Modulation of gut microbiota mediates berberine-induced expansion of immuno-suppressive cells to against alcoholic liver disease

Abstract

Background: Berberine is an isoquinoline alkaloid compound derived from many herbs, which has been used extensively to improve liver function. But action mechanism of its hepatoprotection in alcoholic liver disease (ALD) is far from being clear.

Aim: To investigate the underlying mechanism of berberine's therapeutic effect on ALD associated with gut microbiota-immune system axis.

Method: An animal model fed with ethanol that mimics drinking pattern ideally in ALD patients was established. Liver function was evaluated by biochemical test and histological examination. Immune cells were detected by flow cytometry and feces samples were collected for 16S rRNA gene amplicon sequencing.

Results: We first reported the promising beneficial effect of berberine on ameliorating acute-on-chronic alcoholic hepatic damage and explored the underlying mechanism involving gut microbiota-immune system axis. Notably, berberine activated a population with immune suppressive function, defined as granulocytic- myeloid-derived suppressor cell (G-MDSC)-like population, in the liver of mice with alleviating alcohol-induced hepatic injury. Berberine remarkably enhanced the increase of G-MDSC-like cells in blood and liver and decreased cytotoxic T cells correspondingly. Suppression of G-MDSC-like population significantly attenuated the protective effect of berberine against alcohol. Berberine activated IL6/STAT3 signaling in in vitro culture of G-MSDCs-like population, while inhibition of STAT3 activity attenuated the activation of this population by berberine. Moreover, berberine changed the overall gut microbial community, primarily increased the abundance of Akkermansia muciniphila. Of note, depletion of gut microbiota abolished the inducing effect of berberine on G-MDSC-like population, and attenuated its hepatoprotective effect against alcohol in mice, suggesting intestinal flora might be involved in mediating the expansion of this protective population.

Conclusion: Collectively, this study delivered insight into the role of immunosuppressive response in ALD, and facilitated the understanding of the pharmacological effects and action mechanisms of berberine.

Keywords: alcoholic liver injury; berberine; granulocytic-myeloid-derived suppressor cell-like population; gut microbiota.

FIGURE 1

Berberine treatment significantly reduced alcoholic liver injury. Mice were divided into five groups, control group (N = 15) receiving control Lieber‐DeCarli diet and vehicle treatment, model group (N = 15) receiving ethanol Lieber‐DeCarli diet and vehicle treatment, low‐dose berberine group (N = 15, 10 mg/kg), medium‐dose berberine group (N = 15, 50 mg/kg), high‐dose berberine group (N = 15, 100 mg/kg) receiving ethanol Lieber‐DeCarli diet. Five mice of each group were sacrificed at day 11, day 22, and day 33, respectively. A, Serum ALT and AST level of mice from groups mentioned above at different time points. Berberine treatment significantly reduced the elevated serum ALT and AST levels at checked time points. No statistical differences among different dose groups of berberine were found. Thus, the dose of 10 mg/kg was used in the following experiments (B). Representative H&E staining images of livers from mice of control group, model group, and berberine group (10 mg/kg). The lipid deposition and steatosis are indicated by arrows. C, Scoring of liver histology. D, Serum contents of inflammatory cytokines including IFN‐γ, TNF‐α, IL‐1β, MCP‐1, IFN‐β, IL‐17A, IL‐10, IL‐27, and GM‐CSF determined by using LEGENDplex™ Mouse Inflammation Panel (13‐plex) with flow cytometer in mice from control group, model group, and berberine group. Berberine reduced the alcohol‐induced increased levels of IFN‐γ, TNF‐α, and IL‐1β. ^* P < .05, ^** P < .01, ^*** P < .001, ^**** P < .0001; n.s., not significant

FIGURE 2

Berberine mediated the increase of G‐MDSC‐like cells to protect liver from alcohol‐induced injury. The populations of G‐MDSC‐like cells, M‐MDSC, or T cells in blood, liver, and spleen of mice were determined by flow cytometer. Berberine significantly promoted the increase of G‐MDSC‐like cells in liver and blood. A, The representative histogram images and quantification (b) of flow cytometric analyses of G‐MDSC‐like cells and M‐MDSC in blood and liver of mice of control group, ethanol model group, and berberine group. C, Quantification of flow cytometric analyses of T helper and cytotoxic T cells in blood and spleen of mice. Anti‐ly6G antibody or vehicle was treated to ethanol‐fed mice (N = 5 for each group). D, Representative histogram images and quantification (E) of flow cytometric analyses of G‐MDSC‐like cells in liver of mice treated with anti‐Ly6G or vehicle. F, Serum ALT and AST level of mice treated with anti‐Ly6G or vehicle. G, Representative H&E staining images of liver of mice treated with anti‐Ly6G or vehicle and scoring of histological damage (H). The lipid deposition and steatosis are indicated by arrows. ^* P < .05, ^** P < .01, ^*** P < .001; n.s., not significant; BBR, berberine

FIGURE 3

Berberine sensitized G‐MDSC‐like population by activating IL‐6/STAT3 pathways. Bone marrow derived cells were co‐cultured with berberine (10 µM) for 24 or 48 h in RPMI1640 supplemented with 10% fetal bovine serum, IL‐10, and GM‐CSF. Afterward, cells were collected for population examination, total RNA and protein extraction. A, The representative histogram images, and quantification (B) of flow cytometric analyses of G‐MDSC‐like cells after 24 or 48 h in vitro culture. C, The m‐RNA relative expression level of targets including Oct4, SOX2, Arginase‐1, IL‐10, GM‐CSF, IL‐6, soluable IL‐6 receptor (SIL6R), S100A9, S100A8, and STAT3 in cells cultured in vitro. D, The protein expression level of p‐STAT3 determined by western blotting in cells cultured in vitro. E, The content of IL‐6 in serum of mice. F, The population of G‐MDSC‐like cells in the presence of STAT3 activation inhibitor. ^* P < .05, ^** P < .01, ^*** P < .001, ^**** P < .0001; n.s., not significant

FIGURE 4

Diversity and richness of the gut microbiota in mice and responses of the structure of the gut microbiota to berberine treatment. Chromosomal DNA was extracted from feces sample of mice. After sample quality tests, 13 samples (N = 3 for control group, N = 5 for model group, N = 5 for berberine group) were qualified. Gene amplification and sequencing of the V4 region of 16S rRNA were then performed by The Beijing Genomics Institute. A, Shannon curve. B, Rarefaction OUT estimates. C, Shannon index. D, PCA score plot. E, PCoA score plot based on unweighted and weighted Unifrac metrics. F, Unweighted pair group method with arithmetic mean (UPGMA) based on unweighted Unifrac (F) or weighted Unifrac (G) analysis. ^** P < .01, ^*** P < .001; n.s., not significant

FIGURE 5

Berberine regulated alcohol‐induced disordered gut microbiota. A, The taxonomic composition distribution in samples of phylum‐level. B, Log‐scaled percentage heat map of genus‐level. C, The taxonomic composition distribution in samples of genus‐level. D, The taxonomic composition distribution in samples of species‐level

FIGURE 6

Gut microbiota is actively involved in the beneficial effect of berberine. PGF mice were built up by antibiotics treatment. These PGF mice received ethanol‐ Lieber‐DeCarli diet with berberine treatment (10 mg/kg) or vehicle treatment for 11 days (N = 5 for each group). Then liver injury was evaluated and the population of G‐MDSC‐like cells in mice were examined by flow cytometer. A, Serum ALT and AST level of mice from control group, ethanol model group, berberine group, berberine with antibiotics group, and vehicle with antibiotics group. B, Representative H&E staining images of livers and scoring of histological damage (C) from mice of ethanol model group, berberine group, berberine with antibiotics group, and vehicle with antibiotics group. Moreover, cecal contents were collected from mice of berberine group and then treated to mice receiving ethanol‐Lieber‐DeCarli diet for 11 days (N = 5 for each group). D, ALT and AST level of mice from ethanol model group and feces transplant group. E, Representative H&E staining images of liver and scoring of histological damage (F) from mice of model group and feces transplant group. G, The representative histogram images and quantification (H) of flow cytometric analyses of G‐MDSC‐like cells in liver of mice from ethanol group, berberine group, and berberine with antibiotics group. I, The population of T helper and cytotoxic T cells in liver of mice from ethanol group, berberine group, and berberine with antibiotics group. J, The population of G‐MDSC‐like cells and M‐MDSC in blood of mice from ethanol group, berberine group, berberine with antibiotics group. ^* P < .05, ^** P < .01; n.s., not significant; BBR, berberine