A two-hit model for NLRP3-mediated pyroptosis in human bladder epithelial cells: Hyperglycemia primes, infection triggers

A two-hit model for NLRP3-mediated pyroptosis in human bladder epithelial cells: Hyperglycemia primes, infection triggers | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article A two-hit model for NLRP3-mediated pyroptosis in human bladder epithelial cells: Hyperglycemia primes, infection triggers Maidina Aisihaer, Asimujiang Abula, Yueju Gu, Guanglu Song This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8934076/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Diabetic bladder dysfunction (DBD) affects over half of all diabetics, although its pathogenesis remains unexplained. Animal studies have connected NLRP3 inflammasome-mediated pyroptosis to DBD. It is uncertain whether hyperglycemia directly activates this pathway in human bladder epithelial cells. Immunohistochemistry was used to identify NLRP3 and GSDMD expression in bladder cancer tissues and adjacent normal epithelial tissues from six non-diabetic patients with bladder cancer. Human immortalized bladder epithelial cells (SV-HUC-1) were treated with high glucose (30 mM) or LPS (1 µg/mL) for 24–48 hours. Cell survival, pyroptosis, motility, and expression of NLRP3/Caspase-1/IL-1β were evaluated. In non-diabetic bladder cancer patients, there was no significant difference in NLRP3 or GSDMD expression between the malignant and normal tissues (P > 0.05). Both high glucose and LPS significantly enhanced the expression of NLRP3, Caspase-1, and IL-1β proteins in SV-HUC-1 cells (approximately 1.7- to 2.1-fold increase, all P < 0.001). High glucose by itself showed no discernible effects on these functional characteristics, while LPS treatment alone significantly decreased cell viability (70.8% vs control, P < 0.05), increased pyroptosis rate (9.6% vs 2.7%, P < 0.05), and impeded migratory capability (73.3 vs 165.0, P < 0.05). This study offers the first proof that elevated glucose levels in human bladder epithelial cells are sufficient to trigger the NLRP3 inflammasome pathway but not enough to cause pyroptosis. A crucial "second hit" that converts pathway activation into functional cellular damage is LPS. These findings support a "two-hit" model for DBD etiology and provide fresh insights into early intervention options that target the priming phase of NLRP3 activation. Biological sciences/Cell biology Health sciences/Diseases Health sciences/Endocrinology Health sciences/Urology Diabetic bladder dysfunction NLRP3 inflammasome Pyroptosis High glucose Lipopolysaccharide Figures Figure 1 Figure 2 Figure 3 Figure 4 Introduction Diabetes has become one of the most severe global public health challenges of the 21st century. According to the International Diabetes Federation's 10th edition of the Diabetes Atlas report, the global prevalence of diabetes among individuals aged 20 to 79 was estimated to be 10.5% (537 million people) in 2021. It is expected to grow to 12.2% (783 million people) by 2045 1 . Diabetes and its complications not only jeopardize individuals' quality of life, but they also place a significant burden on global healthcare systems 2 . Among the numerous complications of diabetes, diabetic bladder dysfunction (DBD) is one of the most common yet long-underestimated urinary system complications, affecting approximately 50% of diabetic patients 3 , 4 . DBD exhibits highly heterogeneous clinical manifestations, presenting as overactive bladder, underactive bladder, or mixed symptoms. Some patients ultimately progress to a decompensated state, requiring long-term dependence on intermittent catheterization for urination. Currently, there are no specific therapeutic agents available for DBD, and even strict glycemic control cannot completely prevent or reverse the progression of bladder dysfunction 4 . This strongly suggests that, beyond hyperglycemia itself, other critical pathological mechanisms remain poorly understood. In recent years, pyroptosis—a form of programmed inflammatory necrosis that is mediated by gasdermin family proteins—has gained prominence in the pathogenesis of diabetic complications 2 . The classical pyroptotic pathway initiates upon pattern recognition receptor (PRR) detection of pathogen-associated molecular patterns (PAMPs) or damage-associated molecular patterns (DAMPs). NOD-like receptor family pyrin domain-containing 3 (NLRP3) is the most extensively studied inflammasome sensor molecule and is the one most closely associated with metabolic diseases 4 , 5 . Upon sensing danger signals, NLRP3 undergoes oligomerization, recruiting apoptosis-associated speck-like protein containing a CARD (ASC) and pro-caspase-1 to form a functional inflammasome complex 6 . Notably, the NLRP3 inflammasome is activated in two steps: first, a priming signal (such as TLR ligands or cytokines) increases NLRP3 and pro-IL-1β expression through NF-κB, and then an activation signal (such as ATP, nigericin, or bacterial toxins) triggers inflammasome assembly and caspase-1 activation 7 . This process promotes the self-cleavage and activation of caspase-1. Activated caspase-1 cleaves pro-IL-1β and pro-IL-18 into mature pro-inflammatory cytokines while also cleaving gasdermin D (GSDMD), releasing its pore-forming N-terminal domain (GSDMD-NT) 8 . GSDMD-NT oligomerizes on the plasma membrane to form transmembrane pores, disrupting cellular osmotic homeostasis. This ultimately leads to cell swelling, membrane rupture, and massive release of proinflammatory contents, amplifying both local and systemic inflammatory responses 3 , 9 . In the field of DBD, the pioneering work by the Odom and Hughes teams laid the groundwork. They first showed that NLRP3 inflammasomes in urinary tract epithelial cells are markedly activated in diabetes situations using female type 1 diabetic Akita mouse models. In diabetic mice, the systemic deletion of the NLRP3 gene eliminated the development of bladder inflammation, disruption of the urinary epithelial barrier, and urinary dysfunction 3 , 4 . Subsequent studies further revealed that NLRP3-mediated inflammation contributes to diabetic bladder contraction dysfunction by upregulating prostaglandin F receptor (FP receptor) expression and abnormally regulating prostaglandin release 10 . This evidence from whole-animal models robustly establishes the central role of the NLRP3 inflammasome in DBD pathogenesis. However, it must be noted that the majority of the research that has been done on the DBD mechanism has only looked at the whole animal level. The observed chain of events—"hyperglycemia → NLRP3 activation → urinary epithelial pyroptosis → bladder dysfunction"—represents a terminal phenotype emerging within a complex context involving multiple overlapping factors, including neural, vascular, immune cell, hemodynamic, and systemic metabolic dysregulation. Can hyperglycemia, as a single factor, independently complete the entire process from "NLRP3 pathway activation" to "pyroptosis execution" in human bladder epithelial cells? This question remains unanswered to date. Our initial findings from human tissue samples provided a key starting point for addressing this issue. In tissue samples from non-diabetic bladder cancer patients, NLRP3 and GSDMD expression showed no significant difference between tumor regions and adjacent normal epithelium (P > 0.05). This finding suggests that, when excluding hyperglycemic interference, malignant transformation of bladder epithelium itself is not a sustained driver of this pathway. Conversely, the hyperglycemic metabolic environment may be the key upstream event inducing bladder epithelium into a pyroptosis "susceptibility state." Therefore, this study employed human immortalized bladder epithelial cells (SV-HUC-1), which are derived from benign human ureteral epithelial lining and immortalized with SV40, serving as an in vitro model of normal urinary tract epithelium 11 . Lipopolysaccharide (LPS) was used as a positive control. The aim was to systematically observe the effects of high glucose stimulation on cellular function and the NLRP3 inflammasome pathway. Both high glucose and LPS dramatically increase NLRP3/caspase-1/IL-1β expression, but with differing functional results. LPS causes pyroptosis and functional impairment, while high glucose has no significant effects. This finding indicates in human bladder epithelial cells that the NLRP3 pathway "upregulation" and "pyroptosis execution" are decoupled biological processes; high glucose only completes "priming" without supplying sufficient "activation" signals. Based on this, we propose a "two-hit" model for DBD pathogenesis: persistent hyperglycemia pre-activates the bladder epithelium's NLRP3; subsequent infection (e.g., LPS) serves as the "second hit," causing pyroptosis. This study provides a cellular mechanism that may explain diabetic patients' susceptibility to bladder infections and offers a theoretical basis for early management during the 'priming phase' of NLRP3 activation. We hypothesized that a second hit would be necessary to initiate pyroptosis execution, and hyperglycemia alone could be enough to stimulate but not fully activate the NLRP3 inflammasome in human bladder epithelial cells. Method Clinical Samples and Immunohistochemistry The Ethics Committee of The First Affiliated Hospital of Xinjiang Medical University approved this study involving human bladder tissue samples (Approval No. K202601-103). All procedures involving human participants were performed in accordance with the Declaration of Helsinki and relevant institutional guidelines and regulations. Written informed consent was obtained from all participants and/or their legal guardians prior to inclusion in the study. Six paraffin-embedded bladder cancer tissue specimens archived from 2019 to 2023 in the Department of Pathology, The First Affiliated Hospital of Xinjiang Medical University (bladder cancer group), and six matched adjacent normal tissue specimens (submucosal epithelial group) were collected. All patients were non-diabetic and had not received preoperative radiotherapy or chemotherapy. Tissue sections (4 µm thick) underwent routine dewaxing and rehydration, followed by antigen retrieval in citrate buffer (pH 6.0) via high-pressure autoclaving (heated until steam vented, then removed from heat and immersed for 10 min; cooled to room temperature naturally). Tissues were incubated with 3% H₂O₂ for 10 min to block endogenous peroxidase activity, followed by blocking with 10% normal goat serum at room temperature for 30 min. Rabbit anti-human NLRP3 monoclonal antibody (1:100, Thermo Fisher, MA5-32255) and mouse anti-human GSDMD monoclonal antibody (1:100, Santa Cruz, sc-393581) were applied separately and incubated overnight at 4°C. The following day, add horseradish peroxidase-labeled secondary antibody (Beijing Zhongshan Jinqiao, SP9000) and incubate at room temperature for 20 min. Subsequently, add horseradish peroxidase-labeled streptavidin working solution and incubate at room temperature for 20 min. Developed with DAB (Beijing Zhongshan Jinqiao, DAB-1031), controlling development time under the microscope; stopped with tap water. Counterstained with hematoxylin for 3 min, differentiated with hydrochloric acid in alcohol for several seconds, and counterstained with blue in tap water for 5 min. Dehydrated with graded alcohol, cleared with xylene, and mounted with neutral resin. For each batch, a negative control was prepared by replacing the primary antibody with PBS. Staining results were semi-quantitatively scored by two blinded pathologists using a double-blind method. Scoring criteria: The final immune response score was calculated as the product of staining intensity (0 = negative, 1 = weak positive, 2 = moderate positive, 3 = strong positive) and the percentage of positive cells (0 = < 5%, 1 = 5%–25%, 2 = 26%–50%, 3 = 51%–75%, 4 = ≥ 75%) (0–12 points). 2. Cell Culture and Processing The Human ureteral epithelial immortalized cell line SV-HUC-1 (Wuhan Shang'en Bio, Catalog No. SNL-233, ATCC). Cells were cultured in F-12K complete medium (containing 10% fetal bovine serum and 1% penicillin-streptomycin, Wuhan Shang'en Bio, SNLM-233) at 37°C in a CO₂ incubator maintained at 5% CO₂ saturation. Passaging or experimental procedures were performed when cells reached 80–90% confluence. The experiment was divided into three groups: the control group (standard medium with 5.5 mM glucose concentration), the high-glucose group (supplemented with D-glucose to a final concentration of 30 mM), and the LPS group (standard medium supplemented with 1 µg/mL LPS, Sigma-Aldrich, L4391). Treatment durations for each group were 48 hours (for cell proliferation, pyroptosis, and migration assays) or 24 hours (for Western blot analysis). 3. Cell Proliferation Assay Cell proliferation activity was assessed using the CCK-8 assay kit (Wuhan Shang'en Bio, SNK-010). Log-phase SV-HUC-1 cells were seeded at 5 × 10³ cells/well in a 96-well plate and cultured overnight before undergoing group-specific treatments for 48 hours. Add 10 µL of CCK-8 solution to each well and incubate for an additional 2 hours. Measure the absorbance at 450 nm using a microplate reader. Each experiment was independently repeated three times, with five replicate wells per group. Cell survival rate (%) = (OD value of treated group / OD value of control group) × 100%. 4. Pyroptosis Detection Pyroptosis was detected using the Annexin V-FITC/PI Pyroptosis Detection Kit (Wuhan Shang'en Bio, SNP-005). After 48 h of treatment, cells were collected by digestion with EDTA-free trypsin, washed twice with PBS, and resuspended in 1× binding buffer. Take 100 µL of cell suspension (approximately 1×10⁵ cells), add 5 µL Annexin V-FITC and 5 µL PI, incubate at room temperature in the dark for 15 min, then immediately add 400 µL binding buffer and analyze by flow cytometry (BD FACSCanto II). The experiment was independently repeated three times, with three replicate wells per group. The pyroptosis rate was expressed as the sum of early apoptosis (Annexin V⁺/PI⁻) and late pyroptosis (Annexin V⁺/PI⁺) cell percentages. 5. Cell Migration Assay (Scratch Assay) SV-HUC-1 cells were seeded at 2 × 10⁵ cells/well in a 6-well plate and cultured to confluence. A vertical scratch was made in the center of the monolayer in each well using a 200 µL sterile pipette tip. The plate was gently washed three times with PBS to remove detached cells. Add treatment medium containing 2% FBS (to eliminate proliferation interference). Observe and photograph under an inverted microscope (Nikon E200) at 0 h and 48 h. Measure the scratch area using ImageJ software and calculate the relative migration rate: Migration Rate (%) = (Initial Area − 48 h Area) / Initial Area × 100%. The experiment was independently repeated three times, with three replicate wells per group. 6. Western blot assay After 24 hours of treatment, discard the medium from each cell group, wash twice with PBS, add RIPA lysis buffer containing a protease inhibitor mixture (Wuhan Shang'en Bio, SNL-111), and incubate on ice for 30 min. Centrifuge at 12,000 rpm at 4°C for 15 min, then collect the supernatant. Protein concentration was determined using the BCA Protein Quantification Kit (Wuhan Shang'en Bio, SNB-001). Thirty µg of total protein was separated by 10% SDS-PAGE gel electrophoresis and transferred to a PVDF membrane (Millipore, IPVH00010). Blocked with 5% nonfat milk at room temperature for 1 hour. Primary antibodies added: Rabbit anti-NLRP3 monoclonal antibody (1:100, optimized in preliminary experiments, Thermo Fisher, MA5-32255); Rabbit anti-caspase-1 polyclonal antibody (1:100, optimized in preliminary experiments, Fuzhou Maxine, RAB-0303); Rabbit anti-IL-1β polyclonal antibody (using antibody provided in the Lianke Bio IL-1β ELISA kit, catalog no. EK301B/3–48, lot no. A301B91044), Mouse anti-β-actin monoclonal antibody (1:5000, Beijing Zhongshan Jinqiao, TA-09), and incubated overnight at 4°C. The next day, wash three times with TBST for 10 min each. Add HRP-labeled goat anti-rabbit/anti-mouse secondary antibody (1:5000, Beijing Zhongshan Jinqiao, SP9000) and incubate at room temperature for 1 h. After three TBST washes, develop with ECL chemiluminescent substrate (Millipore, WBKLS0500). Capture images using the ChemiDoc XRS⁺ imaging system (Bio-Rad). Analyze band grayscale values with ImageJ software, expressing relative expression as the ratio of target protein to β-actin grayscale values. Each experiment was independently repeated three times. 7. Statistical Analysis All data are expressed as mean ± standard deviation (SD). Statistical analysis was performed using SPSS 25.0 software. Independent samples t-tests were used for comparisons between two groups, one-way analysis of variance (ANOVA) for comparisons among multiple groups, and LSD post hoc tests for pairwise comparisons within groups. A P value < 0.05 was considered statistically significant. All cellular experiments were independently replicated three times (n = 3). Result Expression of NLRP3 and GSDMD in bladder tissues Immunohistochemistry was performed to mark these proteins in bladder cancer tissues and adjacent normal epithelial tissues (referred to as the normal group) to determine whether bladder cancer induces permanent activation of the NLRP3/GSDMD pyroptosis pathway. NLRP3 was predominantly localised in the cytoplasm of bladder epithelial cells, while GSDMD demonstrated cytoplasmic expression with occasional nuclear positivity (Figs. 1 A-D). Representative images from bladder cancer and adjacent normal tissues are shown in Fig. 1 A-D. Neither the muscle layer nor the epithelium layer showed no significant differences in IHC values between the two groups, according to semi-quantitative analysis (Table 1 ). In the muscle layer, NLRP3 scores were 8.33 ± 2.25 in the bladder cancer group vs. 7.83 ± 0.98 in the adjacent normal epithelial tissues ( P = 0.629), whereas GSDMD ratings were 10.00 ± 1.55 vs. 10.50 ± 1.64 ( P = 0.599). In the epithelial layer, NLRP3 scores were 8.33 ± 2.25 vs. 9.33 ± 1.37 ( P = 0.374), while GSDMD scores were 10.00 ± 1.55 vs. 10.50 ± 1.64 ( P = 0.599) (Figs. 1 E-F). These findings indicate that, under normoglycemic settings, malignant bladder epithelial transition is not associated with significant activation of the NLRP3/GSDMD pyroptosis pathway. Table 1 Comparison of immunohistochemical scores of NLRP3 and GSDMD proteins in bladder tissues between the two groups (Mean ± SD, n = 6) Tissue Layer Group NLRP3 GSDMD Muscle layer Bladder cancer group 8.33 ± 2.25 10.00 ± 1.55 Muscularis propria group 7.83 ± 0.98 10.50 ± 1.64 t value *0.499* *0.542* P value *0.629* *0.599* Epithelial layer Bladder cancer group 8.33 ± 2.25 10.00 ± 1.55 Muscularis propria group 9.33 ± 1.37 10.50 ± 1.64 t value *0.930* *0.542* P value *0.374* *0.599* Note : Data are presented as mean ± SD (n = 6 per group). Immunohistochemical staining was semi-quantitatively scored as the product of staining intensity (0–3) and the percentage of positive cells (0–4), ranging from 0 to 12. Statistical analysis was performed by unpaired t-test. P < 0.05 was considered statistically significant. No significant differences were observed between the bladder cancer group and the muscularis propria group in either the muscle layer or the epithelial layer for both NLRP3 and GSDMD expression. 2. Effects of high glucose and LPS on SV-HUC-1 cell functions We further utilized human immortalized bladder epithelial cells (SV-HUC-1) to observe the effects of high glucose and LPS stimulation on cellular function (Table 2 ). CCK-8 assay results showed that compared with the control group, cell viability in the high-glucose group (95.679 ± 5.395%) did not change significantly ( P > 0.05). In contrast, cell viability in the LPS group (70.828 ± 6.078%) was significantly lower than that in the control and high-glucose groups ( P < 0.05) (Fig. 2 A). Table 2 Effects of high glucose and LPS on SV-HUC-1 cell functions Group n Cell Viability (%) Pyroptosis Rate (%) Migrating Cell Count Control 5/3/3¹ 100.0 ± 7.1 2.66 ± 0.25 165.0 ± 17.5 High glucose 5/3/3¹ 95.7 ± 5.4 3.14 ± 0.54 172.0 ± 13.1 LPS 5/3/3¹ 70.8 ± 6.1*# 9.60 ± 1.01*# 73.3 ± 18.9*# F -value 24.876 108.234 42.567 P -value < 0.001 < 0.001 < 0.001 Note : Data are presented as mean ± SD. ¹n = 5 for cell viability assay, n = 3 for pyroptosis and migration assays. Cell viability was measured by CCK-8 assay after 48 h treatment. Pyroptosis rate was determined by Annexin V-FITC/PI flow cytometry after 48 h treatment. Cell migration capacity was assessed by scratch assay after 48 h treatment, and the number of migrating cells was counted. Statistical analysis was performed by one-way ANOVA followed by post-hoc test. P < 0.05 vs. Control group; #P < 0.05 vs. High glucose group. Annexin V-FITC/PI double-staining flow cytometry was employed to detect pyroptotic cell death. Results indicated that the pyroptosis rate in the high-glucose group (3.137 ± 0.535%) showed no significant difference compared to the control group (2.660 ± 0.252%) ( P > 0.05). The pyroptosis rate in the LPS group (9.600 ± 1.005%) was significantly higher than that in both the control and high-glucose groups ( P < 0.05) (Fig. 2 B). The scratch assay assessed cell migration capacity. Results showed no significant difference in migrating cell counts between the high-glucose group (172.000 ± 13.077) and the control group (165.000 ± 17.521) ( P > 0.05). The number of migrating cells in the LPS group (73.333 ± 18.903) was significantly lower than that in both the control and high-glucose groups ( P < 0.05) (Fig. 2 C). Representative images of the scratch assay are shown in (Fig. 2 D-F). 3. Effects of high glucose and LPS on NLRP3 inflammasome pathway activation Western blot analysis was performed to detect the expression levels of NLRP3, Caspase-1, and IL-1β. As presented in Table 3 , NLRP3 (0.632 ± 0.035 vs. 0.352 ± 0.011), Caspase-1 (0.666 ± 0.032 vs. 0.313 ± 0.044), and IL-1β (0.549 ± 0.022 vs. 0.328 ± 0.049) all showed considerably higher expression levels in the high-glucose group than in the control group (all P < 0.001) (Figs. 3 A). Table 3 Effects of high glucose and LPS on NLRP3 inflammasome pathway protein expression Group NLRP3 Caspase-1 IL-1β High glucose experiment Control 0.35 ± 0.01 0.31 ± 0.04 0.33 ± 0.05 High glucose 0.63 ± 0.04* 0.67 ± 0.03* 0.55 ± 0.02* P-value < 0.001 < 0.001 < 0.001 LPS experiment Control 0.38 ± 0.03 0.41 ± 0.05 0.36 ± 0.04 LPS 0.77 ± 0.05* 0.84 ± 0.09* 0.76 ± 0.04* P-value < 0.001 < 0.001 < 0.001 Note : Data are presented as mean ± SD (n = 3). SV-HUC-1 cells were treated with high glucose (30 mM) or LPS (1 µg/mL) for 24 h. Protein levels of NLRP3, Caspase-1, and IL-1β were measured by Western blot and normalized to β-actin. Statistical analysis was performed by independent samples t-test. P < 0.001 vs. respective Control group. Similarly, the expression levels of Caspase-1 (0.844 ± 0.086 vs. 0.413 ± 0.050), NLRP3 (0.769 ± 0.047 vs. 0.384 ± 0.029), and IL-1β (0.757 ± 0.039 vs. 0.355 ± 0.036) were considerably higher in the LPS group than in the control group (all P < 0.001) (Figs. 3 B). It is noteworthy that high glucose and LPS induced similar upregulation of the aforementioned proteins (approximately 1.8–2.2-fold), yet they led to distinctly different cellular functional outcomes. The quantitative analysis of these blots is presented in ( Fig. 4 A) (high glucose) and (Fig. 4 B) (LPS), confirming the significant upregulation of all three proteins. Discussion This study, through clinical sample analysis and in vitro cell experiments, demonstrates the differential regulatory effects of high glucose and LPS on the NLRP3/GSDMD pyroptosis pathway in human bladder epithelial cells. Our main results include: (1) In non-diabetic bladder cancer patients, there was no significant difference in NLRP3 and GSDMD expression between cancerous and adjacent non-cancerous tissues, indicating that malignant transformation is not the driving mechanism for continuous activation of this pathway. (2) Both high glucose and LPS significantly upregulate NLRP3, Caspase-1, and IL-1β protein expression in SV-HUC-1 cells, with comparable magnitudes of upregulation. (3) However, only LPS stimulation induces cell proliferation inhibition, increased pyroptosis rates, and reduced migration capacity, while high glucose does not cause significant alterations in these functional phenotypes. This finding demonstrates in bladder epithelial cells that “protein expression upregulation” and “pyroptosis execution” within the NLRP3 inflammasome pathway are two distinct biological processes. High glucose merely completes the “priming” step, while LPS provides the “activation” signal required for pyroptosis execution 12 , 13 . The observed dissociation between pathway upregulation and functional execution suggests that, in human bladder epithelial cells, the NLRP3 inflammasome is subject to stringent regulatory checkpoints beyond transcriptional control. In recent years, the NLRP3 inflammasome has garnered significant attention for its role in the pathogenesis of diabetic bladder dysfunction (DBD). Using a female type 1 diabetic Akita mouse model, Odom et al. 10 demonstrated that NLRP3 gene knockout completely prevented bladder inflammation 4 , 14 , urinary epithelial barrier damage 15 – 17 , and voiding dysfunction, confirming NLRP3-mediated inflammation as a key driver of DBD. Subsequent studies further revealed that NLRP3 activation contributes to detrusor dysfunction by upregulating prostaglandin receptor expression and abnormally regulating prostaglandin release 10 . However, these conclusions were derived from whole-animal models, making it difficult to distinguish the contribution of hyperglycemia itself from other in vivo factors (such as neural, vascular, or immune cell involvement). More importantly, no previous studies had addressed whether the observed chain of events—"hyperglycemia → NLRP3 activation → urinary epithelial pyroptosis"—in animal models could be directly replicated in human bladder epithelial cells. This study utilized the human immortalized bladder epithelial cell line SV-HUC-1 to eliminate interference from the complex in vivo environment, directly examining the isolated effect of high glucose and filling this research gap. Notably, we observed that whereas both LPS and high glucose considerably increase the NLRP3 pathway proteins, they produce quite different functional results. This phenomenon closely resembles conventional "two-step model" of NLRP3 activation 18 : Toll-like receptors or cytokines usually initiate the first step, which upregulates the transcriptional expression of NLRP3, pro-IL-1β, and other components via the NF-κB pathway; a second signal (like ATP, uric acid crystals, or bacterial toxins) is needed for the second stage (activation) to induce inflammasome assembly, caspase-1 activation, and GSDMD cleavage. Glucose metabolites, including damage-associated molecular patterns (DAMPs) 5 , 19 – 21 , reactive oxygen species (ROS) 9 , 22 , and advanced glycation end products (AGEs) 19 , 21 , may serve as starting signals in high-glucose situations. However, they lack a secondary signal sufficient to trigger full caspase-1 activation, thus remaining at the stage of protein expression upregulation. In contrast, LPS, a potent pathogen-associated molecular pattern, provides a strong priming signal via TLR4 and, in the context of bladder epithelial cells, appears to also provide sufficient activation signals to drive the entire pyroptosis process 23 , 24 . This conclusion is consistent with previous research showing that high-glucose pretreatment alone increases NLRP3 expression in macrophages but necessitates further stimulation to trigger IL-1β production 25 . The findings of this study provide important insights into understanding bladder pathology in diabetic patients. Diabetic patients often present with asymptomatic bacteriuria or recurrent urinary tract infections, and infection is considered an aggravating factor in the progression of diabetic bladder dysfunction (DBD) 26 , 27 . Our data indicate that prolonged hyperglycemia induces a preactivated state of the NLRP3 pathway in bladder epithelial cells. While this state does not directly cause injury, it triggers pyroptotic damage far exceeding normal levels upon encountering infection. This leads to disruption of the urinary epithelial barrier, amplified inflammatory cascades, and ultimately promotes the onset and progression of DBD. This "two-hit" model explains why enhanced glycemic control alone cannot fully prevent DBD, because the established "preactivation" state may possess metabolic memory effects 28 , 29 , and infection, as the "second hit" operates independently of blood glucose levels. Furthermore, this model offers novel insights for intervention strategies: Targeting the initiation phase of the NLRP3 pathway (e.g., inhibiting the AGEs-RAGE axis or scavenging ROS 30 ) may serve as a primary prevention measure, while interventions focused on the activation phase (e.g., blocking caspase-1 or GSDMD) are suitable for diabetic patients with concurrent infections. It's interesting to note that prior research has documented NLRP3's sex-specific functions in DBD, with NLRP3 deletion shielding female Akita mice from bladder dysfunction but not males 4 , 20 , 31 . Our study was conducted in a human cell line without sex specification; any sex differences in the priming-activation dynamics of the NLRP3 pathway should be considered in future research. The innovation of this study is manifested in three key aspects. First, this study utilizes a human bladder epithelial cell model to directly evaluate the independent effect of high glucose on the NLRP3 pyroptosis pathway, thereby overcoming the confounding factors inherent in animal models. Second, it demonstrates the dissociation between pathway activation and functional execution in bladder epithelial cells, thus extending the two-step NLRP3 model to the urinary system. Third, based on these findings, this study proposes the “two-hit hypothesis” for DBD, providing a new perspective for understanding clinical phenomena. However, this study also presents several limitations. First, the in vitro experimental design failed to simulate the long-term and complex nature of diabetic conditions, including the accumulation of AGEs and the dynamic alterations in oxidative stress. Second, caspase-1 activity and GSDMD cleavage fragments were not directly detected; subsequent validation of pyroptosis pathway activation necessitates ELISA or western blot analysis of activated caspase-1 and GSDMD-NT fragments. Third, experiments combining high glucose with LPS stimulation were not conducted. Fourth, while LPS was employed as a surrogate for bacterial infection, clinical urinary tract infections involve complex pathogen-host interactions that cannot be fully recapitulated by a single PAMP. Fifth, our study did not investigate the upstream signaling events linking high glucose to NLRP3 priming, such as the AGEs-RAGE axis 30 or the ROS-TXNIP pathway 3 , 6 , which warrant further investigation. Future studies could design a "high glucose pretreatment + LPS stimulation" protocol directly to validate the synergistic effects of dual insults. Conclusion In summary, this study demonstrates in human bladder epithelial cells that elevated glucose can activate the NLRP3 inflammasome pathway but is insufficient to initiate the execution of pyroptosis. The second hit is infectious stimulation, which is a prerequisite for pyroptosis and cellular failure. This finding provides a novel mechanistic explanation for diabetic bladder disease and offers a theoretical basis for developing early intervention strategies. Declarations Acknowledgments Not applicable. Author Contribution: M.A. contributed to the research design, conducted the cell experiments, analyzed the data, and wrote the paper. A.A. and Y.G. helped with cell culture, immunohistochemistry studies, and data collection. G.S. gave valuable help in study design, project supervision, and manuscript drafting and revision. All writers read and approved the final manuscript. Funding statement ： The authors declare that no funding was received for this study. Data Availability: The datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request. Competing Interests: The authors have no relevant financial or non-financial interests to disclose. References Pouya, S. et al. Global and regional diabetes prevalence estimates for 2019 and projections for 2030 and 2045: Results from the International Diabetes Federation Diabetes Atlas, 9(th) edition. Diabetes Res. Clin. Pract. 157 https://doi.org:10.1016/j.diabres.2019.107843 (2019). Yichao, C., Riqiu, C., Xiaozhen, J., Zhifu, Z. & Changrong, G. NLRP3 Inflammasome-Mediated Pyroptosis in Diabetic Nephropathy: Pathogenic Mechanisms and Therapeutic Targets. J. Inflamm. Res. 18 https://doi.org:10.2147/jir.S524246 (2025). Odom, M. R., Hughes, F. M. Jr., Jin, H. & Purves, J. T. Diabetes causes NLRP3-dependent barrier dysfunction in mice with detrusor overactivity but not underactivity. Am. J. Physiol. Ren. Physiol. 323 , F616–f632. https://doi.org:10.1152/ajprenal.00047.2022 (2022). Hughes, F. M. Jr. et al. NLRP3 Promotes Diabetic Bladder Dysfunction and Changes in Symptom-Specific Bladder Innervation. Diabetes 68 , 430–440. https://doi.org:10.2337/db18-0845 (2019). Hughes, F. M. Jr., Harper, S. N., Jin, H., Odom, M. R. & Purves, J. T. Strict glucose control and elimination of NLRP3-induced inflammation prevents diabetic bladder dysfunction in the female Akita mouse model. Neurourol. Urodyn. 43 , 2269–2278. https://doi.org:10.1002/nau.25554 (2024). Zahra, H. et al. NLRP3 inflammasome: Its regulation and involvement in atherosclerosis. J. Cell. Physiol. 233 https://doi.org:10.1002/jcp.25930 (2017). Rong-Xin, Z. et al. Inactivation of NLRP3 inflammasome by dephosphorylation at Serine 658 alleviates glial inflammation in the mouse model of Parkinson's disease. Mol. Neurodegener . 20 https://doi.org:10.1186/s13024-025-00818-z (2025). Shi, J. et al. Cleavage of GSDMD by inflammatory caspases determines pyroptotic cell death. Nature 526 , 660–665. https://doi.org:10.1038/nature15514 (2015). Huang, B. et al. Metformin modulates the TXNIP-NLRP3-GSDMD pathway to improve diabetic bladder dysfunction. Sci. Rep. 14 , 23868. https://doi.org:10.1038/s41598-024-72129-0 (2024). Odom, M. R., Hughes, F. M. Jr., Pope, N., Jin, H. & Purves, J. T. Female Type 1 Diabetic Akita Mice Demonstrate Increased Bladder Contractility via FP Receptor Activation due to NLRP3-Mediated Inflammation. Front. Biosci. (Landmark Ed) . 29 , 154. https://doi.org:10.31083/j.fbl2904154 (2024). Ilaria, C., Beatrice, M., Gaetano, M., Eleonora, I. & Barbara, M. Effect of platelet lysate on uterine response of mares susceptible to persistent mating-induced endometritis. Theriogenology 179 https://doi.org:10.1016/j.theriogenology.2021.12.001 (2021). Budden, C. F. et al. Inflammasome-induced extracellular vesicles harbour distinct RNA signatures and alter bystander macrophage responses. J. Extracell. Vesicles . 10 , e12127. https://doi.org:10.1002/jev2.12127 (2021). Broz, P. Recognition of Intracellular Bacteria by Inflammasomes. Microbiol. Spectr. 7 https://doi.org:10.1128/microbiolspec.BAI-0003-2019 (2019). Hughes, F. M. Jr., Allkanjari, A., Odom, M. R., Jin, H. & Purves, J. T. Diabetic bladder dysfunction progresses from an overactive to an underactive phenotype in a type-1 diabetic mouse model (Akita female mouse) and is dependent on NLRP3. Life Sci. 299 , 120528. https://doi.org:10.1016/j.lfs.2022.120528 (2022). Hughes, F. M. Jr., Odom, M. R., Cervantes, A. & Purves, J. T. Inflammation triggered by the NLRP3 inflammasome is a critical driver of diabetic bladder dysfunction. Front. Physiol. 13 , 920487. https://doi.org:10.3389/fphys.2022.920487 (2022). Sylvia, R., Carmen, P., Rabia, J., Johan, L. & Christo, J. F. Intestinal Barrier Function and Immune Homeostasis Are Missing Links in Obesity and Type 2 Diabetes Development. Front. Endocrinol. (Lausanne) . 12. https://doi.org:10.3389/fendo.2021.833544 (2022). Tina, X. & Lawrence, J. Effects of diabetic retinopathy on the barrier functions of the retinal pigment epithelium. Vis. Res. 139 https://doi.org:10.1016/j.visres.2017.02.006 (2017). Karen, V., Meng, S., Jenny, P-Y. & D. & The NLRP3 inflammasome: molecular activation and regulation to therapeutics. Nat. Rev. Immunol. 19 https://doi.org:10.1038/s41577-019-0165-0 (2019). Tao, G., Lei, L., Wei, J. & Rongbin, Z. DAMP-sensing receptors in sterile inflammation and inflammatory diseases. Nat. Rev. Immunol. 20 https://doi.org:10.1038/s41577-019-0215-7 (2019). Livingston, A. J., Purves, J. T., Odom, M. R., Jin, H. & Hughes, F. M. Jr. Male Akita diabetic mice develop underactive bladder independent of NLRP3 that can be prevented with blood glucose control. Cont. (Amst) . 12. https://doi.org:10.1016/j.cont.2024.101690 (2024). Jing, L., Shuo, P., Xiqiang, W., Zhongwei, L. & Yong, Z. Role of advanced glycation end products in diabetic vascular injury: molecular mechanisms and therapeutic perspectives. Eur. J. Med. Res. 28 https://doi.org:10.1186/s40001-023-01431-w (2023). Stanislovas, S. Heart failure in diabetes. Metabolism 125 https://doi.org:10.1016/j.metabol.2021.154910 (2021). Xuan, L. et al. Gram-negative bacterial outer membrane proteins and lipopolysaccharides key factors linking chicken coop environment and oxidative stress. Redox Biol. 89 https://doi.org:10.1016/j.redox.2025.103986 (2026). Xudong, T., Yilin, H., Ying, Z. & Yin, X. Immune dysregulation in ulcerative colitis: pathogenic mechanisms and therapeutic strategies of traditional Chinese medicine. Front. Cell. Dev. Biol. 13 https://doi.org:10.3389/fcell.2025.1610435 (2025). Yiqun, M. et al. High glucose enhances the activation of NLRP3 inflammasome by ambient fine particulate matter in alveolar macrophages. Part. Fibre Toxicol. 20 https://doi.org:10.1186/s12989-023-00552-8 (2023). Mi-Hye, K. et al. Functional and Immunofluorescence Evaluations of Vascular and Neural Integrities in Urinary Bladder of Streptozotocin-Induced Diabetic Mice. Int. Neurourol. J. 26 https://doi.org:10.5213/inj.2244152.076 (2022). Hanan, W. et al. Recurrent Urinary Tract Infection in Diabetics: A Retrospective Analysis. Cureus 17 https://doi.org:10.7759/cureus.87816 (2025). Stephen, K. Effect of intensive glycemic control and diabetes complications on lower urinary tract symptoms in men with type 1 diabetes: Diabetes Control and Complications Trial/Epidemiology of Diabetes Interventions and Complications (DCCT/EDIC) study. Diabetes Care . 32 https://doi.org:10.2337/dc07-2375 (2009). Saul, G. Insights from the diabetes control and complications trial/epidemiology of diabetes interventions and complications study on the use of intensive glycemic treatment to reduce the risk of complications of type 1 diabetes. Endocr. Pract. https://doi.org:10.4158/ep.12.S1.34 (2006). Gaozhi, P. Diabetic Kidney Disease: Disease Progression Driven by Positive Feedback Loops and Therapeutic Strategies Targeting Pathogenic Pathways. Diabetes Metab. Syndr. Obes. 18 https://doi.org:10.2147/dmso.S513080 (2025). Hughes, F. M. Jr. et al. Male Akita mice develop signs of bladder underactivity independent of NLRP3 as a result of a decrease in neurotransmitter release from efferent neurons. Am. J. Physiol. Ren. Physiol. 325 , F61–f72. https://doi.org:10.1152/ajprenal.00284.2022 (2023). Additional Declarations No competing interests reported. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-8934076","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":605321714,"identity":"e9a4570f-9d39-43c5-9051-93ed808f3990","order_by":0,"name":"Maidina Aisihaer","email":"","orcid":"","institution":"The First Affiliated Hospital of Xinjiang Medical University","correspondingAuthor":false,"prefix":"","firstName":"Maidina","middleName":"","lastName":"Aisihaer","suffix":""},{"id":605321715,"identity":"cdf888d0-0b27-4b0e-be02-508bd02db11e","order_by":1,"name":"Asimujiang Abula","email":"","orcid":"","institution":"The First Affiliated Hospital of Xinjiang Medical University","correspondingAuthor":false,"prefix":"","firstName":"Asimujiang","middleName":"","lastName":"Abula","suffix":""},{"id":605321716,"identity":"c6c3956f-85ea-4961-9bee-62b2fdcecb30","order_by":2,"name":"Yueju Gu","email":"","orcid":"","institution":"The First Affiliated Hospital of Xinjiang Medical University","correspondingAuthor":false,"prefix":"","firstName":"Yueju","middleName":"","lastName":"Gu","suffix":""},{"id":605321717,"identity":"5ee9fe0a-b145-4224-99dd-3dcd45121c4d","order_by":3,"name":"Guanglu Song","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAxElEQVRIiWNgGAWjYLCCDz9q7PiZmQ8/IFoH48yeY8mS7WxpBkRrYeZhY2bccJ5HQYIo5fLt6c8keIB6jA/zMBgw1NhEE9RicOaNmYSEhQyf2WHeAw8YjqXlNhDUIpHDJmEAtMXsMF+CAWPDYcJa5GcAHZYA9MvmZh4DCaK0MNxIMJM4API+M7FagH4xtmwEBrLEYWAgJxDjF2CIPbz9BxSV/YcPP/hQY0OEwxgSWBDRkUBYOVgZ8wfiFI6CUTAKRsGIBQDYdjwDJ1ILKwAAAABJRU5ErkJggg==","orcid":"","institution":"The First Affiliated Hospital of Xinjiang Medical University","correspondingAuthor":true,"prefix":"","firstName":"Guanglu","middleName":"","lastName":"Song","suffix":""}],"badges":[],"createdAt":"2026-02-21 14:23:28","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8934076/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8934076/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":104593839,"identity":"a38188ff-3e4f-49c7-9d0a-b2dc84fb6a4d","added_by":"auto","created_at":"2026-03-13 17:46:19","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1498777,"visible":true,"origin":"","legend":"\u003cp\u003eExpression of NLRP3 and GSDMD in human bladder epithelial tissues.\u003c/p\u003e\n\u003cp\u003eRepresentative immunohistochemical images are presented which demonstrate the expression and localization of NLRP3 and GSDMD in bladder epithelial cells from bladder cancer tissues and adjacent normal tissues (adjacent normal epithelial tissues). (A) NLRP3 staining in bladder cancer epithelium; (B) NLRP3 staining in normal epithelium; (C) GSDMD staining in bladder cancer epithelium; (D) GSDMD staining in normal epithelium. Both proteins were predominantly localized in the cytoplasm, with occasional nuclear positivity for GSDMD. Scale bars: 50 μm. (E-F) Quantitative analysis of NLRP3 and GSDMD expression by H-score in the epithelial layer. Data are presented as mean ± SD (n = 6 per group). (E) NLRP3 H-scores showed no significant difference between bladder cancer group (8.33 ± 2.25) and normal group (9.33 ± 1.37) (P = 0.374, unpaired t-test). (F) GSDMD H-scores also revealed no significant difference between bladder cancer group (10.00 ± 1.55) and normal group (10.50 ± 1.64) (P = 0.599, unpaired t-test). ns, not significant.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-8934076/v1/5874e7a621e741103604aeef.png"},{"id":104781901,"identity":"53d8be4c-1c4d-4aea-82dd-2a559ae75b64","added_by":"auto","created_at":"2026-03-17 07:56:32","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":3186861,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of high glucose and LPS on SV-HUC-1 cell functions.\u003c/p\u003e\n\u003cp\u003e(A) Cell viability measured by CCK-8 assay after 48 h treatment. Data are presented as mean ± SD (n = 5). (B) Pyroptosis rate determined by Annexin V-FITC/PI flow cytometry after 48 h treatment. Data are presented as mean ± SD (n = 3). (C) Cell migration capacity assessed by scratch assay after 48 h treatment. The number of migrating cells was counted and expressed as mean ± SD (n = 3). (D–F) Representative images of the scratch assay at 0 h and 48 h for the control group (D), high glucose group (E), and LPS group (F). Scale bars: 100 μm. ***P \u0026lt; 0.001 vs. control group; ***P \u0026lt; 0.001 vs. high glucose group (one-way ANOVA with post-hoc test).\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-8934076/v1/ec2bfc9151988b29edcea6f9.png"},{"id":104808683,"identity":"08dff0b0-0d6f-468f-ac26-896bf8f5ce97","added_by":"auto","created_at":"2026-03-17 12:39:26","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":269808,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of high glucose and LPS on NLRP3 inflammasome pathway protein expression by Western blot.\u003c/p\u003e\n\u003cp\u003eA.Western blot pictures of NLRP3, Caspase-1, and IL-1β protein levels in SV-HUC-1 cells after 24 hours of treatment with high glucose (30 mM) or control. The blots for several proteins were cropped from different regions of the same full-length membrane based on molecular weight and are displayed here for simplicity. Supplementary Figure S1 shows full-length, uncropped blots. β-actin was employed as the loading control.\u003c/p\u003e\n\u003cp\u003eB. Western blot images demonstrate protein levels of NLRP3, Caspase-1, and IL-1β in SV-HUC-1 cells treated with LPS (1 μg/mL) or control after 24 hours. The blots were similarly clipped from different portions of the same full-length membrane and are displayed here. Supplementary Figure S2 shows full-length blots with β-actin as a loading control.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-8934076/v1/f39ad13e1ba4ece9cb13127b.png"},{"id":104593837,"identity":"bc352e22-55df-40ca-8135-e8629dbc0d2a","added_by":"auto","created_at":"2026-03-13 17:46:18","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":355585,"visible":true,"origin":"","legend":"\u003cp\u003eQuantitative analysis of NLRP3 inflammasome pathway protein expression.\u003c/p\u003e\n\u003cp\u003eRelative protein expression levels of NLRP3, Caspase-1, and IL-1β were quantified by densitometry and normalized to β-actin. Data are presented as mean ± SD (n = 3). (A) High glucose group compared to its respective control. (B) LPS group compared to its respective control. ***P \u0026lt; 0.001 vs. control (unpaired t-test).\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-8934076/v1/8beeec8455191fe83c0deca6.png"},{"id":104809222,"identity":"9349727d-9667-4278-b53a-37e9c208bbe6","added_by":"auto","created_at":"2026-03-17 12:48:31","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":8118261,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8934076/v1/0c72c7ab-ce7e-4c5d-8817-28a19b0212db.pdf"},{"id":104593835,"identity":"7bd91d4f-f09f-4024-8a94-ad6ff58e1bb8","added_by":"auto","created_at":"2026-03-13 17:46:18","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":171112,"visible":true,"origin":"","legend":"","description":"","filename":"SupplimentaryFile.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8934076/v1/9817d08f599ea0602040e678.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"A two-hit model for NLRP3-mediated pyroptosis in human bladder epithelial cells: Hyperglycemia primes, infection triggers","fulltext":[{"header":"Introduction","content":"\u003cp\u003eDiabetes has become one of the most severe global public health challenges of the 21st century. According to the International Diabetes Federation's 10th edition of the Diabetes Atlas report, the global prevalence of diabetes among individuals aged 20 to 79 was estimated to be 10.5% (537\u0026nbsp;million people) in 2021. It is expected to grow to 12.2% (783\u0026nbsp;million people) by 2045\u003csup\u003e1\u003c/sup\u003e. Diabetes and its complications not only jeopardize individuals' quality of life, but they also place a significant burden on global healthcare systems\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. Among the numerous complications of diabetes, diabetic bladder dysfunction (DBD) is one of the most common yet long-underestimated urinary system complications, affecting approximately 50% of diabetic patients\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e3\u003c/span\u003e,\u003cspan class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e. DBD exhibits highly heterogeneous clinical manifestations, presenting as overactive bladder, underactive bladder, or mixed symptoms. Some patients ultimately progress to a decompensated state, requiring long-term dependence on intermittent catheterization for urination. Currently, there are no specific therapeutic agents available for DBD, and even strict glycemic control cannot completely prevent or reverse the progression of bladder dysfunction\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e. This strongly suggests that, beyond hyperglycemia itself, other critical pathological mechanisms remain poorly understood.\u003c/p\u003e \u003cp\u003eIn recent years, pyroptosis—a form of programmed inflammatory necrosis that is mediated by gasdermin family proteins—has gained prominence in the pathogenesis of diabetic complications\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. The classical pyroptotic pathway initiates upon pattern recognition receptor (PRR) detection of pathogen-associated molecular patterns (PAMPs) or damage-associated molecular patterns (DAMPs). NOD-like receptor family pyrin domain-containing 3 (NLRP3) is the most extensively studied inflammasome sensor molecule and is the one most closely associated with metabolic diseases\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e4\u003c/span\u003e,\u003cspan class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e. Upon sensing danger signals, NLRP3 undergoes oligomerization, recruiting apoptosis-associated speck-like protein containing a CARD (ASC) and pro-caspase-1 to form a functional inflammasome complex\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e. Notably, the NLRP3 inflammasome is activated in two steps: first, a priming signal (such as TLR ligands or cytokines) increases NLRP3 and pro-IL-1β expression through NF-κB, and then an activation signal (such as ATP, nigericin, or bacterial toxins) triggers inflammasome assembly and caspase-1 activation\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e. This process promotes the self-cleavage and activation of caspase-1. Activated caspase-1 cleaves pro-IL-1β and pro-IL-18 into mature pro-inflammatory cytokines while also cleaving gasdermin D (GSDMD), releasing its pore-forming N-terminal domain (GSDMD-NT)\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e. GSDMD-NT oligomerizes on the plasma membrane to form transmembrane pores, disrupting cellular osmotic homeostasis. This ultimately leads to cell swelling, membrane rupture, and massive release of proinflammatory contents, amplifying both local and systemic inflammatory responses\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e3\u003c/span\u003e,\u003cspan class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eIn the field of DBD, the pioneering work by the Odom and Hughes teams laid the groundwork. They first showed that NLRP3 inflammasomes in urinary tract epithelial cells are markedly activated in diabetes situations using female type 1 diabetic Akita mouse models. In diabetic mice, the systemic deletion of the NLRP3 gene eliminated the development of bladder inflammation, disruption of the urinary epithelial barrier, and urinary dysfunction\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e3\u003c/span\u003e,\u003cspan class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e. Subsequent studies further revealed that NLRP3-mediated inflammation contributes to diabetic bladder contraction dysfunction by upregulating prostaglandin F receptor (FP receptor) expression and abnormally regulating prostaglandin release\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. This evidence from whole-animal models robustly establishes the central role of the NLRP3 inflammasome in DBD pathogenesis. However, it must be noted that the majority of the research that has been done on the DBD mechanism has only looked at the whole animal level. The observed chain of events—\"hyperglycemia → NLRP3 activation → urinary epithelial pyroptosis → bladder dysfunction\"—represents a terminal phenotype emerging within a complex context involving multiple overlapping factors, including neural, vascular, immune cell, hemodynamic, and systemic metabolic dysregulation. Can hyperglycemia, as a single factor, independently complete the entire process from \"NLRP3 pathway activation\" to \"pyroptosis execution\" in human bladder epithelial cells? This question remains unanswered to date.\u003c/p\u003e \u003cp\u003eOur initial findings from human tissue samples provided a key starting point for addressing this issue. In tissue samples from non-diabetic bladder cancer patients, NLRP3 and GSDMD expression showed no significant difference between tumor regions and adjacent normal epithelium (P \u0026gt; 0.05). This finding suggests that, when excluding hyperglycemic interference, malignant transformation of bladder epithelium itself is not a sustained driver of this pathway. Conversely, the hyperglycemic metabolic environment may be the key upstream event inducing bladder epithelium into a pyroptosis \"susceptibility state.\" Therefore, this study employed human immortalized bladder epithelial cells (SV-HUC-1), which are derived from benign human ureteral epithelial lining and immortalized with SV40, serving as an in vitro model of normal urinary tract epithelium\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. Lipopolysaccharide (LPS) was used as a positive control. The aim was to systematically observe the effects of high glucose stimulation on cellular function and the NLRP3 inflammasome pathway. Both high glucose and LPS dramatically increase NLRP3/caspase-1/IL-1β expression, but with differing functional results. LPS causes pyroptosis and functional impairment, while high glucose has no significant effects. This finding indicates in human bladder epithelial cells that the NLRP3 pathway \"upregulation\" and \"pyroptosis execution\" are decoupled biological processes; high glucose only completes \"priming\" without supplying sufficient \"activation\" signals. Based on this, we propose a \"two-hit\" model for DBD pathogenesis: persistent hyperglycemia pre-activates the bladder epithelium's NLRP3; subsequent infection (e.g., LPS) serves as the \"second hit,\" causing pyroptosis. This study provides a cellular mechanism that may explain diabetic patients' susceptibility to bladder infections and offers a theoretical basis for early management during the 'priming phase' of NLRP3 activation. We hypothesized that a second hit would be necessary to initiate pyroptosis execution, and hyperglycemia alone could be enough to stimulate but not fully activate the NLRP3 inflammasome in human bladder epithelial cells.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e\u003c/p\u003e "},{"header":"Method","content":"\u003col\u003e \u003cspan\u003e \u003cli\u003e \u003cp\u003e \u003cb\u003eClinical Samples and Immunohistochemistry\u003c/b\u003e \u003c/p\u003e \u003c/li\u003e \u003c/span\u003e \u003c/ol\u003e\u003cp\u003e The Ethics Committee of The First Affiliated Hospital of Xinjiang Medical University approved this study involving human bladder tissue samples (Approval No. K202601-103). All procedures involving human participants were performed in accordance with the Declaration of Helsinki and relevant institutional guidelines and regulations. Written informed consent was obtained from all participants and/or their legal guardians prior to inclusion in the study. Six paraffin-embedded bladder cancer tissue specimens archived from 2019 to 2023 in the Department of Pathology, The First Affiliated Hospital of Xinjiang Medical University (bladder cancer group), and six matched adjacent normal tissue specimens (submucosal epithelial group) were collected. All patients were non-diabetic and had not received preoperative radiotherapy or chemotherapy. Tissue sections (4 µm thick) underwent routine dewaxing and rehydration, followed by antigen retrieval in citrate buffer (pH 6.0) via high-pressure autoclaving (heated until steam vented, then removed from heat and immersed for 10 min; cooled to room temperature naturally). Tissues were incubated with 3% H₂O₂ for 10 min to block endogenous peroxidase activity, followed by blocking with 10% normal goat serum at room temperature for 30 min. Rabbit anti-human NLRP3 monoclonal antibody (1:100, Thermo Fisher, MA5-32255) and mouse anti-human GSDMD monoclonal antibody (1:100, Santa Cruz, sc-393581) were applied separately and incubated overnight at 4°C. The following day, add horseradish peroxidase-labeled secondary antibody (Beijing Zhongshan Jinqiao, SP9000) and incubate at room temperature for 20 min. Subsequently, add horseradish peroxidase-labeled streptavidin working solution and incubate at room temperature for 20 min. Developed with DAB (Beijing Zhongshan Jinqiao, DAB-1031), controlling development time under the microscope; stopped with tap water. Counterstained with hematoxylin for 3 min, differentiated with hydrochloric acid in alcohol for several seconds, and counterstained with blue in tap water for 5 min. Dehydrated with graded alcohol, cleared with xylene, and mounted with neutral resin. For each batch, a negative control was prepared by replacing the primary antibody with PBS. Staining results were semi-quantitatively scored by two blinded pathologists using a double-blind method. Scoring criteria: The final immune response score was calculated as the product of staining intensity (0 = negative, 1 = weak positive, 2 = moderate positive, 3 = strong positive) and the percentage of positive cells (0 = \u0026lt; 5%, 1 = 5%–25%, 2 = 26%–50%, 3 = 51%–75%, 4 = ≥ 75%) (0–12 points).\u003c/p\u003e\n\u003ch3\u003e2. Cell Culture and Processing\u003c/h3\u003e\n\u003cp\u003eThe Human ureteral epithelial immortalized cell line SV-HUC-1 (Wuhan Shang'en Bio, Catalog No. SNL-233, ATCC). Cells were cultured in F-12K complete medium (containing 10% fetal bovine serum and 1% penicillin-streptomycin, Wuhan Shang'en Bio, SNLM-233) at 37°C in a CO₂ incubator maintained at 5% CO₂ saturation. Passaging or experimental procedures were performed when cells reached 80–90% confluence.\u003c/p\u003e \u003cp\u003eThe experiment was divided into three groups: the control group (standard medium with 5.5 mM glucose concentration), the high-glucose group (supplemented with D-glucose to a final concentration of 30 mM), and the LPS group (standard medium supplemented with 1 µg/mL LPS, Sigma-Aldrich, L4391). Treatment durations for each group were 48 hours (for cell proliferation, pyroptosis, and migration assays) or 24 hours (for Western blot analysis).\u003c/p\u003e\n\u003ch3\u003e3. Cell Proliferation Assay\u003c/h3\u003e\n\u003cp\u003eCell proliferation activity was assessed using the CCK-8 assay kit (Wuhan Shang'en Bio, SNK-010). Log-phase SV-HUC-1 cells were seeded at 5 × 10³ cells/well in a 96-well plate and cultured overnight before undergoing group-specific treatments for 48 hours. Add 10 µL of CCK-8 solution to each well and incubate for an additional 2 hours. Measure the absorbance at 450 nm using a microplate reader. Each experiment was independently repeated three times, with five replicate wells per group. Cell survival rate (%) = (OD value of treated group / OD value of control group) × 100%.\u003c/p\u003e\n\u003ch3\u003e4. Pyroptosis Detection\u003c/h3\u003e\n\u003cp\u003ePyroptosis was detected using the Annexin V-FITC/PI Pyroptosis Detection Kit (Wuhan Shang'en Bio, SNP-005). After 48 h of treatment, cells were collected by digestion with EDTA-free trypsin, washed twice with PBS, and resuspended in 1× binding buffer. Take 100 µL of cell suspension (approximately 1×10⁵ cells), add 5 µL Annexin V-FITC and 5 µL PI, incubate at room temperature in the dark for 15 min, then immediately add 400 µL binding buffer and analyze by flow cytometry (BD FACSCanto II). The experiment was independently repeated three times, with three replicate wells per group. The pyroptosis rate was expressed as the sum of early apoptosis (Annexin V⁺/PI⁻) and late pyroptosis (Annexin V⁺/PI⁺) cell percentages.\u003c/p\u003e\n\u003ch3\u003e5. Cell Migration Assay (Scratch Assay)\u003c/h3\u003e\n\u003cp\u003eSV-HUC-1 cells were seeded at 2 × 10⁵ cells/well in a 6-well plate and cultured to confluence. A vertical scratch was made in the center of the monolayer in each well using a 200 µL sterile pipette tip. The plate was gently washed three times with PBS to remove detached cells. Add treatment medium containing 2% FBS (to eliminate proliferation interference). Observe and photograph under an inverted microscope (Nikon E200) at 0 h and 48 h. Measure the scratch area using ImageJ software and calculate the relative migration rate: Migration Rate (%) = (Initial Area − 48 h Area) / Initial Area × 100%. The experiment was independently repeated three times, with three replicate wells per group.\u003c/p\u003e\n\u003ch3\u003e6. Western blot assay\u003c/h3\u003e\n\u003cp\u003eAfter 24 hours of treatment, discard the medium from each cell group, wash twice with PBS, add RIPA lysis buffer containing a protease inhibitor mixture (Wuhan Shang'en Bio, SNL-111), and incubate on ice for 30 min. Centrifuge at 12,000 rpm at 4°C for 15 min, then collect the supernatant. Protein concentration was determined using the BCA Protein Quantification Kit (Wuhan Shang'en Bio, SNB-001). Thirty µg of total protein was separated by 10% SDS-PAGE gel electrophoresis and transferred to a PVDF membrane (Millipore, IPVH00010). Blocked with 5% nonfat milk at room temperature for 1 hour. Primary antibodies added: Rabbit anti-NLRP3 monoclonal antibody (1:100, optimized in preliminary experiments, Thermo Fisher, MA5-32255); Rabbit anti-caspase-1 polyclonal antibody (1:100, optimized in preliminary experiments, Fuzhou Maxine, RAB-0303); Rabbit anti-IL-1β polyclonal antibody (using antibody provided in the Lianke Bio IL-1β ELISA kit, catalog no. EK301B/3–48, lot no. A301B91044), Mouse anti-β-actin monoclonal antibody (1:5000, Beijing Zhongshan Jinqiao, TA-09), and incubated overnight at 4°C. The next day, wash three times with TBST for 10 min each. Add HRP-labeled goat anti-rabbit/anti-mouse secondary antibody (1:5000, Beijing Zhongshan Jinqiao, SP9000) and incubate at room temperature for 1 h. After three TBST washes, develop with ECL chemiluminescent substrate (Millipore, WBKLS0500). Capture images using the ChemiDoc XRS⁺ imaging system (Bio-Rad). Analyze band grayscale values with ImageJ software, expressing relative expression as the ratio of target protein to β-actin grayscale values. Each experiment was independently repeated three times.\u003c/p\u003e\n\u003ch3\u003e7. Statistical Analysis\u003c/h3\u003e\n\u003cp\u003eAll data are expressed as mean ± standard deviation (SD). Statistical analysis was performed using SPSS 25.0 software. Independent samples t-tests were used for comparisons between two groups, one-way analysis of variance (ANOVA) for comparisons among multiple groups, and LSD post hoc tests for pairwise comparisons within groups. A P value \u0026lt; 0.05 was considered statistically significant. All cellular experiments were independently replicated three times (n = 3).\u003c/p\u003e "},{"header":"Result","content":"\u003col\u003e \u003cspan\u003e \u003cli\u003e \u003cp\u003e \u003cb\u003eExpression of NLRP3 and GSDMD in bladder tissues\u003c/b\u003e \u003c/p\u003e \u003c/li\u003e \u003c/span\u003e \u003c/ol\u003e\u003cp\u003eImmunohistochemistry was performed to mark these proteins in bladder cancer tissues and adjacent normal epithelial tissues (referred to as the normal group) to determine whether bladder cancer induces permanent activation of the NLRP3/GSDMD pyroptosis pathway. NLRP3 was predominantly localised in the cytoplasm of bladder epithelial cells, while GSDMD demonstrated cytoplasmic expression with occasional nuclear positivity (Figs.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eA-D).\u003c/p\u003e\u003cp\u003eRepresentative images from bladder cancer and adjacent normal tissues are shown in Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eA-D. Neither the muscle layer nor the epithelium layer showed no significant differences in IHC values between the two groups, according to semi-quantitative analysis (Table\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e). In the muscle layer, NLRP3 scores were 8.33 ± 2.25 in the bladder cancer group vs. 7.83 ± 0.98 in the adjacent normal epithelial tissues (\u003cem\u003eP\u003c/em\u003e = 0.629), whereas GSDMD ratings were 10.00 ± 1.55 vs. 10.50 ± 1.64 (\u003cem\u003eP\u003c/em\u003e = 0.599). In the epithelial layer, NLRP3 scores were 8.33 ± 2.25 vs. 9.33 ± 1.37 (\u003cem\u003eP\u003c/em\u003e = 0.374), while GSDMD scores were 10.00 ± 1.55 vs. 10.50 ± 1.64 (\u003cem\u003eP\u003c/em\u003e = 0.599) (Figs.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eE-F). These findings indicate that, under normoglycemic settings, malignant bladder epithelial transition is not associated with significant activation of the NLRP3/GSDMD pyroptosis pathway.\u003c/p\u003e\u003cdiv class=\"gridtable\"\u003e\u003cdiv align=\"left\" class=\"colspec\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\"\u003e\u003c/div\u003e\u003ctable id=\"Tab1\" border=\"1\"\u003e \u003ccaption\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eComparison of immunohistochemical scores of NLRP3 and GSDMD proteins in bladder tissues between the two groups (Mean ± SD, n = 6)\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003c/colgroup\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\"\u003e \u003cp\u003eTissue Layer\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\"\u003e \u003cp\u003eGroup\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\"\u003e \u003cp\u003eNLRP3\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\"\u003e \u003cp\u003eGSDMD\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\"\u003e \u003cp\u003e\u003cb\u003eMuscle layer\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\"\u003e \u003cp\u003eBladder cancer group\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\"\u003e \u003cp\u003e8.33 ± 2.25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\"\u003e \u003cp\u003e10.00 ± 1.55\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\"\u003e \u003cp\u003eMuscularis propria group\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\"\u003e \u003cp\u003e7.83 ± 0.98\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\"\u003e \u003cp\u003e10.50 ± 1.64\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\"\u003e \u003cp\u003e\u003cem\u003et value\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\"\u003e \u003cp\u003e*0.499*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\"\u003e \u003cp\u003e*0.542*\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\"\u003e \u003cp\u003e\u003cem\u003eP value\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\"\u003e \u003cp\u003e*0.629*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\"\u003e \u003cp\u003e*0.599*\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\"\u003e \u003cp\u003e\u003cb\u003eEpithelial layer\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\"\u003e \u003cp\u003eBladder cancer group\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\"\u003e \u003cp\u003e8.33 ± 2.25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\"\u003e \u003cp\u003e10.00 ± 1.55\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\"\u003e \u003cp\u003eMuscularis propria group\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\"\u003e \u003cp\u003e9.33 ± 1.37\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\"\u003e \u003cp\u003e10.50 ± 1.64\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\"\u003e \u003cp\u003e\u003cem\u003et value\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\"\u003e \u003cp\u003e*0.930*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\"\u003e \u003cp\u003e*0.542*\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\"\u003e \u003cp\u003e\u003cem\u003eP value\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\"\u003e \u003cp\u003e*0.374*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\"\u003e \u003cp\u003e*0.599*\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"4\"\u003e\u003cb\u003eNote\u003c/b\u003e: Data are presented as mean ± SD (n = 6 per group). Immunohistochemical staining was semi-quantitatively scored as the product of staining intensity (0–3) and the percentage of positive cells (0–4), ranging from 0 to 12. Statistical analysis was performed by unpaired t-test. P \u0026lt; 0.05 was considered statistically significant. No significant differences were observed between the bladder cancer group and the muscularis propria group in either the muscle layer or the epithelial layer for both NLRP3 and GSDMD expression.\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e\n\u003ch3\u003e2. Effects of high glucose and LPS on SV-HUC-1 cell functions\u003c/h3\u003e\n\u003cp\u003eWe further utilized human immortalized bladder epithelial cells (SV-HUC-1) to observe the effects of high glucose and LPS stimulation on cellular function (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). CCK-8 assay results showed that compared with the control group, cell viability in the high-glucose group (95.679\u0026thinsp;\u0026plusmn;\u0026thinsp;5.395%) did not change significantly (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05). In contrast, cell viability in the LPS group (70.828\u0026thinsp;\u0026plusmn;\u0026thinsp;6.078%) was significantly lower than that in the control and high-glucose groups (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eEffects of high glucose and LPS on SV-HUC-1 cell functions\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGroup\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003en\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eCell Viability (%)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003ePyroptosis Rate (%)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eMigrating Cell Count\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eControl\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e5/3/3\u0026sup1;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e100.0\u0026thinsp;\u0026plusmn;\u0026thinsp;7.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e2.66\u0026thinsp;\u0026plusmn;\u0026thinsp;0.25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e165.0\u0026thinsp;\u0026plusmn;\u0026thinsp;17.5\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHigh glucose\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e5/3/3\u0026sup1;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e95.7\u0026thinsp;\u0026plusmn;\u0026thinsp;5.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e3.14\u0026thinsp;\u0026plusmn;\u0026thinsp;0.54\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e172.0\u0026thinsp;\u0026plusmn;\u0026thinsp;13.1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLPS\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e5/3/3\u0026sup1;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e70.8\u0026thinsp;\u0026plusmn;\u0026thinsp;6.1*#\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e9.60\u0026thinsp;\u0026plusmn;\u0026thinsp;1.01*#\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e73.3\u0026thinsp;\u0026plusmn;\u0026thinsp;18.9*#\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eF\u003c/b\u003e\u003cb\u003e-value\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cb\u003e24.876\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cb\u003e108.234\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cb\u003e42.567\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eP\u003c/b\u003e\u003cb\u003e-value\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cb\u003e\u0026lt;\u0026thinsp;0.001\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cb\u003e\u0026lt;\u0026thinsp;0.001\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cb\u003e\u0026lt;\u0026thinsp;0.001\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"5\"\u003e\u003cb\u003eNote\u003c/b\u003e: Data are presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD. \u0026sup1;n\u0026thinsp;=\u0026thinsp;5 for cell viability assay, n\u0026thinsp;=\u0026thinsp;3 for pyroptosis and migration assays. Cell viability was measured by CCK-8 assay after 48 h treatment. Pyroptosis rate was determined by Annexin V-FITC/PI flow cytometry after 48 h treatment. Cell migration capacity was assessed by scratch assay after 48 h treatment, and the number of migrating cells was counted. Statistical analysis was performed by one-way ANOVA followed by post-hoc test. P\u0026thinsp;\u0026lt;\u0026thinsp;0.05 vs. Control group; #P\u0026thinsp;\u0026lt;\u0026thinsp;0.05 vs. High glucose group.\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv description=\"\" class=\"Drawing\" id=\"1487620522\" name=\"图片 2\"\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eAnnexin V-FITC/PI double-staining flow cytometry was employed to detect pyroptotic cell death. Results indicated that the pyroptosis rate in the high-glucose group (3.137\u0026thinsp;\u0026plusmn;\u0026thinsp;0.535%) showed no significant difference compared to the control group (2.660\u0026thinsp;\u0026plusmn;\u0026thinsp;0.252%) (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05). The pyroptosis rate in the LPS group (9.600\u0026thinsp;\u0026plusmn;\u0026thinsp;1.005%) was significantly higher than that in both the control and high-glucose groups (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). The scratch assay assessed cell migration capacity. Results showed no significant difference in migrating cell counts between the high-glucose group (172.000\u0026thinsp;\u0026plusmn;\u0026thinsp;13.077) and the control group (165.000\u0026thinsp;\u0026plusmn;\u0026thinsp;17.521) (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05). The number of migrating cells in the LPS group (73.333\u0026thinsp;\u0026plusmn;\u0026thinsp;18.903) was significantly lower than that in both the control and high-glucose groups (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC). Representative images of the scratch assay are shown in (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD-F).\u003c/p\u003e\n\u003ch3\u003e3. Effects of high glucose and LPS on NLRP3 inflammasome pathway activation\u003c/h3\u003e\n\u003cp\u003eWestern blot analysis was performed to detect the expression levels of NLRP3, Caspase-1, and IL-1β. As presented in Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e, NLRP3 (0.632\u0026thinsp;\u0026plusmn;\u0026thinsp;0.035 vs. 0.352\u0026thinsp;\u0026plusmn;\u0026thinsp;0.011), Caspase-1 (0.666\u0026thinsp;\u0026plusmn;\u0026thinsp;0.032 vs. 0.313\u0026thinsp;\u0026plusmn;\u0026thinsp;0.044), and IL-1β (0.549\u0026thinsp;\u0026plusmn;\u0026thinsp;0.022 vs. 0.328\u0026thinsp;\u0026plusmn;\u0026thinsp;0.049) all showed considerably higher expression levels in the high-glucose group than in the control group (all \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001) (Figs.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e3\u003c/span\u003eA).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eEffects of high glucose and LPS on NLRP3 inflammasome pathway protein expression\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGroup\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eNLRP3\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eCaspase-1\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eIL-1β\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eHigh glucose experiment\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eControl\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.35\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.31\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.33\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHigh glucose\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.63\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.67\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.55\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02*\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eP-value\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003e\u0026lt;\u0026thinsp;0.001\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003e\u0026lt;\u0026thinsp;0.001\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003e\u0026lt;\u0026thinsp;0.001\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eLPS experiment\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eControl\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.38\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.41\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.36\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLPS\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.77\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.84\u0026thinsp;\u0026plusmn;\u0026thinsp;0.09*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.76\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04*\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eP-value\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003e\u0026lt;\u0026thinsp;0.001\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003e\u0026lt;\u0026thinsp;0.001\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003e\u0026lt;\u0026thinsp;0.001\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"4\"\u003e\u003cb\u003eNote\u003c/b\u003e: Data are presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD (n\u0026thinsp;=\u0026thinsp;3). SV-HUC-1 cells were treated with high glucose (30 mM) or LPS (1 \u0026micro;g/mL) for 24 h. Protein levels of NLRP3, Caspase-1, and IL-1β were measured by Western blot and normalized to β-actin. Statistical analysis was performed by independent samples t-test. P\u0026thinsp;\u0026lt;\u0026thinsp;0.001 vs. respective Control group.\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cdiv description=\"\" class=\"Drawing\" id=\"391894321\" name=\"图片 3\"\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eSimilarly, the expression levels of Caspase-1 (0.844\u0026thinsp;\u0026plusmn;\u0026thinsp;0.086 vs. 0.413\u0026thinsp;\u0026plusmn;\u0026thinsp;0.050), NLRP3 (0.769\u0026thinsp;\u0026plusmn;\u0026thinsp;0.047 vs. 0.384\u0026thinsp;\u0026plusmn;\u0026thinsp;0.029), and IL-1β (0.757\u0026thinsp;\u0026plusmn;\u0026thinsp;0.039 vs. 0.355\u0026thinsp;\u0026plusmn;\u0026thinsp;0.036) were considerably higher in the LPS group than in the control group (all \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001) (Figs.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). It is noteworthy that high glucose and LPS induced similar upregulation of the aforementioned proteins (approximately 1.8\u0026ndash;2.2-fold), yet they led to distinctly different cellular functional outcomes. The quantitative analysis of these blots is presented in \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e4\u003c/span\u003eA) (high glucose) and (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e4\u003c/span\u003eB) (LPS), confirming the significant upregulation of all three proteins.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThis study, through clinical sample analysis and in vitro cell experiments, demonstrates the differential regulatory effects of high glucose and LPS on the NLRP3/GSDMD pyroptosis pathway in human bladder epithelial cells. Our main results include: (1) In non-diabetic bladder cancer patients, there was no significant difference in NLRP3 and GSDMD expression between cancerous and adjacent non-cancerous tissues, indicating that malignant transformation is not the driving mechanism for continuous activation of this pathway. (2) Both high glucose and LPS significantly upregulate NLRP3, Caspase-1, and IL-1β protein expression in SV-HUC-1 cells, with comparable magnitudes of upregulation. (3) However, only LPS stimulation induces cell proliferation inhibition, increased pyroptosis rates, and reduced migration capacity, while high glucose does not cause significant alterations in these functional phenotypes. This finding demonstrates in bladder epithelial cells that \u0026ldquo;protein expression upregulation\u0026rdquo; and \u0026ldquo;pyroptosis execution\u0026rdquo; within the NLRP3 inflammasome pathway are two distinct biological processes. High glucose merely completes the \u0026ldquo;priming\u0026rdquo; step, while LPS provides the \u0026ldquo;activation\u0026rdquo; signal required for pyroptosis execution\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e,\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e. The observed dissociation between pathway upregulation and functional execution suggests that, in human bladder epithelial cells, the NLRP3 inflammasome is subject to stringent regulatory checkpoints beyond transcriptional control.\u003c/p\u003e \u003cp\u003eIn recent years, the NLRP3 inflammasome has garnered significant attention for its role in the pathogenesis of diabetic bladder dysfunction (DBD). Using a female type 1 diabetic Akita mouse model, Odom et al.\u003csup\u003e10\u003c/sup\u003e demonstrated that NLRP3 gene knockout completely prevented bladder inflammation\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e,\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e, urinary epithelial barrier damage\u003csup\u003e\u003cspan additionalcitationids=\"CR16\" citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e, and voiding dysfunction, confirming NLRP3-mediated inflammation as a key driver of DBD. Subsequent studies further revealed that NLRP3 activation contributes to detrusor dysfunction by upregulating prostaglandin receptor expression and abnormally regulating prostaglandin release\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. However, these conclusions were derived from whole-animal models, making it difficult to distinguish the contribution of hyperglycemia itself from other in vivo factors (such as neural, vascular, or immune cell involvement). More importantly, no previous studies had addressed whether the observed chain of events\u0026mdash;\"hyperglycemia \u0026rarr; NLRP3 activation \u0026rarr; urinary epithelial pyroptosis\"\u0026mdash;in animal models could be directly replicated in human bladder epithelial cells. This study utilized the human immortalized bladder epithelial cell line SV-HUC-1 to eliminate interference from the complex in vivo environment, directly examining the isolated effect of high glucose and filling this research gap.\u003c/p\u003e \u003cp\u003eNotably, we observed that whereas both LPS and high glucose considerably increase the NLRP3 pathway proteins, they produce quite different functional results. This phenomenon closely resembles conventional \"two-step model\" of NLRP3 activation\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e: Toll-like receptors or cytokines usually initiate the first step, which upregulates the transcriptional expression of NLRP3, pro-IL-1β, and other components via the NF-κB pathway; a second signal (like ATP, uric acid crystals, or bacterial toxins) is needed for the second stage (activation) to induce inflammasome assembly, caspase-1 activation, and GSDMD cleavage. Glucose metabolites, including damage-associated molecular patterns (DAMPs)\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e,\u003cspan additionalcitationids=\"CR20\" citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e, reactive oxygen species (ROS)\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e,\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e, and advanced glycation end products (AGEs)\u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e,\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e, may serve as starting signals in high-glucose situations. However, they lack a secondary signal sufficient to trigger full caspase-1 activation, thus remaining at the stage of protein expression upregulation. In contrast, LPS, a potent pathogen-associated molecular pattern, provides a strong priming signal via TLR4 and, in the context of bladder epithelial cells, appears to also provide sufficient activation signals to drive the entire pyroptosis process\u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e,\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e. This conclusion is consistent with previous research showing that high-glucose pretreatment alone increases NLRP3 expression in macrophages but necessitates further stimulation to trigger IL-1β production\u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThe findings of this study provide important insights into understanding bladder pathology in diabetic patients. Diabetic patients often present with asymptomatic bacteriuria or recurrent urinary tract infections, and infection is considered an aggravating factor in the progression of diabetic bladder dysfunction (DBD)\u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e,\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e. Our data indicate that prolonged hyperglycemia induces a preactivated state of the NLRP3 pathway in bladder epithelial cells. While this state does not directly cause injury, it triggers pyroptotic damage far exceeding normal levels upon encountering infection. This leads to disruption of the urinary epithelial barrier, amplified inflammatory cascades, and ultimately promotes the onset and progression of DBD. This \"two-hit\" model explains why enhanced glycemic control alone cannot fully prevent DBD, because the established \"preactivation\" state may possess metabolic memory effects\u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e,\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e, and infection, as the \"second hit\" operates independently of blood glucose levels. Furthermore, this model offers novel insights for intervention strategies: Targeting the initiation phase of the NLRP3 pathway (e.g., inhibiting the AGEs-RAGE axis or scavenging ROS\u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e) may serve as a primary prevention measure, while interventions focused on the activation phase (e.g., blocking caspase-1 or GSDMD) are suitable for diabetic patients with concurrent infections. It's interesting to note that prior research has documented NLRP3's sex-specific functions in DBD, with NLRP3 deletion shielding female Akita mice from bladder dysfunction but not males\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e,\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e,\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e. Our study was conducted in a human cell line without sex specification; any sex differences in the priming-activation dynamics of the NLRP3 pathway should be considered in future research.\u003c/p\u003e \u003cp\u003eThe innovation of this study is manifested in three key aspects. First, this study utilizes a human bladder epithelial cell model to directly evaluate the independent effect of high glucose on the NLRP3 pyroptosis pathway, thereby overcoming the confounding factors inherent in animal models. Second, it demonstrates the dissociation between pathway activation and functional execution in bladder epithelial cells, thus extending the two-step NLRP3 model to the urinary system. Third, based on these findings, this study proposes the \u0026ldquo;two-hit hypothesis\u0026rdquo; for DBD, providing a new perspective for understanding clinical phenomena. However, this study also presents several limitations. First, the in vitro experimental design failed to simulate the long-term and complex nature of diabetic conditions, including the accumulation of AGEs and the dynamic alterations in oxidative stress. Second, caspase-1 activity and GSDMD cleavage fragments were not directly detected; subsequent validation of pyroptosis pathway activation necessitates ELISA or western blot analysis of activated caspase-1 and GSDMD-NT fragments. Third, experiments combining high glucose with LPS stimulation were not conducted. Fourth, while LPS was employed as a surrogate for bacterial infection, clinical urinary tract infections involve complex pathogen-host interactions that cannot be fully recapitulated by a single PAMP. Fifth, our study did not investigate the upstream signaling events linking high glucose to NLRP3 priming, such as the AGEs-RAGE axis\u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e or the ROS-TXNIP pathway\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e,\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e, which warrant further investigation. Future studies could design a \"high glucose pretreatment\u0026thinsp;+\u0026thinsp;LPS stimulation\" protocol directly to validate the synergistic effects of dual insults.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eIn summary, this study demonstrates in human bladder epithelial cells that elevated glucose can activate the NLRP3 inflammasome pathway but is insufficient to initiate the execution of pyroptosis. The second hit is infectious stimulation, which is a prerequisite for pyroptosis and cellular failure. This finding provides a novel mechanistic explanation for diabetic bladder disease and offers a theoretical basis for developing early intervention strategies.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgments\u0026nbsp;\u003c/strong\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contribution:\u0026nbsp;\u003c/strong\u003eM.A. contributed to the research design, conducted the cell experiments, analyzed the data, and wrote the paper. A.A. and Y.G. helped with cell culture, immunohistochemistry studies, and data collection. G.S. gave valuable help in study design, project supervision, and manuscript drafting and revision. All writers read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding statement\u003c/strong\u003e\u003cstrong\u003e：\u003c/strong\u003eThe authors declare that no funding was received for this study.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability:\u0026nbsp;\u003c/strong\u003eThe datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting Interests:\u0026nbsp;\u003c/strong\u003eThe authors have no relevant financial or non-financial interests to disclose.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003ePouya, S. et al. Global and regional diabetes prevalence estimates for 2019 and projections for 2030 and 2045: Results from the International Diabetes Federation Diabetes Atlas, 9(th) edition. \u003cem\u003eDiabetes Res. Clin. 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Male Akita mice develop signs of bladder underactivity independent of NLRP3 as a result of a decrease in neurotransmitter release from efferent neurons. \u003cem\u003eAm. J. Physiol. Ren. Physiol.\u003c/em\u003e \u003cb\u003e325\u003c/b\u003e, F61\u0026ndash;f72. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org:10.1152/ajprenal.00284.2022\u003c/span\u003e\u003cspan address=\"https://doi.org:10.1152/ajprenal.00284.2022\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2023).\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Diabetic bladder dysfunction, NLRP3 inflammasome, Pyroptosis, High glucose, Lipopolysaccharide","lastPublishedDoi":"10.21203/rs.3.rs-8934076/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8934076/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eDiabetic bladder dysfunction (DBD) affects over half of all diabetics, although its pathogenesis remains unexplained. Animal studies have connected NLRP3 inflammasome-mediated pyroptosis to DBD. It is uncertain whether hyperglycemia directly activates this pathway in human bladder epithelial cells. Immunohistochemistry was used to identify NLRP3 and GSDMD expression in bladder cancer tissues and adjacent normal epithelial tissues from six non-diabetic patients with bladder cancer. Human immortalized bladder epithelial cells (SV-HUC-1) were treated with high glucose (30 mM) or LPS (1 \u0026micro;g/mL) for 24\u0026ndash;48 hours. Cell survival, pyroptosis, motility, and expression of NLRP3/Caspase-1/IL-1β were evaluated. In non-diabetic bladder cancer patients, there was no significant difference in NLRP3 or GSDMD expression between the malignant and normal tissues (P\u0026thinsp;\u0026gt;\u0026thinsp;0.05). Both high glucose and LPS significantly enhanced the expression of NLRP3, Caspase-1, and IL-1β proteins in SV-HUC-1 cells (approximately 1.7- to 2.1-fold increase, all P\u0026thinsp;\u0026lt;\u0026thinsp;0.001). High glucose by itself showed no discernible effects on these functional characteristics, while LPS treatment alone significantly decreased cell viability (70.8% vs control, P\u0026thinsp;\u0026lt;\u0026thinsp;0.05), increased pyroptosis rate (9.6% vs 2.7%, P\u0026thinsp;\u0026lt;\u0026thinsp;0.05), and impeded migratory capability (73.3 vs 165.0, P\u0026thinsp;\u0026lt;\u0026thinsp;0.05). This study offers the first proof that elevated glucose levels in human bladder epithelial cells are sufficient to trigger the NLRP3 inflammasome pathway but not enough to cause pyroptosis. A crucial \"second hit\" that converts pathway activation into functional cellular damage is LPS. These findings support a \"two-hit\" model for DBD etiology and provide fresh insights into early intervention options that target the priming phase of NLRP3 activation.\u003c/p\u003e","manuscriptTitle":"A two-hit model for NLRP3-mediated pyroptosis in human bladder epithelial cells: Hyperglycemia primes, infection triggers","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-03-13 17:46:14","doi":"10.21203/rs.3.rs-8934076/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"e1aacdbb-e5ce-4cac-baf8-ae2d0b62c06c","owner":[],"postedDate":"March 13th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":64422336,"name":"Biological sciences/Cell biology"},{"id":64422337,"name":"Health sciences/Diseases"},{"id":64422338,"name":"Health sciences/Endocrinology"},{"id":64422339,"name":"Health sciences/Urology"}],"tags":[],"updatedAt":"2026-03-17T03:40:27+00:00","versionOfRecord":[],"versionCreatedAt":"2026-03-13 17:46:14","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8934076","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8934076","identity":"rs-8934076","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}