Chlorogenic Acid Ameliorates Experimental Colitis by Promoting Growth of Akkermansia in Mice

Abstract

Chlorogenic acid (ChA)-one of the most abundant polyphenol compounds in the human diet-exerts anti-inflammatory activities. The aim of this study was to investigate the effect of ChA on gut microbiota in ulcerative colitis (UC). Colitis was induced by 2.5% dextran sulfate sodium (DSS) in C57BL/6 mice, which were on a control diet or diet with ChA (1 mM). The histopathological changes and inflammation were evaluated. Fecal samples were analyzed by 16S rRNA gene sequencing. ChA attenuated several effects of DSS-induced colitis, including weight loss, increased disease activity index, and improved mucosal damage. Moreover, ChA could significantly suppress the secretion of IFNγ, TNFα, and IL-6 and the colonic infiltration of F4/80⁺ macrophages, CD3⁺ T cells, and CD177⁺ neutrophils via inhibition of the active NF-κB signaling pathway. In addition, ChA decreased the proportion of Firmicutes and Bacteroidetes. ChA also enhanced a reduction in fecal microbiota diversity in DSS treated mice. Interestingly, ChA treatment markedly increased the proportion of the mucin-degrading bacterium Akkermansia in colitis mice. ChA acted as the intestine-modifying gut microbial community structure, resulting in a lower intestinal and systemic inflammation and also improving the course of the DSS-induced colitis, which is associated with a proportional increase in Akkermansia.

Keywords: Akkermansia; chlorogenic acid; colitis; gut microbiota; inflammation.

Figure 1

The effects of Chlorogenic acid (ChA) on the disease activity index (DAI) in dextran sulfate sodium (DSS) colitis mice. (A) Data for weight changes are expressed as the mean percentage change from the starting body weight; (B) Disease activity index was evaluated as the average of score of clinical parameters as body weight changes, rectal bleeding, and stool consistency or diarrhea; (C) Colon length of each group. The data are expressed as the mean ± SD from 10 mice in each group. * p < 0.05, ** p < 0.01, *** p < 0.001, compared with control group; ^# p < 0.05, ^## p < 0.01, ^### p < 0.001, compared with the DSS group according to one-way analysis of variance (ANOVA) statistical analysis.

Figure 2

The effects of ChA on the histopathological characterization in DSS-colitis mice. (A) Representative HE-stained sections of the distal colonic tissues from the control, DSS, and DSS+ ChA group; (B) Representative alcian blue-stained sections of these different groups. The mucus is stained blue. Formalin fixed, paraffin-embedded 5 μm cross-sections were stained with respective primary antibody. Scale bar: 50 μm.

Figure 3

The effects of ChA on serum cytokines in DSS-colitis mice. A comparison of the serum concentration of (A) IFNγ; (B) TNF α; (C) IL-1β; (D) IL-4; (E) IL-6; and (F) IL-12 were performed by the Mann–Whitney U test. The boxplot represented the values of cytokines from minimum to maximum from 10 mice, *** p < 0.001 compared with control group; ^### p < 0.001, compared with the DSS group.

Figure 4

The effects of ChA on colonic infiltration of inflammatory cells and NF-κB signaling in DSS-colitis mice. Representative images of (A) F4/80; (B) CD3; (C) CD177 immunostaining in the distal colon of mice one week after cessation of DSS treatment. Formalin fixed, paraffin-embedded 5 μm cross-sections were stained with respective primary antibody. Scale bar: 50 μm; (D) Each protein (80 μg) was determined by immunoblot using specific antibodies. ImageJ software was used to quantify the integrated band intensity, which was transferred to relative values using corresponding GAPDH as an internal reference. The data are expressed as the mean ± SD of three separate experiments from each mouse. ** p < 0.01, compared with control group; ^## p < 0.01, compared with the DSS group.

Figure 5

The effects of ChA on microbial composition in DSS-colitis mice. (A) Principal coordinate analysis plot of the fecal microbiota from five cages in each group based on the Bray–Curtis; (B) Relative abundance of bacterial phyla; (C) The ratio of Firmicutes and Bacteroidetes. * p < 0.05, ** p < 0.01, compared with control group; ^# p < 0.05, compared with the DSS group.

Figure 6

Bacterial α-diversity comparison and heatmap of the relative bacterial species abundance. (A) The Shannon and Simpson indices were used to estimate diversity (at a 97% similarity level) of the fecal microbiota in mice; (B) Heatmap indicating genus-level changes among controls, DSS, and DSS+ ChA groups. Consistent with alpha diversity indices, clustering analysis of these genera highlighted the apparent differences in their distributions. The relative abundance of each genus is indicated by a gradient of color from blue (low abundance) to red (high abundance). The data represented five samples from respective groups. * p < 0.05, ** p < 0.01, *** p < 0.001, compared with the control group; ^# p < 0.05, ^## p < 0.01, compared with the DSS group.

Figure 7

The effects of ChA on Akkermansia. (A) Comparison of the relative abundance of Akkermansia in fecal samples of DSS colitis mice analyzed by 16S rRNA gene sequencing; (B) A comparison of the relative abundance of Akkermansia in fecal samples of normal mice that were treated with ChA. Akkermansia was quantified with qPCR by amplifying fecal DNA with primers specific for Akkermansia and universal bacterial primers; (C) The polymerase chain reaction products were analyzed by electrophoresis in a 2% agarose gel. * p < 0.05, ** p < 0.01, *** p < 0.001, compared with the control group; ^# p < 0.05, compared with the DSS group.