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.