Probiotic Bacillus licheniformis ZW3 Alleviates DSS-Induced Colitis and Enhances Gut Homeostasis

Abstract

Despite Bacillus species having been extensively utilized in the food industry and biocontrol as part of probiotic preparations, limited knowledge exists regarding their impact on intestinal disorders. In this study, we investigated the effect of Bacillus licheniformis ZW3 (ZW3), a potential probiotic isolated from camel feces, on dextran sulfate sodium (DSS)-induced colitis. The results showed ZW3 partially mitigated body weight loss, disease activity index (DAI), colon shortening, and suppressed immune response in colitis mice, as evidenced by the reduction in the levels of the inflammatory markers IL-1β, TNF-α, and IL-6 (p < 0.05). ZW3 was found to ameliorate DSS-induced dysfunction of the colonic barrier by enhancing mucin 2 (MUC2), zonula occluden-1 (ZO-1), and occludin. Furthermore, enriched beneficial bacteria Lachnospiraceae_NK4A136_group and decreased harmful bacteria Escherichia-Shigella revealed that ZW3 improved the imbalanced gut microbiota. Abnormally elevated uric acid levels in colitis were further normalized upon ZW3 supplementation. Overall, this study emphasized the protective effects of ZW3 in colitis mice as well as some potential applications in the management of inflammation-related diseases.

Keywords: Bacillus licheniformis; colitis; gut microbiota; intestinal mucosal barrier; purine metabolite; uric acid.

Figure 1

Bacillus licheniformis ZW3 (ZW3) attenuates DSS-induced colitis symptoms. (A) Schematic of experimental design. (B) Body weight loss, (C) DIA, (D) representative colon images, (E) colon length of the mice with free drinking water (control), DSS (DSS), and gavaging ZW3 and DSS (ZW3). (F) Representative images of hematoxylin and eosin-stained colonic sections and histological score. (G) Neutrophil number/HPF and colonic myeloperoxidase (MPO) activity. Immunohistochemical analysis of the colonic inflammation markers MPO (H) and F4/80 (I). Scale bar: 100 μm. * p < 0.05, ** p < 0.01, and *** p < 0.001; ns, not significant.

Figure 2

ZW3 suppressed DSS-induced inflammatory responses in colitis mice. Real-time PCR assay for the pro-inflammation genes (A) IL-1β, (B) TNF-α, and (C) IL-6 in colonic tissues. ELISA assay for the contents of inflammatory cytokines (D) IL-1β, (E) TNF-α, and (F) IL-6 in the colon tissue. * p < 0.05, ** p < 0.01, and *** p < 0.001; ns, not significant.

Figure 3

ZW3 attenuated DSS-induced colonic barrier dysfunction. (A) PAS staining of the colon with its mucin secretion rate; scale bar: 500 μm. Immunofluorescence staining and its relative fluorescence intensity of MUC2 (B), ZO-1 (C), and occludin (D) in the colonic section from with free drinking water (control), DSS (DSS), and ZW3 plus DSS (ZW3). Blue: DAPI; green: MUC2 and occludin; and red: ZO-1. Scale bar: 100 μm. (E) The protein expression and densitometric analysis of MUC2, ZO-1, and occludin. * p < 0.05, ** p < 0.01, and *** p < 0.001.

Figure 4

ZW3 regulated the gut microbiota composition in DSS-induced colitis mice. α-diversity of (A) Chao1 and (B) Shannon index. (C) β diversity analysis of the intestinal microbiota using the PCA method. Bar chart of the bacterial community composition at (D) the phylum level and (E) the genus level. (F) Heatmap of the correlation between the relative abundance of bacteria and inflammatory-related mediators using Spearman’s rank correlation test. Relative abundance of (G) Escherichia−Shigella and (H) Lachnospiraceae_NK4A136_group at the genus level. Data are shown as the means ± SD. * p < 0.05, ** p < 0.01, and *** p < 0.001 (n = 5).

Figure 5

ZW3 altered metabolic profiles in the colitis mice. (A) PLS-DA score plot of the DSS and ZW3 groups. (B) Pathway enrichment analysis of the significantly altered metabolites in colonic colitis mice and ZW3-treated colitis mice. The color indicated the significance of enrichment, and the redder indicated more significance. Concentrations of uric acid in the feces (C) and serum (D) of mice (n = 8). (E) Effect of ZW3 and its CFS (cell-free supernatant) on uric acid concentration in vitro. Data are means ± SD. * p < 0.05, ** p < 0.01, and ns, not significant.