Amomum tsaoko flavonoids attenuate ulcerative colitis by inhibiting TLR4/NF-κB/NLRP3 signaling pathway and modulating gut microbiota in mice

Abstract

Ulcerative colitis (UC) is a global disease for which there is little of effective treatment options, thus creating an urgent need for the development of new drug candidates from natural and functional foods. Amomum tsaoko Crevost et Lemarie is a kind of medicinal and food herb that is rich in flavonoids. However, the pharmacological effects of Amomum tsaoko flavonoids (ATF) on UC have not yet been reported. The present study established a mouse model of UC using 3% dextran sulfate sodium (DSS), and modern molecular biology techniques such as IHC, RT-qPCR, Western blot, and 16S rRNA gene analysis were used to investigate the effect of ATF in UC mice. The results demonstrated that a high dose of ATF (100 mg/kg) led to a significant restoration of body weight, disease activity index score, and colon length, in addition to ameliorating colonic tissue damage in UC mice. ATF reduced the serum levels of lipopolysaccharides (LPS), inhibited the activation of the colonic TLR4/NF-κB/NLRP3 signaling pathway, and increased the mRNA expression of tight junction proteins such as ZO-1, Occludin, and Claudin4. Furthermore, ATF was found to reduce the relative abundance of the DSS-induced conditional pathogenic (Escherichia-Shigella, Colidextribacter, and Oscillibacter), increase the potential probiotic taxa (Akkermansia, Bifidobacterium and unclassified_f__Atopobiaceae). Interestingly, these genera were found to be significantly correlated with the UC core phenotypic indicators. These findings indicated that ATF may alleviate UC symptoms by modulating the gut microbiota-LPS/TLR4/NF-κB/NLRP3 axis. The present study has the potential to serve as a valuable reference point for understanding the role of natural flavonoids in the prevention of inflammatory diseases, and to expand the future applications of ATF in the fields of food and medicine.

Keywords: Akkermansia; Amomum tsaoko; Bifidobacterium; Colidextribacter; Escherichia-Shigella; Oscillibacter; flavonoids; ulcerative colitis.

Figure 1

Amomum tsaoko flavonoids (ATF) is effective in alleviating symptoms of the DSS-induced colitis. (A) Animal experiment design; (B) survival curves; (C) mice body weights were recorded daily; (D) calculate the DAI score; (E,F) bowel images and colon length stats; (G–I) liver, spleen and kidney index; (J) images of colon sections stained with H&E. All data presented in mean ± SEMs, n = 10–12. # compared with the CON group; * compared with the DSS group. ^#p < 0.05, ^##p < 0.01, ^###p < 0.001; *p < 0.05, **p < 0.01, ***p < 0.001.

Figure 2

Amomum tsaoko flavonoids reversed the DSS-induced up-regulation of pro-inflammatory factors and inflammatory cell infiltration. (A,B) Serum levels of TNF-α and IL-1β; (C–O) the mRNA expression of TNF-α, IL-1β, COX-2, MCP-1, IL-6, IFN-γ, IL-18, F4/80, CD68, CD11b, CD14, Ly-6G (Gr-1), and CD115. (P,Q) Percentage of F4/80 positive area in colonic tissue, and represented IHC images in three group. All data presented in mean ± SEMs, n = 8–10. # compared with the CON group; * compared with the DSS group. ^#p < 0.05, ^##p < 0.01, ^###p < 0.001; *p < 0.05, **p < 0.01.

Figure 3

Amomum tsaoko flavonoids alleviated metabolic endotoxaemia and ameliorated intestinal barrier damage. (A) Serum LPS levels. (B) The mRNA expression of TLR4 in colon. (C,D) Percentage of TLR4 positive area in colon, and represented IHC images in three group. (E–H) The mRNA expression of Muc2, ZO-1, Occludin, and Claudin4, respectively. All data presented in mean ± SEMs, n = 8–10. # compared with the CON group; * compared with the DSS group. ^#p < 0.05, ^##p < 0.01, ^###p < 0.001; *p < 0.05, **p < 0.01.

Figure 4

The effect of ATF on the NF-κB and NLRP3 signaling pathway. (A–C) The mRNA expression of NF-κB, MyD88, TRAF6 in colon, respectively. (D,E) The phosphorylation of p65 was detected by Western blotting, and the relative expression of p-p65/p65. (F–H) The mRNA expression of NLRP3, Caspase-1, and ASC in colon, respectively. (I,J) Represented IHC images in three group, and percentage of NLRP3-positive area in colon. All data presented in mean ± SEMs, n = 8–10. # compared with the CON group; * compared with the DSS group. ^#p < 0.05, ^##p < 0.01, ^###p < 0.001; *p < 0.05, **p < 0.01.

Figure 5

Amomum tsaoko flavonoids altered the structure and composition of the gut microbial community in UC mice. (A) Rank-abundance curve; (B) α-diversity index; (C) PCoA based on the Bray–Curtis distance algorithm. (D) Linear discriminant analysis effect size (LEfSe) analyses (LDA score > 2.0). (E) Relative abundance of microbes at the phylum level. (F) Relative abundances of microbes at the family level. (G–J) Relative abundances of microbes at the genus level. All data presented in mean ± SEMs, n = 6. # compared with the CON group; * compared with the DSS group. ^#p < 0.05, ^##p < 0.01, ^###p < 0.001; *p < 0.05, **p < 0.01, ***p < 0.001.

Figure 6

Correlations between the differential genera and core indicators of UC. (A) Correlations between differential genera and conventional pathological phenotypes of UC mice. (B) Correlations between differential genera and inflammatory factors and immune cell markers. (C) Correlations between differential genera and metabolic endotoxaemia and colonic barrier factors. (D) Correlations between differential genera and NF-κB/NLRP3 signaling pathway. BWCR, body weight change rate of mice; CL, colon length; DAI, disease activity index. S-IL-1β, IL-1β in serum; S-TNF-α, TNF-α in serum; LPS, LPS in serum; all other factors are relative expressions of mRNAs. The color at each point of intersection indicates the value of the r coefficient (n = 22–24). The Benjamini–Hochberg (BH) procedures were used to adjust p values for multiple testing. * indicates that there is a significant correlation between these two parameters (p < 0.05). *p < 0.05, **p < 0.01.