Gender-based effect of absence of gut microbiota on the protective efficacy of Bifidobacterium longum-fermented rice bran diet against inflammation-associated colon tumorigenesis

Abstract

Dietary rice bran (RB) has shown capacity to influence metabolism by modulation of gut microbiota in individuals at risk for colorectal cancer (CRC), which warranted attention for delineating mechanisms for bidirectional influences and cross-feeding between the host and RB-modified gut microbiota to reduce CRC. Accordingly, in the present study, fermented rice bran (FRB, fermented with a RB responsive microbe Bifidobacterium longum), and non-fermented RB were fed as 10% w/w (diet) to gut microbiota-intact^spf or germ-free mice^gf to investigate comparative efficacy against inflammation-associated azoxymethane/dextran sodium sulfate (AOM/DSS)-induced CRC. Results indicated both microbiota-dependent and independent mechanisms for RB meditated protective efficacy against CRC that was associated with reduced neoplastic lesion size and local-mucosal/systemic inflammation, and restoration of colonic epithelial integrity. Enrichment of beneficial commensals (such as, Clostridiales, Blautia, Roseburia), phenolic metabolites (benzoate and catechol metabolism), and dietary components (ferulic acid-4 sulfate, trigonelline, and salicylate) were correlated with anti-CRC efficacy. Germ-free studies revealed gender-specific physiological variables could differentially impact CRC growth and progression. In the germ-free females, the RB dietary treatment showed a ∼72% reduction in the incidence of colonic epithelial erosion when compared to the ∼40% reduction in FRB-fed mice^gf . Ex vivo fermentation of RB did not parallel the localized-protective benefits of gut microbial metabolism by RB in damaged colonic tissues. Findings from this study suggest potential needs for safety considerations of fermented fiber rich foods as dietary strategies against severe inflammation-associated colon tumorigenesis (particularly with severe damage to the colonic epithelium).

Keywords: azoxymethane; cancer intervention; colon carcinogenesis; fermentation; germ free mice; metabolomics; microbiome; probiotics; rice bran.

Fig 1.. Effect of RB and FRB-intake on AOM/DSS-induced colon tumorigenesis in gut microbiota-intact^spf (specific pathogen-free) Balb/c mice.

(A) Histopathological grading of AOM/DSS mice^spf colon tissues (with and without RB and FRB-supplemented diets); a score ranging from 0 for normal (or lower) to 3 (extreme events) was assigned. (B) Representative pictographs of distal colon (H&E images) showing histopathological changes; images were acquired at x200 with digitally magnified insets. (C, upper left-and lower-panel) Pictorial and data representation for % Ki-67 positive cells, and immunoreactivity scores for β-catenin, CD44, ZO-1, and claudin-2 expression levels based on immunohistochemical staining. Images were acquired at x400 with digitally magnified insets. % Positive cells were quantified as the number of brown-stained cells x100 per total number of cells counted under ×400 magnification in 5-8 randomly selected fields in each sample. Immunoreactivity (represented by intensity of brown staining) was scored as 0 (no staining), +1 (weak), +2 (moderate), +3 (strong) and +4 (very strong). (C, upper right-panel) Representative pictograph (x400 magnification) of alcian blue-PAS staining in the distal colon tissue depicting changes in mucin composition and distribution. Quantified data is represented as columns (mean for each group); bars represent SEM. ***P ≤0 .001, **P ≤ 0.01, and *P ≤ 0.05. RB, rice bran; FRB, Bifidobacterium longum-fermented RB; AOM, azoxymethane; DSS, dextran sodium sulfate; HG, high grade; MG, moderate grade; LG, low grade dysplasia; PAS, Periodic acid–Schiff; mice^spf, specific pathogen-free mice.

Fig 2.. Effect of RB and FRB-intake on gut microbiome (caecum and colon) composition during inflammation associated colon tumorigenesis in gut microbiota-intact^spf mice.

A) species richness and diversity were analyzed by Shannon/Chao indices, B) clear separation in community composition based upon sample type and diet group by Principal components analysis (PCA) of centred log-ratio transformed abundances for all amplicon sequence variants. Percentage values along each axis indicate the amount of variation explained by each of the first two principal components C) Bifidobacterium longum abundance was not changed between dietary groups in caecum, and had greater expression in RB diet supplemented mice. Letters and colors denote study diet group for each sample type (caecum or distal colon). D) Bar charts of log2 fold differences for 26 differentially abundant (FDR-P<0.1) amplicon sequence variants when comparing rice bran or B. longum-fermented rice bran diets compared to control.

Fig 3.. Effect of RB and FRB-intake on microbial metabolites found locally in gut or systemic circulation following AOM/DSS-induced colon tumorigenesis in microbiota-intact^spf mice.

(A) Principal components analysis (PCA) of colonic and plasma metabolomes from control, rice bran and fermented rice bran diet groups. (B-C) Median scaled relative abundance for selected metabolites in colon tissue and plasma for rice bran or B. longum-fermented rice bran diet groups compared to control. Differential metabolite abundance is reported as relative to values obtained in control diet fed AOM/DSS treated mice^spf. Quantified data is represented as Columns (relative fold change); *P ≤ 0.05. RB, rice bran; FRB, Bifidobacterium longum-fermented RB; AOM, azoxymethane; DSS, dextran sodium sulfate; mice^spf, specific pathogen-free mice.

Fig 4.. Effect of RB and FRB-intake on the expression of inflammatory mediators during AOM/DSS-induced colon tumorigenesis in gut microbiota-intact^spf mice.

Pictorial and data representation for colonic expression of inflammation-associated molecules (A) Cox-2, and (B) NF-κB (p65). Images were acquired at x400 with digitally magnified insets. Immunoreactivity (represented by intensity of DAB-stained brown staining) was scored as 0 (no staining), +1 (weak), +2 (moderate), +3 (strong) and +4 (very strong). Quantified data is represented as Columns (mean for each group); bars represent SEM. ***P ≤0 .001, **P ≤ 0.01, and *P ≤ 0.05. (C) Heat map depicting relative changes in the plasma expression of various cytokine/chemokines and other inflammatory-mediators involved in immune responses, and CRC growth and progression. Plasma collected from untreated (negative controls) and AOM/DSS mice^spf (with and without RB and FRB-supplemented diets) was evaluated for the presence of various cytokines/chemokines using a mouse-Proteome profiler membrane array containing antibodies to 111 different cytokines/chemokines. Colors are assigned according to the relative scale of expression, ranging from ****−2 to 8, and represent fold change compared to negative controls. (D) Densitometric analysis of dot blots from pooled samples in each group are shown as fold changes relative to negative controls (NC). Quantified data is represented as Columns (mean for each group); bars represent SEM. $; p<0.05, ψ; p<0.01, *; p<0.001). RB, rice bran; FRB, Bifidobacterium longum-fermented RB; AOM, azoxymethane; DSS, dextran sodium sulfate; mice^spf, specific pathogen free mice.

Fig 5.. Gender-specific effects of RB and FRB-intake on AOM/DSS-induced colon tumorigenesis in the absence of gut microbiota (germ-free mice^gf).

Histopathological analysis of AOM/DSS mice^gf colon tissues (with and without RB and FRB-supplemented diets) in male (left-panel) and female (right-panel) C57Bl/6^gf mice; (A) % incidence of colonic dysplasia and epithelial erosion, (B) Representative pictographs of distal colon (H&E images) showing histopathological changes; images were acquired at x200, and (C) Pathological scores for inflammatory infiltrates, submucosal inflammation, crypt hyperplasia, decrease in crypt density and loss in goblet cells; a score ranging from 0 for normal (or lower) to 3 (extreme events) was assigned. (D, upper right-panel, male and female) Representative pictograph (x400 magnification) of alcian blue-PAS staining in the distal colon tissue depicting changes in mucin composition and distribution; (D, upper left-and lower-panel, male and female) Pictorial and data representation for % Ki-67 positive cells, and immunoreactivity scores for β-catenin, Cox-2, NF-κB (p65), ZO-1, and claudin-2 expression levels based on immunohistochemical staining. % Positive cells were quantified as the number of brown-stained cells x100 per total number of cells counted under ×400 magnifications in 5-8 randomly selected fields in each sample. Immunoreactivity (represented by intensity of brown staining) was scored as 0 (no staining), +1 (weak), +2 (moderate), +3 (strong) and +4 (very strong). Quantified data is represented as Columns (mean for each group); bars represent SEM. ***P ≤0 .001, **P ≤ 0.01, and *P ≤ 0.05. RB, rice bran; FRB, Bifidobacterium longum-fermented RB; AOM, azoxymethane; DSS, dextran sodium sulfate; HG, high grade; MG, moderate grade; LG, low grade dysplasia; PAS, Periodic acid-Schiff; mice^gf, germ free mice.

Fig 6.. Effect of RB and FRB-intake on colon and plasma metabolite profiles during AOM/DSS-induced colon tumorigenesis in germ-free mice^gf.

(A) Principal components analysis of colon (proximal and distal) and plasma metabolite profiles. Relative abundance of metabolites (B) in proximal colon tissue; (C) in distal colon tissue, and (D) in systemic circulation-plasma. Differential metabolite abundance is reported relative to values obtained in control diet fed AOM/DSS treated mice^gf. Quantified data is represented as Columns (relative fold change); *P ≤ 0.05. RB, rice bran; FRB, Bifidobacterium longum-fermented RB; AOM, azoxymethane; DSS, dextran sodium sulfate; mice^gf, germ free mice.

Fig 6.. Effect of RB and FRB-intake on colon and plasma metabolite profiles during AOM/DSS-induced colon tumorigenesis in germ-free mice^gf.

(A) Principal components analysis of colon (proximal and distal) and plasma metabolite profiles. Relative abundance of metabolites (B) in proximal colon tissue; (C) in distal colon tissue, and (D) in systemic circulation-plasma. Differential metabolite abundance is reported relative to values obtained in control diet fed AOM/DSS treated mice^gf. Quantified data is represented as Columns (relative fold change); *P ≤ 0.05. RB, rice bran; FRB, Bifidobacterium longum-fermented RB; AOM, azoxymethane; DSS, dextran sodium sulfate; mice^gf, germ free mice.