Direct inhibition of the TXNIP-NLRP3-GSDMD pathway reduces pyroptosis in colonocytes and alleviates ulcerative colitis in mice by the small compound PEITC

Abstract

Ulcerative colitis (UC) is a chronic inflammatory bowel disease. The etiology of UC is multifaceted, and the underlying pathogenesis remains incompletely understood. Pyroptosis, programmed cell death mediated by the gasdermins, is a pivotal driver of UC pathology due to its dual role in epithelial barrier disruption and inflammatory amplification. We previously showed that phenethyl isothiocyanate (PEITC), an isothiocyanate derived from cruciferous vegetables, alleviated acute liver injury in mice by suppressing hepatocyte pyroptosis. In this study we evaluated the therapeutic potential of PEITC in the treatment of UC and the underlying mechanisms. UC mouse models were established by administration of 2.5% (w/v) dextran sulfate sodium (DSS) daily for 7 days. PEITC (5, 10, or 20 mg·kg⁻¹·d⁻¹, i.g.) was given 2 days before the start of modeling, and the dosing lasted for a total of 10 days. We showed that during the progression of DSS-induced UC, the pyroptosis pathway was activated accompanied by elevated expression levels of thioredoxin-interacting protein (TXNIP) and NOD-like receptor thermal protein domain associated protein 3 (NLRP3), as well as the activation of caspase-1, gasdermin D (GSDMD) and interleukin-1β (IL-1β). Treatment with PEITC dose-dependently reduced TXNIP and NLRP3 expression while inhibiting the cleavage of proteins associated with the pyroptosis pathway such as caspase-1, GSDMD, and IL-1β. We confirmed the inhibitory effect of PEITC on colonocyte pyroptosis in an in vitro model established in HT29 cells, where PEITC (0.2, 1, 5 µM) dose-dependently inhibited TXNIP and NLRP3 expression and the activation of pro-caspase-1, GSDMD and pro-IL-1β. We revealed that PEITC is directly bound to TXNIP and disrupted the interaction between TXNIP and NLRP3, leading to diminished cellular inflammation and oxidative stress levels. In conclusion, this study demonstrates that PEITC disrupts the interaction of TXNIP and NLRP3 by binding to TXNIP, inhibits NLRP3 activation and colonocyte pyroptosis, and thus effectively alleviates UC symptoms in mice. This study offers novel drug targets along with potential therapeutic candidates for the clinical prevention and treatment of UC.

Introduction

Ulcerative colitis (UC) is a chronic intestinal inflammatory disease characterized by persistent inflammation of the colon [1]. Its clinical manifestations include recurrent abdominal pain, diarrhea, bloody stools, etc., and even various systemic complications, which are difficult to cure and prone to relapses [2, 3]. The pathogenesis of UC is complex and incompletely understood, and it has been reported that inflammation initiated and maintained by epithelial injury and immune or intestinal microenvironment dysregulation are closely related to the occurrence and development of UC; thus, the key physiological process that promotes normal organ function and maintains intestinal homeostasis is the resolution of inflammation [4–6]. Intestinal epithelial cells (IECs) play a critical role in UC because of their anatomical location and complex interactions with the mucosal immune system as well as the local intestinal microenvironment; thus, these cells represent promising therapeutic targets for UC [7, 8]. Although several medications, including antibiotics, 5-aminosalicylic acid (5-ASA) derivatives, corticosteroids, and immunosuppressants, are currently utilized in clinical practice, these treatments have limited efficacy and cause a variety of adverse reactions [9–11]. Therefore, there is an urgent need for low-cost, safe, and effective UC treatment drugs.

Pyroptosis is programmed cell death mediated by the gasdermins, which is highly related to inflammation [12, 13]. Gasdermins are widely expressed in epithelial cells, especially IECs. Although various types of cell death are induced in UC, including pyroptosis, apoptosis, necrosis, etc., pyroptosis is a pivotal driver of UC pathology due to its dual role in epithelial barrier disruption and inflammatory amplification. Its distinct reliance on inflammasomes and GSDMD-mediated cellular lysis differentiates it from other forms of cell death, making it a compelling therapeutic target [14]. During pyroptosis, NOD-like receptor thermal protein domain associated protein 3 (NLRP3) is activated, which then recruits pro-caspase-1 and apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC), initiates self-cleavage of pro-caspase-1, and forms caspase-1 [15, 16]. Subsequently, caspase-1 recognizes pro-interleukin-1β (IL-1β) and pro-IL-18 and converts them into the active inflammatory cytokines IL-1β and IL-18. On the other hand, caspase-1 cleaves GSDMD into an N-terminal fragment (GSDMD-N), which is oligomerized and then translocated to the cell membrane and forms pores in the cell membrane, resulting in the release of pro-inflammatory cytokines and other cytoplasmic contents [17]. The secretion of these pro-inflammatory cytokines can trigger a strong inflammatory response, exacerbate the immune response in the intestine, and lead to more IEC deaths, further damaging the intestinal barrier [18]. This may be one of the key reasons why UC is incurable and recurrent.

Thioredoxin-interacting protein (TXNIP) is highly related to the oxidative stress process and is a key signaling molecule linking oxidative stress and pyroptosis [19, 20]. High levels of reactive oxygen species (ROS) after oxidative stress activate TXNIP and dissociate it from its endogenous ligand thioredoxin (TRX) [21]. After that, the C-terminal carboxyl extension domain of free TXNIP binds to the leucine-rich repeat domain of NLRP3 and then activates caspase-1, which causes pyroptosis and induces a cascade reaction to release more inflammatory factors, ultimately forming a vicious cycle [22–24]. Studies have revealed a significant upregulation of TXNIP and pyroptosis-associated proteins (NLRP3, caspase-1, and GSDMD) in the colonic tissues of UC patients, with similar observations reported in dextran sulfate sodium (DSS)-induced murine UC models [25, 26]. These findings highlight the pivotal role of the TXNIP-NLRP3-GSDMD signaling pathway in UC pathogenesis. As a molecular bridge connecting oxidative stress to pyroptosis, TXNIP emerges as a promising therapeutic target for UC and other inflammatory disorders. However, current strategies to prevent or treat UC by targeting TXNIP to modulate pyroptosis remain limited, underscoring the urgent need for innovative interventions to disrupt this pathway.

Phenethyl isothiocyanate (PEITC) is an isothiocyanate in cruciferous plants that is derived mainly from cruciferous vegetables such as broccoli and cabbage [27]. PEITC is low molecular weight (163.2 g/mol), highly lipophilic, and significantly electrophilic, resulting in rapid absorption, high oral bioavailability, and high reactivity [28]. PEITC, an antioxidant, activates antioxidant defense mechanisms and lowers ROS levels by increasing the expression of detoxifying enzymes [29]. It can also regulate the levels of inflammatory cytokines, such as tumor necrosis factor (TNF)-α, IL-1β, and IL-6, which play key roles in the inflammatory response [30]. In addition, it can exert anti-inflammatory effects by inhibiting nuclear factor kappa-B signaling pathway activation [31]. PEITC can participate in a variety of biological mechanisms to exert antioxidant, anti-inflammatory, antibacterial, and anticancer effects, but its role and specific mechanisms in UC are still unclear. Our previous study demonstrated that PEITC alleviates acute liver injury in mice by suppressing hepatocyte pyroptosis [28]. This work revealed that PEITC can block pyroptosis through GSDMD intervention; however, it does not exclude potential regulatory effects on upstream signaling molecules of GSDMD. Notably, we observed that PEITC suppressed the upregulated level of NLRP3 in damaged tissues. Thereafter, following a screening of the upstream molecules associated with NLRP3, we directed our attention toward TXNIP. We also noticed that sulforaphane (a structurally related isothiocyanate) has been reported to mitigate paraquat-triggered oxidative damage in oocytes by downregulating TXNIP [32]. Given PEITC’s antioxidant activity, these findings collectively suggest that PEITC may modulate pyroptosis via direct interaction with TXNIP, an upstream regulator of pyroptosis. Therefore, in light of the critical role of the TXNIP-NLRP3-GSDMD pathway in UC pathogenesis, investigating the therapeutic efficacy and molecular mechanisms of PEITC in UC holds significant scientific and translational value.

This study revealed that TXNIP plays a key role in the pyroptosis pathway of IECs in UC. PEITC, a natural small molecule, can significantly alleviate DSS-induced UC in mice by binding to TXNIP, disrupting the interaction between TXNIP and NLRP3, reducing the activation of NLRP3, and inhibiting pyroptosis.

Materials and methods

Reagents

PEITC was obtained from JC (Wuxi) Company, Inc. (Wuxi, China). DSS (02160110-CF) was purchased from MP Biomedicals (Aurora, OH, USA). An antibody against β-Tubulin (M30109) was provided by Abmart (Shanghai, China). Antibodies against TXNIP (sc-271237), caspase-1 (sc-56036), and IL-1β (sc-12742) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Anti-NLRP3 (15101) and anti-E-cadherin (3195) antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA). Anti-GSDMD (ab209845, ab210070) were purchased from Abcam (Cambridge, UK). Alexa Fluor 488-conjugated anti-rabbit IgG (A-11008), Alexa Fluor 594-conjugated anti-mouse IgG (A-11005), and Lipofectamine™ 2000 transfection reagents (11668019) were purchased from Thermo Fisher Scientific (Waltham, MA, USA). Malondialdehyde (MDA, A003-1-2) and superoxide dismutase (SOD, A001-1-2) assay kits were obtained from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). A mouse IL-1β enzyme-linked immunosorbent assay (ELISA) kit (1210122) was purchased from Dakewe Biotech Co., Ltd. (Shenzhen, China). Mesalazine (BP1075) was purchased from Nanjing Well-Offer Biotechnology Co., Ltd. (Nanjing, China). LPS (L2880) was purchased from Sigma–Aldrich (St. Louis, MO, USA). ATP (HY-B2176) was purchased from MedChemExpress (Monmouth Junction, NJ, USA). Rabbit IgG (A7016) and mouse IgG (A7028) were purchased from Beyotime Biotechnology (Shanghai, China). Human IL-1β ELISA kit (RK00001) and human IL-18 ELISA Kit (RK00176) were purchased from ABclonal Technology (Wuhan, China).

Animal experiments

C57BL/6 male mice (6–8 weeks old) were procured from SiPeiFu Biotechnology Co. Ltd. (Suzhou, China) and were grown in an SPF-grade animal room at 21 ± 2 °C with a 12:12-h day-night cycle. The animal study was reviewed and approved by the Nanjing University Animal Care and Use Committee (IACUC-D2202115). UC was induced in mice with 2.5% (w/v) DSS [33]. The specific method of model construction and calculation of the mouse disease activity index (DAI) were performed as described previously [34]. We collected tissues such as blood, colon, and spleen from mice on days 0, 3, 5, 7, 9, and 11.

Based on our previous investigations into the efficacy and safety of PEITC, we conducted in vivo experiments using a safe and non-toxic dose of PEITC (5, 10, or 20 mg/kg) to explore its preventive and therapeutic effects on UC [28, 35]. We then constructed a UC mouse model and randomly divided the mice into the following seven groups: the normal group, DSS group, PEITC (5 mg/kg, pre) group, PEITC (10 mg/kg, pre) group, PEITC (20 mg/kg, pre) group, PEITC (20 mg/kg, post) group, and 5-ASA (200 mg/kg, pre) group. The mice in the PEITC (5, 10, or 20 mg/kg, pre) group were treated with 2.5% (w/v) DSS daily for 7 days, and the PEITC (i.g.) treatment was performed 2 days before the start of modeling, and the dosing lasted for a total of 10 days. The mice in the PEITC (20 mg/kg, post) group drank 2.5% (w/v) DSS daily for 7 days, and PEITC (i.g.) treatment was performed the day after the start of modeling; the dosing lasted for a total of 6 days. The mice in the 5-ASA (200 mg/kg, pre) group were treated with 2.5% (w/v) DSS daily for 7 days, and 5-ASA (i.g.) treatment was performed 2 days before the start of modeling; the dosing lasted for a total of 10 days. Finally, we collected tissues such as blood, colon, and spleen from the mice.

Western blot and coimmunoprecipitation

The samples were lysed with lysis buffer containing protease and phosphatase inhibitors, fractionated via SDS-PAGE and electrophoretically transferred to polyvinylidene fluoride membranes (Millipore, Burlington, MA, USA). The membrane was then blocked with 5% BSA for 1.5 h at room temperature and incubated with different antibodies overnight at 4 °C. Next, the membrane was incubated with secondary antibodies for 1.5 h at room temperature. Finally, imaging was performed, and densitometry was performed via Quantity One software (Bio-Rad Laboratories, Hercules, CA, USA).

For the coimmunoprecipitation assay, the cell lysate (1 mg) was incubated with rabbit IgG (2 μg) or anti-TXNIP antibody (2 μg) overnight at 4 °C, after which protein A/G magnetic beads (40 μL) were added for 4 h at 4 °C to capture the antigen-antibody complex. Next, the beads were removed, and the antigen-antibody complex was collected and analyzed via Western blotting.

Real-time quantitative PCR

Real-time quantitative PCR was performed with a Bio-Rad CFX96 Touch™ real-time PCR detection system (Bio-Rad, Hercules, CA, USA). The amplification conditions were 95 °C for 2 min, 1 cycle, denaturation at 95 °C for 10 s, annealing at 60 °C for 30 s, extension at 72 °C for 30 s, and 40 cycles. The sequences of primers used in this study are shown in Supplementary Table S1.

Cell culture

The HT29 cell line was obtained from the National Collection of Authenticated Cell Cultures (Shanghai, China). HT29 cells were cultured in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum, 100 U/mL penicillin, and 100 µg/mL streptomycin under an atmosphere of 5% (v/v) CO₂ at 37 °C. To induce pyroptosis, HT29 cells were treated with LPS (1 μg/mL) and PEITC (0.2, 1, or 5 μM) for 24 h, followed by ATP (5 mM) for 3 h, after which the cells were collected for subsequent experiments.

siRNA transfection

Si-TXNIP was purchased from OBiO Technology (Shanghai) Corp., Ltd. (Shanghai, China). Transfection experiments were performed according to the instructions of the Lipofectamine™ 2000 transfection reagent.

Molecular docking

The dimensional structure of TXNIP (PDB ID: 4LL1) was obtained from the PDB (https://www.rcsb.org/), and the molecular structure of PEITC was mapped via ChemDraw. The CDOCKER algorithm of Discovery Studio was used for the molecular docking of PEITC to TXNIP.

Analysis of oxidative stress indicators and cytokine levels

MDA, SOD, IL-1β, and IL-18 levels were detected according to the instructions of the assay kits.

Immunofluorescence assay

The tissue sections were deparaffinized, subjected to antigen retrieval, peroxidase-free, permeabilized, blocked, incubated with different primary antibodies overnight at 4 °C, incubated with different secondary antibodies for 1.5 h at room temperature, labeled with DAPI, and finally photographed and analyzed via fluorescence microscopy.

Cellular thermal shift assay

The cellular thermal shift assay (CETSA) was performed as previously described [28, 36]. 2 × 10⁶ HT29 cells were incubated with DMSO or PEITC (5 μM) for 2 h, then the cells were collected and resuspended in 500 μL PBS. The cell suspension was divided into 10 aliquots, heated at the indicated temperatures for 3 min, and then left at room temperature for 3 min. Subsequently, the samples were subjected to two cycles of freeze-thaw in liquid nitrogen. The lysates were centrifuged at 20,000 × g for 15 min, and the supernatant was collected. Loading buffer was added to the supernatant, followed by heat denaturation at 100 °C for 5 min. Finally, the samples were analyzed by Western blot.

Human TXNIP WT-EGFP, and human TXNIP Mut (Q81A and G84A)-EGFP plasmids were purchased from General Biology (Hefei, China). HEK293T cells transfected with human TXNIP WT-EGFP or human TXNIP Mut-EGFP plasmid were treated with DMSO or PEITC (5 μM) for 2 h, and the subsequent operations were basically the same as above.

Electron microscopy

After the samples were fixed with fixative (Servicebio, Wuhan, China), they were sent to Wuhan Servicebio Co., Ltd. (Wuhan, China) for postprocessing and electron microscopy scanning.

Microscale thermophoresis assay

The microscale thermophoresis assay was performed as previously described [28, 36]. After human TXNIP WT-EGFP or human TXNIP, Mut-EGFP plasmid was transiently transfected into HEK293T cells for 36 h, the cells were lysed with lysis buffer, and the supernatant was collected after centrifugation. PEITC was gradually diluted from 25 μM and added to an aliquot of cell lysis. They were mixed and sucked into Monolith NT.115 capillary tubes (Nanotemper Technologies, München, Germany). Finally, samples were detected by Monolith NT.115 (Nanotemper Technologies), and data were analyzed by MO.Affinity Analysis.

Statistical analysis

The data are shown as the mean ± SEM, and each experiment included at least triplicate sets. All the statistical analyses were performed via GraphPad Prism 8 software (San Diego, CA, USA). The data were statistically evaluated via Student’s t-test or one-way ANOVA. P < 0.05 was considered significant.

Results

Intestinal inflammation and oxidative stress levels are elevated in DSS-induced UC in mice

The construction process of the DSS-induced UC mouse model is shown in Fig. 1a. After 4 days of drinking 2.5% DSS, the body weights of the mice in the model group began to decrease rapidly (Fig. 1b). The DAI score revealed that the mice in the model group began to experience diarrhea, hematochezia and other symptoms after 3 days of drinking 2.5% DSS, indicating that the UC mice were successfully generated (Fig. 1c). Compared with those in the normal group, the spleen damage in the model group was obvious, the spleen color turned dark red, and the spleen index and volume increased significantly, reflecting a severe inflammatory response in the model group (Fig. 1d). In addition, the model group of mice presented severe intestinal injury, edema of the intestinal tissue, a shortened intestinal length, decreased intestinal contents, and loose stools (Fig. 1e). Hematoxylin and eosin (H&E) staining and colon histopathology scores revealed that the colonic tissue structure of the control group was clear and intact, whereas the intestinal tract of the model group was damaged, with increased inflammatory cell infiltration, severe intestinal ulcers, and partial disappearance of the crypt structure (Fig. 1f). Moreover, real-time fluorescence quantitative polymerase chain reaction (qPCR) revealed that the levels of the inflammatory factors IL-6 and Tnf-α in the colon tissue of the model group were significantly increased (Fig. 1g).

Fig. 1. UC mice presented elevated levels of intestinal inflammation and oxidative stress.

a Schematic overview of the UC model. b Body weight and c DAI of the normal group and DSS group during the experiment. d The spleen was photographed and recorded, and the spleen index was calculated. e The colon was photographed and recorded, and the colon length was measured. f Histological score and hematoxylin and eosin (H&E) staining of colorectal tissue sections. Scale bar, 50 µm. g mRNA expression levels of Il6 and Tnfα. h The contents of MDA and SOD. The data are presented as the mean ± SEM (n = 3–4 per group). *P < 0.05, **P < 0.01, ***P < 0.001 vs. day 0.

The content of malondialdehyde (MDA) can reflect the degree of lipid peroxidation in the body, and its excess can indirectly reflect cell damage. SOD also plays a vital role in the body’s oxidation and antioxidation balance by scavenging superoxide anion radicals (O₂⁻) and protecting cells from damage. Therefore, to explore the changes in oxidative stress levels during the development of UC in mice, we measured the MDA content and SOD activity in colon tissues. The results revealed that the MDA content significantly increased and that SOD activity significantly decreased during the progression of UC, indicating an increased level of oxidative stress in mouse colon tissue (Fig. 1h). These results suggest that intestinal damage, inflammation, and oxidative stress are significantly increased during the development of UC in mice.

The pyroptosis signaling pathway was activated in DSS-induced UC in mice

Since UC is often accompanied by severe inflammation and IEC damage and death, we hypothesized that this process may be related to pyroptosis, a type of programmed cell death highly associated with inflammation and that TXNIP can induce pyroptosis in response to oxidative stress. We found that the expression of TXNIP and NLRP3 was significantly increased and that the levels of the pyroptosis-related proteins caspase-1, GSDMD-N, and IL-1β were increased in the colon tissues of the model group, as shown by Western blot experiments (Fig. 2a–f). The qPCR results also revealed that the levels of Txnip, Nlrp3, Casp1, Gsdmd, Il1β, and Il18 in the colon tissues of the model group were significantly increased (Fig. 2g). Immunofluorescence experiments also verified that the pyroptosis pathway was activated in the colon tissue of UC mice (Supplementary Fig. S1). These results suggest that the pyroptosis pathway is activated during the occurrence and development of UC and that TXNIP plays an important role in this process.

Fig. 2. Pyroptosis signaling was activated in the colon tissue of DSS-induced UC model mice.

a–f Immunoblot analysis of TXNIP, NLRP3, pro-caspase-1, caspase-1, GSDMD, GSDMD-N, pro-IL-1β and IL-1β expression. g mRNA expression levels of Txnip, Nlrp3, Casp1, Gsdmd, Il1β, and Il18. The data are presented as the mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001 vs. day 0.

PEITC relieves DSS-induced UC in mice

The natural small-molecule compound PEITC has antioxidant, anti-inflammatory, antibacterial, and anticancer effects. To explore its function in UC, we treated UC mice with PEITC and mesalazine (5-ASA), a commonly used drug in the clinical treatment of UC, as a positive control, and the administration diagram is shown in Fig. 3a. Compared with those in the model group, the body weights of the mice in the PEITC (10 mg/kg, pre), PEITC (20 mg/kg, pre), PEITC (20 mg/kg, post) and 5-ASA (200 mg/kg, pre) groups were greater, and the DAI scores revealed that PEITC can alleviate symptoms such as diarrhea and bloody stools in a dose-dependent manner (Fig. 3b, c). By assessing the spleen index, we found a significant improvement in spleen lesions in PEITC-treated mice, especially in the PEITC (10 mg/kg, pre), PEITC (20 mg/kg, pre), and PEITC (20 mg/kg, post) groups, indicating that PEITC can attenuate the inflammatory response in UC mice (Fig. 3d). Moreover, PEITC significantly increased the intestinal length of UC model mice (Fig. 3e). Further analysis of H&E-stained mouse colon tissue revealed that compared with model mice, PEITC-treated mice presented reduced colon injury, inflammatory cell infiltration and histopathological scores (Fig. 3f).

Fig. 3. PEITC dose-dependently alleviated UC in mice.

a Schematic overview of UC mouse modeling and administration. b Body weight and c DAI of each group during the experiment. d The spleen was photographed and recorded, and the spleen index was calculated. e The colon was photographed and recorded, and the colon length was measured. f Histological score and hematoxylin and eosin (H&E) staining of colorectal tissue sections. Scale bar, 50 µm. g mRNA expression levels of Il6 and Tnfα. h The contents of IL-1β, MDA, and SOD. The data are presented as the mean ± SEM (n = 8 per group). ^#P < 0.05, ^##P < 0.01, ^###P < 0.001 vs. the normal group. *P < 0.05, **P < 0.01, ***P < 0.001 vs. the DSS group.

Next, we detected the levels of the inflammatory factors Il6 and Tnfα in colon tissues via a qPCR assay and found that PEITC reduced the levels of Il6 and Tnfα (Fig. 3g). The results of the ELISA also revealed that the serum level of IL-1β in the model group was significantly greater than that in the normal group, whereas the content of IL-1β in the PEITC (5 mg/kg, pre), PEITC (10 mg/kg, pre), PEITC (20 mg/kg, pre), PEITC (20 mg/kg, post) and 5-ASA (200 mg/kg, pre) groups was significantly reduced, indicating that PEITC could ameliorate colon tissue inflammation in UC mice in a dose-dependent manner (Fig. 3h). In addition, PEITC significantly decreased the DSS-induced increase in MDA, restored SOD activity, indicatiing itreduced oxidative stress levels in the mouse colon (Fig. 3h). Importantly, PEITC is used at a lower dose and has better therapeutic effects and milder side effects than does 5-ASA.

PEITC prevented the activation of pyroptosis signaling in UC mice

Next, to investigate the specific molecular mechanism by which PEITC alleviates UC in mice, we measured the expression levels of TXNIP, NLRP3, Caspase-1, GSDMD-N, and IL-1β in mouse colon tissues via Western blotting. The results showed that PEITC treatment dose-dependently inhibited the expression of TXNIP and NLRP3 and the activation of pro-caspase-1, GSDMD, and pro-IL-1β (Fig. 4a–f). The qPCR results also revealed that PEITC could reduce the levels of Txnip, Nlrp3, Casp1, Gsdmd, Il1β, and Il18 (Fig. 4g). To further verify the inhibitory effect of PEITC on the pyroptosis pathway in UC mouse colon tissues, we detected the contents of TXNIP, NLRP3, Caspase-1 and GSDMD in mouse colon tissues via immunofluorescence. The results confirmed that the expression levels of TXNIP, NLRP3, Caspase-1, and GSDMD were significantly increased in the model group but decreased after PEITC treatment (Supplementary Fig. S2). These data revealed that PEITC can inhibit the TXNIP-NLRP3-GSDMD signaling pathway and reduce the levels of pyroptosis-related proteins in the colon tissues of UC mice.

Fig. 4. PEITC inhibited the activation of pyroptosis signaling in the colon tissue of UC mice.

a–f Immunoblot analysis of TXNIP, NLRP3, pro-caspase-1, caspase-1, GSDMD, GSDMD-N, pro-IL-1β and IL-1β expression. g mRNA expression levels of Txnip, Nlrp3, Casp1, Gsdmd, Il1β, and Il18. The data are presented as the mean ± SEM. ^#P < 0.05, ^##P < 0.01, ^###P < 0.001 vs. the normal group. *P < 0.05, **P < 0.01, ***P < 0.001 vs. the DSS group.

PEITC inhibited pyroptosis in HT29 cells induced by LPS and ATP in vitro

IECs have become promising therapeutic targets for UC because of their anatomical location and complex interactions with the mucosal immune system and intestinal microenvironment, and the restoration of their normal structure and physiological function is crucial in the prevention and treatment of UC. To determine the expression of the pyroptosis-related proteins TXNIP and NLRP3 in the colon, we labeled colonic epithelial cells with E-cadherin and conducted immunofluorescence experiments. We found that TXNIP colocalized with E-cadherin, indicating that TXNIP was expressed in colonic epithelial cells (Supplementary Fig. S3a). Moreover, TXNIP colocalized with NLRP3, and PEITC treatment reduced TXNIP colocalization (Supplementary Fig. S3b). These results suggest a strong correlation between TXNIP and NLRP3 expression and a significant interaction between them, prompting us to further explore the influence of PEITC on IEC pyroptosis. We used HT29 cells to construct an in vitro pyroptosis model and found that PEITC dose-dependently inhibited the expression of TXNIP and NLRP3 and the activation of pro-caspase-1, GSDMD, and pro-IL-1β in HT29 cells by immunoblotting (Fig. 5a–g). The qPCR results also indicated that PEITC could downregulate the levels of Txnip, Nlrp3, Casp1, Gsdmd, Il1β, and Il18 (Fig. 5h).

Fig. 5. PEITC inhibited pyroptosis in HT29 cells in vitro.

a–g Immunoblot analysis of TXNIP, NLRP3, pro-caspase-1, caspase-1, GSDMD, GSDMD-N, pro-IL-1β and IL-1β expression. h mRNA expression levels of Txnip, Nlrp3, Casp1, Gsdmd, Il1β, and Il18. The data are presented as the mean ± SEM. ^#P < 0.05, ^##P < 0.01, ^###P < 0.001 vs. the control. *P < 0.05, **P < 0.01, ***P < 0.001 vs. LPS plus ATP.

PEITC attenuated pyroptosis by acting on TXNIP to interfere with its binding to NLRP3

Both in vivo and in vitro experiments demonstrated that PEITC could inhibit the pyroptosis pathway, but the specific mode of action is still unclear. On the basis of the current results, we speculate that PEITC may inhibit pyroptosis by directly affecting the functions of proteins upstream of pyroptosis or the transcription factors encoding pyroptosis-related genes. Previous studies using two-dimensional electrophoresis and mass spectrometry analysis identified thioredoxin as a binding protein of PEITC [37]. Therefore, we further investigated the interaction between PEITC and TXNIP and the impact of PEITC on the interaction between TXNIP and NLRP3.

We used coimmunoprecipitation experiments to explore the influence of PEITC on the interaction between TXNIP and NLRP3, which revealed that PEITC inhibited the binding of TXNIP to NLRP3 (Fig. 6a). Next, we predicted the binding of PEITC to TXNIP (PDB ID: 4LL1, –cdocker interaction energy = 30.3116 kcal/mol) via molecular docking (Fig. 6b). CETSA results revealed that, compared with the control, PEITC treatment significantly increased the protein thermostability of TXNIP in HT29 cells, indicating that PEITC interacted with TXNIP (Fig. 6c, d). Then, based on the molecular docking results, which indicated that glutamine at site 81 of TXNIP could form a carbon-hydrogen bond with PEITC and glycine at site 84 of TXNIP could form a conventional hydrogen bond with PEITC, we constructed human TXNIP Mut (Q81A and G84A)-EGFP plasmid. Using the human TXNIP WT-EGFP plasmid as a control, we performed CETSA and MST experiments to analyze the interaction between PEITC and TXNIP. The CETSA results indicated that when glutamine at site 81 and glycine at site 84 of TXNIP was mutated to alanine, PEITC had no significant effect on its thermal stability (Fig. 6e–h). The MST results showed that the K_d value between PEITC and human TXNIP WT was 95.7 nM, and PEITC did not bind to human TXNIP Mut (Fig. 6i). These results illustrated that PEITC can competitively bind to TXNIP, resulting in the disruption of the interaction between TXNIP and NLRP3, and that glutamine at site 81 and glycine at site 84 of TXNIP are important for its interaction with PEITC.

Fig. 6. PEITC bound to TXNIP and attenuated its binding to NLRP3.

a Proteins were immunoprecipitated with IgG or anti-TXNIP antibody. b Molecular docking of PEITC and TXNIP. c, d Effects of PEITC on the thermal stability of TXNIP. e, f Effects of PEITC on the thermal stability of TXNIP WT-EGFP. g, h Effects of PEITC on the thermal stability of TXNIP Mut-EGFP. i The binding ability between PEITC and human TXNIP WT-EGFP or TXNIP Mut-EGFP. j Representative electron microscopy images of cell morphology. Yellow arrow: intracellular vacuole. Red arrow: membrane pore. Scale bar, 1 μm. k mRNA expression levels of Il6 and Tnfα. l The contents of MDA and SOD. The data are presented as the mean ± SEM. ^#P < 0.05, ^##P < 0.01 vs. DMSO or the control. *P < 0.05, **P < 0.01, ***P < 0.001 vs. LPS plus ATP.

We subsequently investigated the effect of TXNIP on pyroptosis by transfecting TXNIP small interfering RNA (siRNA) into HT29 cells to reduce TXNIP levels. The qPCR results revealed high knockdown efficiency for the transfection of 50 nM si-TXNIP in HT29 cells for 48 h (Supplementary Fig. S4). According to the electron microscopy results, HT29 cells were obviously swollen after LPS and ATP stimulation, with cytoplasmic membrane rupture, multiple intracellular vacuoles and membrane pore formation; however, after PEITC treatment or TXNIP knockdown, the morphology of the HT29 cells returned to normal, the number of intracellular vacuoles was reduced, and the cell membrane became intact (Fig. 6j). Additionally, the levels of the inflammatory factors Il6 and Tnf-α were increased after LPS and ATP stimulation in HT29 cells, whereas TXNIP or PEITC treatment significantly decreased the levels of inflammatory factors (Fig. 6k). TXNIP knockdown or PEITC treatment also significantly decreased the content of MDA and restored SOD activity, suggesting it inhibited oxidative stress (Fig. 6l). These results indicate that PEITC can act on TXNIP and inhibit the pyroptosis pathway by weakening its binding to NLRP3.

Knockdown of TXNIP can inhibit the pyroptosis signaling pathway

Finally, we used si-TXNIP to interfere with TXNIP expression in HT29 cells, and Western blot experiments revealed that TXNIP knockdown reduced the levels of downstream pyroptosis-related proteins, such as NLRP3, caspase-1, and GSDMD-N (Fig. 7a, c–f). The immunofluorescence results also indicated that the contents of TXNIP and NLRP3 were significantly increased in the model group and that these proteins colocalized. However, the contents and colocalization of TXNIP and NLRP3 decreased after the administration of PEITC or the knockdown of TXNIP (Fig. 7b). Meanwhile, ELISA results demonstrated that both PEITC treatment and TXNIP knockdown significantly inhibited the increase of IL-1β and IL-18 levels in LPS and ATP-induced HT29 cell culture medium (Fig. 7g, h).

Fig. 7. TXNIP knockdown downregulated pyroptosis signaling.

a Immunoblot analysis of TXNIP, NLRP3, pro-caspase-1, caspase-1, GSDMD, and GSDMD-N expression. b Immunofluorescence staining of TXNIP, NLRP3, and DAPI. Scale bar, 10 µm. c–f Statistical analysis of TXNIP, NLRP3, caspase-1, and GSDMD-N expression. g, h The contents of IL-1β and IL-18. i Immunofluorescence staining of ASC and DAPI. Scale bar, 10 µm. j Immunofluorescence staining of GSDMD and DAPI. Scale bar, 10 µm. k The mRNA expression levels of Txnip, Nlrp3, Casp1, Gsdmd, Il1β, and Il18. The data are presented as the mean ± SEM. ^#P < 0.05, ^##P < 0.01, ^###P < 0.001 vs. the control. *P < 0.05, **P < 0.01, ***P < 0.001 vs. LPS plus ATP.

Given that ASC speck formation is a hallmark feature of NLRP3 inflammasome activation, we conducted immunofluorescence experiments to investigate the effects of PEITC and TXNIP knockdown on ASC speck formation in HT29 cells treated with LPS and ATP. The experimental results demonstrated that both PEITC treatment and TXNIP knockdown significantly suppressed ASC speck formation induced by LPS and ATP (Fig. 7i). This suggests that PEITC disrupts the assembly of the NLRP3 inflammasome complex, a critical step for caspase-1 activation and subsequent GSDMD cleavage and IL-1β maturation. In addition, the GSDMD-N-terminus is translocated to the cell membrane during pyroptosis, resulting in the formation of membrane pores. In the model group, we observed an obvious increase in the expression of GSDMD and its cell membrane localization, whereas the membrane localization and expression of GSDMD decreased significantly after the administration of PEITC or the knockdown of TXNIP (Fig. 7j). Additionally, the qPCR results revealed that TXNIP knockdown inhibited the increase in the mRNA levels of Txnip, Nlrp3, Casp1, Gsdmd, Il1β, and Il18 induced by LPS plus ATP, which was consistent with the inhibitory effect of PEITC (Fig. 7k). These results demonstrated that TXNIP plays a crucial role in IEC pyroptosis by influencing the activation of NLRP3 and subsequently affecting the downstream pyroptosis signaling pathway. Therefore, TXNIP is expected to become a new target for the clinical prevention and treatment of UC, and PEITC is expected to become a new drug candidate for the clinical treatment of UC.

Discussion

UC is a chronic inflammatory bowel disease that clinically manifests as recurrent abdominal pain, diarrhea, mucus and blood in the stool, and even various systemic complications [38]. It is difficult to cure and prone to recurrence. At present, it has become a global health challenge, and its global incidence is increasing annually [39]. The etiology and pathogenesis of UC are complex and poorly understood, and most clinical drugs are expensive and have limited efficacy, so there is an urgent need to find low-cost, safe, and effective therapeutic drugs [40].

Owing to the pronounced inflammatory response in UC, our study focused on pyroptosis, a form of programmed cell death closely linked with inflammation. We first carried out experiments in mice and revealed the significant involvement of pyroptosis in the occurrence and development of UC (Figs. 1 and 2, Supplementary Fig. S1). We also illustrated the temporal dynamics of the TXNIP-NLRP3-GSDMD signaling pathway throughout the process. Targeting TXNIP in colonic epithelial cells may present a novel therapeutic strategy for treating UC due to several potential benefits. High levels of TXNIP can lead to oxidative stress and inflammation, both of which are pivotal in the pathogenesis of UC [19–21]. By targeting TXNIP, it may be possible to restore redox homeostasis in IECs, mitigating oxidative damage, reducing inflammation, blocking pyroptosis of epithelial cells, and contributing to repair of barrier dysfunction in the gut. This could be particularly beneficial in UC, where the epithelial barrier is frequently compromised, contributing to disease pathology.

In our study, PEITC, a small natural compound with lipophilic and electrophilic properties [41] and high bioavailability in vivo [28], was shown to have a good therapeutic effect on DSS-induced UC in mice via its unique effects on TXNIP signaling in the colon. It can alleviate disease symptoms, spleen lesions, intestinal damage, and colon inflammation (Fig. 3). It also significantly inhibits the expression of pyroptosis-related proteins such as NLRP3, caspase-1, GSDMD and IL-1β (Fig. 4, Supplementary Fig. S2). Moreover, while TXNIP accumulated during the progression of UC, PEITC can significantly reduce TXNIP expression in the colon (Fig. 4, Supplementary Figs. S2 and S3).

To elucidate the function of TXNIP and the mechanism of PEITC, we used si-TXNIP or PEITC to intervene in an in vitro pyroptosis model in HT29 cells. The results revealed that PEITC downregulated the levels of LPS and ATP-induced pyroptosis and significantly inhibited the expression of TXNIP (Fig. 5). Further studies revealed that PEITC can interact with the Gln81 and Gly84 of TXNIP and disrupt the binding of TXNIP to NLRP3 (Fig. 6). Moreover, the knockdown of TXNIP levels in HT29 cells via si-TXNIP also significantly decreased pyroptosis-related protein levels and reduced cellular inflammation and oxidative stress levels (Fig. 7, Supplementary Fig. S4). These data suggest that TXNIP plays an important role in pyroptosis and that PEITC can inhibit the activation of NLRP3 by binding to TXNIP and disrupting the interaction between TXNIP and NLRP3, thereby inhibiting pyroptosis and improving UC (Fig. 8).

Fig. 8. Schematic diagram of the molecular mechanism by which PEITC inhibits intestinal epithelial cell pyroptosis.

PEITC can inhibit the activation of NLRP3 by binding to TXNIP and disrupting the interaction between TXNIP and NLRP3, thereby inhibiting pyroptosis and improving UC.

Overall, targeting the TXNIP-NLRP3 pathway and inhibiting pyroptosis in IECs has been demonstrated to be an effective strategy for treating UC in our study. Although small-molecule compounds regulating the activity of NLRP3 inflammasome for the treatment of UC have been reported, there is still a lack of intervention strategies targeting upstream signaling molecules of the inflammasome. This upstream targeting avoids compensatory feedback loops associated with direct NLRP3 inhibition and offers a broader therapeutic window. Compared to direct NLRP3 inhibitors (e.g., MCC950), PEITC can inhibit the full-pathway activation of the TXNIP-NLRP3-GSDMD signaling in UC by directly targeting TXNIP, thereby avoiding issues such as incomplete NLRP3 suppression. However, there remain a number of pressing unanswered questions. We cannot rule out the possibility that PEITC influences pyroptosis by modulating the expression of pyroptosis-related proteins, which warrants further investigation in the future. Then, since NLRP3 inflammasome activation involves multiple upstream regulators (e.g., NEK7), whether PEITC would trigger compensatory mechanisms through these molecules to limit its long-term efficacy might be measured. Moreover, although PEITC has demonstrated therapeutic effects in murine UC models, its efficacy and off-target effects in UC patients remain unclear. These questions will guide our future work to further refine the application of PEITC in inflammatory diseases.

In conclusion, our study confirmed the significant role of TXNIP in pyroptosis within IECs, establishing it as a novel drug target for the prevention and treatment of UC. Additionally, we revealed for the first time that PEITC down-regulates the TXNIP-NLRP3-GSDMD pathway in the context of colitis treatment, thereby offering a new perspective for the management of pyroptosis-related diseases.

Author contributions

JW, XFW, and QX designed research. JW, CZ, JQ, NA, and MB performed research and analyzed data. RHD, YS, XDW, JCC, XFW, and QX contributed new reagents. JW and XFW wrote the paper. All of the authors have approved the final manuscript.

Competing interests

Jing-cai Cheng is the employee of Drug R&D Institute, JC (Wuxi) Company, Inc. He declares no competing interests.

Supplementary information

The online version contains supplementary material available at 10.1038/s41401-025-01549-z.