In this study, we demonstrated that the gut microbiota and intrahepatic cytokine profiles in mice were significantly disturbed by 16-week HFD. However, 8-week FMT intervention corrected the gut microbiota disturbance to a certain degree and increased the production of butyrate in the cecal content along with the correction of the imbalance of pro- and anti-inflammatory cytokines and the reversion of steatohepatitis in mice chronic fed with HFD. These results indicated that FMT may have profound implications in the treatment of NASH.

With NASH becoming more and more prevalent worldwide, it is urgent to find an economical and effective therapeutic approach to prevent the onset and progression of NASH. Rapidly increasing evidence supports the association between NASH and the gut microbiota16. Meanwhile, FMT has emerged as a highly effective treatment for some gastrointestinal diseases such as gastrointestinal infections and inflammatory bowel disease. There is a growing number of studies on the intestinal microbiota or FMT in a wide range of non-gastrointestinal diseases, including metabolic disorders5, 17. A previous clinical study conducted by Vrieze showed that the insulin sensitivity of recipients increased along with the levels of butyrate-producing intestinal microbiota18. Although the internal mechanisms are still unknown, this offers a rationale for novel therapeutic interventions for metabolic diseases, including NASH. In our study, we found that after 8 weeks of FMT intervention, the metabolism-related indicators, such as body weight, liver index, and epididymal fat index, were obviously improved, with a tendency toward improvement in FBG, HOMA-IR and ISI. This may suggest a beneficial effect of FMT in HFD-induced metabolic disturbances. Furthermore, serum levels of transaminases, and the degree of hepatic steatosis, lobular inflammation and hepatocyte ballooning were markedly attenuated after FMT intervention.

To elucidate the underlying mechanisms, we detected the gut microbiota of the different groups at baseline and the 16th week. According to the PCoA, NMDS and the hierarchical cluster analysis, FMT reversed the overall changes of the gut microbiota structure induced by HFD, and the HFD + FMT were distributed between the HFD and control groups at the 16th week. At the phylum level, the Bacteroidetes phylum was decreased and the Firmicutes phylum was increased after FMT intervention compared with the HFD group. Although the Firmicutes/Bacteroidetes ratio was still inconsistent in patients with NAFLD2, bacteria from the Firmicutes phylum could efficiently ferment fiber into butyrate, while bacteria from the Bacteroidetes phylum were involved in fermenting fiber into acetate and propionates. I19. Our results showed that proportion of Lactobacillus from Firmicutes phylum and butyrate level in the gut was both significantly elevated after FMT intervention. This result is supported by the finding that the Bacterial strains from Lactobacillus could increase the proportions of butyrate-producing bacterial strains20, and that lactate from Lactobacillus could promote butyrate production in feces21. Nonetheless, acetate and propionate were not significantly decreased in mice on HFD following a slight decrease in Bacteroidetes. The underlying mechanism was not clear in current study. The fermentation process to produce short-chain fatty acids in the gut is rather complex under different conditions. Bacteroidetes could ferment fiber into acetate and propionate, besides, there are other pathways involved in production of acetate and propionate22. Based on those backgrounds, we speculate that a slight decrease in Bacteroidetes as we observed here may not necessarily lead to a significant decrease in the basic levels of acetate and propionate in the gut, since they are affected by multiple other factors. And Butyrate has multiple beneficial effects on mammals. Previous studies showed that butyrate protected the intestinal barrier function and maintained the integral bifidogenic effect23, 24, which is fundamental in the maintenance of gut health. Our results showed that the tight junction protein ZO-1 was also significantly upregulated after FMT intervention along with improved intestinal morphometry and decreased levels of serum endotoxin. Increased intestinal permeability and metabolic endotoxemia following deleterious composition of the intestinal microbiota contributes to the progression of NASH5. The endotoxin is derived from the gram-negative bacteria. The relative abundance of gram-negative, putative “pro-inflammatory” bacteria in various family and genus level taxa from the phylum Bacteroidetes were also decreased after FMT intervention compared with other groups. Decrease in gram-negative bacteria might contribute to reduced production of endotoxin in the gut, which may be partially responsible for decreased endotoxin level in the FMT group than the healthy controls as we observed here25. In addition, the proportion of Lactobacillus was elevated the most after FMT intervention. Lactobacillus is a well-known probiotic with multiple beneficial effects on body metabolism. Lactobacillus could decrease the endotoxin level, inhibit endotoxin-induced inflammation and regulate immunity. Increase of Lactobacillus after FMT intervention might further reduce opportunistic pathogens and endotoxin level26,27,28,29. Alleviation of endotoxemia was further supported by the decreased expression of TLR4 and its downstream signaling protein Myd88 in the liver after FMT intervention. These results suggest that FMT might attenuate HFD-induced NASH via restoring intestinal health. FMT intervention significantly decreased the TGF-β1 and α-SMA level, to a level even lower than those of the control group. There are several explanations for this finding. First, TLR4 plays a pivotal role in the pro-inflammatory response and liver fibrosis, the downregulation of TLR4 signaling pathway following decreased endotoxin after FMT would lead to the inhibition of TGF-β1 and α-SMA expression30. Second, others showed that NaB could bring the expression of TGF-β1 and α-SMA down to a level lower than those of the control group via inhibiting histone deacetylases31, 32. In the current study, NaB was significantly elevated in HFD + FMT group compared with that of the control group and HFD group. When comparing the taxonomy profile at the genus level, Lactobacillus and Christensenellaceae were increased in the HFD + FMT group, and this increase may be related to the bifidogenic effect of FMT via the fermentation product butyrate33. A previous study showed that Christensenellaceae is positively associated with a low body mass index in humans and reduced weight gain in mice34. Oscillibacter was increased in the HFD group but decreased after FMT intervention at the present study. This finding is consistent with another study that shows a positive and strong association of Oscillibacter with metabolic syndrome and the development of obesity-related metabolic disorders35, 36. Although the mechanisms of interaction between these specific gut microbes and the host metabolism still need further exploration, our results suggest that the modulation of the gut microbiota might be an effective strategy for managing NASH.

Gut microbiota-host immune maladaptation has been implicated in the rising incidence of NASH. The progression of NASH is greatly associated with the impaired immune microenvironment37, 38. The gut microbiota metabolite butyrate, which obviously increased after FMT intervention, is a pivotal immune regulator and anti-inflammatory substance. It improves the hepatic insulin resistance and attenuates HFD-induced steatohepatitis23, 39,40,41. In this study, the pro-inflammatory factors in the liver, such as TNF-α, MCP-1, IL-1 and IL-6, were decreased in the FMT-treated mice. This change would favor an anti-inflammatory immune microenvironment in the liver. Accordingly, the TLR4-Myd88 pathway, which was also depressed by FMT, could aggravate the development of insulin resistance42. In addition, PPAR-α, a gene that mainly participates in fatty acid oxidation, and PPAR-γ, a gene that plays pivotal roles in the regulation of lipid metabolism and inflammation as well as in insulin sensitivity42, 43, were both increased in the liver and epididymal fat tissue, while TG and cholesterol were decreased in the liver after FMT intervention. And hepatic steatosis was attenuated in the absent of improved serum fasting glucose, insulin level or HOMA-IR. The improvement in hepatic steatosis may result from enhanced intrahepatic insulin sensitivity after FMT. Others showed that in mice with NASH, supplementation of sodium butyrate caused increased expression of insulin receptor in the liver, leading to improved liver insulin sensitivity and hepatic steatosis41. Here we also showed that in mice subjected to FMT, the protein level of insulin receptor was upregulated, following a significant increase of butyrate concentration in cecal content after FMT. These findings warrant further studies to reveal other factors and mechanisms in enhancing intrahepatic insulin sensitivity after FMT.

Different subsets of T cells are involved in the progression of NASH. These cells contribute to an impaired liver immune microenvironment. Butyrate has the ability to regulate T cell differentiation23, 40, which suggests that FMT might correct the ratios of T cell subsets via the gut metabolite butyrate. Our results showed that IFN-γ (Th1) and IL-17 (Th17) were decreased and IL-4 (Th2), IL-22 (Th22) and Foxp3 (Treg) were increased by FMT in HFD-fed mice. These changes may result in a preferred T cell subset38, 44,45,46. Existing evidence revealed that the reduction of Treg cells in the liver could promote the transformation of simple hepatic steatosis to steatohepatitis. Animal experiments indicated that adoptive transfer of Treg cells into mice could alleviate HFD-induced steatohepatitis38, 47. Cytokines such as IFN-γ and IL-17 from Th1 and Th17 cells can promote the release of IL-6 and TNF-α, which aggravate liver insulin resistance and steatohepatitis46, 48. Previous work showed that intrahepatic IL-17 was significantly elevated in NASH patients. IL-17 could inhibit the insulin-signaling pathway and aggravate hepatocyte steatosis and the extent of liver inflammation49, 50. IL-22 is known to be a hepatocyte protector that could inhibit liver steatosis and liver damage45, 51. All the above research supports the point that FMT might be beneficial in immunologic balance of the liver.

Our study has the following limitations. First, we did not perform a metabolomic analysis of the cecal content. This analysis would be helpful to elucidate the influences of FMT on mice. Second, we only conducted the 16s rRNA sequencing of the gut microbiota. Whole genome sequencing would be more precise for finding the internal interactions from the perspective of gene function.

In summary, our results showed that FMT attenuated HFD-induced steatohepatitis in mice. until now, patients who undergo FMT are mainly via endoscopy, so it is impossible to conduct FMT on a high frequency. Our result supports a possible way of oral FMT through more economic and convenient methods such as feces capsules, which is under investigation in human. Development of microbiome-targeted therapeutic strategies should be considered to open the door to new ways of prevention and treatment of NASH.