Total fecal microbiota transplantation alleviates high-fat diet-induced steatohepatitis in mice via beneficial regulation of gut microbiota

Abstract

Non-alcoholic steatohepatitis (NASH) is an epidemic metabolic disease with limited therapeutic strategies. Cumulative data support the pivotal role of gut microbiota in NASH. Here, we investigated the hypothesis regarding whether fecal microbiota transplantation (FMT) is effective in attenuating high-fat diet (HFD)-induced steatohepatitis in mice. Mice were randomized into control, HFD and HFD + FMT groups. After an 8-week HFD, FMT treatment was initiated and carried out for 8 weeks. The gut microbiota structure, butyrate concentrations of the cecal content, liver pathology and intrahepatic lipid and cytokines were examined. Our results showed that after FMT, the gut microbiota disturbance was corrected in HFD-fed mice with elevated abundances of the beneficial bacteria Christensenellaceae and Lactobacillus. FMT also increased butyrate concentrations of the cecal content and the intestinal tight junction protein ZO-1, resulting in relief of endotoxima in HFD-fed mice. Steatohepatitis was alleviated after FMT, as indicated by a significant decrease in intrahepatic lipid accumulation (reduced Oli-red staining, decreased intrahepatic triglyceride and cholesterol), intrahepatic pro-inflammatory cytokines, and the NAS score. Accordingly, intrahepatic IFN-γ and IL-17 were decreased, but Foxp3, IL-4 and IL-22 were increased after FMT intervention. These data indicate that FMT attenuated HFD-induced steatohepatitis in mice via a beneficial effect on the gut microbiota.

Figure 1

FMT attenuated HFD-induced obesity, liver injury and metabolic disturbance. (A,B) Body weight changes and body weight at 16th week. (C) Liver index = liver weight/body weight × 100. (D) Epididymal fat index = epididymal fat weight/body weight × 100. (E) Energy intake per mouse per week. (F,I) Fasting serum glucose, fasting serum insulin, HOMA-IR and ISI of the three groups. J,K) Serum ALT and AST. The data represent the mean ± S.E.M. (n = 12 mice per group), ^* P < 0.05, ^** P < 0.01 and ^*** P < 0.001.

Figure 2

Influences of FMT on gut microbiota. (A,B) Scatter plot of the unweighted-unifrac-principal co-ordinates analysis (PCOA) score showing the similarity of the 18 bacterial communities based on the Unifrac distance and Scatter plot of the weighted-PCOA score. (C) Nonmetric multidimensional scaling (NMDS) showing the difference in bacterial communities according to the Bray-Curtis distance. (D) Hierarchical cluster analysis. (E,F) Bacterial composition of the different communities at the genus level (E) and at phylum level (F). Sequences that could not be classified into any known group were assigned as “no rank.” (G,H) Differences were represented in the color of the most abundant group. Key phylotypes of the gut microbiota responding to FMT treatment (G), the histogram (H) showing the lineages with LDA values as determined by LEfSe. (I,K) The acetic acid, propanoic acid and butyrate acid levels of the cecal content. The data represent the mean ± S.E.M. (n = 12 mice per group), ^*** P < 0.001.

Figure 3

Beneficial effects of FMT on the small intestine and the level of serum endotoxin. (A) HE staining of the small intestine showing that FMT restored HFD-induced mucosal damage. (B) Immunohistochemistry for ZO-1 showing that FMT increased ZO-1 expression. (C) ZO-1 mRNA expression in the small intestine. (D) The level of serum endotoxin. The data represent the mean ± S.E.M. (n = 12 mice per group), ^* P < 0.05, ^** P < 0.01 and ^*** P < 0.001.

Figure 4

FMT improved inflammation and lipid metabolism in liver. (A,B) HE and oil red O staining of liver. (C) NAS score. (D,E) the level of TG and cholesterol in the liver. (F) Lipid metabolism-associated PPAR-α and PPAR-γ gene expression in the liver. (G) Masson’s staining. (H) Endotoxin-associated gene expression of TLR4 and Myd88 and fibrosis-associated gene expression of TGF-β, Smad2, Smad7 and α-SMA. (I,J) IR protein expression level in liver. Gene expression levels are expressed as values relative to the control group. The data represent the mean ± S.E.M. (n = 12 mice per group), ^* P < 0.05, ^** P < 0.01 and ^*** P < 0.001.

Figure 5

Immunoregulation of FMT in the liver. (A) Pro-inflammation-associated gene expression in the liver. (B,C) Foxp3, IFN-γ, IL-4, IL-17 and IL-22 protein expression levels in the liver. (D–I) Immunohistochemistry for Foxp3, IFN-γ, IL-4, IL-17 and IL-22 in the liver showed that Foxp3, IL-4, and IL-22 were increased and IFN-γ and IL-17 were decreased after FMT intervention. The data represent the mean ± S.E.M. ^* P < 0.05, ^** P < 0.01 and ^*** P < 0.001.

Figure 6

FMT improved inflammation and lipid metabolism in epididymal fat tissue. (A) Lipid metabolism-associated PPAR-α and PPAR-γ gene expression in the epididymal fat tissue. (B) Gene expression levels of MCP-1 and TNF-α in the epididymal fat tissue. Gene expression levels are expressed as values relative to the control group. The data represent the mean ± S.E.M. (n = 12 mice per group), ^* P < 0.05, ^** P < 0.01 and ^*** P < 0.001.