The Protective Effects of Live and Pasteurized Akkermansia muciniphila and Its Extracellular Vesicles against HFD/CCl4-Induced Liver Injury

Abstract

Akkermansia muciniphila, as a member of the gut microbiota, has been proposed as a next-generation probiotic. Liver fibrosis is the main determinant of liver dysfunction and mortality in patients with chronic liver disease. In this study, we aimed to determine the beneficial effects of live and pasteurized A. muciniphila and its extracellular vesicles (EVs) on the prevention of liver fibrosis. The response of hepatic stellate cells (HSCs) to live and pasteurized A. muciniphila and its EVs was examined in quiescent, lipopolysaccharide (LPS)-activated LX-2 cells. Liver fibrosis was induced in 8-week-old C57BL/6 mice, using a high-fat diet (HFD) and carbon tetrachloride (CCl4) administration for 4 weeks. The mice were concomitantly treated via oral gavage with three forms of bacteria. The relative expression of different fibrosis and inflammatory markers was assessed in the tissues. Histological markers, serum biochemical parameters, and cytokine production were also analyzed, and their correlations with the relative abundance of targeted fecal bacteria were examined. All A. muciniphila preparations exhibited protective effects against HSC activation; however, EVs showed the greatest activity in HSC regression. Oral gavage with A. muciniphila ameliorated the serum biochemical and inflammatory cytokines and improved liver and colon histopathological damages. The relative expression of fibrosis and inflammatory biomarkers was substantially attenuated in the tissues of all treated mice. The composition of targeted stool bacteria in the live A. muciniphila group was clearly different from that in the fibrosis group. This study indicated that A. muciniphila and its derivatives could successfully protect against HFD/CCl4-induced liver injury. However, further studies are needed to prove the beneficial effects of A. muciniphila on the liver. IMPORTANCE Akkermansia muciniphila, as a member of the gut microbiota, has been proposed as a next-generation probiotic. Liver fibrosis is the main determinant of liver dysfunction and mortality in patients with chronic liver disease. In this study, we aimed to determine the beneficial effects of live and pasteurized A. muciniphila and its extracellular vesicles (EVs) on the prevention of liver fibrosis. The results of the present study indicated that oral administration of live and pasteurized A. muciniphila and its EVs could normalize the fecal targeted bacteria composition, improve the intestinal permeability, modulate inflammatory responses, and subsequently prevent liver injury in HFD/CCl4-administered mice. Following the improvement of intestinal and liver histopathology, HFD/CCl4-induced kidney damage and adipose tissue inflammation were also ameliorated by different A. muciniphila treatments.

Keywords: Akkermansia muciniphila; extracellular vesicles; hepatic stellate cells; intestinal bacteria; liver fibrosis.

FIG 1

(A) A heatmap plot of microarray data sets meta-analysis. A P value of <0.05 was considered statistically significant by Mann-Whitney method. (B) PCA plot shows separated cluster between two groups. (C) Scanning electron microscopy shows spherical morphology of EVs (magnification: ×60,000). Inhibition of HSC activation and protective effects of all A. muciniphila supplementations in LX-2 cell line. mRNA level of HSCs activation-related genes, (D) α-SMA, (E) TIMP1, (F) Col1a1, (G) TGF-β, (H) TLR-2, and (I) TLR-4, in quiescence and LPS-activated LX-2 cells. Data are expressed as mean ± standard deviation (SD) (n = 5). *, P < 0.05; **, P < 0.01; and ***, P < 0.001 by post hoc Turkey’s one-way ANOVA.

FIG 2

(A) Study design of the animal experiment. (B) Liver weight and (C) body weight changes were measured at the indicated time points by post hoc two-way ANOVA, and (D) liver/body weight ratio was measured by post hoc Turkey’s one-way ANOVA statistical analysis. Data are expressed as mean ± SD (n = 5). *, P < 0.05 and **, P < 0.01. (E) Representative images of gross specimens of liver, liver specimens stained with hematoxylin & eosin, liver specimens stained with Masson’s trichrome, and colon specimens stained with hematoxylin & eosin. All bacterial treatments improved liver injury and fibrosis in HFD/CCL4 mice.

FIG 3

Serum biochemical and cytokines measurement. Serum levels of (A) TNF-α, (B) IL-6, and (C) IL-10; inflammatory and anti-inflammatory cytokines. Serum liver enzymes (D) ALT and (E) AST. Serum level of (F) Glu, (G) TG, (H) total cholesterol, (I) HDL, (J) VLDL, and (K) LDL. Data are expressed as mean ± SD (n = 5). *, P < 0.05; **, P < 0.01; and ***, P < 0.001 by post hoc Turkey’s one-way ANOVA.

FIG 4

Hepatic mRNA expression of liver fibrosis-related genes (A) TGF-β, and (B) α-SMA. (C) Western blotting of α-SMA. Hepatic mRNA expression of liver fibrosis-related genes (D) TIMP1, (E) Col1a1, and (F) PDGF. Hepatic mRNA expression of inflammatory-related genes in mice liver tissue, (G) TLR-2, (H) TLR-4, and (I) TNF-α. Data are expressed as mean ± SD (n = 5). *, P < 0.05; **, P < 0.01; and ***, P < 0.001 by post hoc Turkey’s one-way ANOVA.

FIG 5

All A. muciniphila supplementation reduced inflammatory- and fibrosis-related mRNA expression levels in colon, adipose, and kidney tissue. Relative mRNA levels of different inflammatory and fibrosis markers in colon tissue, (A) TLR-2, (B) TLR-4, (C) ZO1, (D) TNF-α, (E) TGF-β, (F) α-SMA, and (G) PDGF. Relative mRNA levels of inflammatory and fibrosis markers in adipose tissue, (H) α-SMA, (I) TGF-β, (J) PDGF, (K) TNF-α, (L) IL-6 and (M) IL-10. Relative mRNA levels of fibrosis markers in kidney tissue, (N) α-SMA, (O) TGF-β, (P) PDGF, and (Q) Col1a1. Data are expressed as mean ± SD (n = 5). *, P < 0.05; **, P < 0.01; and ***, P < 0.001 by post hoc Turkey’s one-way ANOVA.

FIG 6

The relative percentage of targeted gut microbiome. Data are expressed as mean ± SD (n = 5). (A) Gut microbiome composition at the phylum level. (B) Ratio of Firmicutes to Bacteroidetes. (C) Gut microbiome composition at the class/family level. (D) Gut microbiome composition at the genus level. *, P < 0.05; **, P < 0.01 by nonparametric Kruskal–Wallis test. (E) Correlation analysis between targeted gut microbiota and biological indices. Spearman’s rho nonparametric correlation was used, and significant relationships with P value of <0.05 and r-rank of >0.5 are shown. Blue nodes: biological indices. Purple nodes: differentially distributed genera between groups; the thickness of the connection represents the correlation.

FIG 7

Oral administration of different forms of A. muciniphila could normalize the fecal targeted bacteria composition, improve the intestinal permeability, modulate inflammatory responses, and subsequently prevent liver injury in the HFD/CCl4 mice model. Following the improvement of intestinal and liver histopathology, HFD/CCl4-induced kidney damage and adipose tissue inflammation were also ameliorated by different A. muciniphila treatments.