DIM20 group tended to Control group.TC and TG appeared to be increased in mice in the HFD group,but were improved in both dose groups after treatment with DIM intervention.After treatment with DIM intervention,hepatic lipid synthesis factors sterol regulatory element-binding protein-1c(SREBP-1),acetyl-coa carboxylase 1(ACC-1),Fatty acid synthase(FAS),stearoyl coa desaturase 1(SCD1),and peroxisome proliferator-activated receptor y(PPARy)gene expression levels were decreased compared with the HFD group.3.Effect of DIM intervention on liver injury and inflammatory response in mice with non-alcoholic fatty liver diseaseMice in the HFD group showed an increase in AST and ALT,which were improved in both dose groups after the DIM intervention.The hepatic inflammatory factors monocyte chemotactic protein-1(MCP-1),interleukin 1β(IL-1β),and interleukin 6(IL-6)gene expression levels also showed a decrease after DIM intervention treatment compared to the HFD group.4.Effect of DIM intervention on intestinal barrier in nonalcoholic fatty liver disease miceThe impaired colonic barrier function in the HFD group of high-fat-induced NAFLD mice was manifested by reduced expression levels of cytoplasmic tight adhesion protein I(Zonula occludens-1,ZO-1),occludin(ocludin)gene in colonic tissues,which were up-regulated and the intestinal barrier function was significantly improved.5.Effect of DIM intervention on intestinal flora in mice with non-alcoholic fatty liver diseaDIM significantly increased the abundance of the Bacteroidetes,and decreased the abundance of the Firmicute,improved the ratio of Firmicute/Bacteroidetes,as well as increased the abundance of beneficial species of the genera Muribaculaceae,Prevotellaceae,and Bacteroidetes,and decreased the relative abundance of the harmful group Firmicute,significantly improving the dysbiosis of the gut microbiota.The KEGG analysis revealed a strong association between the flora and lipid metabolism,glucose metabolism,and amino acid metabolism,and we also performed Spearman correlation analysis,which showed a significant correlation between microbiota and metabolic parameters such as body weight,liver weight,white brown fat,and hepatic TG and TC.ConclusionsOur results showed that DIM significantly improved obesity,hepatic lipid metabolism and inflammatory status in NAFLD mice,and further improved the onset and progression of NAFLD disease by affecting and regulating gut microbiota.