2.4 Empagliflozin ameliorated liver fibrosis in HFD/STZ-induced T2DM mice in association with alteration of gut microbiota
To elucidate the protective mechanism of Empagliflozin, we used 16S rDNA high-throughput sequencing to examine the effect of Empagliflozin on the gut microbiota of HFD/STZ-induced T2DM mice. As shown in Figure 3A, significant changes were observed in the relative abundance at the phylum among three groups. As illustrated in the Venn diagram, 311 OUTs were present in the three groups of mice gut microbiota. Compared with the total number of OUTs in T2DM groups, the total number of OUTs in Control group and Empagliflozin group was significantly higher (Figure 3B). α diversity analysis further demonstrated that T2DM remarkably reduced the abundance and diversity of the gut microbiota in mice whereas Empagliflozin administration reversed this effect (Figure 3C). The principal coordinate analysis (PCoA) that evaluates phylogenetic similarities between microbial communities, is often used to calculate the 𝛽-diversity values. As indicated in Figure 3D, three groups were clearly separated into different clusters (p < 0.05) following PCoA analysis. Similarly, the hierarchical cluster analysis also revealed the phylogenetic separation among three groups, indicating that gut dysbiosis during liver fibrosis and the alleviation by Empagliflozin administration (Figure 3E). Meanwhile, according to the analysis of top twenty abundance on species, the abundance of Lactobacillus was significantly increased, while the abundances of Ruminococcus andAdlercreutzia were significantly reduced in Empagliflozin group comparing to T2DM group (Figure 3F and 3G).
As shown in Figure 3H, we performed a correlation analysis to investigate the relationship between the changes in intestinal flora and the improvement of liver fibrosis and glucose metabolism by Empagliflozin. The results showed that Oscillospira ,Ruminococcus andAdlercreutzia were positively correlated with liver fibrosis score, fasting blood glucose, random blood glucose, AUC (OGTT) AND AUC (IPGTT), while Sutterella and Bifidobacterium were negatively correlated with the above indicators. Correlation analysis suggests that intestinal flora is involved in the pathological processes of DM and liver fibrosis, and Empagliflozin may reverse the above processes by changing the structure and function of the gut microbiota.
DISCUSSION
In this study, we aimed to investigate the effect of Empagliflozin on liver fibrosis in HFD/STZ-induced T2DM mice and explored the relationship between the anti-fibrotic effect of Empagliflozin and the alteration of gut microbiota. Here, we discovered that Empagliflozin attenuated the liver fibrosis induced by HFD/STZ, which was consistent to the reports in previous literatures9,21. Combined with the promoting effect of intestinal flora on liver fibrosis22 and the alleviating effect of Empagliflozin on liver fibrosis23, we further explored the potential mechanism and revealed that Empagliflozin altered the intestinal flora of liver fibrosis. More important, the further data revealed thatLactobacillus ,Ruminococcus , and Adlercreutzia may be the vital microbiota for Empagliflozin to improve liver fibrosis in HFD/STZ-induced T2DM mice.
Liver health is essential for human and animals24. At present, NAFLD has gradually become a global health problem, especially its interaction with DM and other metabolic diseases increases the intractability of NAFLD3,25. Therefore, an effective strategy that can simultaneously improve glucose metabolism and slow down the progression of NAFLD, such as SGLT2 inhibitors, has a good therapeutic prospect26. In recent years, the anti-liver fibrosis effect of SGLT2 inhibitors has been gradually confirmed21,27. However, the mechanisms by which SGLT2 inhibitors attenuate liver fibrosis, especially in addition to their glucose-lowering effects, are still unclear and need to be further explored. In recent years, many studies have found that SGLT2 inhibitors can improve various diseases such as diabetic kidney disease28 and atherosclerosis14 by modulating gut microbiota, suggesting that gut microbiota may also play an important role in their alleviation of liver fibrosis. The intestinal flora is now considered to be a novel virtual metabolic organ that maintains homeostasis in the host. When homeostasis is imbalanced and disrupted, the gut microbiota and its metabolites migrate to the liver, leading to a series of pathological changes in the liver, such as liver fibrosis29,30. Based on the evidence of gut microbiota disorder during liver fibrosis and the ability of Empagliflozin to regulate different human diseases by affecting the composition of gut microbiota, we performed 16S rRNA sequencing of mice feces and found that Empagliflozin significantly increased the abundance ofLactobacillus and decreased the abundance of Ruminococcusand Adlercreutzia . Lactobacillus is a common probiotic and its increased abundance has been shown to have beneficial effects on the liver. For instance, heat-killed Lactobacillus plantarum has been proven to alleviate liver fibrosis31 and Lactobacillusstrain LGG can attenuate liver fibrosis by inhibiting liver bile acid production32. The same to previous studies33,34, Ruminococcus is considered as potential pathogenic bacteria and significantly positively correlated with degree of liver fibrosis. Similarly, the increased abundance ofAdlercreutzia in different diseases is associated with fibrosis35. These evidences suggest that Empagliflozin may alleviate liver fibrosis by reversing the aforementioned gut microbiota disorder.
However, the exact mechanism by which SGLT2 inhibitors attenuates liver fibrosis through gut microbiota remains unclear. Since immune inflammatory mechanisms are one of the main pathological mechanisms of liver fibrosis, and gut microbiota can also promote liver fibrosis through altered abundance and mediated inflammatory responses36, the ameliorative effect of SGLT2 inhibitors may be related to the mediating effect on gut microbiota by reducing liver inflammation. When the intestinal barrier function is damaged and intestinal permeability increases, bacteria and endotoxins in the intestine will enter the liver through the portal vein. Subsequently, macrophages (Kupffer cells) in the liver are activated by lipopolysaccharides (endotoxins), which release a large number of inflammatory factors. Various cytokines interact with inflammatory mediators, further causing liver damage and intestinal mucosal damage37. Lactobacillus , as a probiotic, is able to produce bacteriocins, such as antibiotics that inhibit pathogens of the Enterobacteriaceae family (such as Salmonella orShigella ). And the peroxidase system of Lactobacillus has an inhibitory effect on other bacteria, which can reduce the number of bacteria entering the liver from the intestine and slow down the process of liver fibrosis38. In addition, the promotion of liver fibrosis by Ruminococcus may be related to the production of an inflammatory polysaccharide. The polysaccharide can effectively induce inflammatory cytokines secretion by immune cells and may accelerate the process of liver fibrosis34. Previous study39 has reported an increase in the abundance ofAdlercreutzia during HFD, which is positively correlated with the metabolite glycerophospho-N-palmitoyl ethanolamine (GP-NPEA) and negatively correlated with sanguisorbic acid dilactone. GP-NPEA is the precursor of palmitoyl ethanolamine (PEA) that exerts anti-inflammatory activity40, while sanguisorbic acid dilactone can inhibit lipid peroxidation41. These results suggest that SGLT2 inhibitors may ameliorate liver immune inflammation by reversing the aforementioned alterations in gut microbiota and related metabolites, thereby exerting anti-fibrotic effects. However, further study is needed to explore the above potential mechanisms of SGLT2 inhibitors against liver fibrosis.