Metformin modulates the TXNIP-NLRP3-GSDMD pathway to improve diabetic bladder dysfunction

Abstract

To validate the therapeutic efficacy of metformin on diabetic bladder dysfunction (DBD) and further elucidate whether the TXNIP-NLRP3-GSDMD axis serves as a target for metformin in ameliorating DBD. C57BL/6J mice were induced with diet-induced obesity by being fed a high-fat diet (HFD) for 16 weeks. After establishing the model, the mice were treated with metformin for 4 weeks, and their glucose metabolism-related parameters were assessed. Urine spot assays and urodynamic measurements were conducted to reflect the bladder function and urinary behavior in mice, while histological examination was performed to observe morphological changes. Western blot analysis was employed to measure the expression levels of pyroptotic factors such as TXNIP, NLRP3, GSDMD, and tight junction proteins. Metformin treatment significantly improved glucose tolerance and insulin sensitivity in mice. Moreover, it showed promise in decreasing urinary spot occurrence, reducing urination frequency, alleviating non-voiding contractions, and stabilizing peak urinary pressure. Following metformin therapy, mice displayed restored epithelial fold structure, increased thickness of the muscular layer, substantial decrease in muscle fiber content, notably reduced levels of TXNIP and GSDMD proteins in the metformin-treated group compared to the DBD group, and restored expression of tight junction proteins Zo-1, Claudin-1, and Occludin. Metformin ameliorates urothelial cells damage in DBD mice by inhibiting TXNIP generation and reducing NLRP3 and GSDMD production.

Introduction

Diabetes mellitus (DM) is a prevalent national disease, with approximately 19 million people affected in the United States alone¹. Type 2 diabetes mellitus (T2DM) accounts for 87–91% of diabetes cases². Urinary complications are the most common, affecting over 50% of diabetic patients^3,4. Lower urinary tract symptoms (LUTS) are commonly observed in patients with DBD⁵. Physiologically, chronic inflammation, increased mast cell count, apoptosis, and decreased tight junction protein levels due to a hyperglycemic environment impair bladder epithelial barrier function, leading to excessive detrusor muscle activity and other effects⁶. Current research on DBD primarily focuses on detrusor smooth muscle⁷, bladder vascular system, and neural innervation⁸, while with the physiological effects of T2DM on urothelial tissue largely unknown.

T2DM is recognized as a chronic inflammatory condition that results in substantial production of reactive oxygen species (ROS)⁹. ROS stimulate the generation of the oxidative stress regulator thioredoxin-interacting protein (TXNIP), an endogenous negative regulator of thioredoxin (TXN), which plays a crucial role in maintaining cellular redox balance¹⁰. TXNIP intensifies oxidative stress and triggers the activation of the NOD like receptor protein 3 (NLRP3) inflammasome. This complex comprises the sensor molecule NLRP3, a speck-like protein associated with apoptosis, which includes a caspase recruitment domain (ASC) and pro-caspase-1¹¹. Upon TXNIP stimulation, the NLRP3 inflammasome activates pro-caspase-1 to caspase-1, and activates the gasdermin D (GSDMD) pyroptosis pathway¹², directly affecting bladder epithelium and impairing bladder barrier function. However, the role of TXNIP and NLRP3-induced pyroptosis in DBD remains unclear.

The anti-diabetic drug metformin is widely utilized in the management of type 2 diabetes. Recent evidence suggests its potential in treating diabetic complications as well¹³. Previous research has shown that metformin effectively improves diabetic cardiomyopathy¹⁴, diabetic nephropathy¹⁵, diabetic retinopathy¹⁶, and diabetic vascular disease¹⁷ by inhibiting pyroptosis. However, there is currently no research indicating the effectiveness of metformin in treating DBD, and its specific mechanism remains unclear. Given its potent antioxidant properties through TXNIP reduction, we hypothesized that metformin may offer protective effects against bladder damage induced by type 2 diabetes by TXNIP-NLRP3-GSDMD pathway TXNIP-NLRP3-GSDMD pathway. Thus, this study aims to investigate the protective effects of metformin on DBD and elucidate its underlying mechanism.

Materials and methods

Animals

24 SPF grade 4-week-old wild-type C57 male mice, weighing approximately 20 g ± 3 g each (purchased from the Experimental Animal Center of Ningxia Medical University), were housed under specific pathogen-free conditions (temperature 22 °C, humidity 50%) with a 12-h light–dark cycle and ad libitum access to food and water. Mice were pair-matched for similar weights and then randomly assigned to the Control group (n = 8) and the model group (n = 16). The model group was fed a high-fat diet for 16 weeks (cat. No. D12492, USA, Research Diets General Nutrition Protein, 20%, 20%, and 60% Carbohydrate and Fat, prospectively). The Control group was fed standard chow (Pruddon Biotechnology Co., Ltd.). After modeling, the mice were divided into a model group (n = 8) and a metformin treatment group (n = 8) based on body weight. The treatment group received oral gavage of metformin hydrochloride at 200 mg/kg (Merck Pharmaceuticals) for 4 weeks.Both the Control and DBD groups received an equal volume of saline via oral gavage over a 4-week period.

Weight measurement

From 6 weeks of age to 26 weeks of age (a total of 20 weeks), the weight of all mice was measured and recorded weekly.

Blood glucose testing¹⁸

Oral glucose tolerance test (OGTT) and Oral glucose tolerance test (ITT) assays were conducted on mice after 16 weeks of feeding and prior to tissue collection. Blood from the tail vein was dropped onto glucose test strips. Subsequently, glucose (3 g/kg body weight) was orally administered using gavage, and blood glucose levels were monitored at 0, 15, 30, 60, and 120 min. Prior to ITT treatment, mice fasted for 6 h. Following baseline blood glucose level measurements, insulin (1 IU/kg body weight) was injected into the peritoneal cavity. Glucose concentrations were measured at 0, 15, 30, 60, and 120 min, respectively. The area under the curve (AUC) for both OGTT and ITT was calculated accordingly.

Spontaneous Void Spot Assay (VSA)¹⁹

During the experiment, individual mice were gently placed on filter paper inside an experimental cage, covered with a metal mesh and lid, and placed in a quiet environment to allow urination. The testing period lasted for 4 h, during which water was not provided, only food. To analyze spots larger than 0.02 cm² (equivalent to 0.75 ml of urine) using ImageJ, the following parameters were collected: the number of spots (assessing voiding frequency), total area covered by spots (reflecting urine volume), and the percentage of spots in the central region.

Bladder pressure measurement²⁰

For cystometry experiments, mice were anesthetized using 25% ethyl carbamate (1.8 g/kg, intraperitoneal injection) and surgically exposed through an abdominal incision. A small incision was made at the top of the bladder, and a PE-10 catheter was inserted and secured with sutures. Catheters were secured with sutures, tunneled subcutaneously (to leave an exposed end through an incision in the back of the neck), and heat-sealed. Conscious cystometry was conducted in metabolic cages five days following catheter implantation. During pressure measurement, mice were immobilized on a frame, and a pressure sensor and flow pump were connected to the implanted bladder catheter. The bladder catheter and pressure sensor were connected to a biological signal acquisition system (MD3000). Open the recording program, on a computer to calibrate the system pressure and prepare for Recording. Fill a 20 mL syringe with 10–15 mL of room temperature 0.9% NaCl and load into the infusion pump. Program the pump to infuse at a rate of 0.6 mL/h. After an initial stabilization period of 0.5–1 h, data were collected and analyzed across 4–6 representative micturition cycles. Pressure units were set to cmH₂O to measure intercontractile interval, urinary contraction frequency, Maximum urinary excretion peak, and the count of non-urination contractions.

Masson and Hematoxylin–Eosin staining

Following fixation in a 10% formalin solution, the bladder samples were embedded in paraffin and subsequently sectioned into 5-μm-thick slices. These sections were stained with both hematoxylin and eosin (H&E) as well as Masson stain. The samples were analyzed using ImageJ software.

Immunohistochemistry

The bladder stone paraffin sections were deparaffinized and dehydrated with xylene and ethanol.After microwave heating to repair antigens in the tissue sections and blocked with goat serum. Then the sections were incubated with primary antibodies against GSDMD (1:200, affinity, AF4012, USA), ZO-1 (1:200, affinity, AF5145, USA), Claudin-1 (1:200, affinity, DF6919, USA) and Occludin (1:200, Proteintech, 27260-1-AP, China) overnight at 4 ℃. The following day, the sections were incubated for 20 min with enzyme-labeled goat anti-rabbit IgG polymer.Subsequently, the sections were incubated with DAB chromogen solution (diluted to 1:20) and then counterstained with hematoxylin.

Immunofluorescence

The bladder stone paraffin sections were deparaffinized and dehydrated with xylene and ethanol.After microwave heating to repair antigens in the tissue sections. 0.1% Triton X-100 was added to the tissue to rupture cell membranes. Subsequently, 10% goat serum was applied for 1 h to block non-specific binding. Primary antibodies against NLRP3 (1:200, affinity, DF7438, USA) and TXNIP (1:200, Wanleibio, WL05902, China) were incubated on the sections overnight. The next day, incubated with secondary antibodies for 1 h. After the sections were mounted with fluorescence mounting medium and observed under a fluorescence microscope.

Western blot

Briefly, total protein was extracted using RIPA lysis buffer by centrifugation at 13,000 rpm for 15 min. After determining the protein concentration,Different molecular weight proteins were separated by 10% sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto PVDF membranes. After blocking the membranes with 5% skim milk at room temperature, they were cut into different bands according to the desired molecular weight and incubated overnight with different primary antibodies TXNIP (1:1000, Wanleibio, WL05902, China), NLRP3 (1:500, Abcam, Abcam, USA), GSDMD (1:1000, affinity, AF4012, USA), ZO-1 (1:1000, affinity, AF5145, USA), Claudin-1 (1:1000, affinity, DF6919, USA) and Occludin (1:1000, Proteintech, 27260-1-AP, China). Incubated at room temperature for 1 h with HRP-conjugated goat anti-rabbit/mouse IgG (1:5000, affinity, S0001, USA). Finally, the target protein bands were detected using an ECL chemiluminescence kit.

Statistical treatment

Graph Pad Prism 9.0 software was used to analyze the experimental results. Data were expressed as mean ± standard deviation (mean ± SD). One-way variance analysis was used for comparison of means between groups, and the Dunnett test was used for pairwise comparison of homogeneity of variance. P < 0.05 was considered statistically significant.

Results

Metformin improves glucose metabolism in DBD mice

Following treatment with metformin, mice in the DBD group exhibited a significant decrease in body weight, which gradually recovered to normal levels. Additionally, compared to the DBD group, their glucose tolerance improved. Furthermore, metformin treatment significantly alleviated impaired glucose tolerance and insulin resistance in mice (Fig. 1).

Fig. 1.

Changes in metabolic parameters of mice. (A) Variation in mice body weight; (B) Changes in OGTT values and AUC; (C) Changes in ITT values and AUC. (OGTT Oral Glucose Tolerance Test, ITT Insulin Tolerance Test, AUC Area Under the Curve. All statistical results are presented as mean ± SD n = 8, comparisons between DBD and Control groups, as well as Metformin group, *P < 0.05).

Metformin reduced the frequency of urination in DBD mice and improved bladder function

To assess changes in the urination behavior pattern of DBD mice, VSA and urodynamic testing were conducted on three groups of mice.DBD mice had increased numbers and volumes of urinary spots, with multiple spots appearing in the central area of the filter paper and a decrease in the percentage of main spots compared to the Control group (Fig. 2A–D). The results of urodynamic testing indicated that DBD mice had shortened intercontractile interval, increased urination frequency, a higher incidence of non-urination contractions, along with a decrease in bladder capacity and an increase in the peak pressure of urination. Following treatment with metformin, there was a decrease in the number of urinary spots, a reduction in urination frequency and non-voiding contractions, stabilization of the peak pressure of urination, restoration of urinary control, and improvement in bladder function (Fig. 2E–I).

Fig. 2.

The alterations in bladder functional parameters of mice. (A) Results of VSA; (B) urine spot count; (C) Total area of urine spot; (D) The ratio of urine spot area in central area; (E) Cystometry images in awake mice; (F) The number of urination contraction/20 min (G) The quantity of non-urination contractions/20 min; (H) intercontractile interval; (I) Maximum peak pressure of urination. (All statistical results are presented as mean ± SD, n = 8, comparisons between DBD and Control groups, as well as Metformin group, *P < 0.05). The central black box represents the central area (6 cm*4 cm).

Metformin restored bladder structure in DBD mice

Bladder tissues of the Control group mice revealed normal morphology, with intact bladder wall and undamaged mucosa, displaying clearly defined and tightly arranged epithelial folds by the histological examination. In contrast, bladder tissues from the DBD mice exhibited signs of injury, with loose epithelial folds and muscular layer atrophy. Following metformin treatment, the structural integrity of epithelial folds in mice was restored (Fig. 3A,B). In Masson’s staining, bladder muscle layers of mice in the Control group displayed absence of gaps and comparatively lower distribution of collagen proteins. Conversely, DBD mice exhibited increased interstitial spaces, prominent muscle layer disruptions, reduced thickness, and a significant rise in collagen fiber content. Metformin treatment improve thickening of the muscle layer and a notable reduction of collagen fiber (Fig. 3C,D).

Fig. 3.

Morphological analysis of mice bladder. (A) Representative images of bladder H&E staining; (B) Representative images of bladder Masson's trichrome staining; (C) Thickness of the bladder muscle layer; (D) Proportion of collagen fibers in the bladder muscle layer. (All statistical results are presented as mean ± SD, n = 8, comparisons between DBD and Control groups, as well as Metformin group, *P < 0.05).

Metformin restores tight junction proteins and bladder barrier function in DBD mice

To substantiate the protective effect of metformin, we conducted immunohistochemistry and Western blot experiments. The immunohistochemistry results revealed that the positive staining intensity of epithelial tight junction proteins Zo-1, Claudin-1, and Occludin in the DBD group was significantly lower compared to the control mice, indicating impaired epithelial barrier function. After treatment with metformin, the depth of positive staining was notably lower compared to the DBD group mice (Fig. 4A). The Western blot results demonstrated a significant decrease in the expression of tight junction proteins Zo-1, Claudin-1, and Occludin in the DBD group. Conversely, the expression of these tight junction proteins was noticeably restored in the metformin group (Fig. 4B–E).

Fig. 4.

The expression and localization of tight junction proteins in mice bladder tissue. (A) Immunohistochemistry of Zo-1, Claudin-1, and Occludin in bladder tissue; (B) Western blot expression of Zo-1, Occludin, and Claudin-1; (C–E) Statistical results of Zo-1, Occludin, Claudin-1. (All statistical results are presented as mean ± SD, n = 8, comparisons between DBD and Control groups, as well as Metformin group, *P < 0.05).

Metformin inhibits pyroptosis mediated by TXNIP/NLRP3 in DBD

As shown in Fig. 5A,B, Immunofluorescence results demonstrated that the expression levels and areas of TXNIP and NLRP3 in the bladder epithelial layer were lower in the metformin group compared to the DBD group. Immunohistochemistry results revealed a significantly higher depth of positive staining for the pyroptosis factor GSDMD in the epithelial layer of the DBD group compared to Control mice, indicating primary epithelial damage. After treatment with metformin, the depth of positive staining was notably lower compared to the DBD group mice (Fig. 5C). To further ascertain the expression levels of TXNIP, NLRP3, and GSDMD, we assessed their protein expression by Western blot. Western blot results showed a significant increase in the expression of TXNIP NLRP3, and GSDMD in the DBD group. Conversely, the protein levels of TXNIP NLRP3, and GSDMD in the metformin group were significantly lower than those in the DBD group (Fig. 5D–G).

Fig. 5.

The expression and localization of TXNIP, NLRP3, and GSDMD in mice bladder tissue. (A) Immunofluorescence expression of TXNIP in bladder tissue (green represents TXNIP, blue represents DAPI); (B) Immunofluorescence expression of NLRP3 in bladder tissue (green represents NLRP3, blue represents DAPI); (C) Immunohistochemistry of GSDMD in bladder tissue; (D) Western blot expression of NLRP3, TXNIP, and GSDMD. (E–G) Statistical results of NLRP3, TXNIP, and GSDMD. (All statistical results are presented as mean ± SD, n = 8, comparisons between DBD and Control groups, as well as Metformin group, *P < 0.05).

Discussion

DBD is one of the most common complications of diabetes, significantly impacting individuals' quality of life and health. Recent research indicates that elevated blood glucose levels and the resulting oxidative stress are significant contributors to DBD²¹. However, the precise pathological mechanisms remain unclear. TXNIP and NLRP3 are crucial oxidative stress regulatory factors in the body, and the TXNIP-NLRP3 signaling pathway is closely associated with the onset of various diseases. Despite this knowledge, there remains a paucity of studies regarding the role of the TXNIP-NLRP3 pathway in the pathogenesis of DBD, leaving the specific pathological mechanisms elusive. Consequently, we established a T2DM mice model to further elucidate the role of the TXNIP-NLRP3 pathway in DBD pathogenesis. We conducted assessments of the mice's body weight, blood glucose levels, OGTT and ITT. The results revealed that mice in the DBD group exhibited significantly higher body weight and blood glucose levels compared to the control group. OGTT and ITT experiments demonstrated impaired glucose tolerance and insulin resistance in the DBD group. VSA and Bladder Pressure Measurement are commonly employed to analyze bladder dysfunction, specifically regarding urinary storage or voiding performance. Early-stage DBD primarily presents as bladder overactivity (OAB), characterized by urinary frequency, urgency, and lack of voiding control²². Prolonged hyperglycemia results in long-term compensatory detrusor muscle contractions, causing detrusor muscle hypertrophy, inflammation, and ultimately fibrosis, leading to detrusor underactivity in the late stages of DBD. Underactive bladder (UAB) is a symptom complex characterized by prolonged urination time with or without a sensation of incomplete bladder emptying, usually with hesitancy, reduced sensation on filling, and a slow stream²³. Through VSA and urodynamic studies conducted on awake mice, we observed polyuria, shortened intercontractile interval, increased non-urination contractions, and increased peak voiding pressures, all of which are clinical manifestations of OAB. These findings indicate the successful establishment of a T2DM mice model, wherein the mice exhibit clinical characteristics of DBD.

The bladder epithelium comprises basal, intermediate, and superficial layers, with highly differentiated superficial cells forming an important barrier²⁴. This barrier prevents toxins, ions, and water from flowing between urine and blood²⁵, safeguarding epithelial cells from toxins, bacteria, and pro-inflammatory metabolites in urine. The superficial layer contains abundant tight junction proteins, forming a dense network composed of cytoplasmic proteins, cytoskeletal elements, and transmembrane proteins, such as zonula occludens-1 (ZO-1)²⁶. Which scaffold tight junction-associated transmembrane proteins to the surrounding actomyosin cytoskeleton and may play a crucial role in regulating cell-to-cell signaling and controlling lateral barrier function²⁷. Transmembrane proteins associated with tight junctions include junctional adhesion molecules²⁸, including occludin and claudin family proteins¹⁷. The latter two form continuous transmembrane particle strand fibers that completely encircle the apical surfaces of each cell’s lateral aspects, forming a complex barrier with ion and size selectivity²⁹. Due to the unique properties of tight junction proteins maintaining the normal physiological activities of the bladder, in our study, we observed a significant decrease in tight junction proteins in the DBD group mice through immunohistochemistry and Western blotting. This decline may be linked to the TXNIP-NLRP3 pathway induced by DBD.

Current treatment methods for DBD primarily include behavioral therapy, medication, and injections of type A botulinum toxin or surgery²¹. Many patients with T2DM experience suboptimal treatment outcomes and severe side effects, underscoring the ongoing necessity to pursue more efficacious and safer therapeutic modalities. Metformin, a commonly prescribed antidiabetic medication, has been extensively studied for its potent antioxidative and anti-inflammatory properties, showing promise in mitigating various diseases³⁰. Therefore, metformin may have potential efficacy in treating epithelial damage caused by DBD.

During the course of DBD progression, there are dynamic pathological changes involving smooth muscle, urinary tract epithelium, and their neural innervation. Through the VSA and Bladder Pressure Measurement, we detected changes in mouse voiding patterns, aligning with the pathological variations observed in DBD. Prior research has suggested that diabetes-induced bladder neuropathy may augment bladder maximum capacity and residual urine volume³¹. Our study illustrates that metformin treatment markedly ameliorates epithelial pathological injury and voiding irregularities caused by DBD, thereby affirming its protective impact on the bladder.

This study significantly reveals the presence of epithelial pyroptosis in DBD, and highlights the potent anti-pyroptosis effects of metformin. Pyroptosis, a form of programmed cell death, differs markedly from apoptosis. We observed that DBD-induced injury leads to excessive inflammatory responses both intra- and extracellularly, an area of research that has been underexplored in relation to pyroptosis. Recent studies indicate that pyroptosis is triggered by various stimuli and initiated through the activation of the gasdermin (GSDM) protein family³². The buildup of NLRP3 inflammasomes in DBD initiates the fragmentation of the GSDM protein family, inducing epithelial pyroptosis and consequent disruption of bladder barrier integrity. This investigation confirms the protective role of metformin in DBD. Our findings reveal that metformin therapy lowers the incidence of epithelial pyroptosis in the bladder, alleviates injury to tight junction proteins, maintains the structural integrity of the bladder barrier, and protects against DBD-induced harm. Metformin reduces both extracellular and intracellular cell demise. Furthermore, we propose that metformin may improve DBD by inhibiting the TXNIP-NLRP3-GSDMD axis, thereby reducing epithelial cell pyroptosis.

TXNIP is an oxidative stress regulatory factor involved in diabetes and insulin resistance³³. Additionally, it can inhibit the antioxidant activity of TRX by binding to proteins.Both TRX and TXNIP are expressed in the cytoplasm and mitochondria³⁴, where they maintain redox balance and interact with NLRP3, triggering the inflammatory process known as the TXNIP-NLRP3 axis³⁵. Our present investigation implicates the TXNIP-NLRP3 axis in mediating the protective effects of metformin. We propose that metformin demonstrates significant anti-pyroptosis characteristics and is linked to the TXNIP-NLRP3-GSDMD pathway.

The mechanisms responsible for epithelial damage in DBD are intricate and involve numerous pathways. In our present study, we elucidated the crucial involvement of pyroptosis in bladder epithelial cells in the development of DBD. We discovered that TXNIP mediates pyroptosis activation, leading to increased NLRP3 expression and subsequent GSDMD activation, which generates and releases pyroptosis cytokines. Metformin, a hypoglycemic drug, protects against DBD by inhibiting TXNIP, thus suppressing bladder epithelial cell pyroptosis via the TXNIP-NLRP3 pathway. Furthermore, there are several imitations that need to be addressed in this study numerous prospective clinical trials are needed to validate the effects of metformin on humans, as our findings are solely experimental³⁶. We believe that metformin holds significant promise as a therapeutic agent for managing epithelial damage in DBD (Supplementary Information).

Conclusion

Based on the above analysis, we have found that metformin may protect bladder epithelium and effectively improve detrusor overactivity by potentially inhibiting pyroptosis through the TXNIP-NLRP3-GSDMD pathway.

Author contributions

Bincheng Huang contributed to the research design, conducted animal experiments, and wrote the paper. Jin Zhang, Leyi Yu, Cunling Ma, and Shuzhe Fan assisted in drafting the manuscript. Shuai Ren, Yunshang Tuo, Haifu Tian, Xuehao Wang, Jiajing Feng, Keming Chen, Qingjie Peng, and Xiaojiang Chen participated in animal experiments, data analysis, and manuscript revision. Guangyong Li and Rui He provided substantial guidance in study design, division of labor, and manuscript writing and revision.

Funding

This paper is supported by National Natural Science Foundation of China (81860268, 82201000), Ningxia Natural Science Foundation (2021AAC02025, 2021BEB04034), Ningxia science and technology innovation leading talent training project (2020GKLRLX06, 2020GKLRLX11). Ningxia Medical University research project (XT2019017), Key Research and Development Program of Ningxia Hui Autonomous Region(2021BEB04034 2023BEG03021).

Data availability

The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.

Competing interests

The authors declare no competing interests.

Ethics approval and consent to participate

All experimental procedures were performed in accordance with the guidelines of the Institutional Animal Care and Use Committee of Ningxia Medical University and ethical approval was obtained for the animal experiments conducted in the study (Certificate number: IACUC-NYLAC-2021-054), following the guidelines of the US National Institutes of Health and the Animal Research Reporting In Vivo Experiments (ARRIVE).

Supplementary Information

The online version contains supplementary material available at 10.1038/s41598-024-72129-0.