Hudi enteric-coated capsule and its active constituent polydatin suppress Th1/Th17-mediated intestinal inflammation by modulating the KEAP1-NFE2L2 signaling pathway

Abstract

Objective

To investigate the anti-inflammatory mechanisms of Hudi enteric-coated capsule (HDEC) and its major bioactive constituent, polydatin, in ulcerative colitis (UC).

Methods

Mouse models of colitis were established by transplantation adoptive transfer of CD45RB^highCD4⁺ T cells and treated with or without HDEC/polydatin. Therapeutic efficacy was evaluated by assessing disease activity, colon length, and histopathological damage. The differentiation of Th1, Th17, and Treg cells was analyzed using quantitative real-time polymerase chain reaction and flow cytometry. In vitro cultures of mouse and human CD4⁺ T cells were utilized to assess the immunomodulatory activity. RNA sequencing, Western blotting, and immunofluorescence were used to explore the underlying mechanism. Molecular docking, molecular dynamics, and surface plasmon resonance (SPR) assays were employed to confirm the interaction between polydatin and KEAP1.

Results

Treatment with HDEC and polydatin significantly ameliorated murine colitis and mucosal damage. Mechanistically, polydatin directly binds to KEAP1 to promote NFE2L2 nuclear translocation. This NFE2L2 activation reduces intracellular oxidative stress, thereby inhibiting pathogenic Th1/Th17 differentiation and enhancing Treg generation. Importantly, these effects were consistently validated in human CD4⁺ T cells and UC mucosal tissues.

Conclusion

HDEC and polydatin alleviate UC by targeting the KEAP1-NFE2L2 axis to reduce oxidative stress, thereby restoring the Th1/Th17/Treg balance. This highlights polydatin as a promising KEAP1-targeting agent with strong translational potential.

Graphical Abstract

Graphical Abstract.

By directly binding to KEAP1 to activate the NFE2L2 pathway, polydatin restores intestinal immune homeostasis by restricting pathogenic Th1/Th17 differentiation and facilitating Treg cell development.

Introduction

Ulcerative colitis (UC) is a chronic and idiopathic inflammatory bowel disease characterized by symptoms including recurrent abdominal pain, diarrhea, rectal bleeding, and weight loss, which seriously affects patients’ quality of life. In addition, the chronic and recurrent inflammation increases the risk of cancer [1, 2]. Therefore, timely control of symptoms and the induction of long-term remission are urgent. Commercially available drugs for UC treatment include 5-aminosalicylic acid (5-ASA), azathioprine, corticosteroids, biologics, and small molecule inhibitors [3, 4]. Despite the growing choice of therapeutic approaches, the problems such as drug resistance and side-effects still persist [5, 6]. Therefore, the discovery and investigation on alternative medicines remain urgently needed for the clinical management of UC.

The pathogenesis of UC is multifactorial and involves dysregulation of a complex intestinal immune response to the luminal microbiota [7–10]. Among these immune networks, the abnormal activation of CD4⁺ T cells plays a central role. In particular, excessive differentiation of helper T cell 1 (Th1) and Th17 cells, and the lack of regulatory T (Treg) cells, leads to excessive production of pro-inflammatory cytokine such as IFN-γ and IL-17A, leading to severe and persistent colonic inflammation [11, 12]. The maintenance of CD4⁺ T cell homeostasis is essential for intestinal immune tolerance. Therefore, limiting the abnormal differentiation of Th1 and Th17 cells and promoting Treg cells to restore intestinal immune homeostasis is considered a promising strategy for the treatment of UC.

Natural products have become a major source of new drug development. In Asian countries, especially China, herbal medicines have been used to relieve gut symptoms such as diarrhea for thousands of years [13]. Hudi enteric-coated capsule (HDEC) is a traditional Chinese medicine that has been shown to be effective for various intestinal disorders such as diarrhea, abdominal pain, and tenesmus [14]. However, its clinical application in the treatment of UC remains limited, mainly due to the lack of comprehensive studies on its underlying therapeutic mechanisms. Polydatin, a major bioactive compound extracted from Polygonum cuspidatum, is a key chemical component of HDEC and has shown potential anti-inflammatory properties [15]. Previous studies have shown that polydatin attenuates colitis in mice. However, its efficacy has only been observed in the chemically induced acute colitis models, which may not accurately mimic the chronic and relapsing course of UC patients and the characteristics of immune-mediated colonic disorders. Importantly, the regulatory effects of polydatin on intestinal immune cells, particularly lamina propria CD4⁺ T cells, need to be elucidated.

Here, we established a chronic colitis model in mice induced by adoptive transfer of CD45RB^highCD4⁺ T cells to evaluate the efficacy of HDEC and its principal bioactive constituent, polydatin, in the treatment of colitis. Mechanistically, we demonstrate that polydatin effectively restores intestinal immune homeostasis by directly inhibiting the pathogenic differentiation of mucosal Th1 and Th17 cells. Furthermore, in vitro experiments using cultured CD4⁺ T cells indicated that this process involves the regulation of the KEAP1-NFE2L2 axis, providing valuable insights into the molecular mechanism by which polydatin regulates the immune response. Together, these results demonstrate that polydatin serves as a crucial active compound of HDEC. Although the full therapeutic efficacy of HDEC is mediated by the synergistic effects of its multi-component formula, polydatin plays a central role in correcting the imbalance of CD4⁺ T cell subsets. These findings provide direct preclinical and pharmacological evidence for the clinical application of HDEC and highlight polydatin as a promising targeted therapeutic agent for UC.

Materials and methods

Ethics statement

All animal experiments were conducted in accordance with the Guide for the Care and Use of Laboratory Animals and were approved by the Institutional Animal Care and Use Committee of Tongji University (SHDSYY-2018-3912) and the Ethics Committee of the Shanghai Science and Technology Commission (approval number: SYXK2021-0012). The collection and use of human samples, including peripheral blood samples for naïve CD4⁺ T-cell isolation, were approved by the Ethics Committee of Shanghai Tenth People’s Hospital (approval number: SHSY-IEC-5.0/23K104/P02), and all procedures were conducted in accordance with the Declaration of Helsinki. Informed written consent was obtained from all participants.

Mouse models

Splenic CD4⁺ T cells were purified from C57BL/6 mice using anti-mouse CD4 beads (BD Biosciences; San Diego, CA, USA). CD45RB^highCD4⁺ T cells were sorted using a BD FACSAria III (BD Biosciences) and subsequently transferred intravenously to 8-week-old Rag1^−/− mice at a dosage of 5 × 10⁵ cells per mouse to establish a CD45RB^highCD4⁺ T cell transfer-induced chronic colitis model as described previously [16].

The mice were randomly divided into the control group, HDEC group, or polydatin group, which were administered with HDEC (0.984 g/kg) or polydatin (45 mg/kg) by gavage, respectively.

Isolation of lamina propria mononuclear cells

The isolation of lamina propria mononuclear cells (LPMCs) was performed as described previously [16]. After sacrificing the mice, the intestines were carefully removed and cut into small pieces. These pieces were thoroughly washed to eliminate any remaining blood and feces. Subsequently, the tissue was gently agitated at 37°C for 30 min in a solution of PBS supplemented with 5% fetal bovine serum (FBS) and 1 mM/l EDTA. Following this, the intestines were further cut into pieces and subjected to digestion in a 10 ml solution of 5% FBS-RPMI containing collagenase A (1 mg/ml) and DNase (10 µg/ml). This digestion process was carried out at 37°C for 30 min. LPMCs were then isolated through a process of density gradient centrifugation using 40% and 70% Percoll-RPMI solutions.

T cell differentiation in vitro

To obtain naive CD4⁺ T cells, peripheral blood was collected from healthy subjects and processed using anti-human naive CD4 magnetic beads (BD Biosciences). The isolated cells were then cultured at a concentration of 5 × 10⁵ cells per well and activated with anti-CD3 mAb (5 µg/ml; eBioscience, Shanghai, China) and anti-CD28 mAb (2 µg/ml; eBioscience) in RPMI 1640 medium supplemented with 10% FBS. Subsequently, these cells were directed to differentiate into Th1 cells through an addition of IL-12 (10 ng/ml; R&D, Minneapolis, MN, USA) and anti-IL-4 monoclonal antibody (10 µg/ml; R&D) or into Th17 cells using a Th17 cocktail, which included IL-1β (10 ng/ml; R&D), IL-6 (30 ng/ml; R&D), IL-23 (20 ng/ml; R&D), anti-IFN-γ monoclonal antibody (10 µg/ml; eBioscience), and anti-IL-4 monoclonal antibody (10 µg/ml; R&D). These differentiated cells were then harvested for subsequent experiments.

Flow cytometry

To assess intracellular cytokine production, CD4⁺ T cells obtained from LPMC or those cultured in vitro were cultured in a 10% FBS-RPMI medium with ionomycin (1 μg/ml; Sigma-Aldrich, St Louis, MO, USA), PMA (50 ng/ml; Sigma-Aldrich), and brefeldin A (3 µg/ml; eBioscience) at 37°C for last 5 h. For surface staining, the cells were incubated at 4°C with monoclonal antibodies labeled for cell surface markers for 30 min. Intracellular cytokine staining was conducted using fixation-permeation buffer (eBioscience). Cells were analyzed using a BD FACS Canto II flow cytometer (BD Biosciences), and the data were processed with FlowJo_V10 software. The gating strategy is shown in the Supplementary Fig. 1.

Quantitative real-time polymerase chain reaction

Total RNA was isolated using TRIzol Reagent (Thermo Fisher Scientific; Waltham, MA, USA). Complementary DNA (cDNA) was synthesized using 5 × All-in-one RT Mastermix (abm; Richmond, BC, Canada). The quantitative real-time polymerase chain reaction (qRT-PCR) was conducted using the SYBR green method with the following thermal cycling conditions: 95°C for 1 min, followed by 40 cycles of 95°C for 15 s and 60°C for 30 s. The specific primer sequences were listed in Supplementary Table 1. The expression levels of target genes relative to GAPDH were determined by the 2^−ΔΔCt method.

Immunofluorescence analysis

Naive CD4⁺ T cells were isolated from peripheral blood of healthy subjects and cultured (5 × 10⁵ cells per well) under stimulation with or without polydatin (2 µM) for 6 h. Cells were collected and seeded onto poly-L-lysine pre-treated circular coverslips, followed by incubation at 37°C for 3 h. The coverslips were then gently blotted to remove excess medium, and 50–100 μl of permeabilization working solution was added to each coverslip for 15 min to permeabilize the cells. Subsequently, the coverslips were incubated with 3% BSA for 30 min to block nonspecific binding sites. Next, a mixture of rabbit anti‐human NFE2L2 antibodies (1:100, Proteintech) and mouse anti‐human KEAP1 antibodies (1:100, Invitrogen) was added to the coverslips and incubated overnight at 4°C. Following incubation, the coverslips were washed with PBS and then incubated with Alexa Fluor® 594 conjugated goat anti‐rabbit IgG or Alexa Fluor® 488 conjugated goat anti‐mouse IgG (1:1000, Invitrogen) at room temperature for 50 min. Finally, the coverslips were incubated with DAPI solution at room temperature in the dark for 10 min. The laser confocal microscope was utilized for detailed microscopic examination and image acquisition.

Western blotting analysis

Naive CD4⁺ T cells were isolated from the peripheral blood of healthy donors and cultured for 5 days with or without polydatin. Subsequently, cells were collected to isolate cytoplasmic and nuclear proteins using NE-PER Nuclear and Cytoplasmic Extraction Reagents (Thermo Fisher Scientific). Immunoblotting was conducted as previously described [17]. Antibodies against NFE2L2 (1:1000, Proteintech), H3 (1:10 000, Proteintech), and β-actin (1:50 000, Santa Cruz Biotechnology) were used. Subsequently, it was incubated with HRP-linked secondary antibodies and scanned using the Amsman Imager 600 ECL system (GE Healthcare). Detailed antibody information is provided in Supplementary Table 2.

Statistical analysis

All statistical analyses were performed using GraphPad Prism 9 software (GraphPad Software Inc., San Diego, CA, United States). Unpaired t-test, Wilcoxon rank-sum test and one-way analysis of variance (ANOVA) were used for statistical analysis. All quantitative data were presented as the mean ± standard deviation (SD). Two-sided P-value < 0.05 was considered statistically significant.

Further materials and methods are available in the supplementary material.

Results

HDEC ameliorates chronic colitis in mice

To better simulate the chronic and recurrent characteristics of UC, we established an immune-induced chronic colitis model by intravenous transfer of CD45RB^highCD4⁺ T cells from the spleen of wild-type C57BL/6 mice into Rag1^−/− recipients. From week 4 to week 8 after transfer, HDEC (0.984 g/kg) was orally administered to the mice daily. As expected, the recipient mice presented with severe colitis symptoms, characterized by significant weight loss, diarrhea, and a persistently elevated disease activity index (DAI) score. Macroscopic and histological evaluations further revealed that the placebo-treated mice exhibited severe colonic tissue damage and extensive infiltration of inflammatory cells, resulting in a significant increase in the pathological score. Notably, oral administration of HDEC markedly alleviated these clinical symptoms and reduced severe colonic pathological changes. Flow cytometry analysis showed a reduced proportion of CD4⁺ T cells expressing IL-17A or IFN-γ in the HDEC-treated group compared to controls. Concurrently, mRNA levels of T-bet, Ifn-γ, Rorc, Il-17a, and Tnf-α were markedly downregulated in the colonic tissues of HDEC-treated mice (Fig. 1).

Figure 1.

Intragastric administration of HDEC significantly alleviates CD45RB^highCD4⁺ T cell transfer-induced chronic colitis in mice. (A) Splenic CD45RB^highCD4⁺ T cells were isolated from C57BL/6 mice using flow cytometric sorting and injected intravenously into 8-week-old Rag1^−/− mice (5 × 10⁵ cells/mouse, n = 6/group). (B) Mice were weighed weekly after T cell transfer and expressed as a percentage of baseline. (C) DAI scores were calculated weekly after T cell transfer. (D) Representative colon sections were stained with H&E. (E) Pathological scores of the colon tissues were calculated. (F, G) Intracellular expression of IFN‐γ and IL‐17A was analyzed by flow cytometry in CD4⁺ T cells isolated from LPMCs (numbers in graphs indicate percentage of cells). Percentages of IL‐17A⁺ and IFN‐γ⁺CD4⁺ T cells were shown in the graph. (H–M) Colon tissues of these mice were obtained, and the transcriptional expression of T-bet, Ifn-γ, Il-1β, Rorc, Il-17a, and Tnf-α was analyzed by qRT‐PCR. *P < 0.05, **P < 0.01, ***P < 0.001 by Student’s t-test.

Collectively, these findings suggest that HDEC alleviates intestinal mucosal inflammation by inhibiting the differentiation of Th1/Th17 cells.

Polydatin serves as the primary active component of HDEC

Given the complex composition of HDEC, we attempted to identify the main active components that regulate T cell differentiation. We evaluated the effects of the main chemical components (polydatin, emodin, gallic acid, quercetin, luteolin, formononetin, kaempferol, naringenin, and isorhamnetin) previously identified by HPLC [18]. Naïve CD4⁺ T cells were isolated from the spleens of mice and cultured in vitro for 5 days under Th1- or Th17 cell-polarizing conditions in the presence of each specific compound. Flow cytometry analysis showed that among these candidates, polydatin inhibited the differentiation of Th1 and Th17 cells most significantly (Supplementary Fig. 2). These in vitro results suggest that polydatin is a major active component regulating CD4⁺ T cell differentiation in HDEC.

To determine the optimal dose of polydatin, we first performed a dose-titration experiment using a DSS-induced colitis model. We tested polydatin at low (15 mg/kg), medium (45 mg/kg), and high (90 mg/kg) doses. The results showed that the low dose had a weaker therapeutic effect. However, the medium and high doses showed similar and excellent efficacy, meaning a higher dose did not bring extra benefits. Furthermore, serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT) levels remained normal across all groups, indicating no liver toxicity (Supplementary Fig. 3). Based on these results, we selected the safe and effective dose of 45 mg/kg for our main experiments.

To further verify the anti-inflammatory effects of polydatin in vivo, we administered polydatin (45 mg/kg/day) to Rag1^−/− mice with CD45RB^highCD4⁺ T cell-induced colitis (Fig. 2A). As expected, the recipient mice treated with placebo developed severe colitis 4 weeks after transplantation. In contrast, polydatin treatment significantly reduced disease severity, as evidenced by reduced weight loss, diminished colon shortening, lower DAI, and reduced pathological scores (Fig. 2B–G). Flow cytometry analysis of colonic LPMCs revealed that polydatin significantly reduced the frequencies of IFN-γ⁺, IL-17A⁺, and TNF-α⁺CD4⁺ T cells, while decreased levels of IFN-γ and IL-17A in colon tissue culture supernatants were observed by ELISA (Fig. 2H–L). These results suggest that polydatin alleviates intestinal inflammation by suppressing pathogenic Th1 and Th17 cell responses.

Figure 2.

Administration of polydatin significantly ameliorates CD45RB^highCD4⁺ T cell transfer-induced chronic colitis in mice. (A) Schematic of model establishment. (B) Mice were weighed on a weekly basis after T cell transfer and expressed as a percentage of initial weight. (C) DAI scores were calculated weekly. (D, E) Mice were sacrificed at week 8 after T cell transfer. Colon morphology and length were shown. (F, G) Representative colon sections were stained with H&E, and pathological scores of the colon tissues were calculated. (H, I) CD4⁺ T cells were isolated from LPMCs obtained from the colons of Rag1^–/– mice, and intracellular expression of IFN‐γ, IL‐17A, IL-10, and TNF-α was analyzed by flow cytometry (numbers in graphs indicate percentage of cells). (J) Percentages of IL‐17A⁺CD4⁺, IFN‐γ⁺CD4⁺, and TNF-α⁺CD4⁺ T cells were shown in the chart. (K, L) Detection of IFN-γ and IL-17A levels in supernatants from in vitro cultured colon tissues by ELISA. *P < 0.05, **P < 0.01, ***P < 0.001 by Student’s t-test.

Polydatin activates the NFE2L2 pathway to regulate CD4⁺ T cell differentiation in mice

To explore the potential mechanism by which polydatin inhibits Th1/Th17 cell differentiation, we performed RNA sequencing analysis of CD4⁺ T cells treated with polydatin. Pathway analysis showed that the differentially expressed genes were enriched in multiple immune-related pathways (Fig. 3A). Specific gene analysis showed that the expression of Ifng and Il17a in CD4⁺ T cells treated with polydatin was significantly decreased, whereas the expression of anti-inflammatory cytokines such as Tgfb1, Foxp3, and Il10 was increased after polydatin treatment. Notably, the expression of Nfe2l2 and its downstream factors, including Hmox1, Gclc, and Gclm, was observed to be upregulated in polydatin treated cells (Fig. 3B).

Figure 3.

Polydatin activates the NFE2L2 pathway. Naive CD4⁺ T cells (5 × 10⁵ cells per well) were isolated from the spleen of wild-type C57BL/6 mice and stimulated in vitro as indicated in the presence or absence of polydatin (2 μM) for 3 days. Total RNA was extracted from two groups, and RNA sequencing was performed to detect transcriptome differences. (A) Pathway analysis of the differentially expressed genes in CD4⁺ T cells. (B) Heatmap shows the differentially expressed genes between the two groups. (C) The expression of Nfe2l2 in mouse splenic CD4⁺ T cells cultured under Th1 or Th17 cell-polarizing conditions was analyzed by qRT‐PCR. (D) Nfe2l2 mRNA expression in the inflamed colon of mice that received the adoptive transfer of CD45RB^highCD4⁺ T cells as indicated in Fig. 2 was also analyzed by qRT‐PCR. *P < 0.05, **P < 0.01, ***P < 0.001 by Student’s t-test.

To validate these findings, we analyzed the expression of Nfe2l2 by qRT-PCR in mouse splenic CD4⁺ T cells cultured under Th1- or Th17 cell-polarizing conditions. Our results demonstrated that Nfe2l2 mRNA levels were markedly diminished in Th1 and Th17 cells in comparison to Th0 cells. However, under polydatin stimulation, a significant increase in Nfe2l2 mRNA levels was observed (Fig. 3C). Subsequently, we assessed Nfe2l2 mRNA expression in the inflamed colon of mice that received an adoptive transfer of CD45RB^highCD4⁺ T cells. As shown in Fig. 3D, polydatin upregulated the expression of Nfe2l2 and its downstream genes (e.g. Hmox1, Gclc, Gclm) in vivo.

Polydatin regulates CD4⁺ T cell differentiation in an NFE2L2-dependent manner

To further determine whether the anti-inflammatory effect of polydatin is dependent on the NFE2L2 pathway, we employed ML385, a specific pharmacological inhibitor of NFE2L2. Given that our RNA-seq analysis showed increased expression of Foxp3, a key transcription factor for Treg cells, in polydatin treated CD4⁺ T cells, we extended the analysis to Treg cells. Naïve CD4⁺ T cells isolated from mouse spleens were cultured under Th0-, Th1-, Th17-, or Treg cell-polarizing conditions in the presence of polydatin together with or without ML385 pretreatment. Flow cytometry analysis revealed that polydatin significantly reduced the frequencies of IFN‐γ⁺ and IL-17A⁺ CD4⁺ T cells, while co-treatment with ML385 substantially attenuated this suppressive effect (Supplementary Fig. 4A). Specifically, the proportions of Th1 and Th17 cells in the polydatin plus ML385 treatment group were similar to those in the ML385 alone group, indicating that NFE2L2 inhibition largely reversed the immunomodulatory activity of polydatin. On the contrary, we observed that polydatin treatment enhanced Foxp3⁺ Treg cell differentiation. However, this promoting effect was abolished by co-application with ML385 (Supplementary Fig. 4B and C).

To rigorously corroborate these pharmacological findings, we genetically silenced NFE2L2 in CD4⁺ T cells using lentivirus-mediated RNA interference (RNAi). Consistent with the ML385 intervention, the suppressive effect of polydatin on Th1 (IFN-γ⁺) and Th17 (IL-17A⁺) polarization was remarkably abolished upon NFE2L2 knockdown (Fig. 4). These results suggest that polydatin regulates CD4⁺ T cell differentiation through an NFE2L2 dependent manner.

Figure 4.

Knockdown of Nfe2l2 modulates Th1 and Th17 cell differentiation in vitro. (A) Representative bright-field (top) and fluorescence (bottom) microscopy images of primary CD4⁺ T cells infected with lentivirus. (B) Western blot analysis evaluating the knockdown efficiency of NFE2L2 in CD4⁺ T cells transfected with negative control shRNA (NC) or three independent shRNAs targeting Nfe2l2 (sh1, sh2, and sh3). (C) Representative flow cytometry plots illustrating the percentages of Th1 (IFN-γ⁺) and Th17 (IL-17A⁺) cells. Cells were infected with NC or the most efficient Nfe2l2-targeting shRNA (sh2, designated as sh-Nfe2l2) under Th1 or Th17 polarizing conditions. Cells were treated with or without Polydatin (2 µM). (D) Quantitative statistical analysis of the percentages of Th1 and Th17 cells from panel C. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 by one-way ANOVA with Tukey’s post hoc test.

Polydatin mitigates intracellular oxidative stress via the NFE2L2 pathway during CD4⁺ T cell differentiation

While previous studies have demonstrated that NFE2L2 directly binds to the Rorc promoter [19], its potential direct transcriptional regulation of Tbx21 (Th1) and Foxp3 (Treg) remains unexplored. To address this, we used ChIP-qPCR to assess NFE2L2 binding in the promoter regions of these genes. We did not observe significant enrichment of NFE2L2 in the Tbx21 or Foxp3 promoter regions (Supplementary Fig. 5, Supplementary Table 3), suggesting that NFE2L2 does not directly regulate Tbx21 or Foxp3 transcription.

Considering that NFE2L2 plays a key role in cellular antioxidant responses, we further evaluated intracellular reactive oxygen species (ROS) levels under Th1- and Th17-polarizing conditions. Flow cytometry analysis showed that ROS levels were significantly increased during T cell polarization. Polydatin treatment effectively reduced this intracellular ROS production, whereas the antioxidant effect was significantly reversed by co-treatment with the NFE2L2 inhibitor ML385 (Fig. 5). These data suggest that polydatin suppresses intracellular oxidative stress during T cell differentiation via the NFE2L2 pathway, which provides a mechanistic basis for its immunomodulatory effects.

Figure 5.

Polydatin mitigates intracellular oxidative stress during pathogenic T cell differentiation via the NFE2L2 pathway. (A) Representative flow cytometry histograms evaluating intracellular (ROS) levels. Naïve CD4⁺ T cells isolated from mouse spleens were cultured under Th0, Th1-, or Th17-polarizing conditions for 72 h and treated with polydatin in the presence or absence of ML385 pretreatment. ROS production was measured using the DCFH-DA probe. (B) Quantitative analysis of the mean fluorescence intensity of intracellular ROS in cells across different treatment groups. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 by one-way ANOVA with Tukey’s post hoc test.

Pharmacological inhibition of NFE2L2 attenuates the protective effect of polydatin in vivo

To further determine whether the protective effect of polydatin in vivo also depends on NFE2L2 signaling, we performed an additional pharmacological inhibition experiment in a DSS-induced acute colitis model. Mice treated with ML385 alone developed more severe colitis, and according to animal welfare criteria, these animals reached the humane endpoint and were sacrificed on day 7 when body weight had decreased to 80% of the initial value (Fig. 6A). Representative colon images also showed aggravated intestinal injury in the ML385-treated group (Fig. 6B).

Figure 6.

Pharmacological inhibition of NFE2L2 abolishes the protective effect of polydatin in DSS-induced colitis. (A) Changes in body weight during the experimental period. (B) Representative images of colons collected from each group at the experimental endpoint. (C–E) Flow cytometric analysis of IFN-γ, IL-17A, and intracellular ROS levels in CD4⁺ T cells isolated from LPMCs. (F) Western blot analysis of NFE2L2 protein expression in CD4⁺ T cells isolated from LPMCs. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 by one-way ANOVA with Tukey’s post hoc test.

Importantly, co-treatment with ML385 largely abolished the protective and immunomodulatory effects of polydatin in vivo, resulting in an inflammatory phenotype as severe as that observed in the ML385-alone group. Flow cytometric analysis of CD4⁺ T cells isolated from LPMCs showed that the suppressive effects of polydatin on IFN-γ, IL-17A, and intracellular ROS were markedly reversed by ML385 treatment (Fig. 6C–E). Western blot analysis further showed that the polydatin-induced increase in NFE2L2 protein expression was reduced in the presence of ML385 (Fig. 6F). Consistently, immunofluorescence staining of CD4⁺ T cells isolated from LPMCs revealed that the polydatin-induced accumulation of NFE2L2 in the mucosa was markedly diminished by ML385 administration (Fig. 6G). Collectively, these findings indicate that the therapeutic effect of polydatin in vivo is largely dependent on NFE2L2 signaling.

Polydatin enhances NFE2L2 nuclear translocation and regulates CD4⁺ T cell differentiation in UC

Importantly, these immunomodulatory effects were consistently observed in CD4⁺ T cells derived from both healthy donors (n = 11) and active UC patients (n = 10), indicating that polydatin can effectively rebalance Th1/Th17 cell-driven inflammatory responses in human systems. (Supplementary Fig. 6). Furthermore, we observed an increase in the frequency of Foxp3⁺ regulatory T (Treg) cells following polydatin treatment (Supplementary Fig. 7). Given the hypothesis that polydatin might regulate T cell differentiation in a NFE2L2-dependent manner, we then examined the mRNA and protein levels of NFE2L2. For this purpose, CD4⁺ T cells were isolated from the peripheral blood of healthy volunteers, cultured in vitro for 3 days, and then harvested to extract total RNA for qRT-PCR. Consistently, polydatin treatment resulted in increased mRNA levels of NFE2L2, HO-1, NQO1, and SOD (Supplementary Fig. 8).

It is important to note that under physiological conditions, Kelch-like ECH-associated protein 1 (KEAP1) binds to NFE2L2 and promotes its degradation. Therefore, NFE2L2 activation requires its dissociation from KEAP1 and its nuclear translocation [20]. Therefore, we evaluated the subcellular localization of NFE2L2. Cellular immunofluorescence analysis showed that polydatin treatment increased both the expression and nuclear accumulation of NFE2L2 (Fig. 7A). Furthermore, co-localization analysis demonstrated a reduction in the interaction between KEAP1 and NFE2L2 following polydatin treatment (Supplementary Fig. 9). This was further confirmed by Western blotting of nuclear and cytoplasmic fractions, showing that polydatin enriched NFE2L2 protein levels in the nucleus. (Fig. 7B and C).

Figure 7.

Polydatin promotes NFE2L2 nuclear translocation. CD4⁺ T cells (5 × 10⁵ cells per well) isolated from peripheral blood of healthy donors (n = 11) were stimulated in vitro as indicated in the presence or absence of polydatin (2 μM) for 6 h (A) or 24 h (B). (A) Immunofluorescence analysis of NFE2L2 and KEAP1. (B) The protein levels of NFE2L2 in the cytoplasm and the nucleus, respectively, were detected by Western blotting. (C) The relative intensity of NFE2L2 protein was shown in the bar chart. Statistical analysis was performed using unpaired Student’s t-test. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 by Student’s t-test.

To extend our observations beyond peripheral blood CD4⁺ T cells, we additionally used CD4⁺ T cells isolated from the intestinal mucosa of UC patients for short-term ex vivo culture. Following 6 h of polydatin treatment, immunofluorescence staining showed increased nuclear localization of NFE2L2 in UC mucosal tissues compared with the untreated control, indicating that polydatin activates NFE2L2 signaling in human intestinal mucosal tissues (Supplementary Fig. 10).

Polydatin exerts its effects by binding to KEAP1

Preliminary analysis did not identify any reliable binding sites between polydatin and NFE2L2. Therefore, we further examined whether polydatin exerts its effects by binding to KEAP1. To explore this physical interaction, surface plasmon resonance (SPR) experiments were performed, which showed that polydatin binds to KEAP1 in a dose-dependent manner (Supplementary Fig. 11A, Supplementary Table 4). A molecular docking experiment further elucidated the specific binding mode between polydatin and Keap1, showing that polydatin engages in robust van der Waals interactions with Arg415 and Ala556, and forms hydrogen bonds with Gln530, Ser555, and Val463. Furthermore, the benzene ring of polydatin participates in cation-π interactions with Arg415 (Supplementary Fig. 11B and C). To further validate the stability of the polydatin-KEAP1 interaction beyond static docking, molecular dynamics simulation was performed. The results showed that the polydatin-KEAP1 complex remained structurally stable throughout the simulation process, indicating that polydatin can stably bind to KEAP1 under dynamic conditions. Moreover, binding free energy analysis demonstrated a favorable interaction between polydatin and KEAP1 (Supplementary Fig. 11D to I).

Collectively, these findings indicate that polydatin directly binds KEAP1, thereby disrupting the KEAP1-NFE2L2 interaction and promoting NFE2L2 nuclear translocation, which in turn inhibits Th1/Th17 cell differentiation.

Discussion

Natural herbs encompass a multitude of bioactive compounds, holding great promise for developing novel medications [21]. HDEC is a compound composed of nine different chemical components, which has shown clinical application potential in the treatment of intestinal diseases, but its exact immunological mechanisms require further elucidation. A prominent pathological hallmark of UC involves the dysregulation of the CD4⁺ T cell-mediated immune response within the intestinal mucosa [22–24]. Emerging studies have confirmed the presence of an overactivated Th1/Th17 cell immune response in both intestinal mucosa and peripheral blood of UC patients [25]. To more accurately replicate the chronic intestinal inflammatory state observed in UC patients, we employed a chronic colitis model induced by the adoptive transfer of CD45RB^highCD4⁺ T cells. We found that HDEC exhibited a significant mitigating effect on colonic inflammation in mice. Crucially, we observed a notable reduction in the infiltration of IFN-γ⁺ and IL-17A⁺ CD4⁺ T cells. These data strongly suggest that HDEC alleviates colitis by restraining pathogenic Th1 and Th17 cell responses.

To gain deeper insights into how this herbal compound modulates CD4⁺ T cell differentiation, we conducted a series of in vitro experiments using its primary active chemical components. Our findings revealed that polydatin, serving as one of the crucial bioactive constituents, inhibited Th1 and Th17 cell immune responses. Although the full therapeutic efficacy of HDEC likely relies on the synergistic effects of its multi-component formula, our data highlight that polydatin plays a central role in correcting the T cell subset imbalance. Polydatin is a natural compound with multiple therapeutic potential, including antioxidant, anti-inflammatory, and anti-apoptotic properties. An experimental study of a mouse colitis model has demonstrated that the antioxidant and anti-apoptotic effects of polydatin are mediated by upregulation of the Sonic hedgehog (Shh) signaling pathway [26]. Previous study has shown that polydatin inhibits lipopolysaccharides (LPS)-induced activation of nuclear factor‐κB (NF-κB), c‐Jun N‐terminal kinase, and extracellular signal‐regulated kinase‐1/2 signaling pathways in RAW264.7 cells [27]. Moreover, previous work has confirmed that polydatin effectively restores the tight junctions between intestinal epithelial cells in acetic acid-induced colitis in mice, thus preserving intestinal barrier integrity [28]. However, its effects on CD4⁺ T cell differentiation and the underlying mechanisms remained unclear. In this study, we demonstrated that polydatin effectively restores the balance of CD4⁺ T cell subsets. Specifically, our in vivo and in vitro data showed that polydatin significantly inhibits the differentiation of pathogenic Th1 and Th17 cells, which limits the production of pro-inflammatory cytokines. Concurrently, it promotes the generation of Treg cells to maintain intestinal immune tolerance. Furthermore, to elucidate how polydatin controls these T cell responses, we investigated its intracellular targets and identified the NFE2L2 pathway as a key potential mechanism.

NFE2L2 is a critical regulator of oxidative stress and inflammation, exerting its effects by binding to antioxidant response elements (AREs) in target genes [29, 30]. Previous studies have indicated its involvement in the pathogenesis of UC, as IL-17A expression in the intestinal mucosa of patients with UC is negatively correlated with NFE2L2 expression [31]. Once activated, NFE2L2 regulates CD4⁺ T cell differentiation through multifaceted downstream mechanisms. While previous studies reported direct targeting of the Rorc promoter [19], our ChIP-qPCR data revealed no significant enrichment at the Tbx21 or Foxp3 promoters, suggesting indirect regulatory mechanisms. For example, NFE2L2-mediated metabolic reprogramming, such as inhibition of aerobic glycolysis, can limit the expansion of glycolysis-dependent Th1 and Th17 cells and promote the differentiation of Treg cells that are more dependent on oxidative phosphorylation [32]. Importantly, the cellular antioxidant response mediated by NFE2L2 also critically limits pathogenic T cell subsets. ROS act as important secondary messengers during T cell activation, and excessive ROS accumulation promotes the pathogenic polarization of Th1 and Th17 cells. In this study, we observed a significant increase in ROS levels during T cell polarization, which was effectively reduced by polydatin treatment. Moreover, because a stable redox environment is essential for Treg survival and function, NFE2L2-mediated antioxidant effects contribute to the restoration of Th1/Th17/Treg balance in the intestinal mucosa.

To definitively confirm whether the immunomodulatory effects of polydatin strictly depend on NFE2L2 activation, we performed comprehensive rescue experiments both in vitro and in vivo. In vitro, pharmacological inhibition of NFE2L2 with ML385, as well as genetic knockdown using lentivirus-mediated RNA interference, effectively abrogated the ability of polydatin to suppress the pathogenic differentiation of Th1 and Th17 cells, and reversed its ROS-scavenging effects. In addition, ML385 significantly attenuated the enhancement of Treg cell differentiation induced by polydatin. More importantly, these functions were also consistently validated in vivo in DSS-induced colitis model. In combination with ML385, the therapeutic effect of polydatin was substantially attenuated, resulting in a severe inflammatory phenotype and restoration of mucosal Th1/Th17 cell accumulation. Collectively, these findings demonstrate that polydatin restores immune homeostasis and exerts its therapeutic efficacy in an NFE2L2-dependent manner.

Under physiological conditions, KEAP1 binds to NFE2L2 in the cytoplasm, leading to ubiquitination and degradation of NFE2L2. Known NFE2L2 agonists, such as dimethyl fumarate and tert-butylhydroquinone, promote NFE2L2 release and nuclear translocation by directly binding to Kelch-like ECH-associated protein 1 (KEAP1) [33]. Therefore, we investigated whether polydatin exerts its effect through a similar direct interaction. Indeed, molecular docking and SPR assay confirmed the direct binding of polydatin to KEAP1. The molecular dynamics results further supported the structural stability of the polydatin-KEAP1 complex under dynamic conditions and demonstrated favorable binding free energy. In addition, cellular immunofluorescence analysis and Western blot analyses confirmed that polydatin facilitated the nuclear translocation of NFE2L2. More importantly, in CD4⁺ T cells isolated from the intestinal mucosa of patients with UC, polydatin also enhanced NFE2L2 nuclear translocation, providing strong evidence that this pathway can be effectively activated in the specific human cellular context. These findings strongly suggest that polydatin directly interacts with KEAP1 to activate the NFE2L2 pathway.

However, several limitations should be noted. While polydatin promotes Treg generation, its exact impact on T cell plasticity, such as the phenotypic stability of induced Tregs and potential Th17-to-Treg conversion under inflammatory conditions, remains to be elucidated to fully understand its broader immunomodulatory spectrum. Although clinical samples were included to support the disease relevance of the observed immune alterations, the sample size was relatively limited, and these exploratory findings require further validation in larger independent cohorts. Moreover, although the effective dosages of HDEC and polydatin were established and showed no obvious hepatotoxicity based on AST and ALT levels, the detailed pharmacokinetic and pharmacodynamic properties of polydatin, including oral bioavailability, intestinal tissue distribution, in vivo stability, and PK/PD relationships, were not investigated. Future studies should further optimize the dosing strategy, evaluate long-term safety, and confirm the efficacy and safety of polydatin in rigorous clinical trials.

Conclusion

In conclusion, our study provides robust preclinical evidence that HDEC and its important active constituent, polydatin, effectively ameliorate colitis. Mechanistically, we demonstrated that polydatin directly binds to KEAP1 to promote NFE2L2 nuclear translocation, which significantly attenuates ROS accumulation. Ultimately, this regulation suppresses the pathogenic differentiation of mucosal Th1 and Th17 cells and restores the balance among Th1, Th17, and Treg subsets, thereby maintaining intestinal immune homeostasis. These findings elucidate the pharmacological basis of HDEC and highlight polydatin as a promising targeted therapeutic agent for the clinical management of UC.

Author contributions

Xiaohan Wu (Funding acquisition, Investigation, Validation, Writing – original draft), Lei Shi (Formal Analysis, Supervision, Writing – review & editing), Lei Zhu (Formal Analysis, Writing – review & editing), Tangyou Mao (Visualization, Writing – review & editing), Zhibin Wang (Project administration, Writing – review & editing), Fushun Kou (Software, Visualization, Writing – review & editing), Xiao Han (Investigation, Project administration, Writing – review & editing), Han Gao (Formal Analysis, Validation, Writing – review & editing), Xiaohua Chen (Validation, Writing – review & editing), Xianxin Li (Visualization, Writing – review & editing), Tingmin Chang (Funding acquisition, Supervision, Writing – review & editing), Junxiang Li (Funding acquisition, Supervision, Writing – review & editing), Zhanju Liu (Funding acquisition, Resources, Supervision, Writing – review & editing).

Conflicts of interest

None declared. In addition, as an Editorial Board Member of Precision Clinical Medicine, the corresponding author Zhanju Liu was blinded from reviewing and making decision on this manuscript.