Red Cabbage Juice-Mediated Gut Microbiota Modulation Improves Intestinal Epithelial Homeostasis and Ameliorates Colitis

Abstract

Gut microbiota plays a crucial role in inflammatory bowel diseases (IBD) and can potentially prevent IBD through microbial-derived metabolites, making it a promising therapeutic avenue. Recent evidence suggests that despite an unclear underlying mechanism, red cabbage juice (RCJ) alleviates Dextran Sodium Sulfate (DSS)-induced colitis in mice. Thus, the study aims to unravel the molecular mechanism by which RCJ modulates the gut microbiota to alleviate DSS-induced colitis in mice. Using C57BL/6J mice, we evaluated RCJ's protective role in DSS-induced colitis through two cycles of 3% DSS. Mice were daily gavaged with PBS or RCJ until the endpoint, and gut microbiota composition was analyzed via shotgun metagenomics. RCJ treatment significantly improved body weight (p ≤ 0.001), survival in mice (p < 0.001) and reduced disease activity index (DAI) scores. Further, RCJ improved colonic barrier integrity by enhancing the expression of protective colonic mucins (p < 0.001) and tight junction proteins (p ≤ 0.01) in RCJ + DSS-treated mice compared to the DSS group. Shotgun metagenomic analysis revealed an enrichment of short-chain fatty acids (SCFAs)-producing bacteria (p < 0.05), leading to increased Peroxisome Proliferator-Activated Receptor Gamma (PPAR-γ) activation (p ≤ 0.001). This, in turn, resulted in repression of the nuclear factor κB (NFκB) signaling pathway, causing decreased production of inflammatory cytokines and chemokines. Our study demonstrates colitis remission in a DSS-induced mouse model, showcasing RCJ as a potential modulator for gut microbiota and metabolites, with promising implications for IBD prevention and treatment.

Keywords: Dextran Sodium Sulfate (DSS); colitis; gut microbiota; inflammatory bowel disease (IBD); red cabbage; short-chain fatty acids (SCFAs).

Figure 1

(A) Schematic diagram of the in vivo experimental design and access to water and standard feed. (B) Effect of DSS and RCJ on body weight where time point 1 is the baseline before treatments began. Time point 2 is during the RCJ treatments. Time point 3 is after the DSS administration began. Time point 4 is after a rest period. Error bars are the confidence interval (95%). Data were presented as mean ± SEM (n = 15 per group). (C) kinetics of daily disease activity index (DAI) scores throughout the study duration (D). Effect of RCJ and DSS on colon length. Effect of RCJ and DSS on (E) Blood in feces scores. (F) Diarrhea scores. (G) Survival curve. (H) H&E–stained colon section. (I) Histological scores of the colon (n = 15 per group). Scale bars represent 100 µm. Statistical significance was determined using one-way ANOVA, followed by the Tukey test. ns = non-significant; * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001.

Figure 2

RCJ attenuated oxidative stress and colonic damage. (A) IHC staining for Ki67 in colonic epithelium to assess epithelial cell proliferation and TUNEL-positive nuclei (apoptotic cells) in the colonic epithelium in brown (B) Representative colons IHC for oxidative stress marker from each treatment group;. Error bars in the histograms are the standard error of the mean. Scale bars represent 100 µm. Statistical significance was determined using one-way ANOVA, followed by the Tukey test. ns = non-significant; ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001.

Figure 3

RCJ ameliorates colitis by regulating the intestinal barrier function. (A) Alcian blue staining denoting the mucin-secreting goblet cells in the colonic epithelia and PAS-positive cells denoting acid and neutral mucin. (B) IHC staining for MUC2 and MUC4, stained to understand the protective mucin layer. (C) IHC staining for tight junction markers claudin and occludin. (D) Immunostaining for colonic ZO-1, (green) counterstained with the nuclear stain DAPI (blue) and elative intensity, quantitative analysis of the fluorescence by Image J (Fiji 1.54F). All values represent the means ± SD; error bars in the histograms are the standard error of the mean. Scale bars represent 100 µm for the IHC and 50 µm for IF. Statistical significance was determined using one-way ANOVA, followed by the Tukey test. ns: non-significant * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001.

Figure 4

Effect of RCJ on colonic proinflammatory status (A–L). Graphs show a quantified expression of pro- and anti-inflammatory cytokines and chemokines using a cytokine array. (M) Immunofluorescence staining for iNOS (green), COX-2 (red) and counterstained with the nuclear stain DAPI (blue). Graphs show relative intensity, quantitative analysis of the fluorescence by Image J. All values represent the means ± SD; error bars in the histograms are the standard error of the mean. Scale bars represent 50 µm for IF. Statistical significance was determined using one-way ANOVA, followed by the Tukey test. ns: non-significant, * p ≤ 0.05, ** p ≤ 0.01, **** p ≤ 0.0001.

Figure 5

Effect of RCJ treatment on gut microbiota. (A) Alpha diversity using Shannon indices, a measure of evenness in a sample. (B) NMDS plots representing the samples’ closeness when compared to the control. (C) Relative abundance of taxa. (D) Relative abundance of significant organisms at phylum, genus, and species level. (E) Relative abundance of some reported SCFA–producing taxa. Statistical significance was determined using one-way ANOVA, followed by the Tukey test.

Figure 6

Effect of RCJ on biochemical pathways. (A) NMDS plots for the biochemical pathways in the microbiota. (B) Differentially abundant biochemical pathways across treatment groups. (C) Top significantly regulated pathways that are beneficial for the colon epithelium health. (D) Selected organism-specific pathways with significantly differential abundance. Bacteroides sartorii and Bacteroides caecimuris are the two main species associated with arginine synthesis and L-histidine degradation.

Figure 7

RCJ reversing the dysregulation of immunological responses in DSS-induced colitis mice. (A) IHC staining for F4/80 (total macrophages), and to check inflammation stained for p-IKKβ, p-NF-κB, and p-STAT (B) IHC staining for T cell marker panel with CD3, RORγ, FOXp3, and MPO for the neutrophils. All values represent the means ± SD; error bars in the histograms are the standard error of the mean. Scale bars represent 100 µm for the IHC. Statistical significance was determined using one-way ANOVA, followed by the Tukey test. ns: non-significant, * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001.

Figure 8

(A) IHC staining for anti-inflammatory mediators PPAR-γ indicative of butyrate presence. Scale bars represent 100 µm for the IHC. Statistical significance was determined using one-way ANOVA, followed by the Tukey test. **** p ≤ 0.0001. (B) Zoom image of PPAR-γ expression. (C) Schematic model showing the mechanism by which RCJ alleviated DSS-induced colitis. Intestinal microbiota, oxidative stress, inflammation, and barrier integrity are all affected. RCJ treatment changed the gut microbiota by enriching bacteria such as Butyrivibrio, Roseburia, Ruminococcaceae, Acetatifactor muris, Rosburia Sp. CAG:303, Dorea Sp. 5-2, which subsequently led to increased production of SCFAs such as butyrate, which was evidenced by increased expression of PPAR-γ leading to a cascade of events, including anti-oxidative, anti-inflammatory, and barrier-protective responses. Ultimately, intestinal epithelial homeostasis is attenuated, and colitis is attenuated.