Investigation Of The Mechanism Through Which A High-fructose Diet Induces Hepatic Steatosis By Affecting The Gut Microbiome And Immune Microenvironment

Objective:Metabolic diseases are often associated with high fructose(HF)intake.HF consumption can also alter the gut microbiota,which in turn contributes to the development of non-alcoholic fatty liver disease(NAFLD).However,the underlying mechanisms of gut microbiota in this metabolic disorder remain unclear.Therefore,in this study,we examined the effects of gut microbiota changes in a HF diet mouse model on immune cells in the liver and mesenteric lymph nodes,and further investigated the impact of these altered immune environments on hepatic steatosis.Methods:Methods:Mice fed with a high fructose diet(60%fructose)for 12 weeks were used as the experimental group,while mice fed a normal diet served as the control group.Throughout the experiment,we continuously monitored changes in fasting blood glucose and body weight of mice.Glucose tolerance tests(GTT)and insulin tolerance tests(ITT)were performed at the end of weeks 4,8,and 12.HE staining was used to analyze the effects of HF on the overall structure of the mouse liver,white adipose tissue,brown adipose tissue,and colon.Oil Red O staining was used to observe the effects of HF on lipid deposition in the mouse liver.An automatic biochemical analyzer was used to detect changes in ALT,AST,TG,and T-CHO levels in mouse serum.Serum levels of inflammatory factors TNF-α,IL-1β,and IL-6 were measured by ELISA.The effects of HF on mouse gut microbiota were analyzed using 16S r DNA sequencing.The expression levels of tight junction proteins(ZO-1,occludin,and claudin-1)in the colons of both groups of mice were detected by Western blot.Flow cytometry was used to analyze changes in the number of CD3~+CD4~+T cells,CD3~+CD8~+T cells and M1 macrophages in the mouse liver,as well as Th1 and Treg cell numbers in mesenteric lymph nodes(MLN).IHC technique was used to detect the infiltration of CD4~+T T cells and CD8~+T cells in the ileum.RT-q PCR was used to detect the m RNA levels of FAS,ACC1,CD36,Ch REBP,and SREBP1c in mouse liver,and Western blot was used to detect the protein levels of FASN,ACC1,CD36,Ch REBP,and SREBP1c.Germ-free mice were obtained by administering a combination of antibiotics(ampicillin,neomycin,vancomycin,and metronidazole)and were then subjected to fecal microbiota transplantation(FMT)experiments to observe their effects on the high-fructose-induced metabolic disorder mouse model.Results:From the beginning to the end of the experiment,there was no significant difference in body weight between the HF group and the ND group mice.During the first7 weeks of the experiment,there was no significant difference in fasting blood glucose levels between the two groups of mice.However,starting from the 8th week,the fasting blood glucose levels of the HF group mice significantly increased.At 4 weeks,the HF diet did not cause liver damage but did cause damage to the intestine and adipose tissue.After12 weeks,the livers of the HF group mice showed significant hepatocyte swelling and degeneration,ballooning changes,and lipid droplet aggregation.Further analysis of the intestinal microbiota composition revealed that the HF diet reduced the ratio of Bacteroidetes/Firmicutes in the mouse intestine and increased the levels of Blautia,Lachnospiraceae,and Oscillibacter genera.In addition,feeding a high-fructose diet for 8weeks significantly increased the expression of pro-inflammatory cytokines TNF-α,IL-6,and IL-1βin serum.In the mesenteric lymph nodes(MLN)of the HF group mice,the number of pro-inflammatory Th1 cells significantly increased,while the number of anti-inflammatory Treg cells significantly decreased.After fecal microbiota transplantation(FMT),which effectively reduced the proportion of CD3~+CD4~+T cells,CD3~+CD8~+T cells and M1 macrophages in the liver and Th1 cells in the mesenteric lymph nodes of mice in the HF group,and increased the proportion of Treg cells,thereby reducing the levels of ALT,AST,TG and TCHO in plasma.The histological manifestations of hepatocyte ballooning and lipid deposition were alleviated,thereby improving NAFLD induced by an HF diet.Conclusions:In summary,gut microbiota dysbiosis and impaired intestinal barrier may be early events following a high-fructose diet,while liver inflammation and hepatic steatosis are likely subsequent effects.Hepatic steatosis induced by a high-fructose diet may be caused by immune dysregulation mediated by impaired intestinal barrier and altered gut microbiota.FMT effectively ameliorated hepatic steatosis and systemic metabolic disorder by correcting immune imbalances in the liver and intestine.