Akkermansia muciniphila in the small intestine improves liver fibrosis in a murine liver cirrhosis model

Abstract

Recent evidence indicates that liver cirrhosis (LC) is a reversible condition, but there is no established intervention against liver fibrosis. Although the gut microbiota is considered involved in the pathogenesis of LC, the underlying mechanisms remain unclear. Although the antibiotic, rifaximin (RFX), is effective for hepatic encephalopathy (HE) with LC, the impact of RFX on intestinal bacteria is unknown. We investigated the bacterial compositions along the GI tract under RFX treatment using a murine LC model. RFX improved liver fibrosis and hyperammonemia and altered the bacterial composition in the small intestine. The efficacy of RFX was associated with increases in specific bacterial genera, including Akkermansia. Administration of a commensal strain of Akkermansia muciniphila improved liver fibrosis and hyperammonemia with changing bacterial composition in the small intestine. This study proposed a new concept "small intestine-liver axis" in the pathophysiology of LC and oral A. muciniphila administration is a promising microbial intervention.

Fig. 1. Rifaximin improved liver fibrosis and hyperammonemia in a murine model of liver cirrhosis.

A Quantification of the area of liver fibrosis in the liver cirrhosis (LC) group (n = 10) and control group (n = 10). Representative microscopic images of liver tissues stained with hematoxylin and eosin and Azan from each group are shown. Scale bars: 100 µm. B Blood ammonia concentrations in the LC and control groups. C Study design to assess the impact of rifaximin (RFX) on the gut microbiota. D Quantification of the area of liver fibrosis in the LC-no treatment (NTx) group (n = 19), the LC-RFX group (n = 18), and the control-NTx group (n = 19). Representative microscopic images of liver tissues from the LC-NTx and LC-RFX groups are shown. Scale bars: 100 µm. E Blood ammonia concentrations in the LC-NTx, LC-RFX, and control-NTx groups. F Correlation analysis of the area of liver fibrosis and blood ammonia concentration in the LC-NTx, LC-RFX, and control-NTx groups. G mRNA expression in the liver tissues of the LC-NTx, LC-RFX, and control-NTx groups. H Endotoxin concentrations in the portal veins in the LC-NTx, LC-RFX, and control-NTx groups. Data are means ± standard errors of the means (circle: male; triangle: female). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, Mann–Whitney U test for two groups, Kruskal–Wallis test and Dunn’s test for three groups. Correlation analysis was performed with Spearman’s rank correlation coefficient.

Fig. 2. Analysis of gut microbiota at multiple sites in the gastrointestinal tract and stools.

A Bacterial load evaluated with qPCR of tuf gene in male animals of the liver cirrhosis (LC)-no treatment (NTx) group (n = 9), the LC-rifaximin (RFX) group (n = 8), and the control-NTx group (n = 10). B Shannon diversity indices for the gastrointestinal (GI) sites and stools of male animals in the LC-NTx group, the LC-RFX group, and the control-NTx group. C Principal coordinates analysis (PCoA) plot based on weighted UniFrac distances of bacterial compositions in male animals in the LC-NTx group and LC-RFX group. D Differences in weighted UniFrac distances at each GI site and in stools of the LC-NTx group and the LC-RFX group. E Multivariate association analysis of the clinical features (area of liver fibrosis and blood ammonia concentration) and bacterial compositions at the genus level in the jejunum and ileum using samples from both sexes with adjustment for sex (n = 19 in the LC-NTx group and n = 18 in the LC-RFX group). Data on bacterial load, Shannon index, PCoA plot, and weighted UniFrac distances in female animals are shown in Supplementary Fig. 2. Data are means ± standard errors of the means. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, Kruskal–Wallis test and Dunn’s test.

Fig. 3. Akkermansia muciniphila improved liver fibrosis and hyperammonemia in a murine model of liver cirrhosis.

A Study design to assess the effects of Akkermansia muciniphila. B Quantification of area of liver fibrosis in the liver cirrhosis (LC)-no treatment (NTx)2 group (n = 20) and the LC-Akkermansia (AKK) group (n = 20). Representative microscopic images of liver tissues stained with hematoxylin and eosin and Azan are shown. C Blood ammonia concentrations in the LC-NTx2 and LC-AKK groups. D mRNA expression in the liver tissues of the LC-NTx2 and LC-AKK groups. E Endotoxin concentrations in the portal veins of the LC-NTx2 and LC-AKK groups. Data are means ± standard errors of the means (circle: male; triangle: female). *p < 0.05, **p < 0.01, ***p < 0.001, Mann–Whitney U test.

Fig. 4. Akkermansia muciniphila induced compositional changes in the small-intestinal microbiota.

A Bacterial load evaluated with qPCR of tuf gene in male animals in the liver cirrhosis (LC)-no treatment (NTx)2 group (n = 10) and the LC-Akkermansia (AKK) group (n = 10). B Shannon diversity index in male animals of the LC-NTx2 and LC-AKK groups. C Principal coordinates analysis (PCoA) plots based on weighted UniFrac distances of bacterial composition in the jejunum and ileum in male animals in the LC-NTx2 and LC-AKK groups. D Multivariate association analysis between two groups (the LC-NTx2 and LC-AKK groups) and bacterial composition at the genus level in the jejunum and ileum using samples from both sexes, with adjustment for sex (n = 20 in both the LC-NTx2 and LC-AKK groups). Data on bacterial load, Shannon index, and PCoA plots in female animals are shown in Supplementary Fig. 7. Data are means ± standard errors of the means. *p < 0.05, Mann–Whitney U test. Bacterial composition was compared with permutational analysis of variance.