WKB ameliorates DSS-induced colitis through inhibiting enteric glial cells activation and altering the intestinal microbiota

Abstract

Background

Inflammatory bowel disease (IBD) is a chronic condition influenced by diet, which affects gut microbiota and immune functions. The rising prevalence of IBD, linked to Western diets in developing countries, highlights the need for dietary interventions. This study aimed to assess the impact of white kidney beans (WKB) on gut inflammation and microbiota changes, focusing on their effects on enteric glial cells (EGCs) and immune activity in colitis.

Methods

Male C57BL/6 mice were divided into four groups: normal diet (ND), ND with 2.5% dextran sulfate sodium (DSS) for colitis induction, ND with 20% WKB, and WKB with 2.5% DSS. The dietary intervention lasted 17 weeks, with DSS given in the final week. Colonic inflammation was assessed by body weight, disease activity index, and histopathology. Epithelial barrier integrity was evaluated using immunofluorescence, transmission electron microscopy, and permeability assays. EGCs activity was analyzed via immunofluorescence and quantitative real-time PCR. Immune responses were measured using flow cytometry and cytokine profiling, while gut microbiota changes were examined through metagenomic sequencing.

Results

WKB supplementation significantly alleviated DSS-induced colitis in mice, evidenced by reduced weight loss, disease activity, and improved colonic histology. This effect was linked to enhanced mucosal barrier integrity, seen through increased tight junction protein and Muc2 expression, accompanied by favorable ultrastructural changes. WKB modulated EGCs activity via TNF-like cytokine 1 A inhibition, resulting in reduced glial fibrillary acidic protein expression. Immunologically, it downregulated Th1 and Th17 pro-inflammatory cells, increased Treg cells, and altered cytokine profiles (reduced TNF-α, IFN-γ, IL-17; increased IL-10). Metagenomic analysis showed that WKB restored gut microbiota balance, particularly enhancing beneficial bacteria like Akkermansia. KEGG pathway analysis further indicated that WKB supplementation improved key metabolic pathways, notably those related to phenylalanine, tyrosine, and tryptophan biosynthesis, thereby countering DSS-induced metabolic disruptions.

Conclusions

WKB shows promise for treating IBD by enhancing mucosal barriers, inhibiting EGCs activity, balancing Th1/Th17/Treg cells, and restoring gut microbiota and metabolic homeostasis, thereby alleviating colitis symptoms.

Introduction

Inflammatory bowel disease (IBD) is a complex, chronic inflammatory disorder characterized by abdominal pain, diarrhea and mucoid bloody stools, with multiple genetic and environmental factors contributing to its pathogenesis [1]. Despite the limited understanding of its underlying mechanisms, a growing body of research emphasizes the crucial role of dietary intake in shaping the gut microbiota, epithelial barrier function and mucosal immunity, thereby influencing disease susceptibility [2]. The increasing adoption of Western dietary patterns, rich in animal protein, trans fatty acids and refined sugars, in developing countries has been accompanied by a rising incidence of IBD, suggesting a potential link between Western diets and IBD prevalence [3]. For example, consuming a diet high in salt can worsen gut inflammation by decreasing the levels of lactobacillus and hindering butyrate metabolism [4]. On the other hand, diets rich in fat have been linked to the progression of IBD by triggering pro-inflammatory signals and weakening the epithelial barrier [5]. Conversely, plant-based diets have been demonstrated to promote beneficial gut microbiota, facilitating gut recovery during IBD flares [6, 7], and ketogenic diets have been shown to alleviate colitis by modulating the gut microbiota and reducing colonic group 3 innate lymphoid cells [8]. The cumulative evidence suggests that dietary interventions may offer a promising therapeutic approach for IBD management.

Enteric glial cells (EGCs), an essential part of the enteric nervous system, are known to significantly influence the regulation and maintenance of several gastrointestinal functions. These include local immune responses, the integrity of the mucosal barrier, and gut motility [9,10,11]. EGCs depletion or dysfunction has been linked to the development of severe gut inflammation in murine models [12]. Notably, glial fibrillary acidic protein (GFAP), an indicator of EGCs, was found to be markedly increased in inflamed tissues of individuals with ulcerative colitis and Crohn’s disease. This upregulation was also observed in animal models of colitis induced by trinitrobenzene sulfonic acid and dextran sulfate sodium (DSS) [13, 14]. In the local inflammatory microenvironment, EGCs not only increase in number but also exhibit elevated intracellular GFAP content, a phenomenon reminiscent of “reactive gliosis” in the central nervous system [15]. Accumulating evidence suggests that the intricate regulation and crosstalk between EGCs and immune cells in the gut microenvironment play a critical role in the initiation and exacerbation of gut inflammation. Similar to astrocytes in the central nervous system, EGCs are considered antigen-presenting cells (APCs) that, upon inflammatory stimulation, activate macrophages, mast cells, neutrophils and T cells, amplifying inflammatory signals [16, 17]. Overall, suppressing EGCs reactive proliferation may offer a promising therapeutic strategy for IBD treatment.

TNF-like cytokine 1 A (TL1A), predominantly expressed on APCs, senses intestinal inflammation and triggers a co-stimulatory signal through interaction with death domain receptor 3 (DR3) on lymphocytes, thereby amplifying effector T cell responses. Downstream of this signaling pathway, key pro-inflammatory cytokines such as IFN-γ, TNF-α, IL-6 and IL-17 are produced, which are upregulated in IBD patients [18]. Blockade of the TL1A/DR3 signaling axis has been shown to exert potent anti-inflammatory effects [19]. However, it remains unclear whether TL1A is expressed on EGCs, modulating effector T cell responses in response to inflammatory signals, and thereby contributing to intestinal inflammation.

White kidney beans (WKB), one of the most widely consumed legumes globally, are rich in protein, fiber, polyphenols and various bioactive compounds, which have been proven to possess numerous health benefits, attracting extensive attention in recent years [20, 21]. Notably, the diverse components of whole foods may produce synergistic effects. Most of the bioactive compounds in WKB cannot be fully digested and absorbed in the upper gastrointestinal tract, instead, they reach the colon where they interact with the gut microbiota. The consumption of whole WKB is on the rise. Research has revealed that WKB can target the gut microbiota, reducing blood glucose levels in non-alcoholic fatty liver disease mouse models [22]. Y Feng et al. demonstrated that WKB consumption improves glucose metabolism and alleviates diabetic complications by influencing the composition of gut microbiota [23]. Furthermore, in colitis mouse models, WKB has been observed to lower serum levels of the pro-inflammatory cytokines TNF-α and IL-6, though the exact mechanisms are not fully understood [24]. These findings encourage further exploration of the therapeutic potential of whole WKB on DSS-induced colitis in mice, focusing on the interactions between gut microbiota, EGCs, and immune cells. This study may aid in developing new dietary strategies for combating intestinal inflammation.

Materials and methods

Animals and diets

C57BL/6 male mice (18–22 g, 8–10 weeks old) were sourced from the Laboratory Animal Services Center at the Air Force Medical Center in Beijing, China. They were kept in a controlled environment with free access to food and water, following a 12:12 h light-dark cycle (temperature: 21 ± 2 °C, humidity: 45 ± 10%) under specific pathogen-free conditions. All experimental protocols adhered to the National Institutes of Health guidelines and received approval from the Animal Care and Use Committee of the Air Force Medical Center, Beijing.

WKB (Phaseolus vulgaris L.) was procured from Beijing Yushiyuan Food Co., Ltd. The preparation followed a previously established method [22]: the beans were washed, soaked in distilled water for 4 h at room temperature, steamed for 40 min, freeze-dried, ground into powder, sieved through an 80-mesh screen, and stored at -20 °C until needed.

Experimental design

Following a one-week acclimatization period, mice were randomly divided into four groups: a normal diet (ND)-control group receiving a standard diet and water, an ND-DSS group receiving a standard diet and 2.5% DSS, a WKB-control group receiving a standard diet supplemented with 20% cooked WKB powder and water, and a WKB-DSS group receiving a standard diet supplemented with 20% cooked WKB powder and 2.5% DSS. The 20% WKB inclusion level was chosen based on a physiologically relevant and achievable intake of legumes for humans, as recommended by the Dietary Guidelines for Chinese Residents (2022). To maintain equal caloric contributions from each macronutrient, the nutrient composition of the experimental diets was adjusted according to the proximate nutritional profile of WKB [22]. Consequently, the diets in the ND and WKB groups were isocaloric, allowing for a direct comparison of the effects of WKB supplementation.

Throughout the 17-week experiment, mice in the ND-control and ND-DSS groups were fed a standard diet, while those in the WKB-control and WKB-DSS groups received a diet supplemented with 20% cooked WKB powder. During the initial 16 weeks, all mice had ad libitum access to normal water. In the 17th week, mice in the ND-DSS and WKB-DSS groups were administered 2.5% DSS solution instead of normal water for 7 days, after which they were euthanized by cervical dislocation. The concentration of 2.5% DSS was chosen based on previous studies [25, 26] and our preliminary experiments (Supplementary Fig. 1), which demonstrated that this dose reliably induces moderate colitis in C57BL/6 mice without causing excessive mortality or severe weight loss. In contrast, mice in the ND-control and WKB-control groups continued to receive normal water throughout the experiment (Fig. 1A). The DSS powder was obtained from MP Biomedicals, UK (0216011080, molecular weight: 36–50 kDa). After starting DSS treatment, the mice were monitored daily for weight changes and assessed for disease severity, which included evaluating stool consistency, detecting blood in the stool, and noting body weight loss. The Disease Activity Index (DAI) was calculated each day for each mouse by summing individual scores, with a maximum possible score of 12, using a 0–4 scale for each parameter (Table 1) [27].

Fig. 1

WKB diet alleviated the clinical symptoms of DSS-induced colitis. (A) Schematic of animal experimental procedures. (B) The changes of body weight (%) after DSS treatment. Student’s unpaired t-test. **P < 0.01 vs. ND-DSS group. n = 6. (C) Representative images of bloody diarrhea at the end of week 17. (D) Disease activity index (DAI) scores after DSS treatment. Student’s unpaired t-test. *P < 0.05, **P < 0.01 vs. ND-DSS group. n = 6. (E) The percentage of mouse weight loss at the end of week 17. n = 6. Data are shown as mean ± SEM, P values were calculated using One-way ANOVA with Tukey’s test. **P < 0.01, ***P < 0.001 and ****P < 0.0001. All experiments were performed in two independent biological replicates

Table 1 Scoring system for disease activity index (DAI)

Colon mucosal damage index and histopathology assessments

Mouse colon specimens were collected, and their lengths were recorded. Subsequently, the specimens were dissected along the mesenteric border to remove luminal contents. The extent of colonic mucosal damage was then assessed using a standardized scoring system, as outlined in Table 2 [28], to determine the colonic mucosa damage index.

Table 2 Scoring system for colonic mucosa damage index (CMDI)

Subsequently, colon tissues were processed into paraffin-embedded blocks, from which 5-µm-thick sections were generated for hematoxylin and eosin (H&E) staining. An experienced pathologist, blinded to the group assignments, evaluated the sections, and assessed histological scores using a standardized scoring system (Table 3) [29]. Light microscopy was used to scrutinize the morphological features of the tissue sections (ECLIPSE E-200, Nikon, Tokyo). Quantitative analyses, including goblet cell count, crypt length, and submucosal thickness, were conducted with a fully automated digital slide scanning system and CaseViewer software (3DHISTECH Ltd., Budapest, Hungary). For statistical analysis, three mice per group were included, with three tissue sections analyzed per mouse to ensure robust and representative data.

Table 3 Scoring system for histological scores

Immunofluorescence and immunohistochemistry

For immunofluorescence analysis, tissue sections underwent antigen retrieval by heating to 100 °C in citrate buffer (0.01 M, pH 6.0) for 30 min, followed by three successive washes in phosphate-buffered saline (PBS), each for 5 min [30]. Next, sections were incubated in 5% normal donkey serum for 30 min at room temperature, and then with primary antibodies (Supplementary Table 1) overnight at 4 °C. The following day, the sections were washed three times with PBS, each for 5 min, and then incubated with secondary antibodies (Supplementary Table 2) for 2 h at room temperature. Cell nuclei were stained with 4,6-Diamidino-2-phenylindole (DAPI) for ten minutes. The sections were given a final wash, cover slipped, and imaged using a confocal laser scanning microscope (Leica STELLARIS 8 Falcon, Germany). Positive cells were blindly evaluated in 3 high-power fields (HPF) per Sect. (400 × field) and analyzed using Image-Pro Plus 6.0 software (IPP, Media Cybernetics Inc., Rockville, USA). The measurement of integrated optical density (IOD) data was quantified as averages from 3 independent tissue sections per sample.

For immunohistochemical (IHC) analysis, sections were quenched with 0.3% H₂O₂ to inactivate endogenous peroxidase [31]. Subsequently, primary antibodies were applied and incubated overnight at 4 °C, followed by washing in PBS. The sections were then incubated with a horseradish peroxidase-conjugated secondary antibody for 60 min, washed, and treated with a 3,3’-Diaminobenzidine (DAB) working solution for 2 to 10 min to develop a brown precipitate at the antigen sites. Upon achieving the desired staining intensity, the slides were rinsed in distilled water. The sections were counterstained with hematoxylin for contrast, followed by dehydration through graded alcohols, clearing in xylene, and mounting with a permanent medium. Under light microscope, positive staining appears brown, indicating the location of the antigen. Slides were stained with Alcian Blue (AB) solution (pH 2.5) for 20 min to highlight acidic mucopolysaccharides and glycosaminoglycans, followed by a 5-minute rinse in running tap water. Subsequently, the slides were treated with a 1% Periodic Acid-Schiff (PAS) solution for 5 min to oxidize the tissue, thereby generating aldehyde groups on neutral polysaccharides. After rinsing in distilled water, the slides were immersed in Schiff reagent for 10 min, which stains neutral mucosubstances and glycogen a magenta color. The slides were then washed in running tap water until a pink hue develops. Under light microscope, acidic mucosubstances appear blue, while neutral mucosubstances and glycogen appear magenta, facilitating the clear differentiation of these components within tissue Sect. [32]. Immunostaining utilized PV-9001 and G1285 kits from Beijing Zhong Shan-Golden Bridge Biological Technology Co., Ltd, and Beijing Solarbio Science & Technology Co., Ltd., respectively, incorporating DAB and PAS/AB. A fully automated digital slide scanning system was employed to observe the sections (3DHISTECH Ltd., Budapest, Hungary). Goblet cell numbers were quantified as averages of 3 HPF (400 ×) per section using CaseViewer software, across 3 independent tissue sections per sample.

Transmission electron microscopy (TEM)

Following the established protocol for TEM [33], fresh colon tissues were collected, sectioned into roughly 1.5 mm³ pieces, and fixed in 2.5% glutaraldehyde solution (GA 2607, Sigma-Aldrich, USA). The tissues were then postfixed in 1% osmium tetroxide and embedded in epoxy resin. Ultrathin sections, about 80 nm thick, were prepared using an ultramicrotome (Leica EM UC7, Germany) and placed on 100-mesh copper grids. The sections were stained with uranyl acetate and lead citrate. These sections were analyzed using a Tecnai G2 20 S-TWIN transmission electron microscope (FEI, US), equipped with a high-resolution camera system to examine ultrastructural details .

Quantitative real-time PCR (qRT-PCR)

Total RNA from mouse colon tissues was isolated using the RNA Purification Kit (RC112-01, Vazyme, Nanjing, China). This RNA was reverse-transcribed to complementary DNA with the HiScript III RT SuperMix Kit (R323-01, Vazyme, Nanjing, China), adhering to the manufacturer’s guidelines. qRT-PCR was executed on a CFX96 Detection System (Bio-Rad) using ChamQ SYBR qPCR Master Mix (Q311-02, Vazyme, Nanjing, China). Target gene Ct values were normalized to the housekeeping gene GAPDH, and relative fold changes against controls were determined using the _ΔΔCt method. In EGCs, TL1A expression was inversely related to the _ΔCt: high expression was indicated by _ΔCt ≤ 5, intermediate by _ΔCt 5–15, low or very low by _ΔCt ≥ 15, and undetectable by _ΔCt ≥ 40, as previously described [34].

The primer sequences were as follows:

• claudin-1 (mouse, Gene ID: 12737).

  • forward: 5’-TGCCCCAGTGGAAGATTTACT-3’,

  • reverse: 5’-CTTTGCGAAACGCAGGACAT-3’;

• occludin (mouse, Gene ID: 18260).

  • forward: 5’-TGAAAGTCCACCTCCTTACAGA-3’,

  • reverse: 5’-CCGGATAAAAAGAGTACGCTGG-3’;

• ZO-1 (mouse, Gene ID: 21872).

  • forward: 5’-GCTTTAGCGAACAGAAGGAGC-3’,

  • reverse: 5’-TTCATTTTTCCGAGACTTCACCA-3’;

• GFAP (mouse, Gene ID: 14580).

  • forward: 5’-CGGAGACGCATCACCTCTG-3’,

  • reverse: 5’-AGGGAGTGGAGGAGTCATTCG-3’;

• TL1A (mouse, Gene ID: 326623).

  • forward: 5’-AGTCCCAGTGGAAGTGCTG-3’,

  • reverse: 5’-GTGCTAAGTCCTGCGAGGAT-3’;

• GAPDH (mouse, Gene ID: 14433).

  • forward: 5’-TGGCCAAGGTCATCCATGACAACTT-3’,

  • reverse: 5’-CCAGTGAGCTTCCCGTTCAGCTC-3’.

Intestinal permeability test

Mice were subjected to an overnight fast and subsequently administered an oral gavage of fluorescein isothiocyanate (FITC)-conjugated dextran (molecular weight: 3–5 kDa, FD4, Sigma) at 50 mg per 100 g of body weight on day 7 of DSS treatment. Four hours post-gavage, blood samples were collected through retro-orbital bleeding to quantify the FITC-dextran signal using a microplate reader configured for excitation/emission at 485/520 nm. The fluorescence intensity was then analyzed using a standard curve obtained by serially diluting FITC-dextran in PBS. As a control, serum samples from untreated mice were used to establish a background fluorescence level [35].

Enzyme-linked immunosorbent assay (ELISA)

Colon tissue was homogenized in cell lysis buffer with a protease inhibitor cocktail, then subjected to ultrasonic disruption and centrifuged at 13,000 rpm for 20 min. The resulting colon tissue homogenate was then diluted to a concentration of 500 µg/mL and aliquoted into 100 µL samples. The levels of mucin 2 (Muc2) (RGB-60310 M, RigorBio Technology Development Co., Ltd., China) and various cytokines, including TNF-α (ELM-TNFa, RayBiotech Inc., US), IFN-γ (ELM-IFNg, RayBiotech Inc., US), IL-1β (ELM-IL1b, RayBiotech Inc., US), IL-17 A (ELM-IL17, RayBiotech Inc., US), IL-6 (ELM-IL6, RayBiotech Inc., US) and IL-10 (ELM-IL10, RayBiotech Inc., US), were quantified in the colon tissue homogenate using ELISA kits according to the manufacturers’ instructions.

Isolation of lamina propria lymphocytes (LPLs)

The isolation of LPLs from mouse colon tissue followed a previously established protocol [36]. Freshly isolated colon tissues were first cleansed of lumenal content by rinsing with Dulbecco’s Modified Eagle Medium (DMEM). The tissue was then cut into 3–5 mm pieces and incubated in pre-warmed DMEM containing 5% fetal bovine serum (FBS), 5 mM ethylenediaminetetraacetic acid, and 1 mM dithiothreitol for 20 min. Subsequently, the tissue was digested with DMEM containing 5% FBS, 50 mg/mL deoxyribonuclease (DNase, D4527, Sigma) and 75 mg/mL collagenase II (1148090, Sigma) at 37 °C for 30 min. The resulting cell suspension was collected and filtered through a 100-µm strainer. LPLs were subsequently isolated using an 80%/40% Percoll gradient and washed with PBS before further experiments.

Flow cytometric analysis

The following antibodies were obtained from Biolegend: PerCP/Cyanine5.5 anti-mouse CD45 (103132), APC/Cyanine7 anti-mouse CD4 (100414), PE anti-mouse IFN-γ (505807), PE/Cyanine7 anti-mouse IL-17 A (506922), APC anti-mouse CD25 (102011) and PE anti-mouse/rat/human FOXP3 (320008). To detect intracellular cytokines IFN-γ and IL-17 A, sorted LPLs were initially stimulated with a cell activation cocktail (423303, Biolegend) for 6 h at 37 °C in a 5% CO₂ atmosphere. Afterward, cells were stained with the Zombie Aqua™ Fixable Viability Kit and surface markers for 30 min at 4 °C in the dark, followed by fixation and permeabilization. The cells were then stained with anti-IFN-γ or anti-IL-17 A antibodies. For Foxp3 expression analysis, sorted LPLs were stained with an anti-Foxp3 antibody without the need for stimulation or blocking, and the remaining steps were identical to those for intracellular cytokine staining. The data were acquired using a BD FACSAria™ III Cell Sorter and analyzed with FlowJo software (TreeStar). For fluorescence-activated cell sorting (FACS) analysis, colon tissue underwent enzymatic dissociation in a solution with 0.3 mg/mL bovine serum albumin and 1.3 mg/mL collagenase II for 60 min at 37 °C. The resulting cells were immunolabeled with a GFAP antibody (Invitrogen, 53989282). Subsequently, the stained cells were subjected to FACS using a CytoFLEX platform (Beckman Coulter), and the isolated cells were utilized for detecting TL1A mRNA expression.

Metagenomic sequencing and analysis

Stool samples underwent DNA extraction using the QIAamp^® Fast DNA Stool Mini Kit (Qiagen, Hilden, Germany). OE Biotech Co., Ltd. (Shanghai, China) prepared DNA libraries and performed shotgun metagenomic sequencing, producing 150 bp paired-end reads [37]. Alpha and beta diversity were calculated with R software (v 4.1.2), using Good’s Coverage, Chao1, and Shannon indices for alpha diversity, and principal component analysis (PCA), principal coordinate analysis (PCoA), and non-metric multidimensional scaling (NMDS) for beta diversity. Post host DNA removal and fastp (v 0.20.1) quality filtering, the taxonomic profile of the microbiota was assembled. Species taxonomy relied on the NR Library database, and species abundance was determined from gene abundance. The R package facilitated the analysis of significant group differences using ANOVA and Kruskal-Wallis tests. Further analysis applied the linear discriminant analysis effect size (LEfSe), KEGG, and CAZy approaches.

Statistical analysis

Data were presented as mean ± SEM. Prior to conducting parametric tests, data normality was assessed using the Shapiro-Wilk test, and homoscedasticity (equal variance) was evaluated using Levene’s test. For comparisons between two groups, Student’s unpaired t-test was used, whereas one-way ANOVA with Tukey’s post hoc test was applied for multiple group comparisons when assumptions of normality and homoscedasticity were met. For data that did not meet these assumptions, the Kruskal-Wallis test was used as a non-parametric alternative. Graphical visualizations were generated with GraphPad Prism (version 10.0, San Diego, CA, USA), and R software (version 4.1.1) facilitated metagenomic data visualization.

Results

WKB administration ameliorated DSS-induced colitis through pretreatment

To explore the potential protective effect of WKB on IBD, we utilized a mouse model of DSS-induced colitis fed with a WKB diet. As shown in Fig. 1B, mice in the ND-control and WKB-control groups maintained relatively stable weight, whereas those in the ND-DSS and WKB-DSS groups exhibited noticeable weight loss starting from the fifth day of DSS administration. Compared to the ND-DSS group, WKB administration notably alleviated DSS-induced colitis, as shown by reduced weight loss (Fig. 1B), less hemafecia (Fig. 1C) and a lower DAI (Fig. 1D). By the end of the experiment, the ND-DSS group displayed significantly higher weight loss than the ND-control group, whereas the WKB-DSS group exhibited significantly less weight loss compared to the ND-DSS group (Fig. 1E). Furthermore, the extent of colonic mucosa injury was systematically evaluated, revealing that WKB administration significantly protected against DSS-induced shortening of colon length and decreased colonic mucosa damage index in mice (Fig. 2A-C). Histologically, mice in the WKB-DSS group showed reduced inflammatory cell infiltration, better-preserved colonic architecture, less mucosal damage, and lower histology scores compared to the ND-DSS group, which displayed crypt loss, mononuclear cell infiltration, severe mucosal damage, and higher histology scores (Fig. 2D, E). Higher magnification images of the colon tissue in Fig. 2D, along with an additional set of 200x and 400x magnifications, are provided in Supplementary Fig. 2 for further clarity. Moreover, WKB treatment prior to DSS exposure significantly attenuated DSS-induced changes in submucosal thickness, as evidenced by a reduction in both the minimum and maximum values (Fig. 2F, G). Collectively, these findings suggest that WKB pretreatment effectively alleviated DSS-induced colitis.

Fig. 2

WKB diet alleviated intestinal injury in the colon of DSS-induced colitis. (A) Representative images of the colon at the end of week 17 of the four groups. (B) Colon length. n = 6. (C) Colon mucosal damage index scores at the end of week 17. n = 6. (D) Representative H&E staining (200x magnification) of the colon tissue. Arrows indicate key features of histological damage, including epithelial injury, crypt destruction, goblet cell loss, and inflammatory cell infiltration. Scale bars: 100 μm. (E) Histopathological score. n = 3. (F) The min value of submucosal thickness. n = 3. (G) The max value of submucosal thickness. n = 3. Data are shown as mean ± SEM, P values were calculated using One-way ANOVA with Tukey’s test. *P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001. All experiments were performed in two independent biological replicates

WKB protected mucosal barrier function in mice with DSS-induced colitis

Subsequently, we assessed the impact of the WKB diet on the intestinal barrier function in mice with DSS-induced colitis using a combination of immunofluorescence, western blot, TEM, PAS/AB and DAB staining on colonic tissue. Notably, the WKB-DSS group exhibited significantly upregulated expression of claudin-1, occludin and ZO-1 compared to the ND-DSS group (Fig. 3A; Supplementary Fig. 3A), which was validated by qRT-PCR (Fig. 3B). These tight junction proteins are critical for maintaining intestinal barrier integrity, and their upregulation indicates improved barrier function [38]. Quantification of fluorescence intensity supported these findings (Supplementary Fig. 3B). To complement these observations, western blot analysis was performed, confirming the increased expression of ZO-1, occludin, and claudin-1 in the WKB-DSS group compared to the ND-DSS group (Supplementary Fig. 3C). This additional evidence strengthens the conclusion that WKB enhances tight junction protein expression and improves intestinal barrier integrity. TEM revealed the microstructure of microvilli on the colonic mucosa, demonstrating that control mice (ND-control group and WKB-control group) possessed long, dense and orderly microvilli. In contrast, mice challenged with DSS exhibited shriveled, disorganized and sparse microvilli, which was remarkably alleviated by prior WKB administration (Fig. 3C). Consistently, FITC-dextran analysis showed increased intestinal permeability in DSS-treated mice compared to control mice, while the serum level of FITC-dextran in the WKB-DSS group was lower than that in the ND-DSS group (Fig. 3D). Goblet cells play a critical role in maintaining intestinal barrier integrity by secreting mucins, such as Muc2, which form a protective mucus layer. This layer prevents pathogen invasion, reduces inflammation, and supports overall gut homeostasis [39]. PAS/AB staining demonstrated a significant augmentation of goblet cell populations in the colonic mucosa of mice with DSS-induced colitis pre-treated with WKB (Fig. 3E, F). To aid visualization, goblet cells have been marked with arrows in Fig. 3E. This increase in goblet cell populations was accompanied by a consistent upregulation of Muc2 expression, as shown by immunohistochemical staining (Fig. 3G) and increased Muc2 protein levels in colonic tissue detected by ELISA (Fig. 3H). Muc2 staining reflects the functional status of goblet cells and the integrity of the mucus layer, with increased expression indicating enhanced goblet cell activity and improved intestinal barrier function [40]. Collectively, our findings underscore the protective effect of WKB on intestinal barrier function in DSS-induced colitis.

Fig. 3

WKB diet protected mucosal barrier function in mice with colitis. (A) Representative images of immunofluorescence staining in colon tissue with antibodies against claudin-1, occludin and ZO-1 at 200x and 400x magnification. Scale bars: 75 μm and 25 μm, respectively. (B) Relative mRNA expression of claudin-1, occludin and ZO-1. n = 6. (C) Representative images of the colon tissue scanned using a transmission electron microscope. scale bars: 1 μm. Yellow arrows indicate the microvilli structure in each group. ND-control and WKB-control groups show intact and well-organized microvilli, while ND-DSS group exhibits significant damage, including shortened and disorganized microvilli. WKB-DSS group demonstrates partial restoration of microvilli structure compared to ND-DSS group. (D) Serum concentration of FITC-dextran. n = 6. (E) Representative images of AB-PAS staining in colon tissues at 200x and 400x magnification. Scale bars: 100 μm and 50 μm, respectively. (F) The number of goblet cell under 400x magnification. n = 6. (G) Representative images of IHC staining with antibodies against Muc2 at 200x and 400x magnification. Scale bars: 100 μm and 50 μm, respectively. (H) The Muc2 content in colon tissue with the normalized by total protein. n = 6. Data are shown as mean ± SEM, P values were calculated using One-way ANOVA with Tukey’s test. *P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001. All experiments were performed in two independent biological replicates

WKB decreased enteric glial response through TL1A inhibition

EGCs play a crucial role in maintaining the integrity of the intestinal epithelial barrier, which is essential for restoring injured epithelium in IBD [41]. EGCs activation is closely linked to inflammation, and our analysis of the GSE95095 dataset revealed a significant upregulation of the EGCs marker GFAP in inflamed colonic mucosa from Crohn’s disease patients compared to healthy controls (Supplementary Fig. 4A). To investigate whether EGCs contributed to the protective effect of WKB against DSS-induced colitis, we performed immunofluorescence staining (Fig. 4A, B) and qRT-PCR (Fig. 4C) to assess EGCs activation in the local inflamed microenvironment. Our results showed that both the IOD (IOD = area × density) and mRNA expression of GFAP increased in the colonic mucosa of mice after colitis. However, WKB pretreatment significantly diminished colitis-induced GFAP expression, suggesting that WKB inhibits EGCs reaction. Notably, the interaction between APC-derived TL1A and lymphocytic DR3 is involved in intestinal inflammation. Consistent with previous studies, we observed upregulation of TL1A in the ND-DSS group. Interestingly, WKB pretreatment also decreased colitis-induced TL1A expression (Fig. 4). Furthermore, immunofluorescence staining revealed that TL1A was localized on GFAP-positive EGCs (indicated by the yellow arrow in Fig. 4A). To further confirm this, GFAP-positive cells were sorted via FACS (Supplementary Fig. 4B), and qRT-PCR analysis demonstrated that these cells expressed intermediate levels of TL1A mRNA (Supplementary Fig. 4C). Collectively, our findings suggest that WKB treatment is associated with reduced EGCs activation and TL1A expression, indicating a potential link between TL1A inhibition and EGCs activation.

Fig. 4

WKB diet decreased EGCs response through TL1A inhibition in the colon of DSS-induced colitis. (A) Immunofluorescence staining for GFAP (green) and TL1A (red) in colon tissue of the four groups. The yellow box is a further enlargement of the partial picture (200× and 400x magnification). Scale bars: 75 μm and 25 μm, respectively. The yellow arrow indicates the representative colocalized cells. Negative control without primary antibodies. (B) Quantitative analysis of GFAP and TL1A positive cells relative to total nuclear number under 400x magnification. n = 6. (C) Relative mRNA expression of GFAP and TL1A in colonic mucosa of the four groups. n = 6. Data are shown as mean ± SEM, P values were calculated using One-way ANOVA with Tukey’s test. *P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001. All experiments were performed in two independent biological replicates

WKB modulated intestinal immune responses via Th1/Th17/Treg balance

The TL1A/DR3 axis, a potent co-stimulatory system, plays a role in IBD pathogenesis by modulating CD4⁺ T cells differentiation [42]. To assess WKB’s effect on T cell responses, we performed flow cytometry on T cells in the colonic LP from ND-DSS and WKB-DSS groups. The relative abundance of each cell type was measured as a percentage of total CD4⁺ T cells, forming a community data matrix for ecological analysis. Notably, WKB pretreatment significantly reduced the proportion of CD4⁺IFNγ⁺ Th1 cells (Fig. 5A) and CD4⁺IL17⁺ Th17 cells (Fig. 5B) in colonic tissue compared to ND-DSS. Conversely, the percentage of Treg cells, identified by CD25 and Foxp3 expression, increased 3.43-fold in the WKB-DSS group compared to ND-DSS (Fig. 5C). These results indicate that WKB administration promotes a balanced Th1/Th17/Treg response, thereby maintaining intestinal homeostasis. The detailed gating strategy for flow cytometry analysis is provided in Supplementary Fig. 5.

Fig. 5

WKB diet improved the frequency of Th1, Th17 and Treg cells balance in LP. (A) Representative experiment of CD4⁺IFNγ⁺ (Th1) cells in the colonic LP from ND-DSS group and WKB-DSS group were analyzed by flow cytometry and bar charts of the percentage of Th1 cells. n = 6. (B) Representative experiment of CD4⁺IL-17⁺ (Th17) cells and bar charts of the percentage of Th17 cells. n = 6. (C) Representative experiment of CD4⁺CD25⁺Foxp3⁺ (Treg) cells and bar charts of the percentage of Treg cells. n = 6. Data are shown as mean ± SEM, P values were calculated using One-way ANOVA with Tukey’s test. **P < 0.01. All experiments were performed in two independent biological replicates

WKB downregulated pro-inflammatory cytokines and upregulated anti-inflammatory cytokines in DSS-induced colitis

Our findings imply that the Th1/Th17/Treg balance is vital for gut microecosystem homeostasis, which WKB helps maintain. To explore the mechanisms involved, we analyzed cytokine expression. Figure 6 showed that pro-inflammatory cytokines (TNF-α, IFN-γ, IL-1β, IL-17, and IL-6) were significantly increased, while the anti-inflammatory cytokine IL-10 was decreased in the DSS group compared to controls. WKB pretreatment notably reduced TNF-α, IFN-γ, IL-1β, IL-17, and IL-6 levels and increased IL-10 levels. In conclusion, WKB administration substantially modulated the cytokine profile in DSS-induced colitis, indicating a potential therapeutic approach for restoring immune balance in IBD.

Fig. 6

WKB downregulated pro-inflammatory cytokines and upregulated anti-inflammatory cytokines in the colon of DSS-induced colitis. (A) The level of TNF-α cytokine in colonic mucosa homogenate of the four groups were measured by ELISA. n = 6. (B) IFNγ cytokine level. n = 6. (C) IL-1β cytokine level. n = 6. (D) IL-17 A cytokine level. n = 6. (E) IL-6 cytokine level. n = 6. (F) IL-10 cytokine level. n = 6. Data are shown as mean ± SEM, P values were calculated using One-way ANOVA with Tukey’s test. *P < 0.05, **P < 0.01 and ****P < 0.0001. All experiments were performed in two independent biological replicates

WKB modified gut microbiota composition

Multiple bacteria could activate TL1A expression [43]. To investigate whether WKB modulates the expression of TL1A by altering the gut microbiome composition, we performed metagenomic sequencing analysis on fecal bacterial DNA. Our analysis of alpha diversity metrics, including Goods coverage, Chao1 and Shannon indices, revealed no significant differences in richness and diversity between the ND-DSS and WKB-DSS groups (Fig. 7A). To further understand microbiome diversity, we conducted a beta-diversity analysis. The PCA revealed a clear separation between the fecal microbial communities of the ND-DSS and ND-control groups (Fig. 7B). WKB pretreatment adjusted the gut microbiota composition in DSS-induced dysbiosis, aligning it more closely with the control group. This result was further confirmed by PCoA and NMDS using Bray-Curtis distance algorithms (Fig. 7C, D).

Fig. 7

WKB diet modulated the gut microbiota composition in mice treated with DSS. (A) Alpha diversity index (Good’s coverage, Chao 1 and Shannon) of the fecal microbiome in the four groups. n = 3. (B) Beta diversity of bacterial using principal component analysis (PCA). n = 3. (C) Beta diversity of bacterial using Principal Coordinate analysis (PCoA) based on Bray-Curtis distance. n = 3. (D) Beta diversity of bacterial using non-metric multidimensional scaling analysis (NMDS) based on Bray-Curtis distance. n = 3. (E) Phylum-level distributions of the mouse gut microbiota. n = 3. (F) Significant differentially abundant gut bacterial phyla in the four groups. n = 3. (G) Heatmap of selected most differentially abundant features at the species level. The blue color represents less abundant, white color represents intermediate abundance and red represents the most abundant. n = 3. (H) Taxonomic cladogram generated from LEfSe analysis, illustrating taxonomic association between microbiome communities. Each node represents a specific taxonomic type. n = 3. (I) LDA score computed for differentially abundant features, with a selection criteria of a log LDA score greater than 2.5. n = 3. (J) Bar charts of KEGG pathways display 18 signifcant differences among the four groups. n = 3. Data are shown as mean ± SEM, P values were calculated using One-way ANOVA with Tukey’s test or permutational multivariate analysis of variance (PERMANOVA) or Kruskal-Wallis test. *P < 0.05, **P < 0.01

At the phylum level, the bacterial composition of all four groups exhibited a high degree of similarity, with Bacteroidota and Bacillota being the most abundant taxa (Fig. 7E). Comparisons of taxonomic profiles indicated that the ND-DSS group exhibited decreased relative abundances of Bacteroidota and increased levels of Bacillota, Hofneiviricota, and Synergistota compared to the ND-control group. Notably, WKB supplementation significantly reduced these DSS-induced changes in gut microbiota (Fig. 7F). Furthermore, taxonomic compositions were compared at the order/family/species level (Supplementary Fig. 6A). To explore the specific effects of WKB on individual gut microbes, we analyzed the top thirty significant differences between the ND-DSS and WKB-DSS groups. This analysis revealed that Muribaculum_intestinale, Alphaproteobacteria_bacterium, Paramuribaculum_intestinale, uncultured_Duncaniella_sp., Odoribacter_sp., Helicobacter_sp._UBA3407, and Muribaculaceae_bacterium were more abundant in the WKB-DSS group, whereas Escherichia_coli and Acetatifactor_sp. were less abundant in the WKB-DSS group compared to the ND-DSS group (Fig. 7G).

To identify the specific bacterial taxa that were modulated by WKB pretreatment and contributed to the alleviation of DSS-induced colitis, we performed high-dimensional class comparisons using LEfSe. Our analysis revealed that Enterococcaceae, Oscillospiraceae, Enterobacteriaceae and Moraxellaceae were the primary bacterial families associated with gut microbiota dysbiosis in the DSS group. In contrast, Veillonallaceae, Helicobacteraceae and Akkermansiaceae were enriched in the WKB-DSS group, suggesting a potential link to the WKB-mediated alleviation of colitis (Fig. 7H). In the WKB-DSS group, Helicobacter had the highest linear discriminant analysis (LDA) score of 4.10, followed by Akkermansia at 4.09 (Fig. 7I), consistent with taxonomic unit analysis. These findings suggest that WKB treatment significantly reshaped gut microbiota diversity and composition, enriching specific bacterial taxa that may aid in alleviating colitis.

WKB altered functional profle of the gut microbiome in mice treated with DSS

We conducted KEGG (Fig. 7J) and CAZy (Supplementary Fig. 6B) pathway analyses to investigate potential differences in the functional composition of the microbiome across four groups. In total, 23 differential pathways were identified. Nineteen of these pathways showed significant decreases in enrichment, including those related to metabolism, energy metabolism, folate biosynthesis, and phenylalanine, tyrosine, and tryptophan biosynthesis. In contrast, the remaining four pathways, such as cell motility, flagellar assembly, and K06400 site-specific DNA recombinase, were enriched in the ND-DSS group compared to the ND-control group. Notably, WKB pretreatment significantly alleviated the changes in pathway abundance induced by DSS.

Covariance between clinical parameters and gut microbiome

To explore the relationships among the altered microbiota, colitis-related parameters, tight junction, goblet cells and EGCs, Spearman’s rank correlation analysis was performed. Our study found that bacteria modified by the WKB diet were correlated with inflammation, gut barrier biomarkers, and EGCs biomarkers (Supplementary Fig. 6C). For instance, the histological score, a crucial parameter in colitis, was positively associated with bacterium_0.1xD8-71, Lachnospira_sp., and uncultured_Bilophila_sp., which were predominantly enriched in the ND-DSS group. These harmful bacteria were also positively associated with GFAP. The relative abundance of Ruminococcus_sp., which was reduced by the WKB diet, showed a positive correlation with GFAP and the histological score, but a negative correlation with Muc2.

Discussion

In this study, the effect of WKB diet on colon inflammation was investigated in vivo. Our findings demonstrated that WKB diet offers significant protection against DSS-induced colitis, evident through reduced body weight loss, lowered DAI scores, decreased histological scores, and enhanced integrity of the intestinal mucosal barrier. WKB mitigated colon inflammation primarily by inhibiting EGCs activation and altering the composition of the intestinal microbiota. Notably, WKB pretreatment significantly reduced GFAP expression induced by colitis, along with a reduction in the TL1A/DR3 co-stimulatory pathway. This alteration fosters a balanced Th1/Th17/Treg immune response, facilitating the maintenance of intestinal homeostasis. These results suggested that the WKB diet holds promise as a dietary strategy for IBD prevention and management, through the modulation of the intestinal microbiota, EGCs activity, and immune cell responses (Fig. 8).

Fig. 8

WKB diet’s therapeutic role in DSS-induced colitis. The WKB diet enhances tight junctions (TJs) integrity, reduces pro-inflammatory cytokines (IL-1β, TNF-α, and IL-6), and decreases EGCs activation. By modulating the TL1A/DR3 pathway, the WKB diet fosters a balanced Th1/Th17/Treg immune response, characterized by increased Treg cells and IL-10, and decreased IFN-γ and IL-17A levels. These changes collectively contribute to the significant alleviation of colitis symptoms and the promotion of intestinal homeostasis

The rising incidence of IBD poses a significant threat to human health. Conventional treatments for IBD often come with high risks of side effects and issues with efficacy [44]. The prevalence of Western dietary patterns has been identified as a critical factor in the development and progression of IBD [45]. Consequently, there is a growing body of research focused on exploring the potential of natural foods to alleviate and treat colitis. Emerging evidence suggests that legumes, rich in bioactive compounds, serve as excellent sources of protein, dietary fiber, and polyphenols, offering inherent protective effects against inflammation both in vivo and in vitro through multiple pathways. For instance, adzuki bean extracts exhibited antioxidant and anti-inflammatory properties by reducing NO production and cytokine mRNA expression in LPS-stimulated macrophages [46]. Dietary fiber from mung bean coat alleviated colon inflammation by restoring autophagy-related protein expression and inhibiting excessive apoptosis-related protein expression [47]. Additionally, baked corn and bean snack inhibited myeloperoxidase, thereby mitigating chronic colonic inflammation [48]. In this study, mice fed with a 20% WKB diet for 16 weeks showed a significant reduction in DSS-induced colonic inflammation and improved intestinal mucosal barrier function. This observation aligns with previous findings where white and red kidney beans were shown to lessen the severity of colitis, reduce tissue damage, and lower the expression of inflammatory mediators and systemic inflammatory biomarkers [49]. However, the precise mechanisms remain unclear. For the first time, we examine the role and mechanisms of whole WKB in preventing and alleviating colonic inflammation in mice from the perspectives of gut microbiota, EGCs, and immune cells. This study provides new insights and directions for the dietary management of IBD and suggests broader prospects for the development and utilization of whole WKB.

EGCs are vital in maintaining gut homeostasis by regulating intestinal epithelial barrier function, motility, and secretion in both healthy and diseased states. Inflammatory stimuli, such as LPS, IL-1β, and TNF-α, can elevate GFAP expression and increase the number of GFAP-positive cells, indicating that these stimuli trigger abnormal EGCs activation [50]. This shift to a “reactive glial phenotype” and the subsequent proliferation of EGCs lead to the release of significant amounts of inflammatory cytokines, including IL-1β, TNF-α, and IL-6, thereby exacerbating intestinal inflammation [51]. Therefore, EGCs may exacerbate intestinal inflammation under these conditions. In our study, the WKB diet notably decreased both the number of GFAP-positive cells and GFAP mRNA expression in DSS mice, along with reduced levels of inflammatory cytokines such as IL-1β, TNF-α, and IL-6. This suggests that WKB alleviates colitis in DSS mice by inhibiting EGCs activation. Furthermore, CD4⁺ T cells activated by APCs are involved in IBD pathogenesis. Various gut microbes have been shown to induce TL1A expression in human monocytes and monocyte-derived dendritic cells (DCs), enhancing IFN-γ production by CD4⁺ T cells [52]. Interestingly, we found that EGCs, acting as APCs, express TL1A, a marker found elevated in the intestinal mucosa of IBD patients [53]. Consistently, we observed increased TL1A expression in DSS-treated mice, which was notably reduced after the WKB diet intervention. T cell responses in the colonic LP were assessed via flow cytometry. The WKB-DSS group exhibited significant improvement in the Th1/Treg balance within the intestinal mucosa. This improvement was marked by a higher frequency of CD25⁺Foxp3⁺ cells, fewer CD4⁺IFN-γ⁺ cells, increased IL-10, and reduced IFN-γ levels, compared to the DSS group. Moreover, Th17 cells, which are abundant in the intestine and essential for modulating immune responses [54], were notably decreased, as reflected by lower counts of CD4⁺IL-17⁺ cells and reduced IL-17 A levels. Our findings suggest that WKB may alleviate colonic inflammation through two primary mechanisms: the inhibition of EGCs activation and the suppression of TL1A expression in these cells, thereby improving the Th1/Th17/Treg balance during the progression of IBD. Previous studies have shown that TL1A enhances DCs antigen uptake capacity via the DC-SIGN/RAF1/p65/NF-κB signaling pathway and synergistically promotes toll-like receptor-mediated DC activation. This mechanism is critical for TL1A-driven Th1 and Th17 differentiation [52]. Based on these findings, we hypothesize that the WKB diet may alleviate colitis by inhibiting TL1A expression in EGCs, thereby reducing the activation of the RAF1/p65/NF-κB pathway and downstream inflammatory signaling. However, our current study does not directly investigate these downstream pathways, and this remains a limitation.

Alterations in the gut microbiome are central to initiating the pro-inflammatory environment observed in IBD [53]. Diets rich in fiber and polyphenols, such as those that include fruits and vegetables, have been shown to enhance gut microbial diversity and increase the abundance of beneficial genera like Bifidobacterium and Lactobacillus, potentially reducing the incidence of IBD [55]. Current research increasingly highlights the complex interactions between the gut microbiota and the immune system, which are pivotal in the pathogenesis of IBD. In our study, we employed metagenomic sequencing to assess changes in microbial diversity and composition following a WKB diet. Under non-DSS conditions, WKB supplementation had minimal impact on microbial diversity and composition, suggesting that its effects on the gut microbiota are limited under normal physiological conditions. However, under DSS-induced inflammatory conditions, beta diversity analysis revealed distinct clustering of the WKB-DSS group separate from the DSS group, indicating that the WKB diet significantly altered the microbial community structure in response to inflammatory stress. Notably, the abundance of Escherichia coli was reduced in the WKB-DSS group compared to the ND-DSS group. Given Escherichia coli’s role in activating TL1A, this may provide a plausible explanation for the WKB diet’s suppression of TL1A expression. To identify potential biomarkers and dominant bacteria mediated by the WKB diet, LEfSe analysis was conducted. The genus Akkermansia was relatively enriched in the WKB-DSS group. Akkermansia muciniphila, which thrives on host intestinal mucus, is known to enhance gut barrier function, modulate immune responses, and is associated with metabolic health. It has been recognized as a next-generation probiotic with protective effects against type 2 diabetes, obesity, and bone loss [55]. In our study, we also observed an increase in goblet cell and Muc2 content, which may be linked to the elevated abundance of Akkermansia induced by the WKB diet. KEGG pathway analysis revealed that the WKB diet al.leviates DSS-induced suppression of phenylalanine, tyrosine, and tryptophan synthesis. These amino acids serve as precursors for neurotransmitters such as dopamine and serotonin, which are critical for regulating mood, gut motility, and immune function [56,57,58]. Beyond their roles as neurotransmitter precursors, these amino acids also contribute to immune modulation and gut health through distinct metabolic pathways. For instance, phenylalanine and tyrosine metabolism supports the production of catecholamines, such as dopamine and norepinephrine, which modulate immune cell signaling, cytokine release, and inflammatory responses, thereby helping to restore immune balance in IBD [59, 60]. Similarly, tryptophan metabolism via the kynurenine pathway generates immunomodulatory metabolites, such as kynurenine and kynurenic acid, which regulate T-cell activity and suppress excessive inflammation [61]. Additionally, serotonin, derived from tryptophan, plays a key role in maintaining gut motility, epithelial barrier integrity, and shaping the gut microbiota [62]. Furthermore, the metabolism of cofactors and vitamins, which are essential for enzymatic reactions, also contributes to immune cell function and inflammatory regulation [63]. By restoring these metabolic pathways, the WKB diet may enhance neurotransmitter production, modulate immune responses, and improve gut barrier integrity, collectively mitigating IBD-associated inflammation. While these findings highlight significant shifts in microbial composition and metabolic pathways, the specific functional mechanisms linking these changes to EGCs activation and inflammation control remain unclear. For example, Akkermansia muciniphila may exert its protective effects through the production of short-chain fatty acids or other metabolites, which are known to enhance gut barrier integrity and modulate immune responses, potentially suppressing pro-inflammatory pathways such as TL1A signaling [64]. Similarly, the reduction of Escherichia coli, a known activator of TL1A, may alleviate inflammation by attenuating downstream signaling cascades. Future studies employing metabolomic and functional assays are needed to identify key microbial metabolites and their interactions with host signaling pathways. This will provide deeper insights into how the WKB diet modulates gut microbiota to inhibit EGCs activation, enhance gut barrier integrity, and control inflammation.

While our findings demonstrated the protective effects of WKB in a murine model of colitis, its applicability to human IBD patients requires further investigation. The 20% WKB inclusion level used in our study was selected based on its physiological relevance and feasibility for human diets, as recommended by the Dietary Guidelines for Chinese Residents (2022), which advocate for a diet rich in legumes (50–100 g of dry legumes per day, equivalent to 150–300 g of cooked legumes). To ensure the relevance of this dosage for mice, we used body surface area scaling to translate human dietary intake to an equivalent mouse dose [65]. Using the standard formula for dose conversion: Mouse dose (g/kg) = Human dose (g/kg) × (Human Km / Mouse Km). Where Km is the body surface area normalization factor (Km for humans = 37, Km for mice = 3). Assuming an average human intake of 200–300 g of cooked WKB per day (approximately 3.33–5 g/kg for a 60 kg adult), the equivalent mouse dose is: Mouse dose = (3.33–5 g/kg) × (37 / 3) = 41–62 g/kg/day. In our study, the 20% WKB diet corresponds to approximately 50 g/kg/day for mice, which falls within this range. This confirms that the experimental dosage is physiologically relevant and achievable for human consumption. However, determining the optimal dosage, duration, and composition of WKB for human consumption will require further investigation. Human gut microbiota is highly diverse and individualized, which may lead to variability in the response to WKB intervention. Factors such as dietary habits, genetic predisposition, and disease heterogeneity in IBD patients could influence the efficacy of WKB. Translating these findings into clinical practice will require studies in humanized mouse models or well-designed clinical trials to validate the therapeutic potential of WKB. In this study, DSS was administered for one week at the end of the experiment to evaluate the protective effects of long-term WKB supplementation against acute colitis. This design allowed us to focus on the cumulative effects of WKB on gut health and its ability to mitigate acute inflammatory responses. While an earlier or prolonged DSS administration could better simulate chronic inflammatory conditions, our study was not designed to address these aspects. Future studies will explore the impact of WKB on both the onset and progression of colitis using chronic inflammation models to provide a more comprehensive understanding of its therapeutic potential. In addition, our study was conducted exclusively in male mice to minimize variability and ensure consistency in the experimental results. However, male and female mice are known to exhibit differences in immune responses, gut microbiota composition, and susceptibility to DSS-induced colitis, which may influence the effects of dietary interventions such as WKB supplementation. As such, the findings of this study may not fully capture potential sex-specific differences. Future studies should include both male and female mice to comprehensively evaluate the effects of WKB supplementation and its potential applicability to both sexes. Despite these challenges, our study provides a foundation for exploring WKB as a complementary strategy for IBD management, particularly for patients seeking non-invasive or adjunctive therapies.

In conclusion, our findings suggested that WKB held significant promise for managing IBD by enhancing mucosal barrier function and balancing Th1/Th17/Treg cells, primarily through the modulation of gut microbiota and metabolic pathways. This protective effect involved the inhibition of EGCs activity. However, although WKB showed potential as a modulator for treating colon inflammation, the specific bacterial species influenced by WKB and the precise mechanisms inhibiting glial cells required further elucidation. Additional research was needed to fully understand these pathways.