The anti-inflammatory effects of Akkermansia muciniphila and its derivates in HFD/CCL4-induced murine model of liver injury

Abstract

Inflammation plays a critical role in the promotion of hepatocyte damage and liver fibrosis. In recent years the protective role of Akkermansia muciniphila, a next-generation beneficial microbe, has been suggested for metabolic and inflammatory disorders. In this study, we aimed to evaluate the effects of live and pasteurized A. muciniphila and its extra cellular vesicles (EVs) on inflammatory markers involved in liver fibrosis in a mouse model of a high-fat diet (HFD)/carbon tetrachloride (CCl₄)-induced liver injury. Firstly, the responses of hepatic stellate cells (HSCs) to live and pasteurized A. muciniphila and its EVs were examined in the quiescent and LPS-activated LX-2 cells. Next, the anti-inflammatory effects of different forms of A. muciniphila were examined in the mouse model of HFD/CCl₄-induced liver injury. The gene expression of various inflammatory markers was evaluated in liver, colon, and white adipose tissues. The cytokine secretion in the liver and white adipose tissues was also measured by ELISA. The results showed that administration of live and pasteurized A. muciniphila and its EVs leads to amelioration in HSCs activation. Based on data obtained from the histopathological analysis, an improvement in gut health was observed through enhancing the epithelium and mucosal layer thickness and strengthening the intestinal integrity in all treatments. Moreover, live A. muciniphila and its EVs had inhibitory effects on liver inflammation and hepatocytes damage. In addition, the tissue cytokine production and inflammatory gene expression levels revealed that live A. muciniphila and its EVs had more pronounced anti-inflammatory effects on liver and adipose tissues. Furthermore, EVs had better effects on the modulation of gene expression related to TLRs, PPARs, and immune response in the liver. In conclusion, the present results showed that oral administration of A. muciniphila and its derivatives for four weeks could enhance the intestinal integrity and anti-inflammatory responses of the colon, adipose, and liver tissues and subsequently prevent liver injury in HFD/CCL₄ mice.

Figure 1

(A) Study design of the animal experiment. Anti-inflammatory effects of all A. muciniphila supplementations in LX-2 cell line. The mRNA Level of tlr-5 in quiescence and LPS-activated LX-2 cells treated with (B) Lam (C) Pam and (D) EVs; and tlr-9 in quiescence and LPS-activated LX-2 cells treated with (E) Lam (F) Pam and (G) EVs. Un: untreated cells, Lam: live A. muciniphila, Pam: pasteurized A. muciniphila, EV: extra cellular vesicles of A. muciniphila. 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 statistical analysis.

Figure 2

Anti-inflammatory effects of all A. muciniphila supplementations in LX-2 cell line. The mRNA Level of ppar-α in quiescence and LPS-activated LX-2 cells treated with (A) Lam (B) Pam and (C) EVs; and ppar-γ in quiescence and LPS-activated LX-2 cells treated with (D) Lam (E) Pam and (F) EVs. Un: untreated cells, Lam: live A. muciniphila, Pam: pasteurized A. muciniphila, EV: extra cellular vesicles of A. muciniphila. 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 statistical analysis.

Figure 3

(A) Histological structure of colon in different study groups using H&E staining. The infiltration of inflammatory cells (black arrow) was observed in the mucous membrane of the PBS group. The decrease in thickness of mucosal layer (white arrows) and thickness of the epithelium (white arrowheads) of the colon were seen in histological sections of PBS group. An increase in thickness of the epithelium (white arrowheads) and thickness of mucosal layer (white arrows) was seen in Pam, Lam, and EV groups, compared to that in PBS group. histometric analysis shows the difference of (B) epithelium thickness (Epi.Th) and (C) the mucosal layer thickness (Muc.Th) (D) crypt depth and (E) histopathological score among the study groups. Assessment of a live and pasteurized A. muciniphila and its EVs effects on mRNA expression of tight junction proteins gene in the colon tissue. (F) ocldn, (G) cldn-1 and (H) cldn-2. SD: standard diet, PBS: HFD/CCL4 + PBS, Lam: HFD/CCL4 + A. muciniphila (109 CFU), Pam: HFD/CCL4 + pasteurized A. muciniphila (109 CFU) and EV: HFD/CCL4 + 50 µg EVs. Data are expressed as mean ± SD (n = 5). * p < 0.05, ** p < 0.01, *** p < 0.001 in comparison with PBS group and ## p < 0.01 in comparison with Pam by post hoc Turkey’s one-way ANOVA statistical analysis.

Figure 4

(A) The effect of a live and pasteurized A. muciniphila administration on the histopathological structure of the liver in different study groups (H&E). In the PBS group, infiltration of inflammatory cells and spotty necrosis of hepatocytes (Black arrows), lipid micro and macro vesicles (White arrowheads) were presented in the cytoplasm of hepatocytes. Lam and EV groups: without histopathological changes and the presence of bi-nucleated cells (white arrows). Pam group: infiltration of inflammatory cells and spotty necrosis of hepatocytes (Black arrows), lipid micro and macro vesicles (White arrowheads) and the presence of bi-nucleated cells (white arrows). The number of bi-nucleated hepatocytes in histological structure of liver in different study groups (mean ± SD). (B) Counting of bi-nucleated hepatocytes per mm2 (C) histopathological score. The liver tissue cytokines level (D) IL-6, (E) TNF-α and (F) IL-10. SD: standard diet, PBS: HFD/CCL4 + PBS, Lam: HFD/CCL4 + A. muciniphila (109 CFU), Pam: HFD/CCL4 + pasteurized A. muciniphila (109 CFU) and EV: HFD/CCL4 + 50 µg EVs. Data are expressed as mean ± SD (n = 5). * p < 0.05, ** p < 0.01, *** p < 0.001 in comparison with PBS group and ## p < 0.01 in comparison with Pam by post hoc Turkey’s one-way ANOVA statistical analysis.

Figure 5

Hepatic mRNA expression of inflammatory-related genes, (A) tlr-5; (B) tlr-9; (C) il-1β; (D) il-6; and anti-inflammatory-related genes (E) il-10; (F) igf; (G) ppar-α and (H) ppar-γ. SD: standard diet, PBS: HFD/CCL4 + PBS, Lam: HFD/CCL4 + A. muciniphila (10⁹ CFU), Pam: HFD/CCL4 + pasteurized A. muciniphila (10⁹ CFU) and EV: HFD/CCL4 + 50 µg EVs. Data are expressed as mean ± SD (n = 5). * p < 0.05, ** p < 0.01 and *** p < 0.001 in comparison with PBS group, # p < 0.05 and ## p < 0.01in comparison with EV by post hoc Turkey’s one-way ANOVA statistical analysis.

Figure 6

The adipose tissue cytokines level (A) IL-6; (B) TNF-α and (C) IL-10. Adipose tissue mRNA level of (G) ppar-α, (H) ppar-γ and (C) il-1β. SD: standard diet, PBS: HFD/CCL4 + PBS, Lam: HFD/CCL4 + A. muciniphila (10⁹ CFU), Pam: HFD/CCL4 + pasteurized A. muciniphila (10⁹ CFU) and EV: HFD/CCL4 + 50 µg EVs. 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 statistical analysis.