Kaempferol Alleviates Murine Experimental Colitis by Restoring Gut Microbiota and Inhibiting the LPS-TLR4-NF-κB Axis

Abstract

Intestinal microbiota dysbiosis is an established characteristic of ulcerative colitis (UC). Regulating the gut microbiota is an attractive alternative UC treatment strategy, considering the potential adverse effects of synthetic drugs used to treat UC. Kaempferol (Kae) is an anti-inflammatory and antioxidant flavonoid derived from a variety of medicinal plants. In this study, we determined the efficacy and mechanism of action of Kae as an anti-UC agent in dextran sulfate sodium (DSS)-induced colitis mice. DSS challenge in a mouse model of UC led to weight loss, diarrhea accompanied by mucous and blood, histological abnormalities, and shortening of the colon, all of which were significantly alleviated by pretreatment with Kae. In addition, intestinal permeability was shown to improve using fluorescein isothiocyanate (FITC)-dextran administration. DSS-induced destruction of the intestinal barrier was also significantly prevented by Kae administration via increases in the levels of ZO-1, occludin, and claudin-1. Furthermore, Kae pretreatment decreased the levels of IL-1β, IL-6, and TNF-α and downregulated transcription of an array of inflammatory signaling molecules, while it increased IL-10 mRNA expression. Notably, Kae reshaped the intestinal microbiome by elevating the Firmicutes to Bacteroidetes ratio; increasing the linear discriminant analysis scores of beneficial bacteria, such as Prevotellaceae and Ruminococcaceae; and reducing the richness of Proteobacteria in DSS-challenged mice. There was also an evident shift in the profile of fecal metabolites in the Kae treatment group. Serum LPS levels and downstream TLR4-NF-κB signaling were downregulated by Kae supplementation. Moreover, fecal microbiota transplantation from Kae-treated mice to the DSS-induced mice confirmed the effects of Kae on modulating the gut microbiota to alleviate UC. Therefore, Kae may exert protective effects against colitis mice through regulating the gut microbiota and TLR4-related signaling pathways. This study demonstrates the anti-UC effects of Kae and its potential therapeutic mechanisms, and offers novel insights into the prevention of inflammatory diseases using natural products.

Keywords: NF-κB; TLR4; gut microbiota; kaempferol; lipopolysaccharide; ulcerative colitis.

Figure 1

Kae attenuates the symptoms of DSS-induced mice colitis. (A) Chemical structure of kaempferol (Kae). (B) Experimental design to test the effects of Kae on DSS-induced mice (n = 10/group). (C) Disease activity index of mice during the course of colitis. (D, E) Representative images of colons from mice following euthanization and statistical analysis of colon length in each group. (F, G) Representative images of HE stained colon tissue samples (scale bar, 200 μm) and histological scores of colonic tissues. Data are expressed as the mean ± SEM, n = 10, analyzed using one-way ANOVA with Tukey post-hoc analysis. DSS (vs. NC, ^## P < 0.01; vs. DSS-Kae, *P < 0.05, **P < 0.01); Kae (vs. NC, ^a P < 0.05).

Figure 2

Effects of Kae on inflammatory-associated cytokine levels in DSS- and Kae-treated mice. Serum inflammatory factors, (A) IL-1β, (B) IL-6, and (C) TNF-α, detected using ELISA kits. (D) Relative mRNA expression of inflammatory factors in the colon evaluated by qRT-PCR. All data were log₂ converted and are presented as fold-change in expression level versus the NC group (means for the NC group were set as 1). Data are expressed as the mean ± SEM, n = 4–6, analyzed using one-way ANOVA with Tukey post-hoc analysis. DSS (vs. NC, ^# P < 0.05, ^## P < 0.01; vs. DSS-Kae, *P < 0.05, **P < 0.01).

Figure 3

Kae improves gut permeability and enhances expression of intestinal tight junction proteins. Relative mRNA levels analysis of (A) Quantification of serum FITC-dextran. (B) ZO-1, (C) occludin, and (D) claudin-1. (E) Representative images of immunohistochemical staining of ZO-1, occludin, and claudin-1 in colon samples from different experimental groups (scale bar, 50 μm). Positive protein integral optical density was determined using Image J 1.5.7 software. Data are expressed as the mean ± SEM, n = 5–6, analyzed using one-way ANOVA with Tukey post-hoc analysis. DSS (vs. NC, ^# P < 0.05, ^## P < 0.01; vs. DSS-Kae, *P < 0.05, **P < 0.01); ns, no significant difference.

Figure 4

Kae increased the diversity and richness of gut microbiota in DSS-induced mice. (A) Venn diagram illustrates the numbers of OTUs in the NC, Kae, DSS, and DSS-Kae groups. Alpha diversity is illustrated using a violin plot of the (B) Shannon, (C) Simpson, and (D) Chao indices. Beta-diversity was assessed using (E) PCA, (F) PCoA, and (G) NMDS, based on weighted UniFrac distances. Pairwise comparisons using the Wilcoxon rank sum test for alpha diversity, n = 6, **P < 0.01; ns, no significant difference.

Figure 5

(A) Taxonomic analysis of microbiota in fecal samples at the phylum, class, order, family, and genus levels. (B) Ratio of Firmicutes to Bacteroidetes in the gut microbiota. (C) LDA scores for bacterial taxa significantly enriched in gut microbiota from each group (LDA score > 3). (D) Cladogram illustrating the results of LEfSe analysis. (E) All-against-all algorithm of LDA coupled with LEfSe. Ratios are expressed as the mean ± SEM, n = 6, analyzed using one-way ANOVA with Tukey post-hoc analysis. DSS (vs. DSS-Kae, *P < 0.05); Kae (vs. NC, ^a P < 0.05); ns, no significant difference. The significance of differences in taxonomic groups were assessed using the non-parametric factorial Kruskal-Wallis sum-rank test, n = 6. P < 0.05 was considered to indicate a significant difference between groups.

Figure 6

Kae altered fecal metabolic composition. (A) PLS-DA analysis score plot showing comparisons of the NC vs DSS and DSS vs DSS-Kae group metabolome profiles. (B) OPLS-DA analysis score plots showing comparisons of the NC vs DSS and DSS vs DSS-Kae group metabolome profiles. (C) Heat map showing metabolites differing significantly in abundance between the DSS and DSS-Kae groups. (D) KEGG pathway analysis of the DSS and DSS-Kae groups. Statistical analysis was conducted by calculation of Pearson correlation coefficients (VIP > 1 and P < 0.05).

Figure 7

Kae suppresses the LPS-TLR4-NF-κB signaling pathway. (A) Serum levels of LPS. (B) Immunohistochemical analysis of TLR4, MyD88, p-NF-κB-P65, and NLRP3 expression in colon tissues (scale bar, 50 μm). Positive protein integral optical density was analyzed using Image J 1.5.7 software. Data are expressed as mean ± SEM, n = 5, analyzed using one-way ANOVA with Tukey post-hoc analysis. DSS (vs. NC, ^## P < 0.01; vs. DSS-Kae, *P < 0.05, **P < 0.01), ns, no significant difference.

Figure 8

Transplantation of microbiota altered in response to Kae recapitulates the effects of Kae treatment on DSS-induced colitis. (A) Design of the FMT experiment on DSS-treated mice (n = 10/group). (B) Disease activity index of FMT mice during the course of colitis. (C, D) Representative images of mouse colon at sacrifice and statistical analysis of colon length data from each FMT group. (E, F) Representative images of H&E staining of colon samples (scale bar, 200 μm) and histological scores of colonic tissues. Serum inflammatory factors: (G) IL-1β, (H) IL-6, and (I) TNF-α were measured using ELISA kits. (J) Serum LPS levels indicate the endotoxemia index. Data are expressed as the mean ± SEM, n = 4–10, analyzed using one-way ANOVA with Tukey post-hoc analysis. DSS (vs. NC, ^# P < 0.05; ^## P < 0.01; vs. DSS-Kae, *P < 0.05, **P < 0.01); F-Kae (vs. F-NC, ^aa P < 0.01); F-Kae (vs. F-DSS, ^bb P < 0.01); ns, no significant difference.

Figure 9

Kae exerts excellent anti-UC effects via gut microbiota pathways related to the LPS-TLR4-NF-κB core pathway. Kae reduces LPS levels by inhibiting the proliferation of pathogenic Gram-negative bacilli, thereby altering the metabolic profile, blocking NF-κB pathway activation, improving intestinal tight junction integrity, inhibiting pro-inflammatory factors, and increasing antioxidants, thus decreasing DSS-induced colonic inflammation.