Inhibition of the NF-κB Signaling Pathway Alleviates Pyroptosis in Bladder Epithelial Cells and Neurogenic Bladder Fibrosis

Most children with a neurogenic bladder (NB) have bladder fibrosis, which causes irreversible bladder dysfunction and damage to the upper urinary tract. However, the mechanism of bladder fibrosis remains unclear. This study aimed to investigate the underlying causes of bladder fibrosis. Here, the lumbar 6 (L6) and sacral 1 (S1) spinal nerves of Sprague Dawley rats were severed bilaterally to establish NB models. Using RNA-seq, we discovered that the NF-κB signaling pathway and inflammation were upregulated in spinal cord injury (SCI)-induced bladder fibrosis. Subsequent Western blotting, enzyme-linked immunosorbent assays, immunohistochemical staining, and immunofluorescence staining verified the RNA-seq findings. To further clarify whether the NF-κB signaling pathway and pyroptosis were involved in bladder fibrosis, a TGF-β1-treated urinary epithelial cell line (SV-HUC-1 cells) was used as an in vitro model. Based on the results of RNA-seq, we consistently found that the NF-κB signaling pathway and pyroptosis might play important roles in TGF-β1-treated cells. Further experiments also confirmed the RNA-seq findings in vitro. Moreover, using the NLRP3 inhibitor MCC950 rescued TGF-β1-induced fibrosis, and the NF-κB signaling pathway inhibitor BAY 11-7082 effectively rescued TGF-β1-induced pyroptosis and the deposition of extracellular matrix by SV-HUC-1 cells. In summary, our research demonstrated for the first time that the NF-κB signaling pathway inhibition rescued bladder epithelial cells pyroptosis and fibrosis in neurogenic bladders.


Introduction
Neurogenic bladder (NB) refers to the progressive bladder or urethral dysfunction caused by neurological disease, which is one of the main causes of renal dysfunction [1]. NB often leads to severe bladder dysfunction, including bladder wall thickening, poor compliance, increased intravesical pressure, vesicoureteral reflux, and eventually progression to end-stage renal disease [2]. Around 80% of people with catastrophic spinal cord injuries (SCI) will develop neurogenic bladders and the numerous side effects that accompany them [3]. Loss of coordinated activation of the detrusor and suppression of the urethral sphincter during voiding are further effects of injury to the suprasacral spinal pathways. Instead, a condition known as detrusor-sphincter dyssynergia causes the detrusor and urethral sphincter to contract simultaneously [4]. This leads to increased intravesical pressure, detrusor hyperreflexia, and collagen deposition, which in turn reduces bladder compliance [5], finally causing a multitude of problems including urinary tract infection,

Establishment of the Neurogenic Bladder Fibrosis Model
The L6-S1 spinal nerves of Sprague Dawley (SD) rats were bilaterally severed to understand the underlying causes of bladder fibrosis ( Figure 1A). We performed urodynamic examinations on rats after SCI and found that rats in the sham-operated group showed substantial peak contraction of the detrusor muscle during the voiding phase. However, the bladders of rats in the SCI group did not have a distinct filling and voiding phase and showed intermittent urinary incontinence ( Figure 1B). In addition, there was a great increase in maximum bladder capacity (MCC), bladder leak point pressure (BLPP), and bladder compliance (BC) in the NB group at 8 weeks post-surgery ( Figure 1C), indicating severely impaired bladder function. Then, the bladder tissues of rats were collected for morphological observa-tions and weighing. The findings demonstrated that SCI considerably increased the bladder capacity in the NB group and even caused bladder stones to develop ( Figure 1D). Moreover, the weight of bladder tissue increased dramatically in the NB group ( Figure 1E). group showed substantial peak contraction of the detrusor muscle during the voiding phase. However, the bladders of rats in the SCI group did not have a distinct filling and voiding phase and showed intermittent urinary incontinence ( Figure 1B). In addition, there was a great increase in maximum bladder capacity (MCC), bladder leak point pressure (BLPP), and bladder compliance (BC) in the NB group at 8 weeks post-surgery ( Figure 1C), indicating severely impaired bladder function. Then, the bladder tissues of rats were collected for morphological observations and weighing. The findings demonstrated that SCI considerably increased the bladder capacity in the NB group and even caused bladder stones to develop ( Figure 1D). Moreover, the weight of bladder tissue increased dramatically in the NB group ( Figure 1E).  in the control and NB groups (n = 5 each group). (D) Gross morphological changes in bladder tissues in the different groups of rats. (E) Weight changes in bladder tissues between the two groups (n = 7 each group). (F) Scr and BUN levels in the two groups of rats (n = 5 each group). (G) H&E, Masson, and immunohistochemical staining (scale bar = 100 µm) of bladders in the two groups of rats. (H) Based on five randomly chosen fields of stained bladder tissue slices, the fractional area of fibrosis in the bladder was quantified. (I) Relative protein levels of fibronectin and vimentin in the two groups were analyzed by Western blotting. The data are presented as the mean ± SD; ** p < 0.01, *** p < 0.001, **** p < 0.0001.
To determine whether impaired bladder function further affected upper urinary tract function, we tested renal function. The results showed that Serum creatinine (Scr) and blood urea nitrogen (BUN) levels in rats were elevated in the NB group, indicating impaired renal function ( Figure 1F). Then, we performed histological examinations of bladder tissues. Hematoxylin and eosin (H&E) staining showed that bladder tissues in the NB group had thickened muscle bundles, disorganized arrangement, and obvious inflammatory cell infiltration in the epithelial region. Collagen fibers were colored blue after Masson's staining, while bladder smooth muscle cells were colored red. Measurements were made of the collagen area to total area ratio. In comparison to the control group, the NB group's fractional area of bladder fibrosis was considerably higher ( Figure 1G,H). Subsequently, the immunohistochemical and Western blotting results showed an increase in fibronectin and vimentin expression in bladder tissue in the NB group, indicating increased fibrosis ( Figure 1G,I). Overall, these results suggest that bladder and upper urinary tract dysfunction occurred in rats after SCI and induced fibrosis in the bladder.

The NF-κB Signaling Pathway and Inflammation Were Enriched in Neurogenic Bladder Fibrosis
To investigate the mechanism of bladder fibrosis in the NB group, we subjected the tissue transcriptome sequencing results to differential analysis and enrichment analysis (Figure 2A). Using comparative analysis, we found 1397 genes that were abnormally expressed in the NB group. A total of 913 of their genes were elevated, whereas 484 were downregulated ( Figure 2B). Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis of tissues showed that differentially expressed genes (DEGs) were associated with epithelial cell differentiation, ECM, the NF-κB signaling pathway, and inflammation ( Figure 2C). The GSEA analysis also revealed that DEGs were considerably enriched in the NF-κB signaling pathway, inflammation, and EMT ( Figure 2D). These results suggest that bladder fibrosis may be related to the NF-κB signaling pathway and inflammatory response. Using a volcano plot, we identify the genes that are differentially expressed between the control and NB groups. (C) The NF-κB pathway, epithelial cell differentiation, and ECM were shown to be abundant in the DEGs of tissues according to GO and KEGG analyses (the red underline indicates the focus of attention). (D) Potential enhancement of EMT, the NF-κB pathway, and the inflammatory response in tissues was examined using GSEA.

The NF-κB Signaling Pathway and Pyroptosis-Related Proteins Were Significantly Increased In Vivo
DEGs were enriched in the NF-κB signaling pathway and inflammatory response, according to the RNA-seq analysis. We first concentrated on the NF-κB signaling pathway, since this is a key mediator of NLRP3 inflammasome-mediated pyroptosis [18]. We performed immunohistochemical staining of rat bladder tissue sections and found that the bladder epithelium displayed considerably higher levels of P-P65 expression ( Figure 3A). Subsequent Western blotting confirmed the activation of the NF-κB signaling pathway in bladders in the NB group ( Figure 3B). The inflammatory factors IL-6, IL-1β, and IL-18 were increased in the serum of rats after SCI compared with those in the control group ( Figure 3C). To further investigate whether the inflammatory response of bladder tissue in the NB group was associated with pyroptosis, we subjected bladder tissue to immunofluorescence staining. The results showed an increase in the expression of NLRP3 and ASC, which are proteins associated with pyroptosis, in the bladder epithelium ( Figure  3D-F). Subsequently, using scanning electron microscopy (SEM), we observed disorganized cell arrangement, increased filamentous secretion, and the generation of pyroptotic bodies in the bladder epithelial layer in the NB group ( Figure 3G). Transmission electron microscopy (TEM) showed cell death and abnormal endoplasmic

The NF-κB Signaling Pathway and Pyroptosis-Related Proteins Were Significantly Increased In Vivo
DEGs were enriched in the NF-κB signaling pathway and inflammatory response, according to the RNA-seq analysis. We first concentrated on the NF-κB signaling pathway, since this is a key mediator of NLRP3 inflammasome-mediated pyroptosis [18]. We performed immunohistochemical staining of rat bladder tissue sections and found that the bladder epithelium displayed considerably higher levels of P-P65 expression ( Figure 3A). Subsequent Western blotting confirmed the activation of the NF-κB signaling pathway in bladders in the NB group ( Figure 3B). The inflammatory factors IL-6, IL-1β, and IL-18 were increased in the serum of rats after SCI compared with those in the control group ( Figure 3C). To further investigate whether the inflammatory response of bladder tissue in the NB group was associated with pyroptosis, we subjected bladder tissue to immunofluorescence staining.
The results showed an increase in the expression of NLRP3 and ASC, which are proteins associated with pyroptosis, in the bladder epithelium ( Figure 3D-F). Subsequently, using scanning electron microscopy (SEM), we observed disorganized cell arrangement, increased filamentous secretion, and the generation of pyroptotic bodies in the bladder epithelial layer in the NB group ( Figure 3G). Transmission electron microscopy (TEM) showed cell death and abnormal endoplasmic reticulum morphology in the bladder epithelium in the NB group ( Figure 3H). Western blotting later revealed that the bladder tissue of the NB group had higher levels of NLRP3, GSDMD, ASC, and IL-1β expression ( Figure 3I). These results indicated that the bladder tissue of rat was structurally disturbed by cell death and the inflammatory response after SCI, which might be related to bladder epithelial cell pyroptosis. blotting later revealed that the bladder tissue of the NB group had higher levels of NLRP3, GSDMD, ASC, and IL-1β expression ( Figure 3I). These results indicated that the bladder tissue of rat was structurally disturbed by cell death and the inflammatory response after SCI, which might be related to bladder epithelial cell pyroptosis.  The data are presented as the mean ± SD; ** p < 0.01, *** p < 0.001, **** p < 0.0001, ns, not significant.

TGF-β1-Induced Fibrosis Model of SV-HUC-1 Cells In Vitro
To further explore whether the pyroptosis and NF-κB pathway were involved in regulating bladder epithelial cell fibrosis, we further constructed a cellular model of bladder epithelial cell fibrosis. Previous studies have shown that TGF-β1 is a key regulator of the ECM, cell proliferation and differentiation, and the inflammatory response in fibrotic diseases [19]. TGF-β1 is frequently employed in experimental investigations to induce pulmonary fibrosis [20], renal fibrosis [21], and hepatic fibrosis [22] since it is essential for wound healing and the deposition of matrix molecules. Therefore, we used different concentrations (3, 5, and 10 ng/mL) of TGF-β1 to induce ECM in SV-HUC-1 cells. Western blotting was used to detect the protein expression of fibronectin and vimentin, and the results showed that 5 ng/mL TGF-β1 effectively induced ECM in SV-HUC-1 cells ( Figure 4A). Based on this outcome, we chose 5 ng/mL TGF-β1 for the subsequent experiments. To further demonstrate that ECM accumulation occurred in TGF-β1-treated cells, we performed immunofluorescence assays and found that the expression of fibronectin and vimentin was increased ( Figure 4B-D), indicating that the in vitro cell fibrosis model was established.

TGF-β1-Induced Fibrosis Model of SV-HUC-1 Cells In Vitro
To further explore whether the pyroptosis and NF-κB pathway were involved in regulating bladder epithelial cell fibrosis, we further constructed a cellular model of bladder epithelial cell fibrosis. Previous studies have shown that TGF-β1 is a key regulator of the ECM, cell proliferation and differentiation, and the inflammatory response in fibrotic diseases [19]. TGF-β1 is frequently employed in experimental investigations to induce pulmonary fibrosis [20], renal fibrosis [21], and hepatic fibrosis [22] since it is essential for wound healing and the deposition of matrix molecules. Therefore, we used different concentrations (3, 5, and 10 ng/mL) of TGF-β1 to induce ECM in SV-HUC-1 cells. Western blotting was used to detect the protein expression of fibronectin and vimentin, and the results showed that 5 ng/mL TGF-β1 effectively induced ECM in SV-HUC-1 cells ( Figure 4A). Based on this outcome, we chose 5 ng/mL TGF-β1 for the subsequent experiments. To further demonstrate that ECM accumulation occurred in TGF-β1-treated cells, we performed immunofluorescence assays and found that the expression of fibronectin and vimentin was increased ( Figure 4B-D), indicating that the in vitro cell fibrosis model was established. . The data are presented as the mean ± SD; * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

The NF-κB Signaling Pathway and Inflammation Were Enriched in TGF-β1-Induced SV-HUC-1 Cells
Transcriptome sequencing was performed on cells from the control and TGF-β1treated groups (n = 3). As shown in Figure 5A, the samples from the control and TGFtreated groups were evenly distributed across the groups. Moreover, the samples were fully distinct from one another, indicating that each group had the appropriate level of aggregation. Then, a heatmap analysis of cell sample correlation revealed variations The data are presented as the mean ± SD; * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

The NF-κB Signaling Pathway and Inflammation Were Enriched in TGF-β1-Induced SV-HUC-1 Cells
Transcriptome sequencing was performed on cells from the control and TGF-β1treated groups (n = 3). As shown in Figure 5A, the samples from the control and TGFtreated groups were evenly distributed across the groups. Moreover, the samples were fully distinct from one another, indicating that each group had the appropriate level of aggregation. Then, a heatmap analysis of cell sample correlation revealed variations between the groups and excellent reproducibility within the sample groups ( Figure 5B). About 2942 DEGs were discovered, and the numbers of up-and downregulated genes are shown in Figure 5C. We then performed GO and KEGG analysis of DEGs in both groups of cells, and the results showed that DEGs were upregulated in the pathways associated with epithelial cell migration, ECM, and the inflammatory response in the TGF-β1-treated group ( Figure 5D). GSEA revealed that DEGs were considerably enriched in the NF-κB signaling pathways, EMT, and inflammatory response ( Figure 5E). To demonstrate the activation of the NF-κB signaling pathway in cells, Western blotting was performed, and the results showed that the P-P65/P65 ratio was enhanced in response to TGF-β1, and the expression of IKBα was decreased ( Figure 5F). These results suggest that bladder epithelium cell fibrosis may be related to the NF-κB signaling pathway and inflammatory response. between the groups and excellent reproducibility within the sample groups ( Figure 5B). About 2942 DEGs were discovered, and the numbers of up-and downregulated genes are shown in Figure 5C. We then performed GO and KEGG analysis of DEGs in both groups of cells, and the results showed that DEGs were upregulated in the pathways associated with epithelial cell migration, ECM, and the inflammatory response in the TGF-β1-treated group ( Figure 5D). GSEA revealed that DEGs were considerably enriched in the NF-κB signaling pathways, EMT, and inflammatory response ( Figure 5E). To demonstrate the activation of the NF-κB signaling pathway in cells, Western blotting was performed, and the results showed that the P-P65/P65 ratio was enhanced in response to TGF-β1, and the expression of IKBα was decreased ( Figure 5F). These results suggest that bladder epithelium cell fibrosis may be related to the NF-κB signaling pathway and inflammatory response.

Pyroptosis-Related Proteins Were Increased in TGF-β1-Induced SV-HUC-1 Cells
To investigate the role of the inflammatory response in bladder fibrosis, we intersected the cellular DEGs with a set of inflammation-related differential genes (IRGs) downloaded from the GSEA website. Finally, we made a heatmap of the differential genes obtained from the intersection and found that most of the inflammatory genes were elevated in cells in the TGF-β1 group ( Figure 6A,B). These results suggest that bladder fibrosis may be related to the NF-κB signaling pathway and inflammatory response. Based on the transcriptome sequencing results, we performed immunofluorescence assays of pyroptosisrelated proteins in the cells. The results showed that the TGF-β1-induced expression of NLRP3 and ASC was increased in SV-HUC-1 cells (Figure 6C-E). To further confirm pyroptosis in TGF-β1-induced SV-HUC-1 cells, we examined both groups of cells using SEM. We found that the cells were swollen, and cell membrane pores and pyroptotic bodies appeared in the TGF-β1 group ( Figure 6F). TEM showed that cell membrane integrity and organelle structures, such as mitochondria and endoplasmic reticulum, were disrupted in the TGF-β1 group ( Figure 6G). Western blotting showed that TGF-β1-induced expression of NLRP3, GSDMD, ASC, and IL-1β was increased ( Figure 6H). These findings suggest that pyroptosis may be related to bladder epithelium cell fibrosis.

NF-κB Signaling Pathway Inhibition Rescued TGF-β1-Induced Pyroptosis and Fibrosis In Vitro
To clarify the relationship between pyroptosis and fibrosis, we treated the cells with MCC950 (C 20 H 24 N 2 O 5 S), a specific molecular inhibitor of NLRP3. Western blotting showed that MCC950 reduced epithelial cell expression of NLRP3, GSDMD, ASC, and IL-1β ( Figure 7A). More importantly, MCC950 reduced the expression levels of fibronectin and vimentin in the TGF-β1 group ( Figure 7B). These findings demonstrated that bladder fibrosis might be reduced by decreasing NLRP3 inflammasome-dependent pyroptosis. As the key mediator of NLRP3 inflammasome-mediated pyroptosis, the role of the NF-κB signaling pathway in bladder fibrosis needs further exploration. Subsequently, the elevation of P-P65/P65, fibronectin, and vimentin protein expression levels caused by TGF-β1 was then dramatically reversed after treatment with the NF-κB inhibitor BAY 11-7082 ( Figure 7C). To determine the relationship between the NF-κB signaling pathway and pyroptosis, we performed Western blotting and showed that the protein expression levels of NLRP3, GS-DMD, ASC, and IL1-β were inhibited by the NF-κB inhibitor BAY 11-7082 ( Figure 7D). These findings indicated that the NF-κB signaling pathway promotes NB fibrogenesis via bladder epithelial cell pyroptosis. that MCC950 reduced epithelial cell expression of NLRP3, GSDMD, ASC, and IL-1β ( Figure 7A). More importantly, MCC950 reduced the expression levels of fibronectin and vimentin in the TGF-β1 group ( Figure 7B). These findings demonstrated that bladder fibrosis might be reduced by decreasing NLRP3 inflammasome-dependent pyroptosis. As the key mediator of NLRP3 inflammasome-mediated pyroptosis, the role of the NF-κB signaling pathway in bladder fibrosis needs further exploration. Subsequently, the elevation of P-P65/P65, fibronectin, and vimentin protein expression levels caused by TGF-β1 was then dramatically reversed after treatment with the NF-κB inhibitor BAY 11-7082 ( Figure 7C). To determine the relationship between the NF-κB signaling pathway and pyroptosis, we performed Western blotting and showed that the protein expression levels of NLRP3, GSDMD, ASC, and IL1-β were inhibited by the NF-κB inhibitor BAY 11-7082 ( Figure 7D). These findings indicated that the NF-κB signaling pathway promotes NB fibrogenesis via bladder epithelial cell pyroptosis.

Discussion
PNB is mainly due to progressive bladder dysfunction caused by spinal nerve abnormalities. Therefore, we constructed an animal model of PNB caused by SCI for experimental study. With disease progression, bladder fibrosis leads to severe bladder dysfunction, which in turn impairs the function of the upper urinary tract [23]. However, the underlying mechanisms of bladder fibrosis in NB remain unclear. Therefore, we used RNA-seq to examine the involved signaling pathways. We are the first to demonstrate how a new mechanism involving SCI causes pyroptosis in the rat bladder. In particular, we demonstrated that pyroptosis was involved in the development of SCI-induced bladder fibrosis via the NF-κB signaling pathway.
With the persistence of stress, a chronic inflammatory state develops in the bladder. This leads to the activation of multiple biosynthetic or biodegradation pathways, which is followed by the initiation of wound repair processes and the deposition of excess ECM proteins in the bladder wall, ultimately leading to bladder fibrosis and dyscrasia [24,25]. According to previous studies, the inflammatory response plays a vital role in the development of fibrosis. The balance of collagen synthesis and degradation is disrupted in pulmonary fibrosis as more ECM accumulates in the tissue [26], and increased collagen fragmentation promotes inflammation [27]. Pyroptosis is a form of programmed cell death that accompanies the inflammatory response and can rapidly cause cell expansion, plasma membrane rupture, and the release of cellular contents and large amounts of inflammatory factors [28]. The nonclassical pyroptosis pathway interacts with the classical pathway through NLRP3, which is the most studied inflammasome [29].
Recent studies have shown that fibrosis in a variety of human organs is associated with the NLRP3 inflammasome. Fibrosis in these disorders is mostly influenced by the interaction of the NF-κB/NLRP3/caspase-1/IL-1 axis and TGF-β signaling pathway [30,31]. Bladder outlet obstruction followed by overactivation of the NLRP3 inflammasome pathway in the bladder is closely associated with bladder inflammation, fibrosis, and dysfunction [32]. After treatment of mouse renal tubular epithelial cells (TECs) with TGF-β, NLRP3 induces EMT in TECs, leading to increased expression of α-SMA and MMP9 [33]. In addition, NLRP3 −/− [34] mouse models have reduced levels of liver fibrosis. However, the role of the NLRP3 inflammasome in NB fibrosis is unclear.
To simulate PNB caused by SCI, the bilateral L6 and S1 spinal nerves in SD rats were cut. The ECM, inflammatory response, and NF-κB pathway were then considerably enriched in the RNA-seq results, indicating that these pathways may be involved in bladder fibrosis. To further explore whether the NF-κB pathway and pyroptosis were involved in regulating bladder epithelial cell fibrosis, we further constructed a cellular model of bladder epithelial cell fibrosis. We used 3, 5, and 10 ng/mL TGF-β1 to treat cell cultures for 24 h, which was consistent with the in vivo conditions. Subsequently, we sent the control and TGF-β groups of cells for sequencing, and the results were consistent with the tissue sequencing results. Therefore, we investigated the relationship between fibrosis, the NF-κB pathway, and the inflammatory response. The data in this study demonstrate that NLRP3 inflammasome-dependent pyroptosis leads to bladder fibrosis. This mechanism was later confirmed by the use of MCC950, suggesting that NLRP3 mediates pyroptosis and is a target for the prevention of bladder fibrosis.
The NF-κB pathway has long been considered a typical proinflammatory signaling pathway [35]. Previous studies have shown that activation of the NF-κB signaling pathway initiates NLRP3 in the classical pyroptosis pathway. Further assembly of the NLRP3 inflammasome followed by the cleavage of pro-IL-1β and pro-IL-18 by activated caspase-1 promotes the maturation and release of the inflammatory factors IL-1β and IL-18, thereby amplifying the inflammatory response [36]. However, it is not clear whether the NF-κB pathway plays a role in NB fibrosis. In the present study, we suggested that the NF-κB pathway may be the initial factor in bladder fibrosis induced by SCI and may cause the death of bladder epithelial cells.
We observed an increased ratio of P-P65/P65 and decreased levels of IKBα expression in vivo and in vitro. Immunohistochemical analysis showed that P-P65 was mainly expressed in the bladder epithelium. To demonstrate the role of the NF-κB signaling pathway in NB fibrosis, we treated cells with the NF-κB inhibitor BAY 11-7082 and found that inhibiting the NF-κB pathway rescued TGF-β1-induced bladder epithelial cell pyroptosis and fibrosis. Although previous studies have shown that the NF-κB pathway is associated with fibrosis in multiple organs, no studies have demonstrated a role for the NF-κB pathway in NB fibrosis. These findings demonstrate that the NF-κB pathway promotes NB fibrosis by promoting pyroptosis in bladder epithelial cells and indicate a new therapeutic target.
However, there are some limitations to this study. To demonstrate the relationship between pyroptosis and fibrosis, we added the NLRP3 inflammasome inhibitor MCC950 in vitro, but did not validate our findings in vivo. In bladder fibrosis, lesions in the muscular layer of the bladder are important; however, only the role of bladder epithelium in fibrosis was investigated in this study. When performing urodynamic testing, we increased the bladder perfusion rate in the model group to twice as fast in order to shorten the bladder perfusion time, but this may have affected the observed results. In addition, we used only female rats to facilitate urodynamics, which may cause gender bias.

Animals
The Research Ethics Committee of the Children's Hospital of Chongqing Medical University approved this experimental animal study (protocol code CHCMU-IACUC20210625002). The NB rat model was established as previously described [9]. Once the rats were given 20% urethane (5 mL/kg) anesthesia via intraperitoneal injection, the dorsal and ventral roots of the bilateral lumbar 6 (L6) and sacral 1 spinal nerves (S1) were severed from a dorsal median incision. In the control group, only the nerve roots were exposed to surgical instruments without transaction or any other manipulation. Following surgery, penicillin was constantly given for three days. In order to collect bladder tissue samples for future research, the rats were anaesthetized after 8 weeks using 20% urethane (5 mL/kg), and each rat's serum was taken using glass vials for specimen storage, then frozen at −80 • C for long-term storage. Then, all of the rats were killed via cervical dislocation, and we promptly collected their bladders for additional studies.

Experimental Design
In this study, L6-S1 spinal nerve transection and sham surgery on SD rats were compared. Twenty female specific-pathogen-free (SPF) SD rats (120-140 g, 6 weeks) were purchased from the Experimental Animal Center of Chongqing Medical University. All rats were fed under SPF conditions at a temperature of 25 ± 2 • C and a relative humidity of 50 ± 5% using a 12 h artificial light/dark cycle, and the rats had free access to water and food. Twenty female SD rats were placed into two groups at random: a sham-operated control group, and an NB group with L6-S1 spinal nerve transection. Our experiments were conducted using a completely randomized design method.
Every rat was a separate experiment unit because of the intervention experiment. Based on the analysis of PASS 15.0 and the primary data collected for our prior study and pre-experiments, whose set power and were 0.85 and 0.05, the sample size was determined [14]. There were no predetermined inclusion or exclusion criteria that were stated explicitly. No rats or data points were left out of the analysis. For each analysis, the group sample size is noted in the figure legend and text. The rats were randomly assigned, and the parameters were chosen by researchers who were blind to treatment groups. We adhered to the ARRIVE criteria for animal research (Animal Research: Reporting of In Vivo Experiments). Morphology, immunohistochemistry staining, immunofluorescence, and protein expression analysis were listed as the main outcome measures throughout the article. In this study, the choice of the statistical test took into account whether or not the data were normalized and was based on a specific dataset.

Urodynamic Study
A catheter was placed into the bladder after the rats were given an intraperitoneal injection of 20% urethane (5 mL/kg) to induce anesthesia. The other catheter aperture was connected to a peristaltic pump and an acquisition system (RM-6240, Chengdu Instrument Factory, Chengdu, China) using a tee connector for fluid infusion and pressure recording. Urodynamic tests were performed by infusing saline into the bladder at 20 mL/h for the control group and 40 mL/h for the NB group and recording parameters such as MCC, BLPP, and BC. Cystometry was performed every 30 min for each rat three times in a row to ensure consistent bladder activity.

Scr and BUN Analysis
Blood samples were collected after the rats had been anaesthetized with 10% chloralhydrate. The serum was then extracted from the samples by centrifuging them at 400× g for 10 min at 4 • C. Afterwards, using an autoanalyzer, Scr and BUN in each group were evaluated (Cobas C701, Roche, Basel, Switzerland).

RNA Sequencing Analysis
For the mRNA sequencing analysis, three replicates of each set of SV-HUC-1 cells were used. Moreover, three replicate rat bladders were gathered for the transcriptome study. An Illumina NovaSeq 6000 platform from LC-Bio was used in part for high-throughput sequencing (Hangzhou, China). Using an FDR 0.05 and Log2FC 1 as screening parameters, DEGs between the control and NB groups were analyzed using R software (R version 4.2.0), and genes with low expression levels were eliminated. For a clustering analysis of the DEGs, the R software heatmap was employed, and the R package ggplot2 was used to produce volcanic plots. Then, using R software, the enrichment of these DEGs was examined according to GO and KEGG. IRGs for inflammation were retrieved from the GSEA website (accessed on 9 February 2023 at http://www.gsea-msigdb.org/gsea/index.jsp). The differentially expressed IRGs were then obtained by taking the intersection of DEGs and IRGs.

Histological Analysis
The bladders were embedded in paraffin after being dried and treated in 4% paraformaldehyde. The samples were then divided into sections that were four micrometers thick. Using traditional staining techniques, Masson trichrome and H&E staining were used in the sections to analyze the histomorphology of the rat bladder [14]. The ImageJ programme (V 1.8.0.112) assessed collagen accumulation (% of total area) in Masson's stained sections. Those who scored the slide results were unaware of the group assignments.

Immunohistochemistry
Deparaffinized bladder tissue slices were hydrated in xylene and ethanol. After microwave heating to repair antigens in the sectioned tissue, endogenous peroxidase was quenched with 3% H 2 O 2 . The sections were blocked with 0.5% bovine serum albumin (Biofroxx, 4240GR0250) for 1 h, and then the sections were incubated with primary antibodies against fibronectin (Zenbio, Durham, NC, USA, 250073) and vimentin (Zenbio, R22775) overnight at 4 • C. After that, the appropriate secondary antibody (Zenbio, 511203) was added. After 1 h, the sections were counterstained with hematoxylin after being stained with 3,3 -diaminobenzidine (Abcam, Cambridge, UK).
As previously mentioned, bladder tissue pieces were deparaffinized and hydrated. Primary antibodies against NLRP3 (Zenbio, 381207) and ASC (Zenbio, 340097) were incubated on the sections overnight. The slides were then handled similarly to how immunofluorescence staining is performed. Finally, immunofluorescence pictures were taken using a C2 confocal microscope (Nikon, K10587, Tokyo, Japan).

SEM and TEM
Isolated animal tissues and cells were immersed in 3% glutaraldehyde and fixed. The ion-sputter coating was applied after the samples had been dried using gradient ethanol and hexamethyldisilazane. A scanning electron microscope (JSM-IT700HR, Japan) was used to take pictures of the samples. The samples were then dehydrated in a gradient ethanol series, embedded in epoxy resin, and postfixed in 1% osmium tetroxide. Ultrathin slices (60 nm) of the treated tissue and cell samples were cut, and they were then stained with lead citrate and uranyl acetate. The sections were then seen using a transmission electron microscope (JEM-1400FLASH, Japan).

Enzyme-Linked Immunosorbent Assay (ELISA)
To obtain the serum, rat blood samples were centrifuged at 4 • C for 10 min. According to the manufacturer's instructions, ELISA kits (Elab-science, Wuhan, China) were used to determine the concentrations of IL-6, IL-1, and IL-18 in serum.

Statistical Analysis
Each experiment was run at least three times, and all data are reported as the mean ± standard deviation (SD). The SD is displayed in the error bar. Using the Student's t-test, two groups were compared to one another. Several comparisons were made using a one-way ANOVA. Additionally, experimental data analyses were performed with GraphPad Prism 8.0 (San Diego, CA, USA). A p < 0.05 was considered statistically significant.

Conclusions
In summary, the current study suggests that after severing spinal nerves, the bladder structure was damaged, and function was disturbed. We also showed for the first time that pyroptosis was involved in the development of bladder fibrosis via the NF-κB signaling pathway. Therefore, SCI-induced bladder fibrosis could be ameliorated by inhibiting the activity of the NF-κB/NLRP3 signaling pathway ( Figure 8). Our study proposes a strategy for the effective treatment of patients with NB. However, the underlying mechanism still requires further exploration. ethanol series, embedded in epoxy resin, and postfixed in 1% osmium tetroxide. Ultrathin slices (60 nm) of the treated tissue and cell samples were cut, and they were then stained with lead citrate and uranyl acetate. The sections were then seen using a transmission electron microscope (JEM-1400FLASH, Japan).

Enzyme-Linked Immunosorbent Assay (ELISA)
To obtain the serum, rat blood samples were centrifuged at 4 °C for 10 min. According to the manufacturer's instructions, ELISA kits (Elab-science, Wuhan, China) were used to determine the concentrations of IL-6, IL-1, and IL-18 in serum.

Statistical Analysis
Each experiment was run at least three times, and all data are reported as the mean ± standard deviation (SD). The SD is displayed in the error bar. Using the Student's t-test, two groups were compared to one another. Several comparisons were made using a oneway ANOVA. Additionally, experimental data analyses were performed with GraphPad Prism 8.0 (San Diego, CA, USA). A p < 0.05 was considered statistically significant.

Conclusions
In summary, the current study suggests that after severing spinal nerves, the bladder structure was damaged, and function was disturbed. We also showed for the first time that pyroptosis was involved in the development of bladder fibrosis via the NF-κB signaling pathway. Therefore, SCI-induced bladder fibrosis could be ameliorated by inhibiting the activity of the NF-κB/NLRP3 signaling pathway ( Figure 8). Our study proposes a strategy for the effective treatment of patients with NB. However, the underlying mechanism still requires further exploration.

Data Availability Statement:
The data presented in this study are available on request from the corresponding author. The data are not publicly available due to privacy.