Published online Aug 28, 2026. doi: 10.3748/wjg.120450
Revised: April 23, 2026
Accepted: June 22, 2026
Published online: August 28, 2026
Processing time: 159 Days and 18.9 Hours
Extracts from Gleditsia sinensis thorns (EGST) have demonstrated anti-inflammatory effects. However, the involvement of ferroptosis and bile acid (BA) me
To elucidate the mechanism by which EGST mitigate ulcerative colitis through suppression of enterocyte ferroptosis via the peroxisome proliferator-activated receptor gamma (PPARγ)/glutathione peroxidase 4 (GPX4) axis and modulation of BA metabolism.
A murine ulcerative colitis model was established using 3% dextran sulfate sodium (DSS) and treated with EGST at varying doses over 7 days. In vitro, fer
EGST significantly alleviated DSS-induced colitis by restoring colon length, reducing histopathological damage, and modulating cytokine levels (interleukin-6, tumor necrosis factor α, interleukin-10). In vitro, EGST countered erastin-induced alterations in glutathione, Fe2+, malondialdehyde, and reactive oxygen species, while also restoring mitochondrial membrane potential and mitochondrial ultrastructure. Network pharmacology analysis identified 45 overlapping targets, with core hub proteins including prostaglandin-endoperoxide synthase 2 and PPARγ. Molecular dynamics simulations confirmed stable binding of β-sitosterol, a bioactive compound in EGST, to the PPARγ ligand pocket. Mechanistic studies confirmed that EGST activate the PPARγ/GPX4 axis, upregulating PPARγ and GPX4 expression to levels comparable with the agonist rosiglitazone. Moreover, metabolomic analysis revealed a significant upregulation of taurohyodeoxycholic acid (THDCA), indicating that THDCA enhancement and BA metabolism regulation contribute to the improvement of colonic injury.
EGST significantly alleviate DSS-induced colitis by inhibiting intestinal epithelial cell ferroptosis through activation of the PPARγ/GPX4 signaling pathway and restoration of metabolic balance, particularly through the upregulation of THDCA.
Core Tip: Extracts of Gleditsia sinensis Lam. thorn (EGST) ameliorates dextran sulfate sodium-induced ulcerative colitis. EGST inhibited the ferroptosis of erastin-induced Caco-2 cells. EGST regulates nitric oxide and reactive oxygen species inhibit lipid peroxidation. EGST activated the peroxisome proliferator-activated receptor gamma/glutathione peroxidase 4 signaling pathway and inhibit ferroptosis. We report for the first time that EGST alleviates colitis in mice by regulating taurohyodeoxycholic acid.
- Citation: Zhuo ZY, Guo XY, Liu XM, Shang MM, Xue S, Peng L, Guo ZG. Extracts of Gleditsia sinensis Lam. thorn ameliorates ulcerative colitis through PPARγ/GPX4 pathway-mediated inhibition of enterocyte ferroptosis. World J Gastroenterol 2026; 32(32): 120450
- URL: https://www.wjgnet.com/1007-9327/full/v32/i32/120450.htm
- DOI: https://dx.doi.org/10.3748/wjg.120450
Ulcerative colitis (UC), a subtype of inflammatory bowel disease (IBD), is characterized by recurrent relapses, persistent abdominal pain, diarrhea, and mucous-purulent bloody stools[1]. The precise etiology and pathogenesis of UC remain incompletely understood; however, genetic predisposition, environmental factors, immune dysregulation, and dis
Ferroptosis is a distinct form of regulated cell death, characterized by its dependence on iron ions and the excessive accumulation of lipid peroxides[4]. Key biomarkers of ferroptosis include 4-hydroxy-2-nonenal (4-HNE), malondialdehyde (MDA), glutathione peroxidase 4 (GPX4), and long-chain fatty acyl-CoA ligase 4 (ACSL4), among others. Of these, 4-HNE and MDA are primary products of lipid peroxidation derived from polyunsaturated fatty acids. GPX4 plays a critical role in inhibiting lipid peroxidation via glutathione (GSH)-dependent mechanisms, thereby protecting cells from ferroptotic damage[5,6]. In contrast, arachidonate 15-lipoxygenase (ALOX15) and ACSL4 are key ferroptosis inducers, facilitating lipid peroxide accumulation and the incorporation of polyunsaturated fatty acids into membrane phospho
In the present study, bioactive compounds such as β-sitosterol were identified in EGST. β-sitosterol, a phytosterol, has demonstrated a strong affinity for binding to PPARγ, indicating that EGST may exert their effects by directly targeting this receptor[17]. Additionally, network pharmacology (NP) analysis revealed PPARγ and prostaglandin-endoperoxide synthase 2 (PTGS2) as core hub proteins, linking EGST to the modulation of hypoxia and ROS metabolic pathways. These findings support the hypothesis that EGST may inhibit enterocyte ferroptosis via the PPARγ/GPX4 axis, thereby preserving mitochondrial integrity and reducing lipid peroxidation in the colonic epithelium.
In addition to ferroptosis, BA metabolism is essential for maintaining intestinal homeostasis. BAs, amphipathic metabolites synthesized from cholesterol in hepatocytes, play essential roles in lipid absorption and signaling[18]. In UC patients, disturbances in BA metabolism are typically characterized by excessive accumulation of primary BAs and reduced levels of secondary BAs, which result from intestinal dysbiosis and impaired enterohepatic circulation[19]. Secondary BAs, such as taurohyodeoxycholic acid (THDCA), have been shown to possess anti-inflammatory properties[20]. Although prior studies have highlighted the anti-inflammatory effects of EGST, their role in regulating BA metabolism in the context of colitis remains unexplored. This study aims to investigate the protective role of EGST in UC, specifically by inhibiting enterocyte ferroptosis through the PPARγ/GPX4 axis, and to examine their impact on fecal BA metabolism.
The Gleditsia sinensis thorns used in this study were sourced from Song County, Henan Province (batch number: 121210-202106) and processed by the Institute of Forest Chemistry, Chinese Academy of Forestry Sciences. The sample consisted of 10 kg of soap pod thorn powder and 620 g of extract. After extraction, the sample was analyzed using HR-LC-ESI-Orbitrap-MS. The identification of EGST was carried out by Shanghai OE Biotech Co., Ltd. Detailed preparation and identification methods can be found in the literature[13].
C57BL/6 mice (Jiangsu Union Medical Biotechnology, Jiangsu Province, China) were housed under specific pathogen-free conditions, with a controlled environment set at 20 °C-25 °C, a 12-hour light/dark cycle, and ad libitum access to food and water. A murine colitis model was induced by administering 3% dextran sulfate sodium (DSS) (MP Bio
After a one-week acclimatization period, thirty mice were randomly assigned into five groups (n = 6 per group): Control, model, EGST-L (80 mg/kg), EGST-M (160 mg/kg), and EGST-H (320 mg/kg). Mice in the model and DSS + EGST groups (EGST-L, EGST-M, and EGST-H) received 3% DSS in drinking water, while the control group was given standard drinking water. Mice in the DSS + EGST groups underwent oral gavage of EGST at the designated concentrations for seven consecutive days, while those in the control and model groups received an equivalent volume of normal saline. Throughout the experimental period, body weight and fecal consistency were monitored daily, and the disease activity index (DAI) was calculated for each mouse. On day 8, all animals were provided with regular drinking water. Following anesthesia with sodium amobarbital, blood samples were collected from the orbital sinus. The mice were then euthanized via cervical dislocation, and colon tissues were harvested for further analysis.
Tissue samples from the colon, located about 1 cm above the anal area, were removed, after being fixed in a 4% paraformaldehyde solution for 24 hours, the tissues were embedded in paraffin. These paraffin-embedded blocks were cut into 4 μm thick sections and stained with hematoxylin and eosin. After deparaffinization and washing, Periodic acid-Schiff staining was conducted, then dehydration and mounting were done. The sections prepared were examined under a microscopically for histopathological an analysis.
Blood was drawn from the orbital sinus and left to coagulate at room temperature for an hour. The samples were centrifuged at 3000 rpm for 15 minutes at 4 °C to separate the serum. Inflammatory cytokine levels (IL-6, IL-10, and TNF-α) were assessed with an enzyme-linked immunosorbent assay kit from Shanghai Enzyme-linked Biotechnology Co., Ltd., following the provided instructions.
The concentrations of GSH, MDA, and Fe2+ in mouse serum were determined following the protocols outlined in the respective assay kits (Elabscience, Wuhan, China).
Following deparaffinization and rehydration, colon tissue sections were subjected to antigen retrieval with citrate buffer at pH 6.0. The endogenous peroxidase activity was subsequently inhibited by treating the sections with 3% hydrogen peroxide at ambient temperature in a dark environment. To block nonspecific binding, 3% bovine serum albumin was applied. The sections were left to incubate overnight at 4 °C with these primary antibodies: ACSL4 monoclonal antibody (Affinity, 1:100), GPX4 monoclonal antibody (MedChemExpress, NJ, United States, 1:200), and ALOX15 monoclonal antibody (Beyotime, China; 1:100). The following day, at room temperature, a horseradish peroxidase (HRP)-linked goat anti-rabbit secondary antibody (SeraCare, MA, United States; 1:500) was utilized. After washing, color development was achieved using a 3,3’-diaminobenzidine substrate, followed by dehydration, mounting, and microscopic observation.
For further analysis, tissue sections embedded in paraffin underwent deparaffinization and rehydration, then antigen retrieval was performed using EDTA buffer at pH 9.0. Endogenous peroxidase activity was blocked, and nonspecific binding was blocked by applying 10% goat serum at room temperature for 30 minutes. Subsequently, the sections were treated with a primary antibody against 4-hydroxynonenal (4HNE, 1:200, Biosynthesis, Beijing, China), and then with an HRP-conjugated goat anti-rabbit antibody (1:400) left in the dark at room temperature for 50 minutes. A fluorescein-tyramide conjugate was applied and incubated for 20 minutes at room temperature, with cell nuclei counterstained using 4’,6-diamidino-2-phenylindole, and the sections were mounted for microscopic observation.
Caco-2 cells (Fuheng Cell Bank, Shanghai, China) were grown in Dulbecco’s Modified Eagle Medium with 10% fetal bovine serum, 100 U/mL penicillin, and 100 μg/mL streptomycin, kept in a humidified incubator at 37 °C with 5% CO2. The experiment was conducted with three groups: Control, erastin, and erastin + EGST, with EGST administered at concentrations of 0.5, 2.5, and 12.5 μg/mL. The control group received an equivalent volume of fresh culture medium. All experimental groups were then incubated for an additional 24 hours before the analytical assays.
The supernatant from each group was collected, centrifuged, and analyzed according to the protocols provided with the commercial kits. GSH, MDA, PTGS2, and Fe2+ levels were quantified using an automated biochemical analyzer.
Using a JC-1 assay kit, the mitochondrial membrane potential (MMP) of Caco-2 cells was measured. The culture medium was placed into flow cytometry tubes, and the cells were washed with phosphate-buffered saline (PBS). The JC-1 working solution was added to fresh culture medium, mixed well, and incubated under standard conditions. After incubation, the supernatant was removed, and the cells were washed with 1 × JC-1 buffer prior to flow cytometry analysis.
Fresh colon tissues were rapidly sectioned into approximately 1 mm3 fragments and immediately immersed in electron microscopy fixative for fixation at 4 °C for 2-4 hours. After fixation, the samples were rinsed with PBS to remove residual fixative and then underwent post-fixation, graded dehydration, resin infiltration, embedding, ultrathin sectioning, and staining procedures. The ultrastructural morphology was observed and photographed using a transmission electron microscope (FEI, United States) for further analysis.
Caco-2 cells were treated with 4% DSS prior to analysis. The cells were washed with PBS and then digested using trypsin, and digestion was terminated once complete cell detachment was achieved. The resulting cell suspension was transferred to a centrifuge tube and centrifuged to remove the supernatant. To load the probes, suitable amounts of 2’,7’-dichlorodihydrofluorescein diacetate and 4-amino-5-methylamino-2’,7’-difluorofluorescein diacetate were introduced into the culture medium. After incubation in a humidified chamber, the cells were centrifuged, washed with fresh medium, and then subjected to instrumental detection.
To extract proteins, cells were broken down on ice using radioimmunoprecipitation assay buffer (Biosharp, Hefei, China) with added protease inhibitors (ROCHE, Switzerland), and the supernatant was collected. Protein levels were measured with a bicinchoninic acid protein quantification kit (Servicebio, Wuhan, China). Equal amounts of protein were processed by electrophoresis, transferred to nitrocellulose membranes, and subjected to blocking and incubation with primary antibodies followed by HRP-conjugated secondary antibodies at room temperature. Protein bands were detected using an enhanced chemiluminescence kit (Meilunbio, Dalian, China), and the optical density of target bands was analyzed using IPWIN60 software. All western blot experiments were conducted using parallel gels.
The key components of EGST were sourced from the Traditional Chinese Medicine Systems Pharmacology Database, with initial selection criteria of oral bioavailability of at least 30% and drug-likeness at least 0.18. Potential target genes were predicted through the Swiss Target Prediction and SEA databases. Data related to UC were obtained from OMIM, Genecards, and TTD. Genes linked to ferroptosis were obtained from the FerrDb database. After identifying the intersecting targets among the drug, ferroptosis-related genes, and UC, a “drug-compound-target-disease” interaction network was created in Cytoscape. A network of protein-protein interaction was created utilizing the STRING database. Additionally, the shared targets underwent Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes enrichment analyses of the shared targets were performed using the R package, with results screened at a significance level of P < 0.05 and ranked by P-value.
Protein and compound three-dimensional structures were retrieved from the UniProt and PubChem databases, respectively. In AutoDockTools, hydrogen atoms were added to the protein structures, electrostatic charges were computed, and the processed files were saved in PDBQT format. Drug molecules were converted to ligand PDBQT files using Open Babel. Molecular docking and binding free energy analyses were then performed in AutoDockTools, with docking conformations visualized using PyMOL.
Molecular dynamics simulations were conducted using GROMACS 2022. The AMBER14SB and GAFF force fields were applied to the protein and ligand, respectively. The system was solvated with the TIP3P water model and placed within a periodic boundary box. After equilibration under NVT and NPT ensembles for 100 picoseconds each, a 100 nanoseconds production run was performed at 298 K and 1 bar with a 2 femtoseconds integration step. Long-range electrostatic interactions were calculated using the PME method with a 1.2 nm cutoff, while non-bonded interactions were handled with a 10 Å cutoff. Simulation trajectories were analyzed using VMD and PyMOL, and binding free energies were computed using the g_mmpbsa module with the MMPBSA approach.
Fecal samples were collected and stored at -80 °C until analysis. Ultra-high-performance liquid chromatography-tandem mass spectrometry with multiple reaction monitoring was utilized to analyze fecal metabolites. Metabolite abundance was quantified according to the established protocol and normalized with internal standard controls. Data processing involved least squares regression analysis, with a weighting factor of 1/x, yielding optimal accuracy and correlation coefficient (R) for the calibration curve.
All analyses, except those specific to metabolomics, were performed using GraphPad Prism (Version 10.0). For the time-series data in Figure 1A (body weight) and Figure 1B (DAI score), which involved repeated measurements from the same individuals, statistical differences were assessed using repeated measures two-way analysis of variance (ANOVA) with Bonferroni’s post hoc test. Variations among different groups were analyzed using one-way ANOVA followed by Dunnett’s post hoc multiple comparison test. A P value of < 0.05 was considered statistically significant.
Compared to the control group, DSS-treated mice exhibited significant reductions in body weight (Figure 1A), increased DAI scores (Figure 1B), and shortened colon length (Figure 1C and D). EGST treatment at varying concentrations effectively alleviated these pathological signs, with the high-dose group showing the most substantial improvement. The degree of colonic injury correlates with UC severity. Histological analysis through hematoxylin and eosin staining revealed that DSS exposure led to extensive disruption of the mucosal epithelium, with submucosal edema, crypt loss, severe inflammatory cell infiltration, and depletion of goblet cells, compared to the control group. EGST treatment significantly alleviated these histopathological abnormalities (Figure 1E). Periodic acid-Schiff staining further confirmed that EGST administration increased the goblet cell count in DSS-induced mice (Figure 1F and G), suggesting that EGST restore mucosal integrity and reduces colonic inflammation. To evaluate the anti-inflammatory effects of EGST, serum levels of IL-6, IL-10, and TNF-α were measured. In DSS-treated mice, TNF-α and IL-6 levels were significantly elevated compared to the control group. Following EGST intervention, both pro-inflammatory cytokines (TNF-α and IL-6) were markedly reduced, while the anti-inflammatory cytokine IL-10 was upregulated (Figure 1H). These results indicate that EGST modulate systemic cytokine balance, contributing to the alleviation of DSS-induced colitis.
Previous RNA-seq data (accession number: PRJNA1145599) revealed that EGST are closely associated with the ferroptosis signaling pathway. GO enrichment analysis of transcriptomic sequencing suggested that EGST may exert their effects by downregulating iron ion transmembrane transport, reducing iron-binding activity, and suppressing inflammatory processes (Figure 2A). Ferroptosis-related proteins, including ACSL4, GPX4, and ALOX15 (arachidonate 15-lipoxygenase), were identified ascritical regulators of ferroptotic cell death (Figure 2B). Gene set enrichment analysis showed that ferroptosis-related pathways were significantly downregulated in the EGST-treated group compared to the DSS model group (Supplementary Table 1 and Figure 2C). To verify the roles of ACSL4, GPX4, and ALOX15 in the colon of DSS mice, immunohistochemical staining was performed. The results showed increased expression of ACSL4 and ALOX15 in DSS-treated mice, while GPX4 expression was reduced, consistent with ferroptosis manifestations. After EGST treatment, ACSL4 and ALOX15 expression decreased, while GPX4 expression increased, suggesting that EGST reduce ferroptosis. Furthermore, the key markers of ferroptosis, MDA, Fe2+, and 4HNE, were highly expressed in the DSS group, while GSH levels were reduced, indicating ferroptosis involvement in colitis. After EGST treatment, MDA, Fe2+, and 4HNE expression significantly decreased, while GSH levels increased, further confirming that EGST inhibit ferroptosis in the DSS-induced mouse model by reducing lipid peroxidation (Figure 2D-F).
Erastin was used to induce ferroptosis in Caco-2 cells for in vitro verification. Significant increases in MDA, PTGS2, and Fe2+ levels, along with a decrease in GSH, confirmed the successful induction of ferroptosis. Treatment with EGST reversed these biochemical changes by reducing MDA, PTGS2, and Fe2+ levels while enhancing GSH content (Figure 3A), indicating that EGST inhibit ferroptosis. Additionally, analysis of ACSL4 and GPX4 protein expression revealed that EGST downregulated ACSL4 and upregulated GPX4, with the most prominent effect observed in the erastin + EGST-H group (Figure 3B). As ferroptosis progresses, lipid peroxidation reaches an irreversible stage, leading to significant mitochondrial changes, including a marked reduction or complete loss of MMP. In the erastin-treated group, flow cytometric analysis revealed a notable decline in MMP. Transmission electron microscopy imaging showed severe mi
Cellular oxidative and antioxidant systems play a critical role in ferroptosis. To assess the impact of EGST on the oxidative system, intracellular levels of NO and ROS were quantified using flow cytometry. DSS stimulation significantly elevated NO and ROS levels, indicating an imbalance in redox homeostasis. In contrast, EGST treatment effectively suppressed the accumulation of both NO and ROS (Figure 4A and Figure 4B), suggesting that EGST restore redox balance and may mitigate UC by reducing lipid peroxidation.
To identify the key molecular targets through which EGST modulate UC and ferroptosis, an NP approach was employed. Eleven bioactive compounds were identified from EGST (chemical details in Supplementary Table 2). By integrating data from multiple databases, 375 compound-related targets, 564 ferroptosis-associated genes, and 4165 UC-related targets were retrieved. Venn diagram analysis revealed 45 overlapping targets that may contribute to EGST’s therapeutic effects on UC via ferroptosis modulation (Figure 5A and B). The protein-protein interaction network identified PTGS2, PPARγ, HIF1A, MAPK3, and EGFR as core hub proteins (Figure 5C). GO enrichment analysis revealed that these targets were primarily involved in biological processes such as ROS metabolism, response to oxygen levels, and hypoxia (Figure 5D). Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis further highlighted the “chemical carcinogenesis-ROS” pathway (Figure 5E). These results suggest that the shared targets are primarily involved in ROS metabolic regulation and hypoxia-related signaling, supporting the hypothesis that EGST may suppress ferroptosis. To further evaluate the target-ligand interactions, molecular docking analysis was performed. The binding affinities of EGST with PPARγ, HIF1A, EGFR, PTGS2, andMAPK3 were -7.6, -6.2, -8.3, -8.9, and -8.7 kcal/mol, respectively (Figure 5F), indicating strong and stable interactions between EGST and these proteins.
Molecular dynamics simulations were conducted to assess the binding behavior and structural stability of β-sitosterol in complex with the PPARγ protein. The simulation revealed that β-sitosterol was stably accommodated within the ligand-binding pocket of PPARγ, forming multiple interactions with surrounding amino acid residues (Figure 6A and B). The root mean square deviation of the complex stabilized after approximately 40 nanoseconds, indicating that the system reached equilibrium (Figure 6C). The variation in the distance between the ligand and the binding site further confirmed the stability of the interaction (Figure 6D). Additionally, the radius of gyration of the complex gradually plateaued (Figure 6E), while the solvent accessible surface area fluctuated slightly but remained consistent overall (Figure 6F), suggesting that the binding interface remained structurally intact. The root mean square fluctuation values of the protein residues were generally below 0.2 nm (Figure 6G), reflecting minimal conformational changes. The number of hydrogen bonds between β-sitosterol and PPARγ ranged from 0 to 2 (Figure 6H). The calculated binding free energy was -164.4 kJ/mol (Supplementary Table 3), with van der Waals interactions contributing most significantly, followed by hydrophobic effects, and minor electrostatic interactions (Figure 6I). The energy contributions of key residues are summarized in Figure 6J. Western blot analysis was then employed to validate the regulatory effects of EGST on the PPARγ/GPX4 pathway. The results showed that, compared to the erastin-treated group, PPARγ and GPX4 expression levels were upregulated in a dose-dependent manner following EGST administration. Moreover, the enhancing effect of high-dose EGST on GPX4 expression was comparable to that of the PPARγ activator rosiglitazone (Figure 6K). In addition, administration of the PPARγ inhibitor GW9662 following high-dose EGST treatment resulted in a downregulation of GPX4 and an upregulation of ACSL4, suggesting that the anti-ferroptotic effect of EGST was effectively neutralized (Supplementary Figure 1). These results indicate that EGST mitigate ferroptosis and alleviates UC by activating the PPARγ/GPX4 signaling pathway.
Metabolomics studies have previously highlighted the role of metabolic alterations in UC pathophysiology. To explore changes in fecal BA profiles among different experimental groups, over 50 BA metabolites were quantified using a liquid chromatography-mass spectrometry-based targeted metabolomics approach (Supplementary Table 4). Principal compo
UC is a chronic IBD characterized by recurrent mucosal inflammation and disruption of the intestinal barrier. Ferroptosis, an iron-dependent and lipid peroxidation-driven form of regulated cell death[21,22], has emerged as a significant contributor to mucosal injury, making it a promising therapeutic target in UC. Accumulating evidence suggests that ferroptosis of intestinal epithelial cells exacerbates intestinal damage. PPARγ, an anti-inflammatory protein, is known to suppress inflammatory responses in immune cells[23]. Compounds such as paeonol and a triazole derivative modulate PPARγ activity to alleviate colitis-induced epithelial injury and restore barrier function[24,25]. Additionally, PPARγ plays a key role in ferroptosis and has been shown to alleviate colitis-induced mucosal damage. For instance, pectolinarigenin mitigates 5-fluorouracil-induced intestinal inflammation and ferroptosis by upregulating the PPARγ/GPX4 axis[26]. Transcriptome sequencing has shown that EGST protect against UC through the PPARγ/AMPK/NF-κB pathway, activating PPARγ to inhibit downstream pro-inflammatory factors and ameliorating tissue damage[13]. Thus, PPARγ is a central mediator linking traditional Chinese medicine compounds with GPX4-dependent ferroptosis inhibition. Recent studies have highlighted natural products, such as extracts from Scutellaria baicalensis and compounds like pectolinarigenin and vanillic acid, which ameliorate UC by modulating ferroptosis-related pathways[1,26,27]. Although previous research indicated that EGST alleviate DSS-induced colitis, the precise mechanisms remained unclear. In this study, transcriptomic sequencing and gene set enrichment analysis revealed significant enrichment of ferroptosis-related signaling pathways following EGST treatment. Based on these findings, it is hypothesized that EGST exert their the
To further elucidate the pharmacodynamic mechanisms, NP and molecular docking were employed to identify EGST’s targets. Key targets such as PPARγ, HIF1A, EGFR, and PTGS2 were identified, with PPARγ standing out due to its regulatory role in antioxidant responses and ferroptosis. Molecular dynamics simulations revealed that β-sitosterol, a bioactive component of EGST, stably binds to the ligand-binding pocket of PPARγ with high affinity, maintaining structural stability over time. Western blot analysis confirmed that EGST upregulated both PPARγ and its downstream effector GPX4 in a dose-dependent manner, mirroring the effect of the PPARγ agonist rosiglitazone. These findings indicate that activation of the PPARγ/GPX4 axis is a central mechanism by which EGST inhibit ferroptosis and exerts their anti-inflammatory effects.
Notably, beyond regulating cell death pathways, metabolic reprogramming plays a critical role in the progression of UC. BAs, the end products of hepatic cholesterol catabolism, exhibit dual roles in UC: Some promote inflammation through ferroptosis, while others activate receptors to suppress inflammation and repair the intestinal barrier. Deo
This study is the first to report that EGST significantly increase levels of THDCA, a BA with established anti-inflammatory properties. BA metabolism is intricately linked with gut microbiota composition and intestinal immunity. The gut microbiota facilitates the conversion of primary BAs into secondary BAs, which act as endogenous ligands to regulate intestinal microbial composition and coordinate host immunity[33,34]. While primary BAs can damage the mucosal barrier, secondary BAs such as THDCA have been shown to exert protective effects[35,36]. Our findings suggest that EGST’s ability to restore metabolic balance, particularly through the upregulation of THDCA, may contribute synergistically to the alleviation of colitis by modulating the microbial-metabolite-host axis[20,37,38]. However, the precise molecular mechanism by which EGST regulate THDCA, whether through direct enzymatic modulation or indirect microbiota reshaping, remains to be fully elucidated and warrants further investigation.
In summary, this study provides a preliminary exploration of the multi-target mechanisms underlying EGST’s therapeutic effects in UC. By integrating transcriptomics, NP, and experimental validation, this study demonstrates that EGST activate the PPARγ/GPX4 axis to inhibit ferroptosis and modulates BA metabolism to reduce inflammation. Despite these promising findings, several limitations remain, including the lack of genetic knockout models to defi
In conclusion, EGST significantly improved DSS-induced colitis by restoring intestinal barrier function and modulating systemic cytokine responses. Mechanistically, these protective effects were mediated through the inhibition of intestinal epithelial cell ferroptosis via activation of the PPARγ/GPX4 signaling axis and restoration of redox homeostasis. Furthermore, EGST exerted beneficial regulatory effects on BA metabolism, particularly through the upregulation of the anti-inflammatory metabolite THDCA. Consequently, EGST represent promising multi-target therapeutic agents for the clinical management of UC, modulating both ferroptosis pathways and metabolic balance.
The authors would like to express their sincere gratitude to Dr. Liu Yong from the Institute of Chemical Industry, Chinese Academy of Forestry, for kindly providing the samples used in this study.
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