Gasdermin D(N terminal) Recombinant Rabbit Monoclonal Antibody [0009-1-9]

Background Ulcerative colitis (UC) arises from complex crosstalk between gut microbiota, epithelial barrier integrity, and inflammatory cell death, yet causal mediators along this axis remain poorly defined. We aimed to delineate microbiota–pyroptosis–UC pathways and functionally validate key effectors, with a focus on KLF4. Methods A multistage framework integrating genome-wide association studies of gut microbiota (MiBioGen), plasma proteomics (deCODE), and UC (UK Biobank) was constructed to perform two-sample Mendelian randomization (MR). Pyroptosis-related proteins were screened for causal associations with UC, followed by MR of UC-associated microbial taxa on these proteins and two-step mediation analysis. KLF4 was further evaluated using bulk and single-cell transcriptomic datasets, including virtual knockout network perturbation. Its clinical relevance was tested in two cohorts of UC patients receiving anti-TNF-α therapy. Finally, the KLF4 function was validated in a dextran sulfate sodium (DSS)-induced colitis model with systemic AAV9-mediated KLF4 overexpression. Results MR identified 35 pyroptosis-related plasma proteins and 23 microbial taxa with putative causal effects on UC. Mediation analysis highlighted MAPK11, PTEN, and KLF4 as dominant intermediates linking specific taxa to UC risk. KLF4 was consistently downregulated in UC, and low KLF4 expression was associated with enrichment of pro-inflammatory and immune-activation signatures. Higher mucosal KLF4 levels predicted response to anti-TNF-α therapy with moderate discriminatory performance. In DSS-induced colitis, KLF4 overexpression mitigated weight loss and disease activity, preserved colon length, improved histology, and reduced myeloperoxidase activity. KLF4 restored Claudin-1, Occludin, and Zo-1; suppressed Claudin-2; decreased intestinal permeability; limited gasdermin-D (GSDMD) cleavage; lowered IL-1β/IL-18 levels; and reshaped splenic leukocyte composition. Conclusions Our integrative genetic and experimental data position KLF4 as a central node in a gut microbiota–pyroptosis–barrier axis in UC, supporting KLF4 as a promising biomarker and therapeutic target for the precision management of UC.

BackgroundCrohn’s disease (CD) is characterized by persistent intestinal inflammation, immune dysregulation, and intestinal barrier dysfunction. Inflammasome-mediated pyroptosis is an innate immune mechanism increasingly implicated in inflammatory bowel disease (IBD); however, the upstream molecular signals associated with NLRP3–Caspase-1–GSDMD activation in CD remain insufficiently defined. Here, we explored whether the angiopoietin-1 (ANGPT1)–gamma-aminobutyric acid receptor-associated protein (GABARAP) axis is associated with CD-related pyroptotic signaling.MethodsProtein quantitative trait locus (pQTL)-based two-sample Mendelian randomization (MR) was performed to prioritize pyroptosis-related proteins genetically associated with CD risk. Putative upstream regulators of GABARAP were then examined by two-step MR and mediation analysis. Functional validation was performed using a dextran sulfate sodium (DSS)-induced murine colitis model and LPS plus nigericin-induced cell models of NLRP3 inflammasome activation. ANGPT1–GABARAP signaling and the NLRP3–Caspase-1–GSDMD pathway were evaluated following GABARAP or ANGPT1 knockdown and exogenous recombinant human ANGPT1 (rhANGPT1) supplementation, by qRT-PCR, western blotting, ELISA, and LDH release assays.ResultsMR analysis prioritized GABARAP as a suggestive protective candidate for CD, with genetically predicted higher GABARAP levels associated with a decreased disease risk (OR = 0.563, 95% CI 0.327–0.968, P = 0.038). Two-step MR further suggested a putative genetic association between ANGPT1 and GABARAP, and mediation analysis indicated that GABARAP may partially mediate the genetically predicted ANGPT1–CD association, with an estimated mediation proportion of 22.97%. DSS-induced colitis was associated with reduced ANGPT1 and GABARAP expression, along with increased NLRP3 expression, Caspase-1 processing, GSDMD-N accumulation, and elevated IL-1β, IL-18, and LDH levels. In vitro, silencing either GABARAP or ANGPT1 enhanced NLRP3 inflammasome-associated pyroptotic signaling under LPS plus nigericin stimulation, whereas rhANGPT1 treatment partially attenuated these responses in association with restored GABARAP expression.ConclusionThese findings support a potential role for the ANGPT1–GABARAP axis in NLRP3 inflammasome-mediated pyroptosis associated with intestinal inflammation. Together, these results provide a genetically informed framework for understanding pyroptosis-related inflammatory signaling in CD and support further investigation of the potential therapeutic relevance of this axis.

Hyperlipidemic pancreatitis (HP) is an inflammatory injury of the pancreas triggered by elevated serum triglyceride (TG) levels. The mechanistic target of rapamycin (mTOR) signaling pathway plays a crucial role in regulating lipid homeostasis and inflammation. This study aimed to investigate whether the activity of mTOR complex 2 (mTORC2) affects the progression of HP and its underlying mechanisms. In vivo, a high-fat diet and retrograde administration of sodium taurocholate were employed to establish the HP models in rats, with pancreatic tissue pathology evaluated. The expression of Rictor and peroxisome proliferator-activator receptor (PPAR) was examined. The serum levels of TG, fatty acid metabolites, inflammatory and lipid metabolism-related factors were determined. In vitro, pancreatic acinar cells (PACs) were exposed to palmitic acid and cholecystokinin-8. PAC apoptosis, pyroptosis, and ferroptosis were assessed. In the HP models, rats and PACs exhibited upregulated Rictor and downregulated PPARα, and Rictor knockdown promoted PPARα expression. In vivo, Rictor knockdown decreased the serum levels of TG, α-amylase, total cholesterol, low-density lipoprotein cholesterol, lactate dehydrogenase, and inflammatory factors, while increasing high-density lipoprotein cholesterol levels. Rictor knockdown increased ACOX1 and CPT1α and decreased SREBP-1, CD36, SCD1, ACLY, and ACACA. Rictor knockdown reduced damage to pancreatic tissue structure. In vitro, Rictor knockdown inhibited PAC apoptosis, pyroptosis, and ferroptosis. Treatment with the PPARα antagonist GW6471 abolished the beneficial effects of Rictor knockdown. Rictor/mTORC2 deficiency reduces serum TG levels, maintains lipid homeostasis, and suppresses inflammation by inhibiting PPARα expression. Weakening mTORC2 activity holds promise as a novel therapeutic strategy for HP.

Berberine (BBR), a widely recognized traditional Chinese medicine, has attracted considerable attention for its promising anti-inflammatory effects. The activation of nuclear factor erythroid 2-related factor 2 (Nrf2) effectively safeguards against organ damage stemming from sepsis-induced oxidative stress and inflammatory responses. This study examined the potential of BBR in alleviating sepsis-induced acute gastric injury, with a particular focus on elucidating whether its mechanism of action involves the activation of the Nrf2 signaling pathway. Following intraperitoneal injection of BBR, mice were subjected to the cecal ligation and puncture (CLP) method to induce sepsis. In vitro experiments involved pre-treating the normal gastric epithelial cells (GES-1) with BBR, followed by treatment with lipopolysaccharide (LPS). Functional assays were then performed to assess cell proliferation and apoptosis. To validate the role of Nrf2 in pyroptosis and inflammation, siRNA targeting Nrf2 (si-Nrf2) was transfected into LPS-treated GES-1 cells. Additionally, mice were administered the Nrf2 inhibitor ML385 to confirm the protective effects of BBR in vivo. BBR displayed a dose-dependent effect in mitigating gastric tissue damage, suppressing the release of inflammatory cytokines, and reducing the expression of NLRP3, ASC, and GSDMD-N. In vitro, BBR fostered GES-1 cell proliferation, hindered apoptosis, and suppressed the levels of TNF-α, IL-18, IL-1β, NLRP3, ASC, and GSDMD-N. Further analysis revealed that knocking down Nrf2 reversed BBR's inhibitory effect on pyroptosis in LPS-treated GES-1 cells. Through binding to Keap1, BBR efficiently prevented the ubiquitination and degradation of Nrf2, ultimately promoting its nuclear translocation. In vivo experiments confirmed that ML385 reversed the protective effect of BBR on pyroptosis and inflammation. Our research reveals that BBR interacts with Keap1 to activate the Keap1/Nrf2 signaling pathway in gastric epithelial cells, thereby suppressing pyroptosis and inflammation in sepsis-induced acute gastric injury.

BackgroundCisplatin is an effective anti-cancer drug with limited clinical applications due to ototoxicity. Resveratrol, known for its antioxidant and anti-inflammatory properties, has been reported to mitigate these adverse effects, although the underlying mechanism remains under-researched.ObjectiveThis study aimed to investigate the effects and underlying mechanisms of resveratrol on cisplatin-induced ototoxicity.MethodsOtotoxicity was modeled in House Ear Institute-Organ of Corti 1 (HEI-OC1) cells by cisplatin exposure, followed by interventions using thioredoxin-interacting protein (TXNIP) siRNA transfection, MitoQ, or resveratrol. Apoptosis and proliferation were quantitatively assessed using terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick-end labeling (TUNEL) and Ki67 immunostaining. Quantitative real-time PCR (qRT-PCR) and western blotting were used to measure the changes in mRNA and protein levels. Flow cytometry and enzyme-linked immunosorbent assay (ELISA) were used to analyze pyroptotic cells and inflammatory responses. Reactive oxygen species (ROS) production was tracked using 2', 7'-dichlorofluorescein diacetate (DCFH-DA) staining and flow cytometry. Mitochondrial Membrane Potential (MMP) and mitochondrial permeability transition pore (MPTP) opening levels were analyzed through tetramethylrhodamine ethyl ester (TMRE) staining and specific reagent kits, respectively. Lastly, immunofluorescence staining and co-immunoprecipitation were employed to investigate the co-localization and interactions between TXNIP and thioredoxin (TRX)/NOD-like receptor family pyrin domain-containing 3 (NLRP3) proteins.ResultsCisplatin exacerbated apoptosis, suppressed cell proliferation, and upregulated NLRP3, pro-Caspase-1, cleaved Caspase-1, Gasdermin D (GSDMD), GSDMD-N, and TXNIP expression. Concurrently, cisplatin resulted in increased pyroptotic cells and increased interleukin-6 (IL-6), IL-18, IL-1β, and tumor necrosis factor-α (TNF-α) levels. These effects were mitigated by TXNIP knockdown. Furthermore, cisplatin led to elevated cellular ROS and mitochondrial ROS (mtROS), decreased MMP, and inhibited MPTP opening. Cisplatin reduced the co-localization and interaction between TRX and TXNIP while enhancing those between TXNIP and NLRP3. These changes were attenuated by MitoQ. Resveratrol displayed effects similar to those of TXNIP knockdown and MitoQ treatment.ConclusionResveratrol alleviated the toxic effects of cisplatin on cochlear hair cells by inhibiting cell pyroptosis process mediated by the mtROS/TXNIP/NLRP3 pathway.