BTK Mediates Inflammation, Mast Cell Activation, and Urothelial Barrier Disruption in IC/BPS model

BTK Mediates Inflammation, Mast Cell Activation, and Urothelial Barrier Disruption in IC/BPS model | 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 BTK Mediates Inflammation, Mast Cell Activation, and Urothelial Barrier Disruption in IC/BPS model Guang Wang, Bin-sen Li, Jin-yi Chu, Xian Chen, Tong-xin Yang, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7182790/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 30 Apr, 2026 Read the published version in Scientific Reports → Version 1 posted 10 You are reading this latest preprint version Abstract Background Interstitial cystitis/bladder pain syndrome (IC/BPS) is a chronic urinary disorder with unclear pathogenesis. Previous studies identified BTK as a hub gene potentially involved in IC/BPS. This study investigates BTK’s role in mast cell (MC) activation and bladder inflammation. Methods An LL-37-induced IC/BPS rat model and an in vitro MC model were established. BTK expression was modulated via adenoviral vectors and cell transfection. Bladder inflammation, MC degranulation, and urothelial barrier function were assessed using histology, ELISA, RT-qPCR, Western blot, IHC, TEM, and IF. MC function was evaluated via CCK-8, flow cytometry, Transwell, and TEM. Results LL-37 upregulated BTK in IC/BPS rats, promoting inflammation, cytokine release, collagen deposition, MC degranulation, and urothelial damage. BTK overexpression exacerbated, while knockdown alleviated these effects. In vitro, LL-37 stimulated MC proliferation, invasion, and degranulation, and reduced apoptosis. Co-culture with activated MCs decreased glycosaminoglycan (GAG) and tight junction (TJ) proteins in SV-HUC-1 cells, enhanced by BTK overexpression and reversed by knockdown. Conclusions BTK promotes LL-37-induced MC activation and urothelial barrier disruption by suppressing GAGs and TJ proteins, contributing to IC/BPS pathophysiology. Biological sciences/Cell biology Health sciences/Urology BTK Interstitial cystitis bladder pain syndrome Mast cell Bladder barrier Figures Figure 1 Figure 2 Figure 3 Figure 4 1. Introduction Interstitial cystitis/bladder pain syndrome (IC/BPS) is a chronic bladder disorder with a rising incidence, yet its exact pathogenesis remains unclear. Due to the absence of well-defined etiological mechanisms and diagnostic criteria, IC/BPS is typically diagnosed by exclusion. However, the significant heterogeneity in clinical symptoms and pathophysiological features among patients poses major challenges for accurate diagnosis, effective disease management, and the development of targeted therapies. Mast cells (MCs) are immune cells of hematopoietic origin, derived from the bone marrow and yolk sac, and are widely distributed throughout loose connective tissues in the body. Previous studies have demonstrated that increased proliferation of mast cells (MCs) in bladder tissue may be associated with symptom onset in IC/BPS[ 1 ]. External inflammatory stimuli can activate MCs and trigger degranulation, releasing histamine, cytokines, and proteases that further promote inflammatory cell infiltration into the tissue[ 2 ]. This altered microenvironment reinforces MC activation and degranulation, forming a positive feedback loop that exacerbates IC/BPS symptoms. Aberrant activation and degranulation of mast cells (MCs) are key contributors to the pathogenesis of various diseases. MC stabilizers alleviate disease symptoms by reducing MC activation and degranulation, thereby limiting the release of inflammatory mediators, histamine, and proteases. In IC/BPS, MC activation and degranulation have also been identified as pathological factors involved in disease development, a finding supported by clinical and pathological examinations[ 3 , 4 ]. However, the molecular mechanisms by which MC activation contributes to IC/BPS progression remain poorly understood. Therefore, elucidating the mechanisms that regulate MC proliferation and activation is crucial for developing strategies to suppress MC-mediated degranulation and inflammation in IC/BPS. Bruton’s tyrosine kinase (BTK), a member of the TEC family of non-receptor tyrosine kinases, functions as a key intracellular signaling molecule predominantly expressed in B cells and various hematopoietic lineages. BTK is an essential component of the downstream signaling cascade of the B cell receptor (BCR), regulating B cell maturation, survival, activation, cytokine production, and antigen-dependent stimulation. In addition, BTK also plays roles in modulating the biological behavior of other immune and neuronally-derived cells[ 5 , 6 ]. Emerging evidence has shown that BTK can activate mast cells, promoting the production of eicosanoids and reactive oxygen species (ROS), thereby contributing to allergic responses[ 7 ]. However, whether BTK is involved in the pathogenesis and progression of IC/BPS has not yet been reported. Based on the above findings, we hypothesized that BTK may affect urothelial barrier function by modulating mast cell activation. Therefore, we established an LL-37-induced IC/BPS animal model to investigate the role of BTK expression in bladder inflammation, mast cell activation, urothelial barrier integrity, and its potential downstream signaling pathways. Activated MCs were also co-cultured with immortalized human ureteral epithelial cells (SV-HUC-1) to explore the impact of BTK on MC activation and the subsequent effects of MC activation on urothelial barrier function. 2. Materials and Methods 2.1 Experimental design BTK is expressed in a variety of immune cells, including B cells, macrophages, mast cells, basophils, and platelets, and participates in the regulation of both innate and adaptive immune responses. Based on our previous findings indicating that BTK regulates mast cell proliferation, invasion, and degranulation, an LL-37-induced IC/BPS rat model was established to investigate the functional role of BTK in vivo and to explore its underlying molecular mechanisms. 2.2 Animal model and experimental design To investigate the role of BTK in IC/BPS, an adenovirus-associated virus (AAV)-mediated gene overexpression or knockdown approach was employed in combination with intravesical instillation of LL-37 to induce an IC/BPS model. A total of 36 male Sprague-Dawley (SD) rats (6–8 weeks old, 200 ± 20 g) were randomly assigned into six groups (n = 6 per group): Sham group, IC/BPS group, IC/BPS + OV-NC group, IC/BPS + OV-BTK group, IC/BPS + KD-NC group, and IC/BPS + KD-BTK group. (Table 1 ). Table 1. Grouping of animal models. 1 The injection volume of normal saline in sham group and IC/BPS group was the same as that of AAV in other groups. Groups LL-37 Process AAV Process sham None（normal saline） None（normal saline） IC/BPS Yes None（normal saline） IC/BPS+OV-NC Yes negative control IC/BPS+OV-BTK Yes BTK Overexpression IC/BPS+KD-NC Yes negative control IC/BPS+KD-BTK Yes BTK knock down Pain-related responses were evaluated using Von Frey filaments with forces of 0.07 g, 0.4 g, 1.0 g, and 4.0 g. Pain scores were recorded based on the following criteria (0 = no response; 1 = licking or body twitching; 2 = jumping). After completion of all experimental procedures, rats were euthanized, and bladder tissues were harvested for further analyses, including hematoxylin and eosin (H&E) staining, Masson's trichrome staining, immunohistochemistry (IHC), immunofluorescence (IF), enzyme-linked immunosorbent assay (ELISA), real-time quantitative PCR (RT-qPCR), Western blotting, and transmission electron microscopy (TEM). 2.3 Animal Anesthesia and Euthanasia Procedures Adult male SpragueDawley rats (200 ± 20 g) were anesthetized by intraperitoneal injection of sodium pentobarbital (50 mg/kg), diluted 1:3 in sterile saline. Adequate anesthesia was confirmed by the absence of pedalwithdrawal and palpebral reflexes, and body temperature was maintained at 37°C on a thermostatic pad throughout the procedure. At the experimental endpoint, animals were placed in a sealed chamber filled with CO₂ at a displacement rate of 25–30% of the chamber volume per minute; once respiration ceased, cervical dislocation was performed to ensure death, followed immediately by tissue collection for further analyses. 2.4 Experiment in vitro BMMSCs were counted and seeded at 5 × 10^5 cells mL⁻¹ in 30 mL DMEM/F-12 supplemented with 100 ng mL⁻¹ recombinant human stem cell factor (rhSCF), 100 ng mL⁻¹ recombinant human interleukin-6 (rhIL-6), and 30 ng mL⁻¹ recombinant human interleukin-3 (rhIL-3); rhIL-3 was included only during the first week. After 1 week, the medium was replaced with fresh DMEM/F-12 containing 100 ng mL⁻¹ rhSCF and 50 ng mL⁻¹ rhIL-6, and cultures were maintained with weekly medium changes. From week 3 onward, non-adherent cells were transferred to new flasks at each change to remove adherent contaminants, and debris was discarded. Continuous induction for 7–10 weeks yielded mature mast cells (MCs)[ 8 ]. These cells were allowed to adhere to poly-L-lysine–coated coverslips, fixed, and stained with toluidine blue to visualize characteristic basophilic granules, and their immunophenotype was verified by flow-cytometric analysis of c-kit and CD23 surface expression (Fig. 1 ). Human mast cells (MCs) and SV-HUC-1 human urothelial cells were cultured to investigate the role of BTK in MC activation and its regulatory effects on urothelial transmembrane barrier function. To establish an in vitro IC/BPS model, MCs were stimulated with varying concentrations of LL-37 (0.1, 1, 10, 20, and 100 µg/mL) for 12 hours. Based on preliminary screening, 20 µg/mL was selected as the optimal concentration for subsequent experiments. MCs and SV-HUC-1 cells were then co-cultured in Transwell chambers to assess the impact of MC activation on epithelial barrier function. Six experimental groups were established: untreated control group (NC), LL-37 stimulation group, BTK knockdown group (KD-BTK), BTK overexpression group (OV-BTK), and corresponding negative control groups (KD-NC and OV-NC). These groups were used to evaluate the effects of BTK on MC activation and MC-mediated disruption of the urothelial barrier. 2.5 Molecular and functional experiments MC proliferation was assessed using the Cell Counting Kit-8 (CCK-8) assay. Cell cycle progression and apoptosis rates were analyzed by flow cytometry, while cell invasive capacity was evaluated using Transwell chamber assays. MC degranulation was quantified by measuring tryptase and histamine secretion levels via ELISA, and the release of granule contents was further visualized by transmission electron microscopy (TEM). To assess urothelial barrier function, ELISA was used to quantify the secretion of glycosaminoglycans (GAGs), including hyaluronic acid (HA), chondroitin sulfate (CS), dermatan sulfate (DS), and heparan sulfate (HS). The expression of tight junction proteins—ZO-1, Occludin, and Claudin-1—was measured by RT-qPCR and Western blotting. Transepithelial electrical resistance (TEER) assays were performed to evaluate barrier integrity, and patch-clamp electrophysiology was used to detect changes in membrane potential. Additionally, RT-qPCR and Western blotting were conducted to analyze the expression levels of BTK and downstream signaling proteins including Bcl-2, P21, and c-Myc. 2.6 Statistics All data were analyzed and visualized using GraphPad Prism 8.0 software. Quantitative data with normal distribution were expressed as mean ± standard deviation (SD). Comparisons between two groups were performed using the Student’s t -test, while one-way analysis of variance (ANOVA) was used for comparisons among multiple groups. Tukey’s post hoc test was applied for pairwise comparisons between multiple groups. A P value < 0.05 was considered statistically significant. 3. Results This section may be divided by subheadings. It should provide a concise and precise description of the experimental results, their interpretation, as well as the experimental conclusions that can be drawn. 3.1. Animal model 3.1.1 Pathological effect of BTK expression on bladder tissue of IC/BPS rats Following euthanasia, bladder tissues were harvested from rats for histological analysis. Rat bladder tissue Hematoxylin and eosin (H&E) staining (Fig. 2 A) showed that bladder tissues in the sham group exhibited intact architecture, thick mucosa, and abundant smooth muscle fibers, with no signs of inflammatory cell infiltration. In contrast, the IC/BPS group displayed typical inflammatory features, including submucosal hemorrhage, pronounced interstitial edema, and marked inflammatory cell infiltration. In the BTK overexpression group, inflammatory infiltration was further aggravated, with partial epithelial loss and increased microvascular density in the interstitium. Compared with the KD-NC group, the BTK knockdown group showed reduced inflammatory infiltration and a thickened bladder wall; Masson's trichrome staining (Fig. 2 B–C) revealed well-organized and abundant smooth muscle fibers in the sham group. In the IC/BPS group, there was significant collagen fiber deposition, which was markedly higher than in the sham group ( P < 0.001). BTK overexpression further increased collagen accumulation and disrupted smooth muscle fiber organization ( P < 0.05). In contrast, collagen fiber content was reduced and fibrosis was alleviated in the BTK knockdown group compared to KD-NC ( P < 0.05). These findings indicate that BTK knockdown mitigates LL-37-induced bladder inflammation and fibrosis in IC/BPS rats. 3.1.2 Regulation of BTK on bladder inflammation and urinary epithelial GAGs in IC/BPS rats Inflammatory markers including myeloperoxidase (MPO), interleukin-1β (IL-1β), IL-6, and tumor necrosis factor-α (TNF-α) in bladder tissues were quantified by ELISA (Fig. 2 D(a–d)). Compared to the sham group, levels of MPO (Fig. 2 D(a)), IL-1β (Fig. 2 D(b)), IL-6 (Fig. 2 D(c)), and TNF-α (Fig. 2 D(d)) were significantly elevated in the IC/BPS group. These inflammatory mediators were further upregulated in the BTK overexpression group (MPO: P < 0.05; IL-1β: P < 0.01; IL-6: P < 0.01; TNF-α: P < 0.01). Conversely, BTK knockdown significantly attenuated LL-37-induced upregulation of these inflammatory markers (MPO, IL-1β, IL-6: P < 0.05; TNF-α: P < 0.05). ELISA results of glycosaminoglycan (GAG) levels in urethral tissues showed that concentrations of hyaluronic acid (HA), chondroitin sulfate (CS), dermatan sulfate (DS), and heparan sulfate (HS) were significantly reduced in the IC/BPS group compared to the sham group (HA: P < 0.01, Fig. 2 D(e); CS: Fig. 2 D(f); DS: Fig. 2 D(g); HS: Fig. 2 D(h)). BTK overexpression further decreased HA ( P < 0.05), CS ( P < 0.01), DS, and HS ( P < 0.05), while BTK knockdown significantly increased the levels of HA ( P < 0.05), CS, DS ( P < 0.01), and HS compared to the respective controls. 3.1.3 Regulation of BTK on urinary epithelial barrier in IC/BPS rats The expression of tight junction (TJ)-related markers ZO-1, Occludin, and Claudin-1 in bladder tissues was evaluated by RT-qPCR (Fig. 3 A(a–c)) and Western blotting (Fig. 3 A(d–g)). Compared with the sham group, mRNA and protein levels of ZO-1 ( P < 0.001, Fig. 3 (a, e)), Occludin ( P < 0.001, Fig. 3 A (b, f)), and Claudin-1 ( P < 0.001, Fig. 3 A (c, g)) were significantly decreased in the IC/BPS group. BTK overexpression markedly suppressed the expression of ZO-1, Occludin ( P < 0.01), and Claudin-1 ( P < 0.05) at both the mRNA and protein levels compared to the OV-NC group. In contrast, BTK knockdown significantly upregulated ZO-1, Occludin ( P < 0.01), and Claudin-1 ( P < 0.01) expression. These findings suggest that LL-37 stimulation induces bladder inflammation, disrupts the GAG layer of the urethra, and impairs urothelial barrier function. Inhibition of BTK attenuates LL-37-induced inflammation and preserves urothelial barrier integrity. 3.1.4 Regulation of BTK expression on degranulation of MCs in bladder tissue of IC/BPS rats Immunohistochemical (IHC) staining was performed to detect tryptase-positive cells in bladder tissue sections (Fig. 3 B(a)). Compared with the sham group, the number of tryptase-positive cells was significantly increased in the IC/BPS group. The BTK overexpression group showed more tryptase-positive cells than the OV-NC group, while the BTK knockdown group exhibited a reduction in tryptase-positive cells. Transmission electron microscopy (TEM) was used to evaluate mast cell (MC) degranulation in bladder tissues (Fig. 3 B(b)). MCs in the bladder exhibited diverse morphologies. In the IC/BPS group, cytoplasmic granules—spherical or ovoid in shape—were observed being released into the extracellular space. These granules varied in size and were membrane-bound. The BTK overexpression group displayed increased extracellular granule release compared to controls, whereas fewer granules were observed in the BTK knockdown group. These results indicate that BTK overexpression promotes mast cell activation and degranulation in the bladder tissue of IC/BPS rats, while BTK knockdown reduces mast cell activation in this pathological context. 3.1.5 Expression of BTK in bladder tissue of IC/BPS rats Immunohistochemical (IHC) staining was used to assess BTK expression in rat bladder tissues (Fig. 3 C(a)). Compared to the sham group, the IC/BPS group showed a significant increase in BTK-positive cells. The number of BTK-positive cells was further elevated in the BTK overexpression group compared to the OV-NC group, whereas BTK knockdown led to a marked reduction in BTK-positive cells. Additionally, immunofluorescence (IF) staining was performed to evaluate the expression of BTK and tryptase in bladder tissues (Fig. 3 C(b–d)). Consistent with the IHC findings, fluorescence intensities of both BTK and tryptase were significantly higher in the IC/BPS group compared to the sham group ( P < 0.01). BTK overexpression further enhanced the fluorescence intensities of BTK ( P < 0.05) and tryptase ( P < 0.01), while BTK knockdown resulted in a significant reduction in their fluorescence signals ( P < 0.05). 3.2. BTK regulates MCs activation 3.2.1 Expression of BTK in MCs and its regulation on SV-HUC-1 membrane ions To determine the most effective BTK knockdown target, three BTK-targeting shRNA adenoviral vectors (KD-BTK#1, #2, #3) were transfected into 293T cells. After 48 hours, strong fluorescence indicated successful transfection, and RT-qPCR showed significantly reduced BTK mRNA in all knockdown groups versus KD-NC (P < 0.001, Fig. 4 A(a)). Western blot confirmed reduced BTK protein expression (P < 0.05, Fig. 4 A(b)), with KD-BTK#2 being the most efficient. MCs were then transduced with KD-NC, KD-BTK#2 (KD-BTK), OV-NC, or OV-BTK and stimulated with 20 µg/mL LL-37. LL-37 significantly increased BTK expression in MCs (P < 0.001, Fig. 4 A(c)); BTK was significantly reduced in KD-BTK (P < 0.01) and increased in OV-BTK (P < 0.01) compared to their respective controls. To assess barrier function, SV-HUC-1 cells were co-cultured with treated MCs. LL-37-treated MCs increased SV-HUC-1 whole-cell currents (P < 0.001). Co-culture with LL-37 + KD-BTK MCs reduced current versus LL-37 + KD-NC (P < 0.001), while LL-37 + OV-BTK further increased current compared to LL-37 + OV-NC (P < 0.001, Fig. 4 B). 3.2.2 BTK regulates MCs activation and affects gags levels in SV-HUC-1 cells ELISA revealed significantly elevated LL-37 levels in SV-HUC-1 supernatants co-cultured with LL-37-stimulated MCs compared to control (P < 0.001). BTK knockdown significantly reduced LL-37 levels versus KD-NC (P < 0.05), while BTK overexpression increased them (P < 0.05). ELISA of SV-HUC-1 supernatants showed that co-culture with LL-37-stimulated MCs significantly reduced HA (P < 0.01), CS (P < 0.01), DS (P < 0.001), and HS (P < 0.01) levels. BTK knockdown significantly increased these GAGs compared to KD-NC (P < 0.05), while overexpression significantly decreased them compared to OV-NC (HA: P < 0.05; CS: P < 0.01; DS: P < 0.01; HS: P < 0.05). 3.2.3 BTK regulates MCs activation and affects urothelial barrier RT-qPCR and Western blot analyses were performed to evaluate the expression levels of tight junction (TJ)-related genes ZO-1, Occludin, and Claudin-1 in SV-HUC-1 cells. As shown in Fig. 4 C(a–c), SV-HUC-1 cells co-cultured with LL-37-stimulated MCs exhibited significantly decreased mRNA expression of ZO-1 ( P < 0.001), Occludin ( P < 0.001), and Claudin-1 ( P < 0.001) compared with the SV-HUC-1 monoculture group. Knockdown of BTK in LL-37-stimulated MCs significantly restored the mRNA expression of ZO-1 ( P < 0.05), Occludin ( P < 0.05), and Claudin-1 ( P < 0.05) in SV-HUC-1 cells. Conversely, BTK overexpression in LL-37-stimulated MCs further suppressed ZO-1 ( P < 0.01), Occludin ( P < 0.001), and Claudin-1 ( P < 0.001) expression compared to the LL-37-only co-culture group. Western blot results were consistent with RT-qPCR findings (Fig. 4 C(d–g)). Protein levels of ZO-1 ( P < 0.001, Fig. 4 C(e)), Occludin ( P < 0.001, Fig. 4 C(f)), and Claudin-1 ( P < 0.001, Fig. 4 C (g)) were significantly decreased in SV-HUC-1 cells co-cultured with LL-37-treated MCs. BTK knockdown led to a significant increase in ZO-1 ( P < 0.05), Occludin ( P < 0.05), and Claudin-1 ( P < 0.001) protein expression, while BTK overexpression further suppressed these levels of protein compared with the OV-NC group (ZO-1: P < 0.01; Occludin: P < 0.01; Claudin-1: P < 0.001). Transepithelial electrical resistance (TEER) measurements of SV-HUC-1 monolayers were performed to assess barrier integrity (Fig. 4 C(h)). Co-culture with LL-37-stimulated MCs significantly reduced TEER values compared to the monoculture group ( P < 0.001). BTK knockdown slightly increased TEER, while BTK overexpression led to a marked reduction in TEER values ( P < 0.01). Taken together, these findings suggest that BTK promotes LL-37-induced MC activation, which in turn disrupts the urothelial barrier. Conversely, BTK knockdown attenuates MC activation and helps preserve urothelial barrier function. 3.3. Figures, Tables and Schemes Main Experimental Reagents and Manufacturers Human recombinant IL-3 and human recombinant stem cell factor SCF Gibco™, Thermo Fisher Scientific (Waltham, MA, USA) SV-HUC-1 cells (human urothelial cell line), Human bone marrow mesenchymal stem cells (HBMMSC) Shanghai Zhongqiao Xinzhou Biotechnology Co., Ltd. (Shanghai, China) Annexin V-FITC/PI Apoptosis Detection Kit Absin Bioscience Inc. (Shanghai, China) CCK-8 Assay Kit Beyotime Biotechnology (Shanghai, China) Transwell Chamber Corning Incorporated (USA) Cell Cycle Detection Kit Elabscience Biotechnology Co., Ltd. (Wuhan, China) Human Trypsin and Histamine ELISA Kits MLBIO – Shanghai Enzyme-linked Biotechnology Co., Ltd. FastKing RT Kit (With gDNase), FastKing First-Strand cDNA Synthesis Kit TIANGEN Biotech Co., Ltd. (Beijing, China) Taq Pro Universal SYBR qPCR Master Mix Vazyme Biotech Co., Ltd. (Nanjing, China) Protease Inhibitor Cocktail, PhosSTOP Phosphorylation Protease Inhibitor Roche Diagnostics (Switzerland) BTK Antibody (DF6472) Affinity Biosciences Inc. (USA) Mast Cell Tryptase (Ab2378), Pre-adsorbed Goat Anti-Mouse IgG H&L (CY3) (ab97035) Abcam plc (UK) DyLight 488 Labeled Goat Anti-Rabbit IgG (H + L) (5230 − 0385) KPL (SeraCare Life Sciences, USA) Antibodies: Bcl-2 (bsm-33411M), p21 (bs-55160R), ZO-1 (bs-1329R), Occludin (bs-10011R), Claudin-1 (bs-1428R) Bioss Antibodies (Beijing, China) Antibodies: c-Myc (18583S), Secondary Antibodies (7074, 7076) Cell Signaling Technology, Inc. (USA) GAPDH Antibody (P30008M) Abmart Inc. (Shanghai, China) 4. Discussion Previous studies on the etiology and pathophysiology of IC/BPS have suggested that mast cells (MCs) may play a critical role in disease progression. The impact of MCs on epithelial barrier function has been well documented[ 9 ]. In a mouse model of irritable bowel syndrome, increased paracellular permeability, enhanced MC degranulation, and elevated IFN-γ levels were observed, along with morphological disruption of the colonic epithelial barrier and downregulation of tight junction proteins such as Zona Occludens 2 (ZO-2) and Occludin. In our study, we established a co-culture system of MCs and SV-HUC-1 urothelial cells to evaluate how differently treated MCs influence the expression of glycosaminoglycans (GAGs) and tight junction (TJ)-associated proteins in vitro. The results showed that SV-HUC-1 cells co-cultured with LL-37-stimulated MCs exhibited increased whole-cell currents and intracellular calcium concentrations, along with reduced expression of GAGs (HA, CS, DS, and HS) and TJ proteins (ZO-1, Occludin, and Claudin-1). Co-culture with LL-37-treated and BTK-overexpressing MCs further elevated cellular current and calcium influx, while further suppressing GAG and TJ protein expression. In contrast, BTK knockdown in MCs produced the opposite effect, indicating that BTK plays a regulatory role in MC-mediated disruption of the urothelial barrier. In recent years, an increasing number of studies have utilized in vitro co-culture systems to investigate how mast cell (MC) degranulation and the release of granule-stored mediators regulate physiological processes in neighboring cells. For instance, co-culture with MCs promotes gastric cancer cell proliferation, invasion, migration, and resistance to H₂O₂-induced apoptosis[ 10 ]. MCs also enhance human lung fibroblast contractility in a time- and concentration-dependent manner, an effect not blocked by the tryptase inhibitor bis[5-imidazo(2,1-b)-benzimidazolyl]methane[ 11 ]. Consistent with these findings, our study demonstrated that co-culture of LL-37-activated MCs with SV-HUC-1 urothelial cells resulted in a marked reduction in the expression of glycosaminoglycans (GAGs) and tight junction (TJ) proteins in SV-HUC-1 cells. These results support the concept that activated MCs can disrupt epithelial barrier function through the release of soluble mediators. Mediators secreted by mast cells (MCs) can act as triggers of epithelial barrier dysfunction. Substances such as histamine, tryptase, and prostaglandin D₂ (PGD₂) have been shown to increase epithelial secretion, while other MC-derived products may directly impair epithelial integrity[ 12 ]. Notably, tryptase and chymase can proteolytically cleave tight junction (TJ) proteins, including Claudin-1, Claudin-3, Claudin-5, and junctional adhesion molecule A (JAM-A)[ 13 ]. Groschwitz et al.[ 13 ] demonstrated that MC-derived tryptase can activate protease-activated receptor 2 (PAR2), signaling to colonic epithelial cells in a paracrine manner. PAR2 activation is β-arrestin–dependent and leads to ERK1/2 phosphorylation, which reorganizes perijunctional F-actin and thereby increases epithelial permeability. MC degranulation exerts widespread effects on TJs across various epithelial systems. For example, MCs alter signaling pathways in alveolar epithelial cells, particularly those involved in pro-inflammatory cytokine induction and TJ disruption[ 14 ]. In stress-induced esophageal epithelial dysfunction, MC-derived tryptase activates PAR2, inducing inflammation, TJ disassembly, and cytoskeletal reorganization that contributes to intercellular gap widening[ 15 ]. In our study, co-culture of SV-HUC-1 urothelial cells with LL-37–activated MCs led to a decrease in the expression of GAGs and TJ-related proteins, indicating barrier disruption. The role of mast cells (MCs) in IC/BPS was confirmed in our study; however, the underlying mechanisms warrant further investigation. BTK and its associated signaling pathways play regulatory roles in various immune cells, including B cells, microglia, macrophages, and neutrophils. BTK inhibitors have been reported as potential treatments for central nervous system–related diseases. These inhibitors are capable of crossing the blood–brain barrier, restricting B cell activation, and reducing their antigen-presenting capacity to T cells, thereby attenuating proinflammatory T cell responses both in vivo and in vitro, ultimately contributing to disease suppression[ 16 ]. In our study, BTK overexpression in LL-37–stimulated MCs suppressed the expression of HA, CS, DS, and HS, as well as tight junction–associated proteins ZO-1, Occludin, and Claudin-1 in co-cultured SV-HUC-1 cells. Conversely, BTK knockdown enhanced the expression of GAGs and TJ proteins. However, the broader regulatory effects of BTK on epithelial or endothelial barrier function remain to be elucidated. We found that LL-37–activated MCs reduced the expression of GAGs and TJ proteins in SV-HUC-1 cells when co-cultured in vitro. BTK overexpression in MCs further enhanced LL-37-induced activation, aggravating the disruption of the urothelial barrier, while BTK knockdown mitigated the deleterious effects of MCs on SV-HUC-1 cells. We speculate that LL-37 and exogenous BTK promote MC degranulation, and the mediators released from MC granules subsequently alter GAG and TJ protein levels in SV-HUC-1 cells. However, our current data do not identify which specific granule-derived mediators are responsible for TJ disruption. Further studies are needed to clarify these mechanisms. Moreover, patch-clamp electrophysiology revealed alterations in whole-cell currents in SV-HUC-1 cells co-cultured with LL-37-activated MCs. The specific ion channels responsible for these changes remain unknown and warrant future investigation. 5. Conclusions BTK overexpression promotes mast cell proliferation, invasion, and degranulation, leading to impaired expression of glycosaminoglycans (GAGs) and tight junction (TJ) proteins in SV-HUC-1 cells, thereby compromising urothelial barrier function. This may represent one of the mechanisms by which BTK contributes to the pathogenesis of IC/BPS. In contrast, BTK knockdown attenuates MC activation and alleviates their detrimental effects on epithelial barrier integrity. Declarations Author Contributions: Conceptualization, Yang Tong-xin; methodology, Yang Tong-xin; validation, Wang Guang; formal analysis, Chen Xian; investigation, Li Bin-sen and Chu Jin-yi; writing—original draft preparation, Wang Guang and Li Bin-sen; writing—review and editing, Yang Tong-xin; visualization, Wang Guang; supervision, Fang Ke-wei and Li Jiong-ming; funding acquisition, Yang Tong-xin, Fang Ke-wei and Li Jiong-ming. All authors have read and agreed to the published version of the manuscript. Funding: This research was funded by the National Natural Science Foundation of China, Grant No. 82160150; the Yunnan Provincial Department of Science and Technology Fundamental Research Special Project, Grant No. 202201AT070240; the Yunnan Revitalization Talent Support Program, Grant No. XDYC-QNRC-2022-0308; the Yunnan International Joint R&D Center of Key Technologies in Urological Diagnosis and Treatment, Grant No. 202403AP140016; the Yunnan Provincial Department of Science and Technology Expert Workstation Fund, Grant No. 202405AF140058 and 202505AF350061; the Yunnan Health Training Project of High-Level Talents, Grant No. D-2024027; the Second Affiliated Hospital of Kunming Medical University Talent Echelon Training Project, Grant No. RCTDXS-202306; the Second Affiliated Hospital of Kunming Medical University External Cooperation Projects, Grant No. 2022dwhz10. Institutional Review Board Statement: This study was approved by the Ethics Approval Committee of the second affiliated hospital of Kunming medical university, and the registered number is PJ-2021-70. All animal procedures were conducted in accordance with the ARRIVE guidelines (https://arriveguidelines.org) and approved by the Institutional Animal Care and Use Committee (IACUC) of the second affiliated hospital of Kunming medical university under protocol number kmmu2021153. All methods were performed in accordance with relevant guidelines and regulations. Efforts were made to minimize animal suffering and to reduce the number of animals used. Informed Consent Statement: Not applicable. Data Availability Statement: All data generated or analyzed during this study are included in this published article and its supplementary information files. Conflicts of Interest: The authors declare no conflicts of interest. 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Malik, S.T.; Birch, B.R.; Voegeli, D.; Fader, M.; Foria, V.; Cooper, A.J.; Walls, A.F.; Lwaleed, B.A. Distribution of Mast Cell Subtypes in Interstitial Cystitis: Implications for Novel Diagnostic and Therapeutic Strategies? J. Clin. Pathol. 2018 , 71 , 840–844, doi:10.1136/jclinpath-2017-204881. Carnero Contentti, E.; Correale, J. Bruton’s Tyrosine Kinase Inhibitors: A Promising Emerging Treatment Option for Multiple Sclerosis. Expert Opin. Emerg. Drugs 2020 , 25 , 377–381, doi:10.1080/14728214.2020.1822817. Yu, C.G.; Bondada, V.; Iqbal, H.; Moore, K.L.; Gensel, J.C.; Bondada, S.; Geddes, J.W. Inhibition of Bruton Tyrosine Kinase Reduces Neuroimmune Cascade and Promotes Recovery after Spinal Cord Injury. Int. J. Mol. Sci. 2021 , 23 , 355, doi:10.3390/ijms23010355. Kuehn, H.S.; Swindle, E.J.; Kim, M.-S.; Beaven, M.A.; Metcalfe, D.D.; Gilfillan, A.M. The Phosphoinositide 3-Kinase-Dependent Activation of Btk Is Required for Optimal Eicosanoid Production and Generation of Reactive Oxygen Species in Antigen-Stimulated Mast Cells. J. Immunol. 2008 , 181 , 7706–7712, doi:10.4049/jimmunol.181.11.7706. Bandara, G.; Metcalfe, D.D.; Kirshenbaum, A.S. Growth of Human Mast Cells from Bone Marrow and Peripheral Blood-Derived CD34+ Pluripotent Hematopoietic Cells. In Mast Cells ; Hughes, M.R., McNagny, K.M., Eds.; Methods in Molecular Biology; Springer New York: New York, NY, 2015; Vol. 1220, pp. 155–162 ISBN 978-1-4939-1567-5. Kortekaas Krohn, I.; Seys, S.F.; Lund, G.; Jonckheere, A.; Dierckx De Casterlé, I.; Ceuppens, J.L.; Steelant, B.; Hellings, P.W. Nasal Epithelial Barrier Dysfunction Increases Sensitization and Mast Cell Degranulation in the Absence of Allergic Inflammation. Allergy 2020 , 75 , 1155–1164, doi:10.1111/all.14132. Zhong, B.; Li, Y.; Liu, X.; Wang, D. Association of Mast Cell Infiltration with Gastric Cancer Progression. Oncol. Lett. 2017 , doi:10.3892/ol.2017.7380. Foley, T.T.; Ehrlich, H.P. Mast Cells Prevent Dexamethasone-Induced Cell Death of Cultured Fibroblasts: Relationship to Gap Junctional Intercellular Communications. Plast. Reconstr. Surg. 2014 , 133 , 638e–644e, doi:10.1097/PRS.0000000000000103. Groschwitz, K.R.; Ahrens, R.; Osterfeld, H.; Gurish, M.F.; Han, X.; Åbrink, M.; Finkelman, F.D.; Pejler, G.; Hogan, S.P. Mast Cells Regulate Homeostatic Intestinal Epithelial Migration and Barrier Function by a Chymase/Mcpt4-Dependent Mechanism. Proc. Natl. Acad. Sci. 2009 , 106 , 22381–22386, doi:10.1073/pnas.0906372106. Groschwitz, K.R.; Wu, D.; Osterfeld, H.; Ahrens, R.; Hogan, S.P. Chymase-Mediated Intestinal Epithelial Permeability Is Regulated by a Protease-Activating Receptor/Matrix Metalloproteinase-2-Dependent Mechanism. Am. J. Physiol.-Gastrointest. Liver Physiol. 2013 , 304 , G479–G489, doi:10.1152/ajpgi.00186.2012. Wu, M.-L.; Liu, F.-L.; Sun, J.; Li, X.; He, X.-Y.; Zheng, H.-Y.; Zhou, Y.-H.; Yan, Q.; Chen, L.; Yu, G.-Y.; et al. SARS-CoV-2-Triggered Mast Cell Rapid Degranulation Induces Alveolar Epithelial Inflammation and Lung Injury. Signal Transduct. Target. Ther. 2021 , 6 , 428, doi:10.1038/s41392-021-00849-0. Zhong, C.J.; Wang, K.; Zhang, L.; Yang, C.Q.; Zhang, K.; Zhou, S.P.; Duan, L.P. Mast Cell Activation Is Involved in Stress‐induced Epithelial Barrier Dysfunction in the Esophagus. J. Dig. Dis. 2015 , 16 , 186–196, doi:10.1111/1751-2980.12226. Li, R.; Tang, H.; Burns, J.C.; Hopkins, B.T.; Le Coz, C.; Zhang, B.; De Barcelos, I.P.; Romberg, N.; Goldstein, A.C.; Banwell, B.L.; et al. BTK Inhibition Limits B-Cell–T-Cell Interaction through Modulation of B-Cell Metabolism: Implications for Multiple Sclerosis Therapy. Acta Neuropathol. (Berl.) 2022 , 143 , 505–521, doi:10.1007/s00401-022-02411-w. Additional Declarations No competing interests reported. Supplementary Files Supplementaryfullblot.pdf Cite Share Download PDF Status: Published Journal Publication published 30 Apr, 2026 Read the published version in Scientific Reports → Version 1 posted Editorial decision: Revision requested 08 Dec, 2025 Reviews received at journal 21 Sep, 2025 Reviewers agreed at journal 12 Sep, 2025 Reviews received at journal 28 Aug, 2025 Reviewers agreed at journal 19 Aug, 2025 Reviewers invited by journal 14 Aug, 2025 Editor assigned by journal 14 Aug, 2025 Editor invited by journal 14 Aug, 2025 Submission checks completed at journal 06 Aug, 2025 First submitted to journal 06 Aug, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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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-7182790","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":502565353,"identity":"3092ac54-1b61-4044-a84e-e890566aa6d8","order_by":0,"name":"Guang Wang","email":"","orcid":"","institution":"The Second Affiliated Hospital of Kunming Medical University","correspondingAuthor":false,"prefix":"","firstName":"Guang","middleName":"","lastName":"Wang","suffix":""},{"id":502565354,"identity":"4507bf27-6f54-4248-ae93-a20ba031eac7","order_by":1,"name":"Bin-sen Li","email":"","orcid":"","institution":"The Second Affiliated Hospital of Kunming Medical University","correspondingAuthor":false,"prefix":"","firstName":"Bin-sen","middleName":"","lastName":"Li","suffix":""},{"id":502565355,"identity":"ef226fbb-df4f-445c-8977-24989788565b","order_by":2,"name":"Jin-yi Chu","email":"","orcid":"","institution":"The Second Affiliated Hospital of Kunming Medical University","correspondingAuthor":false,"prefix":"","firstName":"Jin-yi","middleName":"","lastName":"Chu","suffix":""},{"id":502565356,"identity":"31baff9a-795c-47f5-864a-89bd70eb0502","order_by":3,"name":"Xian Chen","email":"","orcid":"","institution":"The Second Affiliated Hospital of Kunming Medical University","correspondingAuthor":false,"prefix":"","firstName":"Xian","middleName":"","lastName":"Chen","suffix":""},{"id":502565357,"identity":"5933f0ec-2933-49a0-b2fe-3abf5178e6e0","order_by":4,"name":"Tong-xin Yang","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA+klEQVRIiWNgGAWjYFADZgbGhw0QpgFh1QcgWpgNSdTCwMAmSZQWg+NnD7/+UHHHbsNx3mOVM3O2JTawN2+TYKi5g1vLmbw0iwNnniXPbOZLu7lx2+3EBp5jZRIMx57h1nIgx8zgYNvhZH5mHrObD0FaJHLMJBgbDuPWcv4NRAsbUEshWIv8GwJabuQYPwBqsQPZwgh2mAQPfi2SN96YMZw5czhBspnHWHLmttvGbTxpxRYJx3Br4TufY/yhouKwvcH5M4Yfe7fdlu1nP7zxxoca3FoUDjCwSQDpxAaYCBuISMCpgYFBvoGB+QOQtsejZhSMglEwCkY6AAAFm17IdlqCFgAAAABJRU5ErkJggg==","orcid":"","institution":"The Second Affiliated Hospital of Kunming Medical University","correspondingAuthor":true,"prefix":"","firstName":"Tong-xin","middleName":"","lastName":"Yang","suffix":""},{"id":502565358,"identity":"b0309d88-c0e1-492f-93a9-6f2c0a580f4f","order_by":5,"name":"Ke-wei Fang","email":"","orcid":"","institution":"The Second Affiliated Hospital of Kunming Medical University","correspondingAuthor":false,"prefix":"","firstName":"Ke-wei","middleName":"","lastName":"Fang","suffix":""},{"id":502565359,"identity":"aff35e76-fd63-492c-9207-14c7a2018537","order_by":6,"name":"Jiong-ming Li","email":"","orcid":"","institution":"The Second Affiliated Hospital of Kunming Medical University","correspondingAuthor":false,"prefix":"","firstName":"Jiong-ming","middleName":"","lastName":"Li","suffix":""}],"badges":[],"createdAt":"2025-07-22 05:23:24","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7182790/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7182790/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41598-026-50443-z","type":"published","date":"2026-04-30T15:57:16+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":89590787,"identity":"4a665638-5971-4e62-bd91-3d071b7c1148","added_by":"auto","created_at":"2025-08-21 16:06:22","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1666306,"visible":true,"origin":"","legend":"\u003cp\u003eA. Induced MCs TB staining, with a scale bar of 50 μm.\u003c/p\u003e\n\u003cp\u003eB. Flow cytometry detects the proportion of c-kit positive cells.\u003c/p\u003e\n\u003cp\u003eC. Flow cytometry detects the proportion of CD23 positive cells.\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-7182790/v1/21bfe9031ef0f3bd8fcccef2.png"},{"id":89590791,"identity":"42b5fba5-39a7-474b-9a2f-dbfe1036011b","added_by":"auto","created_at":"2025-08-21 16:06:23","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":15909135,"visible":true,"origin":"","legend":"\u003cp\u003eA. Rat Bladder Tissue (H\u0026amp;E Staining);\u003c/p\u003e\n\u003cp\u003eB-C. Rat Bladder Tissue (Masson Staining) \u0026amp; Statistics of collagen fibers in bladder tissue (Blue: collagen fiber, red: muscle fiber);\u003c/p\u003e\n\u003cp\u003eD. Detection of Inflammation-Related Marker Myeloperoxidase by ELISA.\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-7182790/v1/84390cc6aa2d1bbec230e1c8.png"},{"id":89593512,"identity":"28d3b15b-ebd3-414d-8772-f005e2cc64f6","added_by":"auto","created_at":"2025-08-21 16:22:23","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":12386017,"visible":true,"origin":"","legend":"\u003cp\u003eA. (a)-(c) Expression of TJ-Related Markers ZO-1, Occludin, and Claudin-1 in Bladder Tissue by RT-qPCR. \u0026nbsp;(d)-(g) Expression of TJ-Related Markers ZO-1, Occludin, and Claudin-1 in Bladder Tissue by Western blot;\u003c/p\u003e\n\u003cp\u003eB. (a): Immunohistochemical (IHC) Staining of Tryptase-Positive Cells in Bladder Tissue (Brown-yellow positive cells indicated; 10× magnification). (b): Transmission Electron Microscopy (TEM) Analysis of Mast Cell (MC) Degranulation in Rat Bladder Tissue\u003c/p\u003e\n\u003cp\u003eC. (a): The positive rate of BTK in bladder tissue cells was detected by IHC staining (the positive cells were brown) (10 ×). (b): If staining was used to detect the fluorescence intensity of BTK and tryptase in bladder tissue (20 ×). \u0026nbsp;(c): Fluorescence intensity statistics of BTK. \u0026nbsp;(d): Fluorescence intensity statistics of tryptase. *P\u0026lt;0.05, **P\u0026lt;0.01.\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-7182790/v1/1ac9b6fcaefad9d0419a6352.png"},{"id":89592952,"identity":"f5fa2179-c08c-4cd9-b476-b698a78067d1","added_by":"auto","created_at":"2025-08-21 16:14:23","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":6562649,"visible":true,"origin":"","legend":"\u003cp\u003eA．Expression of BTK in MCsand its regulation on membrane ions of sv-huc-1 cells\u003c/p\u003e\n\u003cp\u003e(a): The expression of BTK mRNA was analyzed by RT qPCR; (b)and (c): the relative expression of BTK protein was detected by Western blot; \u0026nbsp;(d): The whole cell current was measured by patch clamp technique. *P\u0026lt;0.05, **P\u0026lt;0.01, ***P\u0026lt;0.001.\u003c/p\u003e\n\u003cp\u003eB. \u0026nbsp;Detection of membrane changes of sv-huc-1 cells by calcium channel fluorescent probe\u003c/p\u003e\n\u003cp\u003eC. \u0026nbsp;BTK regulates urothelial barrier injury by activating MCs\u003c/p\u003e\n\u003cp\u003eThe mRNA expressions of ZO1 (a), occludin (b) and claudin-1 (c) were detected by RT qPCR. \u0026nbsp;(d): The relative expressions of ZO1 (e), occludin (f) and claudin-1 (g) proteins were detected by Western blot. \u0026nbsp;(h): TEERdetection of sv-huc-1 cells* P\u0026lt;0.05, **P\u0026lt;0.01, ***P\u0026lt;0.001.\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-7182790/v1/014a74f9b9f7eeb4a1564574.png"},{"id":108437619,"identity":"82396cf8-9550-4d42-9f85-5da6bc521728","added_by":"auto","created_at":"2026-05-04 16:00:38","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":33394230,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7182790/v1/ed318c5c-4b18-403b-acf9-d87191a2c484.pdf"},{"id":89590788,"identity":"90e4a752-f806-4246-9312-9f1d5ae1c469","added_by":"auto","created_at":"2025-08-21 16:06:22","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":377767,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementaryfullblot.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7182790/v1/6c756ad1a62cb0a162e0122c.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"BTK Mediates Inflammation, Mast Cell Activation, and Urothelial Barrier Disruption in IC/BPS model","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eInterstitial cystitis/bladder pain syndrome (IC/BPS) is a chronic bladder disorder with a rising incidence, yet its exact pathogenesis remains unclear. Due to the absence of well-defined etiological mechanisms and diagnostic criteria, IC/BPS is typically diagnosed by exclusion. However, the significant heterogeneity in clinical symptoms and pathophysiological features among patients poses major challenges for accurate diagnosis, effective disease management, and the development of targeted therapies.\u003c/p\u003e\u003cp\u003eMast cells (MCs) are immune cells of hematopoietic origin, derived from the bone marrow and yolk sac, and are widely distributed throughout loose connective tissues in the body. Previous studies have demonstrated that increased proliferation of mast cells (MCs) in bladder tissue may be associated with symptom onset in IC/BPS[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. External inflammatory stimuli can activate MCs and trigger degranulation, releasing histamine, cytokines, and proteases that further promote inflammatory cell infiltration into the tissue[\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. This altered microenvironment reinforces MC activation and degranulation, forming a positive feedback loop that exacerbates IC/BPS symptoms.\u003c/p\u003e\u003cp\u003eAberrant activation and degranulation of mast cells (MCs) are key contributors to the pathogenesis of various diseases. MC stabilizers alleviate disease symptoms by reducing MC activation and degranulation, thereby limiting the release of inflammatory mediators, histamine, and proteases. In IC/BPS, MC activation and degranulation have also been identified as pathological factors involved in disease development, a finding supported by clinical and pathological examinations[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. However, the molecular mechanisms by which MC activation contributes to IC/BPS progression remain poorly understood. Therefore, elucidating the mechanisms that regulate MC proliferation and activation is crucial for developing strategies to suppress MC-mediated degranulation and inflammation in IC/BPS.\u003c/p\u003e\u003cp\u003eBruton\u0026rsquo;s tyrosine kinase (BTK), a member of the TEC family of non-receptor tyrosine kinases, functions as a key intracellular signaling molecule predominantly expressed in B cells and various hematopoietic lineages. BTK is an essential component of the downstream signaling cascade of the B cell receptor (BCR), regulating B cell maturation, survival, activation, cytokine production, and antigen-dependent stimulation. In addition, BTK also plays roles in modulating the biological behavior of other immune and neuronally-derived cells[\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Emerging evidence has shown that BTK can activate mast cells, promoting the production of eicosanoids and reactive oxygen species (ROS), thereby contributing to allergic responses[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. However, whether BTK is involved in the pathogenesis and progression of IC/BPS has not yet been reported.\u003c/p\u003e\u003cp\u003eBased on the above findings, we hypothesized that BTK may affect urothelial barrier function by modulating mast cell activation. Therefore, we established an LL-37-induced IC/BPS animal model to investigate the role of BTK expression in bladder inflammation, mast cell activation, urothelial barrier integrity, and its potential downstream signaling pathways. Activated MCs were also co-cultured with immortalized human ureteral epithelial cells (SV-HUC-1) to explore the impact of BTK on MC activation and the subsequent effects of MC activation on urothelial barrier function.\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e"},{"header":"2. Materials and Methods","content":"\u003ch2\u003e2.1 Experimental design\u003c/h2\u003e\n\u003cp\u003eBTK is expressed in a variety of immune cells, including B cells, macrophages, mast cells, basophils, and platelets, and participates in the regulation of both innate and adaptive immune responses. Based on our previous findings indicating that BTK regulates mast cell proliferation, invasion, and degranulation, an LL-37-induced IC/BPS rat model was established to investigate the functional role of BTK in vivo and to explore its underlying molecular mechanisms.\u003c/p\u003e\n\u003ch2\u003e2.2 Animal model and experimental design\u003c/h2\u003e\n\u003cp\u003eTo investigate the role of BTK in IC/BPS, an adenovirus-associated virus (AAV)-mediated gene overexpression or knockdown approach was employed in combination with intravesical instillation of LL-37 to induce an IC/BPS model. A total of 36 male Sprague-Dawley (SD) rats (6\u0026ndash;8 weeks old, 200\u0026thinsp;\u0026plusmn;\u0026thinsp;20 g) were randomly assigned into six groups (n\u0026thinsp;=\u0026thinsp;6 per group): Sham group, IC/BPS group, IC/BPS\u0026thinsp;+\u0026thinsp;OV-NC group, IC/BPS\u0026thinsp;+\u0026thinsp;OV-BTK group, IC/BPS\u0026thinsp;+\u0026thinsp;KD-NC group, and IC/BPS\u0026thinsp;+\u0026thinsp;KD-BTK group. (Table \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 1.\u003c/strong\u003e Grouping of animal models.\u003c/p\u003e\n\u003cp\u003e\u003csup\u003e1\u003c/sup\u003e The injection volume of normal saline in sham group and IC/BPS group was the same as that of AAV in other groups.\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"524\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 33.3333%;\"\u003e\n \u003cp\u003eGroups\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 33.3333%;\"\u003e\n \u003cp\u003eLL-37 Process\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 33.3333%;\"\u003e\n \u003cp\u003eAAV Process\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 33.3333%;\"\u003e\n \u003cp\u003esham\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 33.3333%;\"\u003e\n \u003cp\u003eNone（normal saline）\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 33.3333%;\"\u003e\n \u003cp\u003eNone（normal saline）\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 33.3333%;\"\u003e\n \u003cp\u003eIC/BPS\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 33.3333%;\"\u003e\n \u003cp\u003eYes\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 33.3333%;\"\u003e\n \u003cp\u003eNone（normal saline）\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 33.3333%;\"\u003e\n \u003cp\u003eIC/BPS+OV-NC\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 33.3333%;\"\u003e\n \u003cp\u003eYes\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 33.3333%;\"\u003e\n \u003cp\u003enegative control\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 33.3333%;\"\u003e\n \u003cp\u003eIC/BPS+OV-BTK\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 33.3333%;\"\u003e\n \u003cp\u003eYes\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 33.3333%;\"\u003e\n \u003cp\u003eBTK Overexpression\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 33.3333%;\"\u003e\n \u003cp\u003eIC/BPS+KD-NC\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 33.3333%;\"\u003e\n \u003cp\u003eYes\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 33.3333%;\"\u003e\n \u003cp\u003enegative control\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 33.3333%;\"\u003e\n \u003cp\u003eIC/BPS+KD-BTK\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 33.3333%;\"\u003e\n \u003cp\u003eYes\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 33.3333%;\"\u003e\n \u003cp\u003eBTK knock down\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003ePain-related responses were evaluated using Von Frey filaments with forces of 0.07 g, 0.4 g, 1.0 g, and 4.0 g. Pain scores were recorded based on the following criteria (0\u0026thinsp;=\u0026thinsp;no response; 1\u0026thinsp;=\u0026thinsp;licking or body twitching; 2\u0026thinsp;=\u0026thinsp;jumping). After completion of all experimental procedures, rats were euthanized, and bladder tissues were harvested for further analyses, including hematoxylin and eosin (H\u0026amp;E) staining, Masson\u0026apos;s trichrome staining, immunohistochemistry (IHC), immunofluorescence (IF), enzyme-linked immunosorbent assay (ELISA), real-time quantitative PCR (RT-qPCR), Western blotting, and transmission electron microscopy (TEM).\u003c/p\u003e\n\u003ch2\u003e2.3 Animal Anesthesia and Euthanasia Procedures\u003c/h2\u003e\n\u003cp\u003eAdult male SpragueDawley rats (200\u0026thinsp;\u0026plusmn;\u0026thinsp;20 g) were anesthetized by intraperitoneal injection of sodium pentobarbital (50 mg/kg), diluted 1:3 in sterile saline. Adequate anesthesia was confirmed by the absence of pedalwithdrawal and palpebral reflexes, and body temperature was maintained at 37\u0026deg;C on a thermostatic pad throughout the procedure. At the experimental endpoint, animals were placed in a sealed chamber filled with CO₂ at a displacement rate of 25\u0026ndash;30% of the chamber volume per minute; once respiration ceased, cervical dislocation was performed to ensure death, followed immediately by tissue collection for further analyses.\u003c/p\u003e\n\u003ch2\u003e2.4 Experiment in vitro\u003c/h2\u003e\n\u003cp\u003eBMMSCs were counted and seeded at 5 \u0026times; 10^5 cells mL⁻\u0026sup1; in 30 mL DMEM/F-12 supplemented with 100 ng mL⁻\u0026sup1; recombinant human stem cell factor (rhSCF), 100 ng mL⁻\u0026sup1; recombinant human interleukin-6 (rhIL-6), and 30 ng mL⁻\u0026sup1; recombinant human interleukin-3 (rhIL-3); rhIL-3 was included only during the first week. After 1 week, the medium was replaced with fresh DMEM/F-12 containing 100 ng mL⁻\u0026sup1; rhSCF and 50 ng mL⁻\u0026sup1; rhIL-6, and cultures were maintained with weekly medium changes. From week 3 onward, non-adherent cells were transferred to new flasks at each change to remove adherent contaminants, and debris was discarded. Continuous induction for 7\u0026ndash;10 weeks yielded mature mast cells (MCs)[\u003cspan class=\"CitationRef\"\u003e8\u003c/span\u003e]. These cells were allowed to adhere to poly-L-lysine\u0026ndash;coated coverslips, fixed, and stained with toluidine blue to visualize characteristic basophilic granules, and their immunophenotype was verified by flow-cytometric analysis of c-kit and CD23 surface expression (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e\n\u003cp\u003eHuman mast cells (MCs) and SV-HUC-1 human urothelial cells were cultured to investigate the role of BTK in MC activation and its regulatory effects on urothelial transmembrane barrier function. To establish an in vitro IC/BPS model, MCs were stimulated with varying concentrations of LL-37 (0.1, 1, 10, 20, and 100 \u0026micro;g/mL) for 12 hours. Based on preliminary screening, 20 \u0026micro;g/mL was selected as the optimal concentration for subsequent experiments. MCs and SV-HUC-1 cells were then co-cultured in Transwell chambers to assess the impact of MC activation on epithelial barrier function.\u003c/p\u003e\n\u003cp\u003eSix experimental groups were established: untreated control group (NC), LL-37 stimulation group, BTK knockdown group (KD-BTK), BTK overexpression group (OV-BTK), and corresponding negative control groups (KD-NC and OV-NC). These groups were used to evaluate the effects of BTK on MC activation and MC-mediated disruption of the urothelial barrier.\u003c/p\u003e\n\u003ch2\u003e2.5 Molecular and functional experiments\u003c/h2\u003e\n\u003cp\u003eMC proliferation was assessed using the Cell Counting Kit-8 (CCK-8) assay. Cell cycle progression and apoptosis rates were analyzed by flow cytometry, while cell invasive capacity was evaluated using Transwell chamber assays. MC degranulation was quantified by measuring tryptase and histamine secretion levels via ELISA, and the release of granule contents was further visualized by transmission electron microscopy (TEM).\u003c/p\u003e\n\u003cp\u003eTo assess urothelial barrier function, ELISA was used to quantify the secretion of glycosaminoglycans (GAGs), including hyaluronic acid (HA), chondroitin sulfate (CS), dermatan sulfate (DS), and heparan sulfate (HS). The expression of tight junction proteins\u0026mdash;ZO-1, Occludin, and Claudin-1\u0026mdash;was measured by RT-qPCR and Western blotting. Transepithelial electrical resistance (TEER) assays were performed to evaluate barrier integrity, and patch-clamp electrophysiology was used to detect changes in membrane potential. Additionally, RT-qPCR and Western blotting were conducted to analyze the expression levels of BTK and downstream signaling proteins including Bcl-2, P21, and c-Myc.\u003c/p\u003e\n\u003ch2\u003e\u003cstrong\u003e2.6 Statistics\u003c/strong\u003e\u003c/h2\u003e\n\u003cp\u003eAll data were analyzed and visualized using GraphPad Prism 8.0 software. Quantitative data with normal distribution were expressed as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (SD). Comparisons between two groups were performed using the Student\u0026rsquo;s \u003cem\u003et\u003c/em\u003e-test, while one-way analysis of variance (ANOVA) was used for comparisons among multiple groups. Tukey\u0026rsquo;s post hoc test was applied for pairwise comparisons between multiple groups. A \u003cem\u003eP\u003c/em\u003e value\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered statistically significant.\u003c/p\u003e"},{"header":"3. Results ","content":"\u003cp\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eThis section may be divided by subheadings. It should provide a concise and precise description of the experimental results, their interpretation, as well as the experimental conclusions that can be drawn.\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e\u003ch2\u003e3.1. Animal model\u003c/h2\u003e\u003cdiv id=\"Sec11\" class=\"Section3\"\u003e\u003ch2\u003e3.1.1 Pathological effect of BTK expression on bladder tissue of IC/BPS rats\u003c/h2\u003e\u003cp\u003e\u003cul\u003e\u003cli\u003e\u003cp\u003eFollowing euthanasia, bladder tissues were harvested from rats for histological analysis. Rat bladder tissue Hematoxylin and eosin (H\u0026amp;E) staining (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA) showed that bladder tissues in the sham group exhibited intact architecture, thick mucosa, and abundant smooth muscle fibers, with no signs of inflammatory cell infiltration. In contrast, the IC/BPS group displayed typical inflammatory features, including submucosal hemorrhage, pronounced interstitial edema, and marked inflammatory cell infiltration. In the BTK overexpression group, inflammatory infiltration was further aggravated, with partial epithelial loss and increased microvascular density in the interstitium. Compared with the KD-NC group, the BTK knockdown group showed reduced inflammatory infiltration and a thickened bladder wall;\u003c/p\u003e\u003c/li\u003e\u003cli\u003e\u003cp\u003eMasson's trichrome staining (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB\u0026ndash;C) revealed well-organized and abundant smooth muscle fibers in the sham group. In the IC/BPS group, there was significant collagen fiber deposition, which was markedly higher than in the sham group (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001). BTK overexpression further increased collagen accumulation and disrupted smooth muscle fiber organization (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). In contrast, collagen fiber content was reduced and fibrosis was alleviated in the BTK knockdown group compared to KD-NC (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). These findings indicate that BTK knockdown mitigates LL-37-induced bladder inflammation and fibrosis in IC/BPS rats.\u003c/p\u003e\u003c/li\u003e\u003c/ul\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section3\"\u003e\u003ch2\u003e3.1.2 Regulation of BTK on bladder inflammation and urinary epithelial GAGs in IC/BPS rats\u003c/h2\u003e\u003cp\u003e\u003cul\u003e\u003cli\u003e\u003cp\u003eInflammatory markers including myeloperoxidase (MPO), interleukin-1β (IL-1β), IL-6, and tumor necrosis factor-α (TNF-α) in bladder tissues were quantified by ELISA (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD(a\u0026ndash;d)). Compared to the sham group, levels of MPO (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD(a)), IL-1β (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD(b)), IL-6 (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD(c)), and TNF-α (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD(d)) were significantly elevated in the IC/BPS group. These inflammatory mediators were further upregulated in the BTK overexpression group (MPO: \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05; IL-1β: \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01; IL-6: \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01; TNF-α: \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01). Conversely, BTK knockdown significantly attenuated LL-37-induced upregulation of these inflammatory markers (MPO, IL-1β, IL-6: \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05; TNF-α: \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/p\u003e\u003c/li\u003e\u003cli\u003e\u003cp\u003eELISA results of glycosaminoglycan (GAG) levels in urethral tissues showed that concentrations of hyaluronic acid (HA), chondroitin sulfate (CS), dermatan sulfate (DS), and heparan sulfate (HS) were significantly reduced in the IC/BPS group compared to the sham group (HA: \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01, Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD(e); CS: Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD(f); DS: Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD(g); HS: Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD(h)). BTK overexpression further decreased HA (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05), CS (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01), DS, and HS (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05), while BTK knockdown significantly increased the levels of HA (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05), CS, DS (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01), and HS compared to the respective controls.\u003c/p\u003e\u003c/li\u003e\u003c/ul\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec13\" class=\"Section3\"\u003e\u003ch2\u003e3.1.3 Regulation of BTK on urinary epithelial barrier in IC/BPS rats\u003c/h2\u003e\u003cp\u003e\u003cul\u003e\u003cli\u003e\u003cp\u003eThe expression of tight junction (TJ)-related markers ZO-1, Occludin, and Claudin-1 in bladder tissues was evaluated by RT-qPCR (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA(a\u0026ndash;c)) and Western blotting (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA(d\u0026ndash;g)). Compared with the sham group, mRNA and protein levels of ZO-1 (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001, Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e(a, e)), Occludin (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001, Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA (b, f)), and Claudin-1 (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001, Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA (c, g)) were significantly decreased in the IC/BPS group.\u003c/p\u003e\u003c/li\u003e\u003cli\u003e\u003cp\u003eBTK overexpression markedly suppressed the expression of ZO-1, Occludin (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01), and Claudin-1 (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) at both the mRNA and protein levels compared to the OV-NC group. In contrast, BTK knockdown significantly upregulated ZO-1, Occludin (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01), and Claudin-1 (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01) expression.\u003c/p\u003e\u003c/li\u003e\u003cli\u003e\u003cp\u003eThese findings suggest that LL-37 stimulation induces bladder inflammation, disrupts the GAG layer of the urethra, and impairs urothelial barrier function. Inhibition of BTK attenuates LL-37-induced inflammation and preserves urothelial barrier integrity.\u003c/p\u003e\u003c/li\u003e\u003c/ul\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec14\" class=\"Section3\"\u003e\u003ch2\u003e3.1.4 Regulation of BTK expression on degranulation of MCs in bladder tissue of IC/BPS rats\u003c/h2\u003e\u003cp\u003e\u003cul\u003e\u003cli\u003e\u003cp\u003eImmunohistochemical (IHC) staining was performed to detect tryptase-positive cells in bladder tissue sections (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB(a)). Compared with the sham group, the number of tryptase-positive cells was significantly increased in the IC/BPS group. The BTK overexpression group showed more tryptase-positive cells than the OV-NC group, while the BTK knockdown group exhibited a reduction in tryptase-positive cells.\u003c/p\u003e\u003c/li\u003e\u003cli\u003e\u003cp\u003eTransmission electron microscopy (TEM) was used to evaluate mast cell (MC) degranulation in bladder tissues (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB(b)). MCs in the bladder exhibited diverse morphologies. In the IC/BPS group, cytoplasmic granules\u0026mdash;spherical or ovoid in shape\u0026mdash;were observed being released into the extracellular space. These granules varied in size and were membrane-bound. The BTK overexpression group displayed increased extracellular granule release compared to controls, whereas fewer granules were observed in the BTK knockdown group.\u003c/p\u003e\u003c/li\u003e\u003cli\u003e\u003cp\u003eThese results indicate that BTK overexpression promotes mast cell activation and degranulation in the bladder tissue of IC/BPS rats, while BTK knockdown reduces mast cell activation in this pathological context.\u003c/p\u003e\u003c/li\u003e\u003c/ul\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec15\" class=\"Section3\"\u003e\u003ch2\u003e3.1.5 Expression of BTK in bladder tissue of IC/BPS rats\u003c/h2\u003e\u003cp\u003e\u003cul\u003e\u003cli\u003e\u003cp\u003eImmunohistochemical (IHC) staining was used to assess BTK expression in rat bladder tissues (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC(a)). Compared to the sham group, the IC/BPS group showed a significant increase in BTK-positive cells. The number of BTK-positive cells was further elevated in the BTK overexpression group compared to the OV-NC group, whereas BTK knockdown led to a marked reduction in BTK-positive cells.\u003c/p\u003e\u003c/li\u003e\u003cli\u003e\u003cp\u003eAdditionally, immunofluorescence (IF) staining was performed to evaluate the expression of BTK and tryptase in bladder tissues (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC(b\u0026ndash;d)). Consistent with the IHC findings, fluorescence intensities of both BTK and tryptase were significantly higher in the IC/BPS group compared to the sham group (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01). BTK overexpression further enhanced the fluorescence intensities of BTK (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) and tryptase (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01), while BTK knockdown resulted in a significant reduction in their fluorescence signals (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/p\u003e\u003c/li\u003e\u003c/ul\u003e\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\u003ch2\u003e3.2. BTK regulates MCs activation\u003c/h2\u003e\u003cdiv id=\"Sec17\" class=\"Section3\"\u003e\u003ch2\u003e3.2.1 Expression of BTK in MCs and its regulation on SV-HUC-1 membrane ions\u003c/h2\u003e\u003cp\u003e\u003cul\u003e\u003cli\u003e\u003cp\u003eTo determine the most effective BTK knockdown target, three BTK-targeting shRNA adenoviral vectors (KD-BTK#1, #2, #3) were transfected into 293T cells. After 48 hours, strong fluorescence indicated successful transfection, and RT-qPCR showed significantly reduced BTK mRNA in all knockdown groups versus KD-NC (P\u0026thinsp;\u0026lt;\u0026thinsp;0.001, Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA(a)). Western blot confirmed reduced BTK protein expression (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05, Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA(b)), with KD-BTK#2 being the most efficient.\u003c/p\u003e\u003c/li\u003e\u003cli\u003e\u003cp\u003eMCs were then transduced with KD-NC, KD-BTK#2 (KD-BTK), OV-NC, or OV-BTK and stimulated with 20 \u0026micro;g/mL LL-37. LL-37 significantly increased BTK expression in MCs (P\u0026thinsp;\u0026lt;\u0026thinsp;0.001, Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA(c)); BTK was significantly reduced in KD-BTK (P\u0026thinsp;\u0026lt;\u0026thinsp;0.01) and increased in OV-BTK (P\u0026thinsp;\u0026lt;\u0026thinsp;0.01) compared to their respective controls.\u003c/p\u003e\u003c/li\u003e\u003cli\u003e\u003cp\u003eTo assess barrier function, SV-HUC-1 cells were co-cultured with treated MCs. LL-37-treated MCs increased SV-HUC-1 whole-cell currents (P\u0026thinsp;\u0026lt;\u0026thinsp;0.001). Co-culture with LL-37\u0026thinsp;+\u0026thinsp;KD-BTK MCs reduced current versus LL-37\u0026thinsp;+\u0026thinsp;KD-NC (P\u0026thinsp;\u0026lt;\u0026thinsp;0.001), while LL-37\u0026thinsp;+\u0026thinsp;OV-BTK further increased current compared to LL-37\u0026thinsp;+\u0026thinsp;OV-NC (P\u0026thinsp;\u0026lt;\u0026thinsp;0.001, Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB).\u003c/p\u003e\u003c/li\u003e\u003c/ul\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec18\" class=\"Section3\"\u003e\u003ch2\u003e3.2.2 BTK regulates MCs activation and affects gags levels in SV-HUC-1 cells\u003c/h2\u003e\u003cp\u003e\u003cul\u003e\u003cli\u003e\u003cp\u003eELISA revealed significantly elevated LL-37 levels in SV-HUC-1 supernatants co-cultured with LL-37-stimulated MCs compared to control (P\u0026thinsp;\u0026lt;\u0026thinsp;0.001). BTK knockdown significantly reduced LL-37 levels versus KD-NC (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05), while BTK overexpression increased them (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/p\u003e\u003c/li\u003e\u003cli\u003e\u003cp\u003eELISA of SV-HUC-1 supernatants showed that co-culture with LL-37-stimulated MCs significantly reduced HA (P\u0026thinsp;\u0026lt;\u0026thinsp;0.01), CS (P\u0026thinsp;\u0026lt;\u0026thinsp;0.01), DS (P\u0026thinsp;\u0026lt;\u0026thinsp;0.001), and HS (P\u0026thinsp;\u0026lt;\u0026thinsp;0.01) levels. BTK knockdown significantly increased these GAGs compared to KD-NC (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05), while overexpression significantly decreased them compared to OV-NC (HA: P\u0026thinsp;\u0026lt;\u0026thinsp;0.05; CS: P\u0026thinsp;\u0026lt;\u0026thinsp;0.01; DS: P\u0026thinsp;\u0026lt;\u0026thinsp;0.01; HS: P\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/p\u003e\u003c/li\u003e\u003c/ul\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec19\" class=\"Section3\"\u003e\u003ch2\u003e3.2.3 BTK regulates MCs activation and affects urothelial barrier\u003c/h2\u003e\u003cp\u003e\u003cul\u003e\u003cli\u003e\u003cp\u003eRT-qPCR and Western blot analyses were performed to evaluate the expression levels of tight junction (TJ)-related genes ZO-1, Occludin, and Claudin-1 in SV-HUC-1 cells. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC(a\u0026ndash;c), SV-HUC-1 cells co-cultured with LL-37-stimulated MCs exhibited significantly decreased mRNA expression of ZO-1 (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001), Occludin (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001), and Claudin-1 (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001) compared with the SV-HUC-1 monoculture group. Knockdown of BTK in LL-37-stimulated MCs significantly restored the mRNA expression of ZO-1 (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05), Occludin (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05), and Claudin-1 (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) in SV-HUC-1 cells. Conversely, BTK overexpression in LL-37-stimulated MCs further suppressed ZO-1 (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01), Occludin (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001), and Claudin-1 (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001) expression compared to the LL-37-only co-culture group.\u003c/p\u003e\u003c/li\u003e\u003cli\u003e\u003cp\u003eWestern blot results were consistent with RT-qPCR findings (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC(d\u0026ndash;g)). Protein levels of ZO-1 (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001, Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC(e)), Occludin (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001, Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC(f)), and Claudin-1 (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001, Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC (g)) were significantly decreased in SV-HUC-1 cells co-cultured with LL-37-treated MCs. BTK knockdown led to a significant increase in ZO-1 (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05), Occludin (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05), and Claudin-1 (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001) protein expression, while BTK overexpression further suppressed these levels of protein compared with the OV-NC group (ZO-1: \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01; Occludin: \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01; Claudin-1: \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001).\u003c/p\u003e\u003c/li\u003e\u003cli\u003e\u003cp\u003eTransepithelial electrical resistance (TEER) measurements of SV-HUC-1 monolayers were performed to assess barrier integrity (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC(h)). Co-culture with LL-37-stimulated MCs significantly reduced TEER values compared to the monoculture group (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001). BTK knockdown slightly increased TEER, while BTK overexpression led to a marked reduction in TEER values (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01).\u003c/p\u003e\u003c/li\u003e\u003cul\u003e\n \u003cli\u003e\n \u003cp\u003eTaken together, these findings suggest that BTK promotes LL-37-induced MC activation, which in turn disrupts the urothelial barrier. Conversely, BTK knockdown attenuates MC activation and helps preserve urothelial barrier function.\u003c/p\u003e\n \u003c/li\u003e\n\u003c/ul\u003e\n\n\u003cdiv id=\"Sec20\" class=\"Section2\"\u003e\n \u003ch2\u003e3.3. Figures, Tables and Schemes\u003c/h2\u003e\n \u003cdiv class=\"BlockQuote\"\u003e\n \u003cp\u003e\u003cstrong\u003eMain Experimental Reagents and Manufacturers\u003c/strong\u003e\u003c/p\u003e\n \u003c/div\u003e\n \u003cdiv class=\"gridtable\"\u003e\n \u003ctable id=\"Taba\" border=\"1\"\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eHuman recombinant IL-3 and human recombinant stem cell factor SCF\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eGibco\u0026trade;, Thermo Fisher Scientific (Waltham, MA, USA)\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSV-HUC-1 cells (human urothelial cell line), Human bone marrow mesenchymal stem cells (HBMMSC)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eShanghai Zhongqiao Xinzhou Biotechnology Co., Ltd. (Shanghai, China)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eAnnexin V-FITC/PI Apoptosis Detection Kit\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eAbsin Bioscience Inc. (Shanghai, China)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCCK-8 Assay Kit\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eBeyotime Biotechnology (Shanghai, China)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eTranswell Chamber\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCorning Incorporated (USA)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCell Cycle Detection Kit\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eElabscience Biotechnology Co., Ltd. (Wuhan, China)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eHuman Trypsin and Histamine ELISA Kits\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eMLBIO \u0026ndash; Shanghai Enzyme-linked Biotechnology Co., Ltd.\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eFastKing RT Kit (With gDNase), FastKing First-Strand cDNA Synthesis Kit\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eTIANGEN Biotech Co., Ltd. (Beijing, China)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eTaq Pro Universal SYBR qPCR Master Mix\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eVazyme Biotech Co., Ltd. (Nanjing, China)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eProtease Inhibitor Cocktail, PhosSTOP Phosphorylation Protease Inhibitor\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eRoche Diagnostics (Switzerland)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eBTK Antibody (DF6472)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eAffinity Biosciences Inc. (USA)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eMast Cell Tryptase (Ab2378), Pre-adsorbed Goat Anti-Mouse IgG H\u0026amp;L (CY3) (ab97035)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eAbcam plc (UK)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eDyLight 488 Labeled Goat Anti-Rabbit IgG (H\u0026thinsp;+\u0026thinsp;L) (5230\u0026thinsp;\u0026minus;\u0026thinsp;0385)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eKPL (SeraCare Life Sciences, USA)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eAntibodies: Bcl-2 (bsm-33411M), p21 (bs-55160R), ZO-1 (bs-1329R), Occludin (bs-10011R), Claudin-1 (bs-1428R)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eBioss Antibodies (Beijing, China)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eAntibodies: c-Myc (18583S), Secondary Antibodies (7074, 7076)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCell Signaling Technology, Inc. (USA)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eGAPDH Antibody (P30008M)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eAbmart Inc. (Shanghai, China)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n \u003c/div\u003e\n\u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003ePrevious studies on the etiology and pathophysiology of IC/BPS have suggested that mast cells (MCs) may play a critical role in disease progression. The impact of MCs on epithelial barrier function has been well documented[\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. In a mouse model of irritable bowel syndrome, increased paracellular permeability, enhanced MC degranulation, and elevated IFN-γ levels were observed, along with morphological disruption of the colonic epithelial barrier and downregulation of tight junction proteins such as Zona Occludens 2 (ZO-2) and Occludin.\u003c/p\u003e\u003cp\u003eIn our study, we established a co-culture system of MCs and SV-HUC-1 urothelial cells to evaluate how differently treated MCs influence the expression of glycosaminoglycans (GAGs) and tight junction (TJ)-associated proteins in vitro. The results showed that SV-HUC-1 cells co-cultured with LL-37-stimulated MCs exhibited increased whole-cell currents and intracellular calcium concentrations, along with reduced expression of GAGs (HA, CS, DS, and HS) and TJ proteins (ZO-1, Occludin, and Claudin-1). Co-culture with LL-37-treated and BTK-overexpressing MCs further elevated cellular current and calcium influx, while further suppressing GAG and TJ protein expression. In contrast, BTK knockdown in MCs produced the opposite effect, indicating that BTK plays a regulatory role in MC-mediated disruption of the urothelial barrier.\u003c/p\u003e\u003cp\u003eIn recent years, an increasing number of studies have utilized in vitro co-culture systems to investigate how mast cell (MC) degranulation and the release of granule-stored mediators regulate physiological processes in neighboring cells. For instance, co-culture with MCs promotes gastric cancer cell proliferation, invasion, migration, and resistance to H₂O₂-induced apoptosis[\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. MCs also enhance human lung fibroblast contractility in a time- and concentration-dependent manner, an effect not blocked by the tryptase inhibitor bis[5-imidazo(2,1-b)-benzimidazolyl]methane[\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eConsistent with these findings, our study demonstrated that co-culture of LL-37-activated MCs with SV-HUC-1 urothelial cells resulted in a marked reduction in the expression of glycosaminoglycans (GAGs) and tight junction (TJ) proteins in SV-HUC-1 cells. These results support the concept that activated MCs can disrupt epithelial barrier function through the release of soluble mediators.\u003c/p\u003e\u003cp\u003eMediators secreted by mast cells (MCs) can act as triggers of epithelial barrier dysfunction. Substances such as histamine, tryptase, and prostaglandin D₂ (PGD₂) have been shown to increase epithelial secretion, while other MC-derived products may directly impair epithelial integrity[\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Notably, tryptase and chymase can proteolytically cleave tight junction (TJ) proteins, including Claudin-1, Claudin-3, Claudin-5, and junctional adhesion molecule A (JAM-A)[\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Groschwitz et al.[\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e] demonstrated that MC-derived tryptase can activate protease-activated receptor 2 (PAR2), signaling to colonic epithelial cells in a paracrine manner. PAR2 activation is β-arrestin\u0026ndash;dependent and leads to ERK1/2 phosphorylation, which reorganizes perijunctional F-actin and thereby increases epithelial permeability.\u003c/p\u003e\u003cp\u003eMC degranulation exerts widespread effects on TJs across various epithelial systems. For example, MCs alter signaling pathways in alveolar epithelial cells, particularly those involved in pro-inflammatory cytokine induction and TJ disruption[\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. In stress-induced esophageal epithelial dysfunction, MC-derived tryptase activates PAR2, inducing inflammation, TJ disassembly, and cytoskeletal reorganization that contributes to intercellular gap widening[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eIn our study, co-culture of SV-HUC-1 urothelial cells with LL-37\u0026ndash;activated MCs led to a decrease in the expression of GAGs and TJ-related proteins, indicating barrier disruption. The role of mast cells (MCs) in IC/BPS was confirmed in our study; however, the underlying mechanisms warrant further investigation.\u003c/p\u003e\u003cp\u003eBTK and its associated signaling pathways play regulatory roles in various immune cells, including B cells, microglia, macrophages, and neutrophils. BTK inhibitors have been reported as potential treatments for central nervous system\u0026ndash;related diseases. These inhibitors are capable of crossing the blood\u0026ndash;brain barrier, restricting B cell activation, and reducing their antigen-presenting capacity to T cells, thereby attenuating proinflammatory T cell responses both in vivo and in vitro, ultimately contributing to disease suppression[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eIn our study, BTK overexpression in LL-37\u0026ndash;stimulated MCs suppressed the expression of HA, CS, DS, and HS, as well as tight junction\u0026ndash;associated proteins ZO-1, Occludin, and Claudin-1 in co-cultured SV-HUC-1 cells. Conversely, BTK knockdown enhanced the expression of GAGs and TJ proteins. However, the broader regulatory effects of BTK on epithelial or endothelial barrier function remain to be elucidated.\u003c/p\u003e\u003cp\u003eWe found that LL-37\u0026ndash;activated MCs reduced the expression of GAGs and TJ proteins in SV-HUC-1 cells when co-cultured in vitro. BTK overexpression in MCs further enhanced LL-37-induced activation, aggravating the disruption of the urothelial barrier, while BTK knockdown mitigated the deleterious effects of MCs on SV-HUC-1 cells. We speculate that LL-37 and exogenous BTK promote MC degranulation, and the mediators released from MC granules subsequently alter GAG and TJ protein levels in SV-HUC-1 cells. However, our current data do not identify which specific granule-derived mediators are responsible for TJ disruption. Further studies are needed to clarify these mechanisms.\u003c/p\u003e\u003cp\u003eMoreover, patch-clamp electrophysiology revealed alterations in whole-cell currents in SV-HUC-1 cells co-cultured with LL-37-activated MCs. The specific ion channels responsible for these changes remain unknown and warrant future investigation.\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e"},{"header":"5. Conclusions","content":"\u003cp\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eBTK overexpression promotes mast cell proliferation, invasion, and degranulation, leading to impaired expression of glycosaminoglycans (GAGs) and tight junction (TJ) proteins in SV-HUC-1 cells, thereby compromising urothelial barrier function. This may represent one of the mechanisms by which BTK contributes to the pathogenesis of IC/BPS. In contrast, BTK knockdown attenuates MC activation and alleviates their detrimental effects on epithelial barrier integrity.\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp dir=\"LTR\"\u003e\u003cstrong\u003eAuthor Contributions:\u003c/strong\u003e Conceptualization, Yang Tong-xin; methodology, Yang Tong-xin; validation, Wang Guang; formal analysis, Chen Xian; investigation, Li Bin-sen and Chu Jin-yi; writing\u0026mdash;original draft preparation, Wang Guang and Li Bin-sen; writing\u0026mdash;review and editing, Yang Tong-xin; visualization, Wang Guang; supervision, Fang Ke-wei and Li Jiong-ming; funding acquisition, Yang Tong-xin, Fang Ke-wei and Li Jiong-ming. All authors have read and agreed to the published version of the manuscript.\u003c/p\u003e\n\u003cp dir=\"LTR\"\u003e\u0026nbsp;\u003cstrong\u003eFunding:\u003c/strong\u003e This research was funded by the National Natural Science Foundation of China, Grant No. 82160150; the Yunnan Provincial Department of Science and Technology Fundamental Research Special Project, Grant No. 202201AT070240; the Yunnan Revitalization Talent Support Program, Grant No. XDYC-QNRC-2022-0308; the Yunnan International Joint R\u0026amp;D Center of Key Technologies in Urological Diagnosis and Treatment, Grant No. 202403AP140016; the Yunnan Provincial Department of Science and Technology Expert Workstation Fund, Grant No. 202405AF140058 and \u0026nbsp;202505AF350061; the Yunnan Health Training Project of High-Level Talents, Grant No. D-2024027; the Second Affiliated Hospital of Kunming Medical University Talent Echelon Training Project, Grant No. RCTDXS-202306; the Second Affiliated Hospital of Kunming Medical University External Cooperation Projects, Grant No. 2022dwhz10.\u003c/p\u003e\n\u003cp dir=\"LTR\"\u003e\u003cstrong\u003eInstitutional Review Board Statement:\u0026nbsp;\u003c/strong\u003eThis study was approved by the Ethics Approval Committee of the second affiliated hospital of Kunming medical university, and the registered number is PJ-2021-70. All animal procedures were conducted in accordance with the ARRIVE guidelines (https://arriveguidelines.org) and approved by the Institutional Animal Care and Use Committee (IACUC) of the second affiliated hospital of Kunming medical university under protocol number kmmu2021153. All methods were performed in accordance with relevant guidelines and regulations. Efforts were made to minimize animal suffering and to reduce the number of animals used.\u003c/p\u003e\n\u003cp dir=\"LTR\"\u003e\u003cstrong\u003eInformed Consent Statement:\u0026nbsp;\u003c/strong\u003eNot applicable.\u003c/p\u003e\n\u003cp dir=\"LTR\"\u003e\u0026nbsp;\u003cstrong\u003eData Availability Statement:\u003c/strong\u003e All data generated or analyzed during this study are included in this published article and its supplementary information files.\u003c/p\u003e\n\u003cp dir=\"LTR\"\u003e\u003cstrong\u003eConflicts of Interest:\u003c/strong\u003e The authors declare no conflicts of interest.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eTheoharides, T.C.; Sant, G.R.; El-Mansoury, M.; Letourneau, R.; Ucci, A.A.; Meares, E.M. Activation of Bladder Mast Cells in Interstitial Cystitis: A Light and Electron Microscopic Study. \u003cem\u003eJ. 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Dis.\u003c/em\u003e \u003cstrong\u003e2015\u003c/strong\u003e, \u003cem\u003e16\u003c/em\u003e, 186\u0026ndash;196, doi:10.1111/1751-2980.12226.\u003c/li\u003e\n\u003cli\u003eLi, R.; Tang, H.; Burns, J.C.; Hopkins, B.T.; Le Coz, C.; Zhang, B.; De Barcelos, I.P.; Romberg, N.; Goldstein, A.C.; Banwell, B.L.; et al. BTK Inhibition Limits B-Cell\u0026ndash;T-Cell Interaction through Modulation of B-Cell Metabolism: Implications for Multiple Sclerosis Therapy. \u003cem\u003eActa Neuropathol. (Berl.)\u003c/em\u003e \u003cstrong\u003e2022\u003c/strong\u003e, \u003cem\u003e143\u003c/em\u003e, 505\u0026ndash;521, doi:10.1007/s00401-022-02411-w.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"BTK, Interstitial cystitis, bladder pain syndrome, Mast cell, Bladder barrier","lastPublishedDoi":"10.21203/rs.3.rs-7182790/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7182790/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e\u003cp\u003eInterstitial cystitis/bladder pain syndrome (IC/BPS) is a chronic urinary disorder with unclear pathogenesis. Previous studies identified BTK as a hub gene potentially involved in IC/BPS. This study investigates BTK\u0026rsquo;s role in mast cell (MC) activation and bladder inflammation.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e\u003cp\u003eAn LL-37-induced IC/BPS rat model and an in vitro MC model were established. BTK expression was modulated via adenoviral vectors and cell transfection. Bladder inflammation, MC degranulation, and urothelial barrier function were assessed using histology, ELISA, RT-qPCR, Western blot, IHC, TEM, and IF. MC function was evaluated via CCK-8, flow cytometry, Transwell, and TEM.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e\u003cp\u003eLL-37 upregulated BTK in IC/BPS rats, promoting inflammation, cytokine release, collagen deposition, MC degranulation, and urothelial damage. BTK overexpression exacerbated, while knockdown alleviated these effects. In vitro, LL-37 stimulated MC proliferation, invasion, and degranulation, and reduced apoptosis. Co-culture with activated MCs decreased glycosaminoglycan (GAG) and tight junction (TJ) proteins in SV-HUC-1 cells, enhanced by BTK overexpression and reversed by knockdown.\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e\u003cp\u003eBTK promotes LL-37-induced MC activation and urothelial barrier disruption by suppressing GAGs and TJ proteins, contributing to IC/BPS pathophysiology.\u003c/p\u003e","manuscriptTitle":"BTK Mediates Inflammation, Mast Cell Activation, and Urothelial Barrier Disruption in IC/BPS model","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-08-21 16:06:18","doi":"10.21203/rs.3.rs-7182790/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-12-08T16:54:43+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-09-21T21:02:49+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"211982490246643232127633924582269471802","date":"2025-09-12T07:43:39+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-08-28T06:51:51+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"273875921136342539693976947708147622161","date":"2025-08-19T10:52:38+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-08-14T06:41:12+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-08-14T06:38:39+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2025-08-14T04:00:51+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-08-06T11:30:34+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2025-08-06T11:26:41+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"268293cd-3789-4a27-9774-c8cd83c01da7","owner":[],"postedDate":"August 21st, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":53386001,"name":"Biological sciences/Cell biology"},{"id":53386002,"name":"Health sciences/Urology"}],"tags":[],"updatedAt":"2026-05-04T15:59:38+00:00","versionOfRecord":{"articleIdentity":"rs-7182790","link":"https://doi.org/10.1038/s41598-026-50443-z","journal":{"identity":"scientific-reports","isVorOnly":false,"title":"Scientific Reports"},"publishedOn":"2026-04-30 15:57:16","publishedOnDateReadable":"April 30th, 2026"},"versionCreatedAt":"2025-08-21 16:06:18","video":"","vorDoi":"10.1038/s41598-026-50443-z","vorDoiUrl":"https://doi.org/10.1038/s41598-026-50443-z","workflowStages":[]},"version":"v1","identity":"rs-7182790","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7182790","identity":"rs-7182790","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}