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. 2022 Dec 21;10(6):e0297922.
doi: 10.1128/spectrum.02979-22. Epub 2022 Oct 11.

Alginate Alleviates Dextran Sulfate Sodium-Induced Colitis by Promoting Bifidobacterium animalis and Intestinal Hyodeoxycholic Acid Synthesis in Mice

Yu Pi  1   2 , Xiangyu Zhang  1 , Yujun Wu  1 , Zhenyu Wang  1 , Yu Bai  1 , Xiaoyi Liu  1 , Dandan Han  1 , Jinbiao Zhao  1 , Isabel Tobin  3 , Jiangchao Zhao  4 , Guolong Zhang  3 , Junjun Wang  1
Affiliations

Alginate Alleviates Dextran Sulfate Sodium-Induced Colitis by Promoting Bifidobacterium animalis and Intestinal Hyodeoxycholic Acid Synthesis in Mice

Yu Pi et al. Microbiol Spectr. .

Abstract

Alginate (ALG) is known to alleviate intestinal inflammation in inflammatory bowel disease, but its mechanism of action remains elusive. In the present study, we studied the involvement of the intestinal microbiota and bile acid (BA) metabolism in ALG-mediated anti-inflammatory effects in mice. A combination of 16S rRNA gene amplicon sequencing, shotgun metagenomic sequencing, and targeted BA metabolomic profiling was employed to investigate structural and functional differences in the colonic microbiota and BA metabolism in dextran sulfate sodium (DSS)-treated mice with or without dietary supplementation of ALG. We further explored the role of the intestinal microbiota as well as a selected ALG-enriched bacterium and BA in DSS-induced colitis. Dietary ALG alleviated DSS-mediated intestinal inflammation and enriched a small set of bacteria including Bifidobacterium animalis in the colon (P < 0.05). Additionally, ALG restored several bacteria carrying secondary BA-synthesizing enzymes such as 7α-hydroxysteroid dehydrogenase and BA hydrolase to healthy levels in DSS-treated mice. Although a majority of BAs were suppressed by DSS, a few secondary BAs such as hyodeoxycholic acid (HDCA) were markedly enriched by ALG. Furthermore, ALG significantly upregulated the expression of a major BA receptor, the farnesoid X receptor, while suppressing NF-κB and c-Jun N-terminal kinase (JNK) activation. Depletion of the intestinal microbiota completely abrogated the protective effect of ALG in DSS-treated mice. Similar to ALG, B. animalis and HDCA exerted a strong anti-inflammatory effect in DSS-induced colitis by downregulating inflammatory cytokines (interleukin-1β [IL-1β], IL-6, and tumor necrosis factor alpha [TNF-α]). Taken together, these results indicated that ALG achieves its alleviating effect on intestinal inflammation through regulation of the microbiota by enriching B. animalis to promote the biosynthesis of specific secondary BAs such as HDCA. These findings have revealed intricate interactions among the intestinal microbiota, BA metabolism, and intestinal health and further provided a novel strategy to improve intestinal health through targeted manipulation of the intestinal microbiota and BA metabolism. IMPORTANCE ALG has been shown to ameliorate inflammatory bowel disease (IBD), but little is known about the mechanism of its anti-inflammatory action. This study was the first to demonstrate that ALG provided a preventive effect against colitis in an intestinal microbiota-dependent manner. Furthermore, we confirmed that by selectively enriching intestinal B. animalis and secondary BA (HDCA), ALG contributed to the attenuation of DSS-induced colitis. These findings contribute to a better understanding of the mechanism of action of ALG on the attenuation of colitis and provide new approaches to IBD therapy by regulating gut microbial BA metabolism.

Keywords: alginate; bile acid; hyodeoxycholic acid; inflammatory bowel disease; microbiome.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

FIG 1
FIG 1
Dietary supplementation of alginate (ALG) alleviates DSS-induced colitis. (A) Experimental scheme of ALG supplementation in a mouse model of DSS-induced acute colitis. (B and C) While DSS (3%) was provided in drinking water for a week to induce colitis, body weight (B) and the disease activity index (DAI) score (C) were recorded daily in the final week of DSS treatment for three groups of mice (n = 8). The colon was collected from each mouse at the end of the experiment. (D to G) Representative images of the colon (D), the colon length (E), H&E staining (magnification of ×50), and TEM of the colon (F) and colonic histology score (G) are shown. (H and I) The mRNA (H) and protein (I) expression levels of three inflammatory cytokines in the colon were also measured. (J) Phosphorylation of p38, ERK1/2, JNK1/2, and p65 NF-κB in the colon. (K) The mRNA gene expression levels of two BA receptors in the colon. (L) Expression of FXR at the protein level. Data are presented as means ± SEM. *, P < 0.05; **, P < 0.01; ***, P < 0.001; #, P < 0.05; compared with the DSS plus ALG group.
FIG 2
FIG 2
Dietary supplementation with ALG alters the composition of the intestinal microbiota in DSS-treated mice. Mice supplemented with or without 5% ALG for 4 weeks, followed by induction of colitis with 3% DSS in drinking water for another week (n = 8). The colonic digesta was collected from each mouse at the end of the experiment and subjected to DNA isolation and 16S rRNA gene sequencing. (A) The α-diversity of the colonic microbiota among three groups of mice. (B) PCoA plot depicting β-diversity of the colonic microbiota based on the Bray-Curtis distance. (A to C) The colonic microbiota composition is shown at the phylum (C), genus (D), and ASVs levels (E). (F) LEfSe analysis of differential enrichment of the colonic bacteria at the ASV level (linear discriminant analysis [LDA] > 4). (G and H) Bar graphs of the top five differentially enriched bacteria are shown at the genus (G) and ASV levels (H). Data are presented as means ± SEM. *, P < 0.05.
FIG 3
FIG 3
Dietary supplementation with ALG modulates the function of the intestinal microbiota in DSS-treated mice. Mice supplemented with or without 5% ALG for 4 weeks, followed by induction of colitis with 3% DSS in drinking water for another week (n = 8). The colonic digesta was collected from each mouse at the end of the experiment and subjected to DNA isolation and shotgun metagenomic sequencing. (A) PCoA plot based on the KEGG pathways of identified bacterial genes. (B) LEfSe analysis of differential enrichment of microbial KEGG pathways (LDA > 2.5). (C) LEfSe analysis of differential enrichment of microbial carbohydrate-active enzymes (CAZymes) (LDA > 3). (D) Functional contribution of top 10 differentially enriched CAZymes. (E) Relative abundances (%) of the major enzymes involved in the biosynthesis of primary and secondary bile acids (BAs). (F) Quantitative PCR analysis of the copy number of total bacteria and specific bacterial groups carrying BA hydrolase (bsh), BA 7-dehydroxylase (baiJ), 7α-hydroxysteroid dehydrogenase (7α-HSDH), and 7β-hydroxysteroid dehydrogenase (7β-HSDH). Data are presented as means ± SEM. *, P < 0.05; **, P < 0.01; ***, P < 0.001.
FIG 4
FIG 4
Dietary supplementation with ALG alters the intestinal BA profile in DSS-treated mice. Mice supplemented with or without 5% ALG for 4 weeks, followed by induction of colitis with 3% DSS in drinking water for another week (n = 8). The colonic digesta was collected from each mouse at the end of the experiment and subjected to targeted BA profiling. (A) PCA plot of the BA profile among three groups of mice. (B) Relative abundances (%) of different BAs among three groups. (C) Z-score plot of all BAs identified among three groups. (D) Alterations of representative BAs among three groups. (E) Spearman correlation between the relative abundance of the intestinal microbiota and BA concentrations. Data are presented as means ± SEM. *, P < 0.05; **, P < 0.01; ***, P < 0.001.
FIG 5
FIG 5
Microbiota depletion abrogates the anti-inflammatory effect of ALG in DSS-treated mice. (A) Experimental scheme to show depletion of the intestinal microbiota by a cocktail of antibiotics and timing of ALG and DSS treatment (n = 10). (B and C) Body weight (B) and disease activity index (DAI) score (C) were recorded daily in the final week of DSS treatment for both groups of mice. The colon was collected from each mouse at the end of the experiment. (D to F) Representative images of the colon (D), the colon length (E), H&E staining (magnification, ×50 ) of the colon (F), and colonic histology score (G) are shown. (H and I) The mRNA (H) and protein (I) expression levels of three inflammatory cytokines in the colon were also measured. (J) The mRNA gene expression levels of two BA receptors in the colon.
FIG 6
FIG 6
Oral gavage of B. animalis alleviates DSS-induced acute colitis in mice. (A) Experimental scheme for DSS-induced colitis and B. animalis treatment (n = 8). (B) Body weight was recorded daily in the final week of DSS treatment for three groups of mice. The colon was collected from each mouse at the end of the experiment. (C to F) Representative images of the colon (C), the colon length (D), and H&E staining (magnification, ×50) and TEM image of the colon (E) and colonic histology score (F) are shown. (G and H) The mRNA gene expression levels of three inflammatory cytokines (G) and two BA receptors (H) in the colon were also measured by RT-qPCR. Data are presented as means ± SEM. *, P < 0.05; **, P < 0.01; ***, P < 0.001; #, P < 0.05; compared with the DSS plus B. animalis group.
FIG 7
FIG 7
Oral gavage of B. animalis alters the intestinal BA profile in DSS-treated mice. Mice supplemented with or without B. animalis for 4 weeks, followed by induction of colitis with 3% DSS in drinking water for another week (n = 8). The colonic digesta was collected from each mouse at the end of the experiment and subjected to targeted BA profiling. (A) PCA plot of the BA profile among three groups of mice. (B) Relative abundances (%) of different BAs among three groups. (C) Z-score plot of all BAs identified among three groups. (D) Alterations of representative BAs among three groups. Data are presented as the mean ± SEM. *, P < 0.05; **, P < 0.01.
FIG 8
FIG 8
Dietary supplementation with HDCA ameliorates DSS-induced acute colitis. (A) Experimental scheme for DSS-induced colitis and HDCA treatment (n = 12). (B) Body weight was recorded daily in the final week of DSS treatment for three groups of mice. The colon was collected from each mouse at the end of the experiment. (C to F) Representative images of the colon (C), the colon length (D), and H&E staining (magnification, ×50), TEM image of the colon (E), and colonic histology score (F) are shown. (G and H) The mRNA gene expression levels of three inflammatory cytokines (G) and two BA receptors (H) in the colon were also measured by RT-qPCR. Data are presented as the mean ± SEM. *, P < 0.05; **, P < 0.01; ***, P < 0.001; #, P < 0.05; ##, P < 0.01; compared with the DSS plus HDCA group.
FIG 9
FIG 9
Schematic model showing the potential mechanism of feeding sodium alginate (ALG) that alleviated DSS-induced colitis. Dietary ALG induced an alteration in the gut microbiota to enrich probiotic bacteria such as Bifidobacterium animalis, which subsequently led to an increased production of hyodeoxycholic acid (HDCA), which is biotransformed from β-muricholic acid (βMCA) by BA 7-dehydroxylase (baiJ), triggering a cascade of anti-inflammatory responses by activating bile acid receptor, farnesoid X receptor (FXR), and the NF-κB signaling pathway. Ultimately, intestinal epithelial homeostasis is attenuated and colitis was attenuated.
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