Natural Dietary Flavonoid Apigenin Mitigates Ulcerative Colitis via Modulating the AMPK/NF-κB/NLRP3 Signaling Axis.

To assess whether Api exhibits an anticolitis effect in vivo , mice received 2.5% (w/v) DSS in their drinking water for 7 days to induce acute colitis. Simultaneously, they received Api via gavage at doses of 75 mg/kg (low dose) and 150 mg/kg (high dose) ( Figure B). Compared with the normal control group, DSS-treated mice exhibited obvious colitis symptoms, including body weight loss ( Figure C), increase in DAI scores ( Figure D) and marked colon shortening ( Figure E-F). Meanwhile, H and E staining revealed severe destruction of the colonic mucus layer, a reduction in goblet cells, and disrupted crypt architecture, accompanied by marked infiltration of inflammatory cells ( Figure G). As a positive control, 5-ASA (300 mg/kg) treatment significantly mitigated above symptoms ( Figure B-F). When Api was administered to DSS-challenged mice, it significantly alleviated colitis-related symptoms, as evidenced by reversed body weight loss ( Figure C), decreased DAI scores ( Figure D), and restored colon length ( Figure E-F). Notably, DSS caused pathological damage of colonic tissue was also reversed by Api, including the enhancement of crypts and goblet cells, as well as the diminished the inflammatory infiltration ( Figure G). Api effectively attenuates DSS-induced colitis in mice. (A) Chemical structure of Api. (B) Experimental timeline. (C) Body weight changes in the Control, DSS, DSS + 5-ASA, DSS + L-Api, and DSS + H-Api groups during modeling. (D) Disease activity index (DAI) scores of each group during modeling. (E, F) Colon length measurements for each group. (G) Representative H&E staining of colonic sections from each group. Upper panels, scale bar = 100 μm; lower panels (magnified views), scale bar = 50 μm. Data are expressed as mean ± SD ( n = 6). ## p < 0.01, ### p < 0.001 vs Control; * p < 0.05, ** p < 0.01, *** p < 0.001 vs DSS; ns, not significant. To further explore the protective role of Api, we evaluated intestinal barrier integrity in DSS- and Api-treated mice. Immunofluorescence staining was first performed to evaluate the distribution of tight junction proteins in colon tissues. DSS administration markedly disrupted the localization of ZO-1 and occludin at the intercellular junctions, whereas both low- and high-dose Api treatments effectively restored their continuous distribution along the epithelial cell borders ( Figure A). PAS staining was subsequently employed to assess goblet cell integrity. DSS challenge resulted in a marked depletion of goblet cells and severe epithelial damage in colonic tissues. These pathological changes were notably ameliorated by Api treatment ( Figure B). Western blot analysis further confirmed the protective effect of Api on epithelial barrier components and apoptosis regulation. DSS exposure downregulated the protein expression of ZO-1 and occludin, which was significantly reversed by both low- and high-dose Api treatments. Additionally, Api markedly increased the Bcl-2/Bax ratio, suggesting a shift toward an antiapoptotic phenotype ( Figure C). Consistently, TUNEL staining revealed extensive apoptosis in colonic epithelial cells following DSS administration, whereas Api treatment substantially reduced the number of TUNEL-positive cells, indicating its protective role against epithelial cell death ( Figure D). Taken together, these findings demonstrate that Api enhances intestinal barrier integrity by restoring tight junction protein expression, preserving goblet cell function, and inhibiting epithelial apoptosis, thereby contributing to its anticolitic efficacy. Api significantly mitigates DSS-induced intestinal barrier damage. (A) Representative immunofluorescence staining of colonic sections from the Control, DSS, DSS + L-Api, and DSS + H-Api groups showing expression of occludin (green) and ZO-1 (red); scale bar, 50 μm. (B) PAS staining illustrating goblet-cell distribution and mucin secretion in each group; scale bar, 100 μm. (C) Western blot analysis of ZO-1, occludin, Bcl-2, and Bax in colonic tissues with densitometric quantification on the right. (D) TUNEL staining for apoptotic cells in colonic sections; scale bar, 50 μm. Data are expressed as mean ± SD ( n = 3). ### p < 0.001 versus Control; * p < 0.05, ** p < 0.01, *** p < 0.001 versus DSS. As neutrophil infiltration and inflammation can be indicated by the activity of colonic myeloperoxidase (MPO), we carried out immunohistochemistry using an MPO antibody across all groups. DSS treatment markedly increased the number of MPO-positive cells in colonic tissue, indicating significant inflammatory infiltration. In contrast, both low- and high-dose Api treatments substantially reduced MPO-positive cell accumulation ( Figure A). Given the close link between inflammation and oxidative stress, we next measured oxidative stress markers in colonic tissue. DSS exposure elevated the levels of malondialdehyde (MDA) while reducing levels of reduced glutathione (GSH) and total antioxidant capacity (T-AOC), indicating redox imbalance. High-dose Api treatment effectively reversed these alterations, suggesting its potent antioxidant capacity ( Figure B). To further confirm the anti-inflammatory effects of Api, the mRNA expression levels of TNF-α, IL-1β, and IL-6 were quantified in colonic tissues. DSS markedly increased their expression, whereas Api treatment significantly suppressed these proinflammatory cytokines ( Figure C). In addition, Western blot analysis was used to evaluate the protein levels of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2), two key enzymes involved in inflammation and oxidative stress. Api administration resulted in a notable downregulation of both iNOS and COX-2 in DSS-treated mice ( Figure D). Collectively, these findings demonstrate that Api exerts anticolitic effects by alleviating colonic inflammation and oxidative stress through multiple pathways, including cytokine inhibition, redox balance restoration, and downregulation of stress-associated enzymes. Api suppresses colonic inflammation and oxidative stress in DSS-induced mice. (A) Representative myeloperoxidase (MPO) immunohistochemistry of colonic sections from Control, DSS, DSS + L-Api, and DSS + H-Api groups ( n = 3). Upper panels, scale bar = 100 μm; lower panels (magnified views), scale bar = 25 μm. (B) Oxidative-stress indices in colonic homogenates: malondialdehyde (MDA), reduced glutathione (GSH), and total antioxidant capacity (T-AOC) ( n = 6). (C) qPCR analysis of pro-inflammatory cytokine transcripts ( Tnf-α, Il-1β, Il-6 ) in colonic tissue ( n = 5). (D) Western blot detection of inducible nitric-oxide synthase (iNOS) and cyclo-oxygenase-2 (COX-2) in colonic protein extracts with β-actin as the loading control. ### p < 0.001 versus Control; * p < 0.05, ** p < 0.01, *** p < 0.001 versus DSS. To investigate the molecular mechanism by which Api ameliorates DSS-induced colitis, transcriptomic analysis was performed on colonic tissue. Unsupervised PCA revealed that DSS treatment altered the global gene expression profile compared with the control group, while Api treatment partially restored the transcriptomic pattern toward that of the normal group ( Figure A). Differential expression analysis showed that, compared with the control group, 2,131 genes were upregulated and 1,070 were downregulated in the DSS group. Api treatment resulted in 939 upregulated and 1,175 downregulated genes versus the control group, and 491 upregulated and 2,509 downregulated genes versus the DSS group ( Figure B). The Venn diagram illustrated the overlap and specificity of differentially expressed genes (DEGs) among the three pairwise comparisons, identifying 415 genes shared across all groups ( Figure C). With a threshold of |log2 FC| > 1 and a P < 0.05, DSS treatment resulted in 2,297 significantly upregulated and 1,171 downregulated genes compared to controls ( Figure D). In contrast, Api treatment specifically upregulated 583 genes and downregulated 2,793 genes in DSS-induced mice ( Figure E). Specifically, several inflammation-related genes, such as Il1a , Cxcl10 , Cxcl1 , Ptgs2 and Ccl2 , as well as oxidative stress-related genes, like Cybb and Ncf1 , were significantly downregulated following Api administration ( Figure F). RT-qPCR analysis further validated that Api markedly inhibited the expression of oxidative stress-related genes Cybb and Ncf1 , along with inflammation-related genes Cxcl1 , Ccl2 , and Il1a . In contrast, it promoted the expression of antioxidant gene Aqp8 ( Figure G). Together, these findings suggest that Api could alleviate UC with anti-inflammatory and antioxidant effects. Api suppresses the activation of the inflammatory signaling pathway to attenuate UC. (A) PCA of RNA-seq profiles from colonic tissues of the Control, DSS, and DSS + H-Api groups ( n = 5 per group). PC1 and PC2 account for 48.16% and 31.01% of the total variance, respectively. (B) Numbers of DEGs detected in each pairwise comparison (|log 2 fold-change| ≥ 1, adjusted p < 0.05). Red, up-regulated genes; blue, down-regulated genes. (C) Venn diagram illustrating the overlap of DEGs among DSS vs Control, DSS + Api vs DSS, and DSS + Api vs Control. (D, E) Volcano plots of DEGs in DSS vs Control (D) and DSS + Api vs DSS (E). Red dots, significantly up-regulated genes; blue dots, significantly down-regulated genes; gray dots, nonsignificant genes. (F) Heatmap of 36 preselected genes relevant to inflammatory and oxidative-stress pathways across the three groups ( n = 5 per group); colors denote Z-score-scaled expression levels. (G) qPCR validation of representative genes ( Cybb, Ncf1, Ccl2, Cxcl1, Il1a, Aqp8 ) in colonic tissue ( n = 5). (H) KEGG enrichment analysis of genes down-regulated by Api relative to DSS, highlighting repression of cytokine-cytokine receptor interaction, MAPK, NF-κB, NOD-like receptor, Toll-like receptor, and related pathways. ### p < 0.001 versus Control; * p < 0.05, ** p < 0.01, *** p < 0.001 versus DSS. To further elucidate the signaling pathways potentially underlying the anticolitis effects of Api, we performed Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses. GO over-representation analysis revealed that Api-downregulated genes were significantly enriched in: (i) molecular function terms, such as extracellular matrix structural constituent and protein binding; (ii) cellular component terms, including the extracellular region, extracellular space, and collagen-containing extracellular matrix; and (iii) biological process terms related to cell adhesion and inflammatory response ( Figure S1 ). KEGG analysis further demonstrated that the Api-downregulated genes were significantly in multiple inflammatory and immune-related signaling pathways, including the MAPK signaling pathway, TNF signaling pathway, NOD-like receptor (NLR) signaling pathway, Toll-like receptor signaling pathway, and the NF-κB signaling pathway ( Figure H). Notably, genes involved in the inflammatory bowel disease (IBD) pathway were also significantly enriched, suggesting that Api mitigates DSS-induced colitis by suppressing key pro-inflammatory signaling cascades and modulating extracellular matrix-associated responses. Given the central role of the NLRP3 inflammasome in intestinal inflammation, we further examined whether it serves as a direct target of Api. Transcriptomic profiling and RT-qPCR analysis revealed that Api significantly reduced Nlrp3 mRNA expression in colonic tissues of DSS-treated mice ( Figure A). Consistently, Western blot analysis showed that Api downregulated the protein levels of NLRP3, cleaved caspase-1, GSDMD, and both pro- and cleaved IL-1β ( Figures B, S2 ), indicating suppressed inflammasome activation and pyroptotic signaling in vivo . Api inhibits both in vivo and in vitro activation of NLRP3 inflammasome. (A) Relative mRNA expression of Nlrp3 in colonic tissues from Control, DSS, and DSS + Api groups (qPCR; n = 5). (B) Western-blot analysis of inflammasome-related proteins in colonic tissue: cleaved and pro-IL-1β, cleaved and pro-caspase-1, NLRP3, ASC, GSDMD; β-actin was used as a loading control. (C) ELISA quantification of IL-1β in BMDM supernatants after LPS + nigericin stimulation with or without Api ( n = 3). (D) qPCR analysis of IL-1β , Nlrp3 , and GSDMD transcripts in BMDMs under the same conditions as in (C) ( n = 3). (E) SPR sensorgram confirming the direct interaction between Api and recombinant NLRP3. (F) Molecular-docking model showing the predicted binding of Api to the NACHT domain of NLRP3 (PDB: 7ALV); calculated binding energy, −8.6 kcal/mol. ### p < 0.001 versus Control; * p < 0.05, ** p < 0.01, *** p < 0.001 versus DSS. To confirm these findings, we established an inflammasome activation model in BMDMs using LPS priming followed by nigericin stimulation. Api exhibited no cytotoxicity up to 40 μM and dose-dependently inhibited IL-1β secretion ( Figures S3, C). In parallel, RT-qPCR showed that Api markedly decreased the mRNA levels of IL-1β, NLRP3, and GSDMD ( Figure D), supporting its inhibitory effect on inflammasome-related gene expression in vitro. To investigate whether Api directly interacts with NLRP3, we performed surface plasmon resonance (SPR) analysis, which confirmed a specific binding interaction between Api and recombinant NLRP3 with a dissociation constant of 18.7 μM ( Figure E). Molecular docking further revealed that Api docks into the NACHT domain of NLRP3 with a binding free energy of – 8.6 kcal/mol, forming a hydrogen bond with the Thr169 residue at a distance of 2.2 Å ( Figure F). These findings provide direct evidence that Api targets NLRP3 to inhibit its activation both in vivo and in vitro . In both colonic tissues and BMDMs, Api treatment not only suppressed NLRP3 and IL-1β expression but also significantly reduced TNF-α and IL-6 levels, indicating anti-inflammatory effects beyond NLRP3 inhibition ( Figures C, A). To further validate this, we combined Api with the NLRP3 inhibitor MCC950 in BMDMs. Notably, Api further enhanced IL-1β suppression even in the presence of MCC950 ( Figure B), supporting the notion that Api modulates additional upstream targets apart from NLRP3 to exert its anticolitis effects. Api activates AMPK signaling and attenuates NF-κB–mediated inflammatory responses. (A) ELISA quantification of TNF-α and IL-6 in BMDM supernatants after LPS + nigericin stimulation with or without Api. (B) IL-1β production in LPS-primed, nigericin-stimulated BMDMs treated with MCC950 (500 nM) or Api (10 μM), alone or in combination, as determined by ELISA. (C) Intracellular reactive-oxygen species in RAW264.7 macrophages detected with CellROX Green dye at the manufacturer-recommended concentration after LPS challenge and Api pretreatment (2.5–10 μM). Scale bar = 50 μm. (D) Western blot of P-AMPK and total AMPK in colonic tissue from Control, DSS, DSS + L-Api, and DSS + H-Api mice, with densitometric analysis. (E) Western blot analysis of NF-κB pathway proteins (P-IκBα, IκBα, P-NF-κB p65, NF-κB p65) in the same tissues; quantitative data shown on the right. Data are expressed as mean ± SD ( n = 3). ### p < 0.001 versus Control; * p < 0.05, ** p < 0.01, *** p < 0.001 versus DSS. Since ROS is crucial for NLRP3 inflammasome activation, we sought to determine whether LPS can lead to the generation of ROS and whether it is involved in the NLRP3 inflammasome. Based on CellROX Green staining, we found the ROS level was dramatically elevated following LPS stimulation, while Api substantially inhibited the accumulation of intracellular ROS in a dose-dependent manner ( Figures C, S4 ). Given that AMPK activation is known to protect against oxidative stress, we next investigated whether Api exerts regulatory effects on AMPK signaling. Western blot analysis demonstrated that Api treatment markedly increased the phosphorylation of AMPK in colonic tissues, as indicated by the elevated p-AMPK/AMPK ratio ( Figure D), suggesting Api activates AMPK in vivo . Since AMPK activation can inhibit NF-κB signaling, we subsequently assessed key proteins involved in this pathway. Api treatment significantly reduced the phosphorylation of NF-κB p65 and IκBα in colonic tissues, as reflected by decreased p-IκBα/IκBα and p-NF-κB p65/NF-κB p65 ratios ( Figure E). Collectively, these findings suggest that Api activates AMPK, thereby inhibiting NFκB signaling and subsequent NLRP3 inflammasome activation, ultimately alleviating oxidative stress and inflammation in UC. To verify the necessity of AMPK in Api’s protective effects, this study treated BMDMs with Api together with either the AMPK activator AICAR or the AMPK inhibitor BAY-3827. The results showed that, Api treatment markedly decreased the levels of TNF-α and IL-6, whereas cotreatment with BAY-3827 substantially reversed these inhibitory effects. Conversely, AICAR mimicked Api’s action, further suppressing TNF-α and IL-6 production ( Figure A). Moreover, knockdown of AMPKα1 via siRNA markedly attenuated Api’s inhibition of TNF-α in activated macrophages, confirming that AMPK activation is a key upstream mechanism for Api’s suppression of NF-kB-mediated inflammatory cytokines ( Figure C). Notably, Api continued to partially inhibit IL-1β secretion even in the presence of BAY-3827 ( Figure B), suggesting that Api’s blockade of IL-1β release is only partially AMPK-dependent. In support of this notion, Api still reduced NLRP3 expression following AMPKα1 knockdown in BMDMs ( Figure C). These results collectively indicate that Api suppresses inflammation through both AMPK-dependent and AMPK-independent pathways, the latter likely involving direct inhibition of the NLRP3 inflammasome. Api alleviates colitis by coordinately modulating AMPK and NLRP3. (A-B) ELISA quantification of TNF-α, IL-6 (A), and IL-1β (B) in BMDM supernatants following LPS + nigericin stimulation with or without Api, the AMPK agonist AICAR, or the AMPK inhibitor BAY-3827 ( n = 4). (C) Effect of AMPKα1 knockdown on the mRNA expression of Prkaa1 , TNF-α , and Nlrp3 in BMDMs subjected to LPS + nigericin stimulation with or without Api treatment ( n = 6). (D) SPR sensorgram confirming the direct interaction between Api and the recombinant AMPK α1/β2/γ1 complex. (E) Molecular-docking models depicting Api bound to multiple reported AMPK allosteric or activator-related pockets, including AMPKα1 (PDB: 6C9F), AMPKα1β1γ1 (PDB: 6C9F), AMPKα1β1γ1 (PDB: 4QFR), and AMPKα2β1γ1 (PDB: 4CFF). To explore how Api activates AMPK, we performed SPR and molecular docking analyses. SPR showed that Api binds directly to the AMPK α1/β2/γ1 complex (KD = 6.52 μM), indicating a specific physical interaction ( Figure D). Furthermore, the molecular docking showed that Api can stably fit into several known allosteric activator binding pockets (PDB: 4QFR, 6C9F, 4CFF), displaying favorable binding energies (approximately – 8.5 to – 8.7 kcal/mol), and that Api binds more favorably to AMPKα1 than AMPKα2 in these models ( Figure E).

Eight-week-old male C57BL/6 mice were purchased from the Beijing Weitong Lihua Experimental Animal Technology Co., Ltd. (Beijing, China). Animals were maintained in a specific pathogen-free facility under a 12 h light/12 h dark cycle with ad libitum access to standard chow and water. Experiments involving animals were carried out following the guidelines set by the Regulations for the Administration of Affairs Concerning Experimental Animals in China. Ethical clearance for the animal experiments was received from the Animal Ethics Committee at Tianjin University of Traditional Chinese Medicine with the approval number of TCM-LAEC2025247Z2096. After one week of acclimation, the mice were randomly allocated (six per group) to the following groups: (A) Control, (B) DSS, (C) DSS + 5-ASA (300 mg/kg), (D) DSS + low-dose Api (L-Api, 75 mg/kg), and (E) DSS + high-dose Api (H-Api, 150 mg/kg). Api (MedChemExpress), 5-ASA (MedChemExpress), and DSS (Yeasen Biotechnology, Shanghai, China) were prepared according to the respective manufacturers’ instructions. Experimental colitis was induced by supplying 2.5% (w/v) DSS in the drinking water for 7 days, as described previously. On day 8, the DSS solution was replaced with distilled water. During this period, the Control group received distilled water, whereas the remaining groups received the DSS solution; treatment groups were additionally gavaged once daily with the designated dose of Api or 5-ASA. Api and 5-ASA were prepared in 0.5% CMC-Na in distilled water, and the mixtures were homogenized by sonication before oral gavage. Mice in the Control and DSS groups were also gavaged daily with 0.5% CMC-Na. The gavage volume for each mouse was adjusted according to its body weight to ensure accurate mg/kg dosing. Body weight, stool consistency, and other clinical signs were monitored daily, and the disease activity index (DAI) score was recorded. On day 9, the mice were euthanized, colon length was measured, and colonic tissues were harvested for subsequent analyses. Distal colon segments were fixed in 4% paraformaldehyde for 24 h, dehydrated, embedded in paraffin, and sectioned for subsequent analyses. Depending on experimental requirements, tissue sections were processed for hematoxylin and eosin (H&E), periodic acid-Schiff (PAS), immunohistochemistry (IHC), immunofluorescence (IF), or TUNEL staining. H&E and PAS staining were performed using standard protocols to assess tissue morphology and mucus secretion. For IHC and IF, sections underwent antigen retrieval and were incubated with the primary antibodies MPO (Proteintech, Rosemont, IL, USA), ZO-1 (Proteintech), and Occludin (Abcam, Cambridge, UK) at the specific working dilutions described in Supplementary Table S3 . Apoptosis was detected by TUNEL staining using a commercial kit (cat. C1088; Beyotime Biotechnology, Shanghai, China) following the manufacturer’s instructions. All sections were dehydrated, coverslipped, and imaged on an inverted fluorescence microscope (Nikon, Japan) for imaging analysis. On day 7 of differentiation, BMDMs were seeded at 5 × 10 4 cells per well in 96-well plates. After 24 h attachment, the medium was replaced with medium containing only the specified concentrations of Api and incubated for 4 h. Cell viability was then determined using the CCK-8 kit (cat. CK04; Dojindo Laboratories, Japan), and absorbance was measured at 450 nm with a microplate reader (Thermo Fisher Scientific). Bone marrow-derived macrophages (BMDMs) were prepared as previously described. Briefly, bone marrow was harvested from the femurs and tibias of C57BL/6 mice and cultured in RPMI-1640 medium (C22400500BT, Gibco, USA) supplemented with 10% FBS and 20 ng/mL M-CSF (MedChemExpress) for 7 days to promote differentiation into macrophages. RAW264.7 cells were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA) and maintained in high-glucose DMEM (C11995500BT, Gibco, USA) supplemented with 10% fetal bovine serum (FBS; 10099141C, Gibco, USA). Cells were cultured at 37 °C in a humidified atmosphere containing 5% CO 2 . For NLRP3 inflammasome activation in BMDMs, cells were seeded at 1 × 10^6 cells/mL in 6-well plates. After 24 h, the medium was replaced with Opti-MEM containing 50 ng/mL LPS (L5293, Sigma-Aldrich, USA), and Api was added at the indicated concentrations for 3 h. Nigericin (MedChemExpress) was then added to a final concentration of 10 μM, and cells were incubated for 40 min to activate the NLRP3 inflammasome. To induce an in vitro inflammatory response, RAW264.7 cells were stimulated with 100 ng/mL LPS and treated with Api at 2.5, 5, or 10 μM for 24 h. Additionally, to further confirm that the anti-inflammatory effects of Api are mediated via the NLRP3 inflammasome pathway, during the LPS priming step, BMDMs were treated with 500 nM MCC950 (MedChemExpress), 10 μM Api, or a combination of both. To assess the involvement of AMPK signaling in BMDMs, cells were pretreated with the AMPK agonist AICAR (1 mM; MedChemExpress) or the AMPK inhibitor BAY-3827 (500 nM; MedChemExpress) for 2 h prior to LPS priming. Following pretreatment, cells were primed with 50 ng/mL LPS in Opti-MEM and then subjected to NLRP3 inflammasome activation exactly as described above. BMDMs were also transfected with AMPKα1-specific siRNA, and the siRNA sequences are listed in Supplementary Table S1 . The levels of IL-1β, IL-6, and TNF-α in mouse serum or cell culture supernatants were measured by ELISA, following the manufacturer’s instructions, using the following kits: Mouse IL-1β OneStep ELISA Kit (SOC3029, STARTER, China), Mouse IL-6 OneStep ELISA Kit (SOC3019, STARTER, China), and Mouse TNF-α OneStep ELISA Kit (SOC3023, STARTER, China). Intracellular ROS levels in RAW264.7 cells were assessed using CellROX Green reagent (cat. C10444 ; Invitrogen, USA) at the concentration recommended by the manufacturer following treatment. After incubation, cells were gently washed three times with PBS and stained with NucBlue Live ReadyProbes nuclear stain (Hoechst 33342; cat. R37605 ; Invitrogen, USA) in the dark for 5 min at 37 °C in a 5% CO 2 incubator. Fluorescent signals were then visualized and imaged using an inverted fluorescence microscope (Thermo Fisher Scientific). Malondialdehyde (MDA; cat. BC0025), glutathione (GSH; cat. BC1175), and total antioxidant capacity (T-AOC; cat. BC1315) levels in mouse colon tissues were measured using Solarbio assay kits (Solarbio, China) according to the manufacturer’s instructions. Standard curves were generated, optical densities were measured at the specified wavelengths, and concentrations were calculated from these curves. All assays were performed in triplicate. To elucidate the molecular mechanisms by which Api ameliorates ulcerative colitis, transcriptomic profiling was performed on five biological replicates per group from the Control, DSS, and DSS+H-Api groups. Total RNA extraction, library construction, and sequencing were carried out by Shanghai Ouyi Biomedical Technology Co., Ltd. RNA purity and integrity were assessed using a NanoDrop 2000 spectrophotometer (Thermo Scientific, USA) and an Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA), respectively. Sequencing libraries were prepared with the VAHTS Universal V10 RNA-seq Library Prep Kit (Premixed Version) according to the manufacturer’s protocol, and paired-end sequencing was performed on an Illumina NovaSeq 6000 platform. Raw sequencing data were subjected to quality control and adapter trimming using fastp, and the resulting clean reads were aligned to the reference genome using HISAT2. Principal component analysis (PCA) of the gene expression count matrix was conducted in R (v3.2.0) to assess biological reproducibility among samples. Differentially expressed genes were identified using a threshold of |log 2 FC| > 1 and P < 0.05. GO and KEGG enrichment analyses of the identified genes were subsequently conducted in R (v3.2.0) based on a hypergeometric distribution algorithm. To evaluate the interactions between Api and AMPK and NLRP3, molecular docking was performed. The crystal structures of AMPK (PDB ID: 4QFR, 6C9F, 4CFF) and NLRP3 (PDB ID: 7ALV) were downloaded from the RCSB Protein Data Bank, and the structure of Api in SDF format was obtained from PubChem. Protein and ligand files were prepared using AutoDock Tools, and potential binding sites were identified based on the protein structures. Docking was carried out with AutoDock Vina (v1.1.2) using a binding affinity threshold of −5.0 kcal/mol (lower values indicate more stable binding). The top-scoring conformations were visualized in PyMOL (v3.1.0). SPR measurements were performed on a Biacore 8K instrument (GE Healthcare, Piscataway, NJ, USA). Commercially available recombinant human NLRP3 protein (CSB-EP822275HU7) and AMPK α1/β2/γ1 heterotrimer protein (HY- P76143 , MCE) were immobilized (∼3,000 RU) on a CM5 Chip (GE Healthcare, Piscataway, NJ, USA) according to a standard amine coupling procedure. Following immobilization, Api was serially diluted in running buffer to appropriate concentration ranges based on the anticipated affinity of each target. For NLRP3, a nine-point concentration series (500 μM, 166.67 μM, 55.56 μM, 18.52 μM, 6.17 μM, 2.06 μM, 0.69 μM, 0.23 μM, and 0 μM) was used, while a seven-point series (25 μM, 12.5 μM, 6.25 μM, 3.13 μM, 1.56 μM, 0.78 μM, and 0 μM) was applied for AMPK, with the flow rate of 65 μL/min. The injection time was 120 s and the dissociation time was 90 s. The final graphs were obtained by subtracting blank sensorgrams. Experimental data were collected with the Biacore 8K manager software and were analyzed by fitting to an appropriate binding model to obtain the equilibrium dissociation constant (KD). Total RNA was extracted from mouse colonic tissues and cells using TRIzol Reagent (Invitrogen, USA) following the manufacturer’s instructions. RNA purity and concentration were determined with a NanoDrop 2000 (Thermo Fisher Scientific, USA). First-strand cDNA was synthesized using TransScript All-in-One First-Strand cDNA Synthesis SuperMix for qPCR (AT341–02, TransGen Biotech, China). qPCR was performed with PerfectStart Green qPCR SuperMix (AQ601–04-V2; TransGen Biotech, China) on a LightCycler 480 II (Roche, Switzerland). β-actin served as the internal reference gene, and relative mRNA expression levels were calculated using the 2 –  ΔΔCt method. Primer sequences are listed in Table S2 . Total protein was extracted from mouse colonic tissues and cells using cell lysis buffer containing protease and phosphatase inhibitors (#9806; Cell Signaling Technology [CST], Danvers, MA, USA). Samples were incubated on ice with intermittent vortexing for 10 min, then centrifuged at 12,000 × g for 15 min at 4 °C. Protein concentration was measured by BCA assay (#7780, CST). Equal masses of protein were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a nitrocellulose membrane (HATF00010; Millipore, Burlington, MA, USA). Membranes were blocked in 5% nonfat milk (or 5% BSA for phosphorylated proteins) and incubated overnight at 4 °C with primary antibodies. After three washes in TBST, membranes were incubated with HRP-conjugated secondary antibodies at room temperature for 2 h. Protein bands were visualized using ECL reagent (WBKLS0500; Millipore) and imaged on a WD-9423C chemiluminescence system (Liuyi Instrument, China). Band intensities were quantified with ImageJ software. All primary antibodies were diluted according to the manufacturer’s recommended dilution ratios. Detailed information of antibodies used in this study is listed in Supplementary Table S3 . All experiments were performed at least three times independently, and data are presented as the mean ± standard deviation (SD). Statistical analyses were conducted using GraphPad Prism 8.0 (GraphPad Software, La Jolla, CA, USA). Comparisons between two groups were made with two-tailed Student’s t -tests, and multiple group comparisons were performed by one-way analysis of variance (ANOVA) followed by Tukey’s post hoc test. Values of p < 0.05 were considered statistically significant.

This study systematically demonstrates that the edible natural compound apigenin (Api) exerts multidimensional health-promoting effects against DSS-induced ulcerative colitis (UC). Api markedly alleviates classical UC symptoms, including weight loss, colon shortening, and elevated disease activity index scores, and simultaneously mitigates mucosal damage, inflammation, and oxidative stress. Mechanistically, Api acts through a dual-layered regulatory mechanism. It directly binds to and suppresses the proinflammatory NLRP3 inflammasome, thereby interrupting the activation of downstream inflammatory cascades. In parallel, it activates AMPK, a central regulator of metabolic and redox homeostasis, which further attenuates NF-κB–driven inflammatory signaling. This coordinated modulation of the NLRP3 inflammasome and AMPK pathway underlies the functional benefits of Api and highlights its potential as a safe, multitarget bioactive dietary ingredient. Among the multiple factors contributing to UC, dysregulated immune and redox signaling converge on aberrant activation of the NLRP3 inflammasome, a critical driver of intestinal inflammation. , Genetic ablation of NLRP3 or caspase-1 markedly alleviates DSS-induced colitis in mice, underscoring the pathogenic role of inflammasome-mediated IL-1β maturation. , Here, we report for the first time that Api, a dietary flavonoid with established anti-inflammatory effects, acts as a direct NLRP3 inflammasome inhibitor. SPR analysis confirmed a specific binding affinity (KD ≈ 18.7 μM) between Api and recombinant NLRP3. Functionally, Api substantially suppressed canonical markers of NLRP3 activation in both DSS-induced colonic tissues and LPS-primed macrophages, concomitant with decreased IL-1β secretion upon diverse NLRP3 stimuli. Notably, this inhibitory effect was independent of upstream modulators such as CD38 blockade, demonstrating that Api directly targets the NLRP3 inflammasome complex. Distinct from previous studies emphasizing Api’s regulation of gut microbiota or mast cell-mediated responses, our findings uncover macrophage-associated NLRP3 suppression as a central and novel mechanism underlying Api’s beneficial effects in UC. While specific NLRP3 inhibitors such as MCC950 selectively suppress IL-1β maturation without broadly affecting cytokine networks, Api exhibited a markedly wider anti-inflammatory spectrum. In addition to inhibiting IL-1β production, Api significantly reduced the expression of TNF-α and IL-6 at both the transcriptional and protein levels, suggesting the involvement of an upstream regulatory mechanism beyond NLRP3 inhibition. Considering the central role of oxidative stress and energy imbalance in UC pathology, we next investigated the AMPK pathway, a master regulator of cellular metabolism and redox homeostasis. Our data demonstrate for the first time that Api acts as a direct AMPK activator in the context of colitis. Pharmacological modulation experiments showed that the inhibitory effects of Api on TNF-α and IL-6 were reversed by the AMPK inhibitor BAY-3827 and recapitulated by the AMPK agonist AICAR, establishing a causal relationship between Api’s anti-inflammatory effects and AMPK activation. Consistently, Api treatment enhanced AMPK phosphorylation in DSS-induced colonic tissues and directly bound to the AMPK α1/β2/γ1 heterotrimer with measurable affinity (KD ≈ 6.52 μM), as confirmed by SPR analysis. Molecular docking further revealed that Api fits into the allosteric binding pocket of AMPK, preferentially interacting with the α1 isoformthe predominant catalytic subunit in macrophagessuggesting an allosteric activation mode similar to that of classical activators such as A-769662. We therefore propose that Api stabilizes the phosphorylated conformation of AMPK, thereby maintaining its active state and amplifying its downstream anti-inflammatory signaling. Collectively, these findings identify AMPK as a second and functionally synergistic target of Api, mediating metabolic and redox reprogramming that complements its direct inhibition of the NLRP3 inflammasome. In this study, we demonstrated that Api effectively mitigates intestinal inflammation and oxidative stress by coordinately regulating metabolic and immune pathways. Activated AMPK is widely recognized as a key negative regulator of NF-κB signaling and oxidative stress. , Consistent with this, Api-induced AMPK activation was accompanied by marked suppression of p65 and IκBα phosphorylation, decreased oxidative stress markers (ROS, MDA), and enhanced antioxidant capacity (GSH, T-AOC). The NLRP3 inflammasome is activated through a well-characterized two-signal model, in which NF-κB–mediated transcription provides the priming signal, and oxidative stress serves as the activation trigger. Our findings indicate that Api directly inhibits the priming step via AMPK-mediated NF-κB suppression and may also attenuate the activation step through its antioxidant effects. In addition, Api directly interacts with NLRP3 to inhibit its activation. Collectively, these results support a dual and synergistic mechanism in which Api targets both AMPK and NLRP3, forming a self-reinforcing metabolic–immune regulatory loop that underlies its potent functional benefits in UC. Despite the promising functional food potential of Api revealed in this study, several important research gaps remain. Emerging evidence suggests that the crosstalk between AMPK signaling and NLRP3 inflammasome activation may extend beyond classical inflammatory regulation. Although interactions between AMPK and NLRP3 have been reported in the context of aging and metabolic disorders, their coordinated roles in intestinal inflammation remain poorly defined. , In this regard, AMPK–NLRP3 signaling may converge on the regulation of cell death–associated processes, which are increasingly recognized as critical determinants of epithelial integrity and inflammatory amplification in ulcerative colitis. Further investigation of this regulatory network may provide deeper insight into the synergistic protective effects of Api. While our data identify NLRP3 inflammasome and AMPK as key targets underlying Api’s anti-inflammatory and antioxidant effects, the precise mode of AMPK activation remains to be fully elucidated. AMPK is classically phosphorylated at Thr172 by upstream kinases such as LKB1 or CaMKKβ, and previous studies in keratinocytes suggest that Api-induced activation may depend on CaMKKβ. , It will therefore be important to determine whether Api activates AMPK in colonic macrophages primarily through CaMKKβ-dependent phosphorylation or through direct allosteric stabilization. Moreover, once activated, it remains unclear whether AMPK can directly modulate NLRP3 inflammasome activation in this context. Addressing these questions represents a meaningful direction for future investigation. In addition to these mechanistic uncertainties, translating Api into a functional dietary component faces several challenges. Like many dietary flavonoids, Api exhibits modest bioavailability, rapid metabolism, and low water solubility, with considerable interindividual variability in local colonic exposure. , In this study, the in vitro concentrations of Api were selected based on CCK-8 assays to ensure safety and efficacy, while the in vivo doses were determined within the reported safe oral range and validated by preliminary experiments for effectiveness. Consequently, developing targeted delivery strategies, such as hyaluronic acid–modified nanoparticles or phosphate-based formulations, may be necessary to enhance stability, mucosal retention, and functional efficacy. , Furthermore, the acute DSS model employed in this study primarily reflects epithelial injury and innate immune activation, and does not fully capture the chronic, relapsing, and adaptive immune dysregulation observed in human UC. Future studies using more physiologically relevant animal models will be essential to validate the effects of Api as a functional dietary component. Finally, given the critical role of the gut microbiota in intestinal health, it will be valuable to explore whether Api’s beneficial effects involve modulation of the microbiota–metabolite–immune axis, potentially acting synergistically with its direct cellular targets.

Ulcerative colitis (UC) is a chronic, nonspecific intestinal inflammatory disorder of the intestine characterized by disruption of the intestinal mucosal barrier, dysbiosis of the gut microbiota, and excessive immune responses. As of 2024, UC affects more than 5 million people globally, with the global incidence rate continuing to rise. Beyond typical intestinal pathologies, UC patients often manifest extraintestinal manifestations such as primary sclerosing cholangitis and arthritis, along with increased risks of colorectal cancer and coronary heart diseases, leading to substantial medical and economic burdens worldwide. Current therapeutic options for UC mainly consist of 5-aminosalicylic acids (5-ASA), corticosteroids, immunosuppressants, biologics, and small-molecule inhibitors. Although these agents are effective in inducing and maintaining remission, their long-term remission rates are only 30–60% due to adverse effects and loss of responsiveness, highlighting the urgent need to explore additional effective complementary therapeutic strategies. Although the etiology and pathogenesis of UC remain unclear, mounting evidence suggests that inflammation and oxidative stress act in concert to drive disease progression. A bidirectional feedback loop exists, wherein oxidative stress and inflammatory signaling pathways mutually reinforce one another. On the one hand, abnormal immune reactions and disordered gut microbiota in UC patients lead to the secretion of multiple inflammatory factors, such as lipopolysaccharide (LPS) and tumor necrosis factor-alpha (TNF-α), which activate the inhibitor of κB (IκB) kinase (IKK) complex, triggering phosphorylation and ubiquitination of IκB proteins. These modified IκB proteins are ultimately degraded by the proteasome to release nuclear factor-kappaB (NF-κB) dimers (such as p50/RelA) and activate the NF-κB signaling pathway. NF-κB activation also provides a priming signal for the assembly of the NOD-like receptor family pyrin domain containing 3 (NLRP3) inflammasome, further amplifying inflammation and tissue injury. , On the other hand, reactive oxygen species (ROS) levels are generally elevated in the colitis tissues, contributing to damage to the intestinal mucosa. Studies are increasingly showing that the activation of AMPK can not only decrease ROS-caused oxidative stress but also inhibit NF-κB and NLRP3-mediated inflammation. The crosstalk between these signaling pathways underscores the substantial involvement of inflammation and oxidative stress in UC development. Recent studies have drawn attention to the therapeutic potential of natural edible products and their bioactive compounds in regulating intestinal inflammation and oxidative injury. For example, Houttuyniae Herba dramatically alleviated colitis indicators via bolstering the proliferation of Bacteroides thetaiotaomicron . Ginsenoside Rb1 successfully attenuates UC symptoms by inhibiting intestinal inflammation and maintaining the intestinal barrier’s integrity via the vitamin D receptor (VDR), peroxisome proliferator-activated receptor gamma (PPARγ), and NF-κB signaling pathways. Apigenin (Api), a naturally occurring flavonoid abundant in a wide range of fruits and vegetables, such as Petroselinum crispum Mill. (Curly-leaved parsley), chamomile and celery. Numerous studies have reported that Api exhibits anticancer, antioxidant, anti-inflammatory, antidiabetic, cardioprotective and neuroprotective activities. With its bioactive features, Api holds promise for UC management. In DSS-induced UC mice, Api markedly alleviates colon injury via remodeling the composition of gut microbiota and promoting its beneficial metabolites short-chain fatty acids (SCFAs). Additionally, Radulovic et al. found that Api regulates an inflammasome-independent pathway involving NLRP6, which reprograms the gut microbiota to protect mice from colitis. Api can also bind to IRAK4 to interfere extracellular signaling of NF-κB and MAPK pathways, thus reducing pro-inflammatory factors in LPS-induced acute inflammation and DSS-induced UC mouse models. However, most existing studies have predominantly focused on systemic inflammatory diseases or cancer-related contexts, while its specific roles and underlying mechanisms in intestinal inflammation, particularly ulcerative colitis, remain insufficiently explored. Moreover, the molecular pathways through which Api integrates metabolic regulation and inflammatory signaling in the intestinal microenvironment are not yet fully clarified. These limitations highlight the need for further mechanistic investigation to better define the potential of Api in the prevention and intervention of intestinal inflammatory disorders. In this study, a dextran sulfate sodium (DSS)-induced acute colitis mouse model was established to evaluate the therapeutic effects of Api and to explore its underlying mechanisms in modulating inflammation and oxidative stress via transcriptomic analysis. A range of pathological and molecular indicators were employed to comprehensively assess the anticolitic effects of Api. This work provides complementary evidence that Api protects against colitis through dual regulation of inflammatory and oxidative pathways. A deeper understanding of these mechanisms may offer new insights into the biological activities of Api and its potential as a functional dietary compound in UC management.