Daikenchuto ameliorates dextran sulfate sodium-induced acute and chronic ulcerative colitis by regulating gut microbiota-derived indoles to activate AhR signaling
Rui Liang, Xue Liu, Qinhua Chen, Menggai Zhang, Yinyue Xu, Hehe Shi, Sicen Wang, Wanghui Jing
TL;DR
Daikenchuto, a traditional Chinese medicine, helps treat ulcerative colitis in mice by balancing gut bacteria and boosting a key signaling pathway.
Contribution
This study reveals that Daikenchuto alleviates colitis by modulating gut microbiota and activating AhR/IL-22/STAT3 signaling.
Findings
Daikenchuto reduced inflammation and improved intestinal barrier function in mouse models of colitis.
The treatment enriched beneficial gut bacteria like Ligilactobacillus murinus and Lactobacillus species.
Activation of the AhR/IL-22/STAT3 pathway was identified as a key mechanism for the therapeutic effects.
Abstract
Ulcerative colitis (UC), a chronic-relapsing inflammatory disease with rising prevalence worldwide, is primarily driven by intestinal epithelial barrier dysfunction resulting from gut microbial dysbiosis and metabolic disturbances. Daikenchuto (DKT), a traditional Chinese medicine formulation, is commonly used for digestive disorders. Although DKT has demonstrated therapeutic potential for gut inflammation by modulating gut microbiota, its therapeutic effects on chronic ulcerative colitis (CUC) and the related mechanisms remain elusive. The main components of DKT were tentatively identified using ultra-performance liquid chromatography-quadrupole-time of flight-mass spectrometry (UPLC-Q-TOF-MS), and the therapeutic effects of DKT were evaluated in the mouse models of acute colitis (AC) and CUC induced using dextran sulfate sodium. The models were validated based on alterations in the…
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Figure 9- —https://doi.org/10.13039/501100001809National Natural Science Foundation of China
- —https://doi.org/10.13039/501100007128Natural Science Foundation of Shaanxi Province
- —Outstanding Young and Middle-Aged Scientific and Technological Talents of Shaanxi Administration of Traditional Chinese Medicine
- —https://doi.org/10.13039/100016067Shaanxi Administration of Traditional Chinese Medicine
- —Science and Technology Planning Project of Shenzen Municipality, China中国神禅市科技规划项目
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Taxonomy
TopicsGut microbiota and health · Metabolomics and Mass Spectrometry Studies · Inflammatory Bowel Disease
Introduction
Inflammatory bowel disease (IBD), encompassing ulcerative colitis (UC) and Crohn's disease (CD), is characterized by chronic colonic inflammation, which drives pathological alterations and leads to chronic, relapsing disorders [1]. UC typically presents with diarrhea, hematochezia, weight loss, and ulcerations [2]. Due to its lifelong, intermittent clinical course, UC imposes significant psychological and economic burdens on patients. As of 2023, its global prevalence has been estimated at approximately 5 million cases, with continuously increasing incidence rates [3–5]. Owing to limited insight into UC pathogenesis, current pharmacological treatments fail to induce complete remission [6].
Intestinal barrier dysregulation is implicated as a key risk factor for UC development [7, 8]. UC progression is accompanied by inflammatory responses and other pathogenic stimuli that induce intestinal epithelial injury [9]. The subsequent translocation of gut microbiota into the lamina propria activates immune signaling cascades, which may perpetuate colonic dysbiosis [8]. UC patients exhibit impaired mucosal barrier function, characterized by reduced tight junction proteins and increased intestinal permeability [10]. These pathological alterations amplify local inflammation and contribute to disease recurrence. Thus, targeting the restoration of intestinal barrier integrity is crucial for preventing UC initiation and progression.
The gut microbiota is pivotal for preserving the integrity of the intestinal mucosal barrier [11]. Gut dysbiosis, a common feature of intestinal disorders, is a key driver in UC pathogenesis [12]. UC patients exhibit reduced gut microbial biodiversity across all disease stages [13, 14]. Dextran sulfate sodium (DSS)-induced colitis mouse models exhibit reduced abundance of Bacteroidetes and Firmicutes alongside increased abundance of Proteobacteria [15]. Clinical studies have shown that the administration of probiotics, such as Bifidobacterium longum and Escherichia coli Nissle 1917, or fecal microbiota transplantation can alleviate UC [16–18]. Thus, targeted modulation of the gut microbiota offers a promising strategy for the treatment of UC.
Gut microbiota-derived metabolites, particularly short-chain fatty acids (SCFAs), bile acids, and tryptophan (Trp) derivatives, regulate host immune responses and metabolic homeostasis [19, 20]. Trp metabolites contribute to intestinal barrier maintenance and mitigate epithelial inflammation [21, 22]. Levels of Trp-derived indole metabolites are significantly depleted in UC patients, concurrent with a decreased abundance of Trp-metabolizing bacterial taxa [13]. Disrupted Trp metabolism directly impairs the microbial synthesis of these indole derivatives, which act as endogenous aryl hydrocarbon receptor (AhR) ligands [23]. Trp supplementation in DSS-induced colitis models can restore AhR ligands and increase interleukin (IL)-22, thereby reducing intestinal inflammation [24, 25].
Traditional Chinese medicine (TCM) is regarded as a valuable complementary and alternative therapeutic approach in clinical practice [26]. TCM formulations can effectively alleviate UC symptoms through multi-faceted mechanisms involving gut microbiota modulation, intestinal barrier restoration, and inflammatory suppression [27–29]. Daikenchuto (DKT), a classical formulation listed in the Catalogue of Ancient Classic and Famous Prescriptions, comprises zanthoxylum (Zanthoxyli pericarpium), dried ginger rhizome (Zingiberis rhizoma), ginseng (Ginseng radix), and maltose. DKT is employed clinically in the management of gastrointestinal disorders [30, 31].
Previous studies have demonstrated that dietary supplementation with 5% DKT extract attenuates UC by suppressing eosinophil infiltration, modulating gut microbiota, maintaining Lactobacillaceae abundance to elevate colonic propionate levels, and restoring the proportion of group 3 innate lymphoid cells (ILC3s) [32, 33]. However, these pharmacological studies on DKT have significant limitations. Most studies have focused primarily on acute colitis (AC) models, neglecting its effects on recurrent inflammation. This narrow focus limits a comprehensive understanding of its therapeutic efficacy. Additionally, while Trp metabolism plays a key role in UC pathogenesis, its specific contribution to the therapeutic mechanism of DKT remains unclear.
This study aimed to evaluate the effects of orally administered DKT in AC and chronic UC (CUC) models, with a particular focus on the Trp metabolic pathway. We found that DKT significantly attenuated inflammation in the AC model, preserved intestinal barrier integrity, and restored gut microbial homeostasis. Given the recurrent and often chronic nature of colitis, this study also validated the therapeutic efficacy of DKT in a CUC model. Mechanistically, DKT ameliorated colitis by promoting microbial Trp metabolism and activating the AhR/IL-22/STAT3 signaling pathway. In conclusion, these findings provide a scientific basis for the application of DKT in colitis management.
Materials and methods
Reagents
Zanthoxylum, dried ginger rhizome, ginseng, and maltose were purchased from Beijing Tongrentang Co. Ltd. (Beijing, China). Reference standards, including skimmianine, 6-gingerol, 6-shogaol, ginsenoside Rb_1_, ginsenoside Re, and ginsenoside Rg_1_, were obtained from Chengdu Push Bio-Technology Co. Ltd. (Sichuan, China). DSS (MW 36,000–50,000) was purchased from MP Biomedicals (Santa Ana, CA, USA), and sulfasalazine (SASP) was purchased from MedChemExpress Co. Ltd. (NJ, USA). Methanol, acetonitrile, and formic acid were sourced from Macklin Biochemical Co. Ltd. (Shanghai, China).
Animals
Male C57BL/6J mice (18–22 g, 6–8 weeks) were obtained from the Experimental Animal Center of Xi'an Jiaotong University (Xi'an, China; License No. SCXK (Shan) 2018–001). AC was induced by administering 2.5% (w/v) DSS in drinking water for 7 days, followed by 1.5% (w/v) DSS for 2 days to maintain the inflammatory state. The mice were randomly assigned to the control, AC, DKT-L (2.3 g/kg), DKT-H (6.8 g/kg), and SASP (0.2 g/kg; positive control) groups. The control and AC group mice were orally administered phosphate-buffered saline (PBS), while the other groups received the corresponding drugs (Fig. 1A). Body weight, stool consistency, and hematochezia were used to determine the disease activity index (DAI) scores.Fig. 1DKT ameliorated the pathological phenotype of DSS-induced AC. A Experimental design and treatment strategy. B Percentage of body weight changes and daily assessment of DAI scores in each experimental group during disease progression (n = 8). C Representative images of colons from the indicated groups and the colon weight to length ratio (n = 5). D MPO activity in colon tissues (n = 3). E Concentrations of inflammatory cytokines (TNF-α, IL-17A, IL-6, IL-1β, and IL-22) in colon tissues (n = 3). F Assessment of intestinal permeability in different groups (n = 3). G Immunoblot showing the expression of ZO-1 and Occludin proteins in the indicated groups (n = 3). Data are presented as the mean ± SEM. Statistical significance was determined using one-way or two-way ANOVA with Dunnett's test for multiple-group comparisons. P < 0.05, ^^P < 0.01, and ^^P < 0.001 vs. control group; ^#^P < 0.05, ^##^P < 0.01, and ^###^P < 0.001 vs. AC group
The CUC model was induced by cyclical administration of DSS, with intervening 7-day recovery periods on normal drinking water. The DSS dosage was 2.5% (w/v) for the first cycle and reduced to 2.0% (w/v) for the second and third cycles to achieve an optimal balance between disease severity and animal survival [34]. The mice were randomly allocated to the control, CUC, and DKT (6.8 g/kg) groups. The dosage was selected based on the AC model, where it demonstrated superior efficacy compared to the low dosage. The experimental design is shown in Fig. 3A. Upon completion of the study, mice were euthanized, followed by measurement of colon length and spleen weight, and collection of colonic tissues and fecal samples for subsequent analysis.
Preparation of DKT
DKT was prepared by mixing dried zanthoxylum fruit, processed ginger, and ginseng at a ratio of 1:2:1 (w/w/w). The mixture was steeped in distilled water at a ratio of 1:10 (w/v) for 2 h and decocted twice; each decoction was concentrated to half of its initial volume. The filtrates from both decoctions were mixed, followed by the addition of maltose syrup (1:5, w/w). The solution was concentrated and lyophilized to a powdered extract. The tentative identification of chemical constituents in DKT was performed using the lyophilized powder. Detailed descriptions of data acquisition, chemical library construction, and data analysis are provided in the Supplementary Material.
Evaluation of myeloperoxidase (MPO) activity and inflammatory factors in colon tissue
The colonic tissues were homogenized in ice-cold lysis buffer, and the homogenates were centrifuged to collect the supernatants for cytokine analysis. An enzyme-linked immunosorbent assay (ELISA) was employed to quantify colonic MPO activity, using a commercial kit (J&L Biological, Shanghai, China) per the manufacturer's protocol. The colonic levels of tumor necrosis factor-α (TNF-α), IL-17A, IL-6, IL-1β, and IL-22 were also measured using their respective ELISA kits (eBioscience, CA, USA). The samples were diluted to fall within the linear range of the standard curves, and absorbance was measured using a microplate reader (BioTek, USA) to calculate cytokine concentrations.
Reverse transcription and real-time quantitative polymerase chain reaction (RT-qPCR)
Total RNA was extracted from colon tissues, reverse transcribed into cDNA, and analyzed using RT-qPCR as per established methodology [35]. Relative mRNA expression levels were calculated using the 2^−ΔΔCt^ method with Gapdh as the internal control. The primer sequences for Tjp1, Ocln, Cldn2, Ahr, Il22, Reg3β, Reg3γ, Cyp1a1, and Gapdh are listed in Supplementary Table S1.
Western blotting
Following extraction from colonic tissues, total protein was quantified and analyzed by Western blotting using standard protocols [36]. The following primary antibodies were used: anti-ZO-1 (1:1000, Cat #ab276131; Abcam, Cambridge, MA, USA), anti-Occludin (1:1000, Cat #ab216327; Abcam), anti-Claudin-2 (1:1000, Cat #A14085; Abclonal, Boston, MA), anti-p-STAT3 (1:1000, Cat #ab32143; Abcam), anti-STAT3 (1:1000, Cat #ab68153; Abcam), and anti-GAPDH (1:5000, Cat #ab8245; Abcam) antibodies.
Intestinal permeability assay
Measurement of intestinal permeability was performed with fluorescein isothiocyanate (FITC)-dextran (average MW 3000–5000, Sigma-Aldrich, MO, USA) according to a standard method [29]. Intestinal permeability was assessed by measuring serum FITC-dextran levels. Briefly, the mice were orally administered FITC-dextran (60 mg/100 g). Their blood samples were collected after 5 h, and serum fluorescence (485/525 nm) was quantified (BioTek, VT, USA). A standard curve generated from serial dilutions of the compound was used for quantification.
Histopathological analysis
Following overnight fixation in 4% paraformaldehyde, distal colon tissues were paraffin-embedded, sectioned, and stained with hematoxylin and eosin (H&E) as previously described [37]. The severity of inflammation was scored based on established protocols [36]. Additionally, colonic tissues were processed for periodic acid-Schiff (PAS) histochemistry using Carnoy's fixative.
Immunofluorescence and immunohistochemistry
Immunostaining was performed according to the standard protocols [29]. Sections were probed with anti-IL-22 (1:100, Cat #ab227033; Abcam) or anti-proliferating cell nuclear antigen (PCNA; 1:100, Cat #ab92552; Abcam) primary antibodies. After rinsing with PBS, the sections were then probed with fluorochrome-conjugated secondary antibodies, followed by counterstaining with 4′,6-diamidino-2-phenylindole (DAPI) for subsequent imaging.
16S rRNA gene sequencing and analysis
Genomic DNA was extracted from fecal samples using the GeneJET Genomic DNA Purification Kit (Thermo Fisher Scientific, Waltham, MA, USA) and assessed for integrity using agarose gel electrophoresis. Negative (blank tube) and positive (mock microbial community) controls were included to monitor extraction efficiency and potential contamination. The V3–V4 hypervariable regions of the 16S rRNA gene were amplified and sequenced on an Illumina MiSeq platform (Illumina, San Diego, CA, USA). After quality control, adapter trimming, and read merging, operational taxonomic units (OTUs) were clustered at 97% similarity using VSEARCH within QIIME 2 (v2020.6). Taxonomy was assigned against the SILVA (v138.2) database with Mothur. β-diversity was evaluated via principal coordinates analysis (PCoA) based on Bray-Curtis distances in R (v4.0.3). Differential abundance was identified using linear discriminant analysis effect size (LEfSe) with a linear discriminant analysis (LDA) score threshold > 4.
Metagenomic sequencing and analysis
Genomic DNA was extracted from fecal samples using a Mag-Bind Stool DNA Kit (Omega Biotek, USA), and integrity was verified via agarose gel electrophoresis (positive and negative controls were included). For library preparation, DNA was fragmented to ~ 300-bp fragments using a Covaris M220 ultrasonicator (Covaris, Inc., USA). Paired-end libraries were constructed using the NEXTFLEX Rapid DNA-Seq kit (Bioo Scientific, USA). Sequencing was conducted on an Illumina NovaSeq platform (Illumina, San Diego, CA, USA). Raw reads were quality-controlled using fastp (v0.23.2) to trim adapters and remove low-quality sequences. Taxonomic classification was performed with Kraken2 (v2.1.2) against the RefSeq database. Functional profiling and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway annotation were conducted using HUMAnN3 (v3.0). Differential abundance analysis of microbial taxa and metabolic pathways was carried out using DESeq2 (v1.30.1). All sequencing procedures were performed by Majorbio Bio-Pharm Technology Co. Ltd. (Shanghai, China).
Targeted metabolomics analysis
The SCFAs and Trp metabolites in fecal samples were quantified using targeted metabolomics as described previously [29]. Targeted metabolomic analysis was conducted on an ultra-performance liquid chromatography-quadrupole-time of flight-mass spectrometry (UPLC-Q-TOF-MS) system (Agilent 1290 Infinity LC coupled to a 6545 iFunnel Q-TOF mass spectrometer; Agilent Technologies, Santa Clara, CA, USA). Separation was performed on an ACQUITY UPLC HSS T3 column (2.1 × 100 mm, 1.8 μm; Waters) with 0.1% formic acid in water (A) and 0.1% formic acid in acetonitrile (B). The gradient elution program was as follows: 0–2 min, 98% A; 2–10 min, 98%−65% A; 10–12 min, 65%−20% A and 12–14 min, 20%−2% A.
The MS source parameters were set as follows: sheath gas temperature at 380 ℃ with a flow rate of 12 L/min; dry gas temperature at 250 ℃ with a flow rate of 16 L/min; and nebulizer pressure at 20 psig. The nozzle voltage was 1500 V, while capillary voltages were maintained at 4000 V for positive and negative modes.
Statistical analyses
Statistical analyses were conducted using GraphPad Prism 8.0 (GraphPad, San Diego, CA, USA). Data are expressed as mean ± standard error of the mean (SEM). The normality of data distribution was assessed using the Shapiro–Wilk test. For normally distributed data, a two-tailed unpaired Student's t-test was used for comparisons between two groups, while comparisons among multiple groups were conducted using one-way or two-way analysis of variance (ANOVA), followed by Dunnett's post hoc test (for comparisons against the model group). When data did not meet normality assumptions, the Kruskal–Wallis test followed by Dunn's post hoc test was applied. For 16S rRNA gene and metagenomic sequencing data, a two-tailed Wilcoxon rank-sum test was applied in R. A P-value < 0.05 was considered statistically significant.
Results
Tentative identification of DKT components by UPLC-Q-TOF-MS
Under optimized conditions, the chemical constituents of DKT were profiled. Data acquisition in both positive and negative ionization modes ensured broad detection (Fig. S1A and S1B). A total of 74 constituents were identified or tentatively characterized, as detailed in Supplementary File S1. The predominant compounds included saponins, gingerols, flavonoids, peppermint amides, and other substances. Specifically, six core compounds, including skimmianine, 6-gingerol, 6-shogaol, ginsenoside Rb_1_, ginsenoside Re, and ginsenoside Rg_1_, were identified by comparison with reference standards (Fig. S1C). The corresponding chemical structures are provided in Fig. S2.
DKT ameliorated symptoms of experimental AC and colonic inflammation in a murine model
To evaluate the therapeutic efficacy of DKT on AC, a mouse model was established. DSS-induced mice exhibited marked weight loss and higher DAI scores, along with characteristic colitis symptoms, such as diarrhea and hematochezia (Fig. 1B). DKT administration attenuated DSS-induced weight loss and DAI elevation in a dose-responsive manner. The therapeutic potency of low-dose DKT was equivalent to that of SASP. As shown in Fig. 1C, mice with AC exhibited marked colon shortening, while high-dose DKT treatment significantly reduced the colon weight-to-length ratio. DKT also attenuated DSS-induced increase in MPO levels, indicating lower neutrophil infiltration (Fig. 1D). Additionally, DKT treatment significantly reduced TNF-α, IL-17A, IL-6, and IL-1β levels and increased the IL-22 level (Fig. 1E).
The intestinal mucosal barrier serves as the first line of defense against pathogenic invasion. The AC group showed significantly higher serum fluorescence intensity compared to the controls in the FITC-dextran permeability assay, confirming the intestinal barrier dysfunction. DKT administration significantly attenuated levels of FITC-dextran in serum compared to those of the AC group, demonstrating its protective effect on intestinal barrier integrity (Fig. 1F). Consistently, DSS administration significantly downregulated Occludin and ZO-1 expression, which were effectively restored by high-dose DKT treatment (Fig. 1G). Altogether, DKT alleviated disease symptoms, mitigated colonic inflammation, and restored intestinal barrier function in DSS-induced AC mice.
DKT regulated dysbiosis of gut microbiota in AC mice
Given the crucial role of gut microbiota in intestinal homeostasis and overall health, the impact of DKT on microbial dysbiosis was evaluated using 16S rRNA gene sequencing. Analysis of microbial α-diversity indicated that DSS administration significantly reduced the gut microbial diversity, which was partially restored after DKT treatment (Fig. 2A). In Fig. 2B, the distribution of microbial species followed distinct patterns, demonstrating both unique and shared OTUs among the three experimental groups. Furthermore, PCoA showed a clear separation of gut microbiota across experimental groups, with the DKT-H group showing greater similarity to the control group (Fig. 2C). Microbial composition was further characterized at multiple taxonomic levels. The Firmicutes (Bacillota)/Bacteroidetes (F/B) ratio is a key indicator of intestinal homeostasis [38]. Consistent with clinical observations in UC patients, the AC group exhibited a reduced F/B ratio, which was restored to near-normal levels by DKT treatment (Fig. 2D).Fig. 2DKT altered the composition of gut microbiota associated with DSS-induced AC. A α-Diversity, measured using the Shannon index (n = 5). B Venn diagram of unique and common species-level phylotypes in different groups. C β-Diversity presented as PCoA based on Bray–Curtis dissimilarity matrices (n = 8). D Distribution of dominant phyla and the Firmicutes/Bacteroidetes (F/B) ratio in different groups. E Relative abundance of gut microbiota at the family level. F Relative abundance of gut microbiota at the genus level (n = 5). G Cladogram and linear discriminant analysis (LDA) scores from LEfSe analysis. Data are presented as the mean ± SEM. Statistical significance was determined using a two-tailed Wilcoxon rank-sum test. ^^P < 0.05, ^^P < 0.01, and ^^P < 0.001 vs. control group; ^#^P < 0.05, ^##^P < 0.01, and ^###^P < 0.001 vs. AC group
The predominant bacterial families in the control group were Lactobacillaceae, Bacillaceae, and Saccharimonadaceae. In the DSS-treated mice, DKT administration effectively reversed the DSS-induced elevated abundances of Erysipelotrichaceae and Streptococcaceae and restored those of Lactobacillaceae and Bacillaceae (Fig. 2E). DSS-treated mice exhibited a decrease in the relative abundances of Lactobacillus and Bacillus but an increase in Streptococcus. However, DKT treatment reversed these DSS-induced changes (Fig. 2F). LEfSe analysis revealed discriminative taxa that were significantly enriched in the AC and DKT treatment groups (Fig. 2G). The predominant bacteria in the AC group belonged to the phylum Bacteroidetes (specifically Prevotellaceae UCG-001), while the DKT group was dominated by the phylum Firmicutes (primarily Lactobacillaceae and Lactobacillus). Taken together, DKT regulated the gut microbiota of mice and increased the abundances of Lactobacillaceae and Lactobacillus.
DKT ameliorated symptoms of experimental CUC and colonic inflammation in a murine model
Since short-term DSS administration cannot simulate the recurrent inflammation and tissue remodeling characteristic of UC, a CUC model was developed to comprehensively assess the therapeutic potential of DKT. As shown in Fig. 3B, CUC mice exhibited three cycles of body weight loss and persistently lower body weight compared to controls. DKT treatment significantly attenuated this weight loss. Additionally, DKT intervention markedly reduced the severity of clinical symptoms (diarrhea and bloody stools) and DAI scores, ultimately reaching the levels of controls. The CUC group exhibited significant colon shortening compared to controls, which was effectively attenuated by DKT (Fig. 3C, D). CUC also resulted in splenomegaly, as indicated by a marked elevation in the spleen index (spleen-to-body weight ratio). As a key immune organ, the spleen frequently enlarges in response to the systemic inflammatory burden associated with UC [39]. DKT intervention significantly reduced splenomegaly and normalized the spleen indices (Fig. 3C, D). In the control group, histopathological analysis revealed intact colonic mucosa without inflammatory infiltrates. In contrast, the CUC group exhibited severe architectural disruption with mucosal thinning, crypt destruction, and extensive inflammatory infiltration. DKT treatment ameliorated these changes and significantly lowered the histological scores (Fig. 3E). Furthermore, the CUC group showed increased MPO activity and TNF-α levels, and reduced IL-10 and IL-22 levels. DKT treatment significantly restored TNF-α and IL-22 levels, while MPO activity and IL-10 levels exhibited non-significant restorative trends (Fig. 3F). These findings indicate that the therapeutic potential of DKT against CUC might be driven by the modulation of inflammatory responses.Fig. 3DKT ameliorated the pathological phenotype of DSS-induced CUC. A Experimental design and treatment strategy. B Percentage of body weight changes and daily assessment of DAI scores in different groups (n = 8). C Representative images of spleen and colon from the indicated groups. D Spleen index (spleen-to-body weight ratio) and colon weight-to-length ratio in the indicated groups (n = 6). E Representative images of H&E-stained colonic sections from the indicated groups and the corresponding histological scores (scale bar = 100 μm, n = 3). F MPO activity and the levels of inflammatory cytokines (TNF-α, IL-10, and IL-22) in colon tissues (n = 3). Data are presented as the mean ± SEM. Statistical significance was determined using one-way or two-way ANOVA with Dunnett's test for multiple-group comparisons. ^^P < 0.01 and ^*^P < 0.001 vs. control group; ^#^P < 0.05, ^##^P < 0.01, and ^###^P < 0.001 vs. CUC group
DKT improved the intestinal barrier dysfunction in CUC mice
Mucins are critical for preserving intestinal barrier integrity and inhibiting bacterial translocation. PAS staining showed lower mucin expression in CUC mice compared to the controls, which was restored to normal levels by DKT treatment (Fig. 4A). Furthermore, compared to the CUC group, DKT intervention significantly upregulated Ocln and Tjp1, whereas Cldn2 exhibited a trend towards downregulation without reaching statistical significance (Fig. 4B). Consistently, DSS-induced CUC exhibited increased Claudin-2 protein expression and reduced protein levels of Occludin and ZO-1, which were significantly reversed by DKT treatment (Fig. 4C). CUC is characterized by impaired structural integrity of the intestinal mucosa, largely resulting from epithelial barrier damage. DKT facilitated intestinal repair in the CUC model by regulating tight junction and mucin expression.Fig. 4DKT treatment restored the compromised intestinal barrier and preserved its structural integrity. A Representative images of periodic acid-Schiff (PAS)-stained colonic tissues from the indicated groups (scale bar = 100 μm). B mRNA levels of Tjp1, Ocln, and Cldn2 in colitis tissues (n = 3). C Immunoblot showing the protein expression of ZO-1, Occludin, and Claudin-2 in the indicated groups. Data are presented as the mean ± SEM (n = 3). Statistical significance was determined using the Kruskal–Wallis test followed by Dunn's post hoc test. ^*^P < 0.05 and ^**^P < 0.01 vs. control group; ^#^P < 0.05, ^##^P < 0.01, and ^###^P < 0.001 vs. CUC group
DKT restored gut microbiota in CUC mice
Next, it was hypothesized that DKT could modulate the gut microbiota in CUC mice similar to that observed in the AC model. Metagenomic sequencing was used to test this hypothesis. α-Diversity analysis demonstrated that DKT significantly reversed the reduction in microbial biodiversity compared to the CUC group (Fig. 5A). The overall structural differences in microbial communities were assessed using PCoA. As shown in Fig. 5B, the DKT group was closer to controls compared to the CUC group, indicating that DKT treatment partially restored gut microbiota to a state resembling that of the controls. Firmicutes, Bacteroidetes, and Actinomycetota were the predominant phyla. DSS intervention increased Bacteroidetes and reduced Firmicutes and Actinomycetota. However, DKT treatment partially reversed these changes and showed a trend towards restoring the F/B ratio, although this effect did not reach statistical significance (Fig. 5C).Fig. 5DKT altered the gut microbial structure associated with DSS-induced CUC. A α-Diversity, measured using the Shannon index. B β-Diversity presented as PCoA based on Bray–Curtis dissimilarity matrices. C Circos diagram of dominant phyla and the F/B ratio in different groups. D Relative abundance of gut microbiota at the family level. E Relative abundance of gut microbiota at the genus level. F Relative abundance of gut microbiota at the species level. Data are presented as the mean ± SEM (n ≥ 3). Statistical significance was determined using a two-tailed Wilcoxon rank-sum test. ^^P < 0.05, ^^P < 0.01, and ^^P < 0.001 vs. control group; ^#^P < 0.05, ^##^P < 0.01, and ^###^P < 0.001 vs. CUC group
At the family level, DSS administration significantly reduced Lactobacillaceae and increased Bacteroidaceae and Enterobacteriaceae. The DKT treatment reversed these dysbiosis trends (Fig. 5D). At the genus level, the CUC mice exhibited decreased Ligilactobacillus and Lactobacillus and increased Bacteroides, Phocaeicola, and Escherichia abundances (Fig. 5E). DKT treatment effectively restored these genera to near-baseline levels. Further analysis at the species level revealed that Ligilactobacillus murinus, Lactobacillus taiwanensis, and Lactobacillus johnsonii were downregulated in the CUC group, while Bacteroides acidifaciens, Bacteroidales bacterium, and E. coli were markedly upregulated (Fig. 5F). DKT intervention reduced the abundance of harmful species, thus demonstrating its ability to ameliorate dysbiosis of gut microbiota in CUC.
LEfSe identified discriminative taxa, revealing a significant increase in Lactobacillaceae following DKT treatment. Notably, genera such as Lactobacillus and Limosilactobacillus displayed elevated LDA scores following DKT treatment, implicating their involvement in the DKT's protective mechanisms against CUC (Fig. 6A, B). The KEGG pathway analysis is shown in Fig. 6C. Spearman correlation analysis further established significant correlations between alterations in the gut microbiota and colitis-related parameters. Notably, the abundance of L. taiwanensis (upregulated by DKT) and E. coli (downregulated by DKT) correlated significantly with the major indicators of intestinal barrier integrity (Fig. 6D). Collectively, DKT modulated the gut microbiota in CUC mice, with L. taiwanensis showing significant correlations with intestinal barrier-related parameters.Fig. 6. Specific gut microbiota linked to the development of CUC. A Diagram of LEfSe analysis. B LDA of LEfSe analysis. C Bubble chart showing the enriched KEGG pathways based on differential gut microbiota. D Spearman correlation heatmap between gut microbiota and physicochemical indicators associated with colitis. ^*^P < 0.05 and ^**^P < 0.01
DKT enriched gut microbiota-derived Trp but not SCFAs
Alterations in the gut microbiota frequently correlate with shifts in microbial metabolite profiles. To determine whether the therapeutic effects of DKT on CUC were mediated by gut bacterial metabolites, targeted metabolomic analysis was conducted on the fecal samples, with particular focus on Trp metabolites and SCFAs. The metabolic profiles of the different groups were analyzed using partial least squares-discriminant analysis (PLS-DA). As shown in Supplementary Fig. S3A, DKT treatment subtly shifted the overall composition of SCFA metabolites. Although DKT increased hexanoic acid, isovaleric acid, and propionic acid, the total SCFA content showed no significant difference. Moreover, the changes in acetic acid, butyric acid, isobutyric acid, and pentanoic acid remained limited (Fig. S3B). This suggested that SCFAs were not the key mediators of the anti-inflammatory and therapeutic effects of DKT in CUC model. The Trp metabolites were also quantified. As shown in Fig. 7A, PLS-DA demonstrated clear separation among the groups, suggesting significant differences in Trp metabolite profiles. DKT administration markedly altered the levels of indole-related metabolites, while exerting limited effects on kynurenine (Kyn) and 5-hydroxytryptamine (5-HT) (Fig. 7B, C and D). Specifically, Trp levels were markedly lower in the CUC group. In addition, a notable reduction in indole and its derivatives, including indole-3-acetic acid (IAA), indole-3-propionic acid (IPA), indole-3-acrylic acid (IA), and indole-3-carboxylic acid (ICA), was observed. In contrast, DSS treatment elevated the indole-3-lactic acid (ILA) levels. DKT intervention increased tryptamine, IPA, and IAA levels while decreasing ILA levels. Notably, DKT treatment increased the fecal levels of indole, IA, and ICA by almost twofold compared to the CUC group. Spearman correlation analysis also revealed significant associations between gut microbiota and Trp metabolites. L. murinus and L. taiwanensis correlated positively with Trp metabolites, particularly indole and its derivatives, while E. coli exhibited a strong negative correlation with 5-HT (Fig. 7E). Taken together, these results demonstrated that DKT intervention altered Trp metabolism, with significant increases in IA and ICA, which may serve as key potential bioactive compounds.Fig. 7DKT reversed the metabolic disorders in mice with CUC. A PLS-DA analysis of targeted tryptophan (Trp) metabolites in the feces from the indicated groups. B Fecal Kyn levels and the Kyn/Trp ratio in the indicated groups. C Levels of Trp and indole pathway metabolites in the feces from the indicated groups. D Levels of 5-HT pathway metabolites in the feces from the indicated groups. E Spearman correlation heatmap between gut microbiota and indole pathway metabolites. Data are presented as the mean ± SEM (n = 5). Statistical significance was determined using one-way or two-way ANOVA with Dunnett's test for multiple-group comparisons. ^^P < 0.05, ^^P < 0.01, and ^^P < 0.001 vs. control group; ^#^P < 0.05 and ^##^P < 0.01 vs. CUC group
DKT ameliorated CUC by modulating the AhR/IL-22/STAT3 pathway
Given that most indole metabolites affected by DKT are established ligands of AhR, the downstream mechanisms of DKT's protective effects on intestinal barrier homeostasis were elucidated. The mRNA levels of Ahr markedly decreased in the CUC group, which were upregulated following DKT treatment. Although Il22 did not reach statistical significance, its expression trend was consistent with that of Ahr. Moreover, the mRNA level of Ahr downstream target gene, Cyp1a1, was significantly reduced in the CUC group and markedly upregulated following DKT intervention. Consistently, DKT increased the Reg3β and Reg3γ mRNA levels compared to those in the CUC group (Fig. 8A). Immunofluorescence staining confirmed a decrease in IL-22 protein levels in the DSS-treated mice, which was restored following DKT intervention (Fig. 8B, C). Furthermore, the p-STAT3/STAT3 ratio significantly increased in the DKT group, suggesting enhanced STAT3 activation through the AhR/IL-22 axis (Fig. 8D). CUC group also exhibited fewer PCNA-positive proliferative cells, which were restored by DKT treatment (Fig. 8E). In summary, DKT enhanced epithelial proliferation and facilitated barrier repair in the CUC model by modulating the AhR/IL-22/STAT3 pathway.Fig. 8DKT improved CUC by regulating the AhR/IL-22/STAT3 pathway. A mRNA expression levels of Ahr, Il-22, Reg3β, Reg3γ, and Cyp1a1 in the colon tissues from the indicated groups. B Representative immunofluorescence photomicrographs of the colon showing in situ expression of IL-22 (red). Nuclei were counterstained with DAPI (blue) (scale bars = 100 μm). C Quantification of the immunofluorescence. D Expression levels of p-STAT3 and STAT3 proteins and the p-STAT3/STAT3 ratios in the indicated groups. E Immunohistochemical analysis of PCNA-positive cells in the colon tissues from the indicated groups (scale bar = 100 μm). Data are presented as the mean ± SEM (n = 3). Statistical significance was determined using the Kruskal–Wallis test followed by Dunn's post hoc test. ^*^P < 0.05, ^**^P < 0.01 vs. control group; ^#^P < 0.05, ^#^P < 0.001 vs. CUC group
Discussion
UC is a prevalent gastrointestinal disorder involving chronic inflammation of the intestinal epithelium [40]. Given the regulatory effects of TCM on gut microbiota and the intestinal barrier function, TCM represents a promising alternative therapeutic option for UC. DKT has been used for the clinical management of various gastrointestinal disorders [41, 42]. In this study, the therapeutic efficacy of DKT was evaluated in both AC and CUC models to gain insights into the underlying mechanisms. DKT administration effectively attenuated weight loss, colonic edema, and inflammation while restoring barrier integrity. Furthermore, DKT modulated gut microbiota, regulated the Trp metabolic pathway, and elevated indole derivatives, such as IA and ICA. Indole derivatives facilitate IL-22 secretion through the AhR and JAK/STAT3 signaling cascade, which in turn stimulates the proliferation of colonic crypts and enhances the production of antimicrobial peptides (AMPs). In this study, the 6.8 g/kg dose was optimized based on efficacy in the AC model. Future clinical studies should evaluate a broader dose range to establish the optimal therapeutic window.
Alterations in gut microbial composition are closely linked to the pathogenesis of UC; thus, gut microbiota can be targeted for therapeutic interventions [43]. UC patients exhibit reduced gut microbiota diversity and significant dysbiosis compared to healthy individuals [44]. In this study, DKT intervention restored the gut microbiota in AC and CUC mice by increasing beneficial bacteria, such as Lactobacillus, and reducing harmful bacteria. Lactobacillus is a common probiotic that is present in fermented foods and may relieve colitis symptoms [45, 46]. At the species level, DKT significantly reduced abundances of B. acidifaciens, B. bacterium, and E. coli, while increasing those of L. murinus, L. taiwanensis, and L. johnsonii. This suggested that these species might serve as potential probiotics for the treatment of colitis. Although the direct contribution of these specific strains cannot be definitively confirmed in the present study, previous studies have demonstrated their beneficial effects. For instance, L. murinus can increase the anti-inflammatory metabolites, such as itaconic acid and cis-11,14-eicosadienoic acid [47]. L. taiwanensis exhibits antimicrobial and immunomodulatory activities and is considered a potential probiotic. [48, 49]. In addition, L. johnsonii can alleviate colitis by restoring the regulatory T cell (Treg)/Th17 balance and increasing butyrate and propionate levels in the colon [50, 51]. Future studies should employ germ-free mice and specific strain isolation to elucidate the functional contributions of the gut microbiota and identify potential probiotic candidates.
Metabolites derived from the gut microbiota play a vital role in preserving epithelial barrier function [52]. UC patients exhibit metabolic dysregulation, which is characterized by reduced levels of SCFAs, amino acids, and sphingolipids in feces [53]. Although DKT administration did not significantly increase the total SCFA content, it markedly increased propionate levels. This finding aligns with previous studies demonstrating that dietary DKT supplementation enhances propionic acid production in colitis mice [32]. In the current study, DKT modulated Trp metabolism and increased levels of indole derivatives in the CUC model. The absence of Kyn alteration indicates that DKT specifically remodels microbiota-driven Trp metabolism rather than host-driven Kyn metabolism. Indole derivatives can upregulate anti-inflammatory cytokines and restore immune homeostasis by modulating the differentiation of intestinal immune cells [54]. Furthermore, these compounds induce mucus secretion from goblet cells and upregulate tight junction proteins in the intestinal epithelial cells. Collectively, these effects promote the repair of intestinal barrier tissue and maintain gut microbial homeostasis, thereby preventing UC progression [55].
Since DKT enriched Ligilactobacillus and Lactobacillus, which can activate AhR and alleviate colitis by metabolizing Trp into indole derivatives, we focused on the Trp metabolic pathway despite its lower ranking in the KEGG analysis [56]. Future studies should employ transcriptomic analysis or targeted functional assays to substantiate the functional predictions derived from KEGG. Additionally, AhR antagonists can be used to confirm the protective role of AhR activation in intestinal barrier function. Indole derivatives have demonstrated anti-inflammatory, barrier-restorative, and anti-tumor effects in colitis models. These effects are achieved through activation of AhR and modulation of nuclear factor-kappa B (NF-κB) and hypoxia-inducible factor (HIF) pathways [29, 57, 58]. In addition, IA serves as a ligand for AhR, thereby maintaining intestinal barrier integrity and attenuating intestinal inflammation [59]. Furthermore, ICA stimulates ILC3s via AhR to secrete IL-22, which is fundamental to the regulation of intestinal homeostasis [60]. Given the distinct spatial distribution of gut microbiota and indole metabolites, fecal concentrations may not fully reflect the higher local levels at the epithelial surface, where metabolite accumulation is likely sufficient to trigger AhR signaling [61–63]. The current study did not assess the impact of direct intervention with IA or ICA; however, DKT significantly reversed the colitis-induced reduction in fecal IA and ICA levels. This suggests that these metabolites may be key active molecules underlying the therapeutic efficacy of DKT.
Although this study aimed to evaluate the overall therapeutic effects of DKT on colitis, identifying its specific bioactive components is essential for future studies. Moreover, the pharmacological activities of the major constituents within DKT have been well documented. For instance, 6-gingerol can reduce inflammation, modulate ferroptosis, and activate adenosine monophosphate-activated protein kinase (AMPK) [64, 65]; 6-shogaol can regulate gut microbiota, with its metabolites contributing to the amelioration of intestinal inflammation [66]; ginsenoside Rb_1_ can alleviate colitis through the vitamin D receptor (VDR), peroxisome proliferator-activated receptor gamma (PPARγ), and NF-κB signaling pathways [67, 68]; and ginsenoside Rg_1_ can modulate gut microbiota and microbial Trp metabolism while restoring the T follicular helper (Tfh)/Treg balance to ameliorate colitis [69, 70]. Future studies can focus on investigating the interactions between these components and potential intestinal probiotics to elucidate the deeper mechanistic basis for DKT's therapeutic effects against UC.
The JAK2/STAT3 pathway plays a key role in regulating immune function, inflammation, and cellular growth, and it is frequently dysregulated in various cancers and inflammatory disorders [71]. STAT3 activation is influenced by several factors, including IL-6, IL-17, and the IL-23/Th17 axis; all these are crucial in regulating inflammation and maintaining intestinal homeostasis [72–74]. Furthermore, the IL-22-mediated activation of JAK2/STAT3 signaling through STAT3 phosphorylation stimulates crypt proliferation, accelerates epithelial regeneration, and enhances the secretion of the AMPs (Reg3β and Reg3γ). This, in turn, promotes epithelial repair and intestinal barrier homeostasis [75]. DKT significantly elevated IL-22 levels in the colons of CUC mice, indicating that its therapeutic effects against CUC might be driven by activation of the IL-22/JAK2/STAT3 signaling axis.
In summary, DKT improved intestinal barrier function and ameliorated colitis by modulating gut microbiota, restoring Trp metabolism, elevating indole derivatives, and activating the AhR/IL-22/STAT3 signaling pathway (Fig. 9). All the findings provide a scientific rationale for the application of DKT in the treatment of UC.Fig. 9A schematic diagram of the mechanism by which DKT might alleviate DSS-induced colitis. AhR, aryl hydrocarbon receptor; DSS, dextran sulfate sodium; IA, indole-3-acrylic acid; ICA, indole-3-carboxylic acid; IPA, indole-3-propionic acid; PCNA, proliferating cell nuclear antigen; TJs, tight junctions. This image was created using BioRender
Conclusions
DKT attenuated DSS-induced AC and CUC in mice by remodeling the gut microbiota and Trp metabolism. The efficacy of DKT can be largely attributed to the engagement of the AhR/IL‑22/STAT3 signaling axis by gut microbiota-derived Trp metabolites. The results identified DKT as an innovative and potential therapeutic approach for UC.
Supplementary Information
Supplementary Material 1.Supplementary Material 2.
The reference list from the paper itself. Each links out to its DOI / PubMed record.
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