Bacillus coagulans-Pectin Synbiotic Modulates Gut Microbiota Composition and Attenuates Ethanol-Induced Alcoholic Liver Disease in Mice

Abstract

Alcohol abuse and alcoholic liver diseases (ALD) are globally prevalent, with alcohol-induced gut microbiota dysbiosis playing a key role in ALD pathogenesis. Synbiotic (combinations of probiotics and prebiotics) are recognized as effective in reducing inflammation in ALD. Bacillus coagulans, a probiotic with favorable industrial and functional traits (e.g., sporulation, lactic acid production), shows potential in treating intestinal diseases. Here, we investigated the effects of B. coagulans, alone or combined with pectin, on ethanol-induced ALD in mice. Synbiotic supplementation (B. coagulans + pectin) more significantly alleviated ethanol-induced ALD severity than B. coagulans or pectin alone. Relative to the ethanol group, synbiotic treatment significantly reduced hepatic inflammatory injury and lipid accumulation, downregulated proinflammatory factors (TNF-α, IL-1β, myeloperoxidase [MPO]), and upregulated tight junction proteins and mucins-enhancing intestinal barrier function. Moreover, these supplements modulated gut microbiota composition and enhanced short-chain fatty acids (SCFAs) production by increasing the abundance of beneficial SCFA-producing bacteria (Muribaculaceae, Akkermansia). In summary, changes in tight junction proteins, cytokines and hepatic injury markers indicate that the synbiotic alleviated overall inflammation in the experimental ALD model and exerted a greater therapeutic effect than B. coagulans or pectin alone.

Keywords: Bacillus coagulans; alcoholic liver diseases; intestinal barrier; microbiota; pectin.

Figure 1

Treatment effect of B. coagulans, pectin and the synbiotic on ethanol-induced ALD mice. (A) Growth curves of B. coagulans in YPD, medium with pectin as carbon source (YP + Pectin) and medium without carbon source (YP). (B) Schematic representation of the ethanol-induced ALD mouse model utilized in this study. Arrows denote intragastric administration of phosphate-buffered saline (PBS) or B. coagulans treatments. (C) Ratio of liver weight to body weight. (D) Serum AST level. (E) Serum ALT level. Data are presented as means ± SEM. Values with distinct superscript letters (a, b and c) indicate significant differences (p < 0.05) as determined by one-way ANOVA followed by Tukey’s test. Ctrl: healthy control group; EtOH: ethanol-induced group; B. co: supplementation of B. coagulans group; Pectin: supplementation of pectin group; B. co + P: supplementation of B. coagulans and pectin group.

Figure 2

B. coagulans, pectin and the synbiotic reduced histological damage on ethanol-induced ALD mice. (A) Liver triglyceride concentration. (B) Histological assessment of steatosis in liver sections, with representative micrographs of H&E staining (top) and Oil Red O staining (bottom). Scale bars = 50 μm. Data are presented as means ± SEM. Values with distinct superscript letters (a, b and c) indicate significant differences (p < 0.05) as determined by one-way ANOVA followed by Tukey’s test. Ctrl: healthy control group; EtOH: ethanol-induced group; B. co: supplementation of B. coagulans group; Pectin: supplementation of pectin group; B. co + P: supplementation of B. coagulans and pectin group.

Figure 3

Regulatory effects of B. coagulans, pectin and their synbiotic on immune markers in ethanol-induced mice. ELISA was used to analyze the protein levels of hepatic cytokines, including (A) IL-1β, (B) TNF-α and (C) MPO. Data are presented as means ± SEM. Values with distinct superscript letters (a, b and c) indicate significant differences (p < 0.05) as determined by one-way ANOVA followed by Tukey’s test. Ctrl: healthy control group; EtOH: ethanol-induced group; B. co: supplementation of B. coagulans group; Pectin: supplementation of pectin group; B. co + P: supplementation of B. coagulans and pectin group.

Figure 4

B. coagulans, pectin and their synbiotic formulation ameliorated the impaired barrier function in ethanol-induced mice. (A) Serum lipopolysaccharide levels. (B) The mRNA levels of tight junction proteins (Occludin, Claudin and ZO-1) and mucin protein Muc2 were detected by RT-qPCR analysis in colon. Data are presented as means ± SEM. Values with distinct superscript letters (a, b and c) indicate significant differences (p < 0.05) as determined by one-way ANOVA followed by Tukey’s test. Ctrl: healthy control group; EtOH: ethanol-induced group; B. co: supplementation of B. coagulans group; Pectin: supplementation of pectin group; B. co + P: supplementation of B. coagulans and pectin group.

Figure 5

B. coagulans, pectin and the synbiotic regulated the composition of gut microbiota. (A) Venn diagram representing the common species between all five group. (B) Principal Components analysis of Bray–Curtis dissimilarities between all five group. (C) Fecal bacterial relative abundance at the phylum level. (D) The relative abundance of fecal bacterial in genus. (E) The heatmap of different species for B. coagulans, pectin and the synbiotic group. (n = 6 per group); Ctrl: healthy control group; EtOH: ethanol-induced group; B. co: supplementation of B. coagulans group; Pectin: supplementation of pectin group; B. co + P: supplementation of B. coagulans and pectin group.

Figure 6

The SCFAs expression levels in fecal samples. Concentrations of fecal acetate (A), propionate (B), and butyrate (C). (n = 6 per group). Data are presented as means ± SEM. Values with distinct superscript letters (a, b and c) indicate significant differences (p < 0.05) as determined by one-way ANOVA followed by Tukey’s test. Ctrl: healthy control group; EtOH: ethanol-induced group; B.co: supplementation of B. coagulans group; Pectin: supplementation of pectin group; B.co + P: supplementation of B. coagulans and pectin group.