Mild magnetic hyperthermia-activated immuno-responses for primary bladder cancer therapy.

Surgical intervention followed by chemotherapy is the principal treatment strategy for bladder cancer, which is hindered by significant surgical risks, toxicity from chemotherapy, and high rates of recurrence after surgery. In this context, a novel approach using mild magnetic hyperthermia therapy (MHT) for bladder cancer treatment through the intra-bladder delivery of magnetic nanoparticles is presented for the first time. This method overcomes the limitations of low magnetic thermal efficiency, inadequate tumor targeting, and reduced therapeutic effectiveness associated with the traditional intravenous administration of magnetic nanoparticles. Core-shell Zn-CoFe2O4@Zn-MnFe2O4 (MNP) nanoparticles were developed and further modified with hyaluronic acid (HA) to enhance their targeting ability toward tumor cells. The application of controlled mild MHT using MNP-HA at temperatures of 43-44 °C successfully suppressed the proliferation of bladder tumor cells and tumor growth, while also decreasing the expression levels of heat shock protein 70 (HSP70). Crucially, this therapeutic approach also activated the body's innate immune response involving macrophages, as well as the adaptive immune responses of dendritic cells (DCs) and T cells, thereby reversing the immunosuppressive environment of the bladder tumor and effectively reducing tumor recurrence. This study uncovers the potential immune-activating mechanism of mild MHT in the treatment of bladder cancer and confirms the effectiveness and safety of this strategy, indicating its promising potential for the clinical management of bladder cancer with a high tendency for relapse.

Acupressure bladder meridian alleviates anxiety disorder via HMGB1.

The aim of this study was to investigate the effects of acupressure bladder meridian (ABM) on anxiety in rats with chronic stress. METHODS: The sugar water preference (SPF), tail suspension time (TST) and forced swimming time (FST) of rats were measured. The levels of reactive oxygen species (ROS), myeloperoxidase (MPO) in hippocampus tissue, oxidative stress parameters and inflammatory cytokines were detected. Underlying mechanisms of ABM on anxiety were detected. lipopolysaccharide (LPS) stimulated PC12 cells were adopted in vitro. HMGB1 knockdown were used in PC12 cells, and related signaling was further detected. RESULTS: ABM significantly increased SPF, decreased TST and FST. ABM decreased ROS, MPO levels, decreased the levels of inflammatory cytokines. Furthermore, ABM decreased the levels of oxidative stress index. ABM reduced the expression of inflammation-related proteins mediated by HMGB1, increased nuclear factor erythroid2-related factor 2 (Nrf-2) and hemeoxygenase-1 (HO-1). In vitro PC12 cells, Rat serum (RS-ABM) treated with ABM significantly decreased LPS induced inflammation-related proteins and increased Nrf-2/HO-1 pathway. HMGB1 knockdown inhibited LPS-induced PC12 cell inflammatory signaling pathway and increased Nrf-2/HO-1 pathway. CONCLUSION: Our results demonstrated that ROS-dependent HMGB1 plays an important role in anxiety, and ABM exhibits inhibited inflammation in anxiety.

Effect of Yiyuan moxibustion on urodynamics and TRPV4/ATP/P2X5 signal pathway of bladder tissue in rats with neurogenic bladder after sacral cord injury. / ç›Šå ƒç¸å¯¹éª¶é«“æŸä¼¤åŽç¥žç»æºæ€§è†€èƒ±å¤§é¼ å°¿æµåŠ¨åŠ›å­¦åŠè†€èƒ±ç»„ç»‡TRPV4/ATP/P2X5信号通路的影响.

OBJECTIVES: To observe the effect of Yiyuan moxibustion on urodynamics and the expressions of transient receptor potential vanilloid 4 (TRPV4), adenosine triphosphate (ATP), tyrosine protein kinase KIT (C-Kit) and adenosine triphosphate receptor P2X5 in bladder tissue of rats with detrusor reflex-free neurogenic bladder (NB) after sacral cord injury (SCI), so as to explore its mechanism in promoting the recovery of urination function of NB rats. METHODS: Female SD rats were randomly divided into sham operation, model, Yiyuan moxibustion, Yiyuan moxibustion+inhibitor (combination) and inhibitor groups, with 12 rats in each group. The model of detruser reflex-free NB after sacral SCI was established by modified Hassan Shaker spinal cord transection method. The behavioral score of Basso Beasttie Bresnahan (BBB) and urodynamic indexes were used to evaluate the model of rats after operation. Fifteen days after modeling, Yiyuan moxibustion was applied to "Shenque" (CV8) and "Guanyuan" (CV4) for 20 min, once daily for 14 days. Rats of the inhibitor and combination groups were given intravesical instillation of HC067047 (1 mL, 1 µmol/L, 30 min). After the interventions, urodynamics was used to evaluate the bladder function of rats. HE staining was used to observe the morphology of bladder tissue. ATP content in bladder tissue was detected by colorimetric method. The positive expression rates of C-Kit and their receptor P2X5 in bladder tissue were observed by immunofluorescence double labeling method, and TRPV4, C-Kit, and P2X5 protein expression levels in bladder tissue were detected by Western blot. RESULTS: Compared with the sham operation group, the maximum bladder capacity and bladder compliance of rats in the model group were increased (P<0.01), the leak point pressure, ATP content, the possitive expression rates of C-Kit and P2X5, and the protein expression levels of TRPV4, C-Kit, P2X5 in bladder tissue were decreased (P<0.01). In comparison with the model and combination groups, the Yiyuan moxibustion group showed a decrease in maximum bladder capacity and bladder compliance (P<0.01), an increase in leakage point pressure, ATP content, the possitive expression rates of C-Kit and P2X5, and TRPV4, C-Kit, and P2X5 protein expression levels (P<0.01, P<0.05)；However, these indicators showed opposite trends in the inhibitor group (P<0.01, P<0.05). CONCLUSIONS: Yiyuan moxibustion can improve the urodynamics and bladder function in rats with bladder detrusor nonreflective after SCI, which may be related to its effect in activating the TRPV4 channel in bladder tissue, promoting the release of ATP from bladder epithelium, thus increasing the expression of bladder Cajal interstitial cells and their purinergic P2X5 receptors.

Therapeutic effects of p38 mitogen-activated protein kinase inhibition on hyperexcitability of capsaicin sensitive bladder afferent neurons in mice with spinal cord injury.

AIMS: Nerve growth factor (NGF) has been implicated as a key molecule of pathology-induced changes in C-fiber afferent nerve excitability, which contributes to the emergence of neurogenic detrusor overactivity due to spinal cord injury (SCI). It is also known that the second messenger signaling pathways activated by NGF utilize p38 Mitogen-Activated Protein Kinase (MAPK). We examined the roles of p38 MAPK on electrophysiological properties of capsaicin sensitive bladder afferent neurons with SCI mice. MAIN METHODS: We used female C57BL/6 mice and transected their spinal cord at the Th8/9 level. Two weeks later, continuous administration of p38 MAPK inhibitor (0.51 µg/h, i.t. for two weeks) was started. Bladder afferent neurons were labelled with a fluorescent retrograde tracer, Fast-Blue (FB), injected into the bladder wall three weeks after SCI. Four weeks after SCI, freshly dissociated L6-S1 dorsal root ganglion neurons were prepared and whole cell patch clamp recordings were performed in FB-labelled neurons. After recording action potentials or voltage-gated K+ currents, the sensitivity of each neuron to capsaicin was evaluated. KEY FINDINGS: In capsaicin-sensitive FB-labelled neurons, SCI significantly reduced the spike threshold and increased the number of action potentials during 800 ms membrane depolarization. Densities of slow-decaying A-type K+ (KA) and sustained delayed rectifier-type K+ (KDR) currents were significantly reduced by SCI. The reduction of KA, but not KDR, current density was reversed by the treatment with p38 MAPK inhibitor. SIGNIFICANCE: P38 MAPK plays an important role in hyperexcitability of capsaicin-sensitive bladder afferent neurons due to the reduction in KA channel activity in SCI mice.

Melatonin induces cell cycle arrest and suppresses tumor invasion in urinary bladder urothelial carcinoma.

Urinary bladder urothelial carcinoma (UBUC) encompasses about 90% of all bladder cancer cases, and the mainstream treatment is the transurethral resection of the bladder tumor followed by intravesical instillation. High rates of mortality, recurrence, and progression in bladder cancer have stimulated the search for alternative adjuvant therapies. The aim of this study was to investigate the potential of melatonin as adjuvant therapy in bladder cancer. Cell viability and clonogenic ability were assessed by an MTT assay and colony formation. Cell cycle and apoptosis analysis were performed by flow cytometry and Hoechst 33342 staining, while cell metastasis capacity was measured by wound healing and transwell assays. Potential mechanisms were investigated by an oncology array and verified via western blotting. The melatonin treatment significantly reduced T24 and UMUC3 bladder cancer cell proliferation and clonogenic ability. G1 arrest and sub-G1 accumulation in the T24 and UMUC3 cells led to cell proliferation suppression and cell death, and Hoechst 33342 staining further verified the apoptosis induction directly by melatonin. Moreover, melatonin weakened cell motility and invasiveness. Based on the oncology array results, we demonstrated that melatonin exerts its anti-cancer effect by down-regulating the HIF-1α and NF-κB pathways and downstream pathways, including Bcl-2, leading to cell cycle arrest and apoptosis induction in the UBUC cells. Overall, these findings support the potential of melatonin as adjuvant therapy in bladder cancer.

Dose-dependant preventive effect of a herbal compound on crystal formation in rat model.

INTRODUCTION: To analyze the dose-dependent preventive effect of a plant-based herbal product on the new crystal formation in a rat model. MATERIALS AND METHODS: A total of 42 rats were divided into 7 groups and zinc discs were placed into the bladder of rats to provide a nidus for the development of new crystal formation: Group 1: control, Group 2: 0.75 percent ethylene glycol (EG); Group 3: 0.75 percent EG plus 0.051 ml of the compound; Group 4: 0.75 percent EG plus 0.179 ml of the compound; Group 5: 0.75 percent EG plus 0.217 ml of the compound; Group 6: 0.75 percent EG plus 0.255 ml of the compound; Group 7 0.75 percent EG plus 0.332 of the compound). The analysis and comparison focused on the disc weights, changes in urinary oxalate and calcium levels, urinary pH, and the histopathologic evaluation of the inflammatory changes in the bladder after 14 days. RESULTS: According to the evaluation of discs placed in the bladders of the animals, animals receiving the herbal compound on a dose-dependent basis showed a limited increase in the disc weights values after 14 days, despite a considerable increase in animals receiving EG alone (p = 0.001). Further evaluation of the increase in disc weights on a dose-dependent basis in different subgroups (from Groups 3 to 7) demonstrated that the limitation of crystal deposition began to be more prominent as the dose of herbal compound increased. This effect was more evident particularly in comparisons between group 7 and others, according to LSD multiple comparison tests (p = 0.001). As anticipated, there has been no discernible change in the weight of the discs in the control group. Although urinary calcium levels in animals of Groups 2, 6, and 7 were significantly higher than the other groups, we were not able to demonstrate a close correlation between urinary oxalate levels and the increasing dose levels. Even though mean urine pH levels were statistically considerably higher in Group 3, there was no statistically significant correlation between the oxalate and calcium levels between all groups, and no association was seen with the administration of herbal agents. The transitional epithelium between the three groups of animals' bladder samples did not exhibit any appreciable difference according to pathological analysis. CONCLUSIONS: In this animal model, the treatment of the compound was successful in lowering the amount of crystal deposition surrounding the zinc discs, most noticeably at a dosage of 0.332 ml, three times per day.

Massage of Bladder Meridian Relieved Anxiety Induced by Chronic Stress in Rats.

The aim of this paper was to explore the mechanism of bladder meridian massage (BMM) on anxiety in rats with chronic stress. Chronic stress induced rats to establish rat anxiety model. The sugar water preference (SPF), tail suspension time (TST), and forced swimming time (FST) of rats were measured. The levels of superoxide dismutase (SOD), malondialdehyde (MDA), and inflammatory cytokines in serum and hippocampus of rats were detected. Brain neurotransmitters (dopamine (DA), 5- hydroxytryptamine (5-HT), and norepinephrine (NE)) were detected by enzyme-linked immunosorbent assay (ELISA) kits. Immunohistochemistry and western blotting were used to detect autophagy protein expression in hippocampus of rats. BMM significantly increased SPF, decreased TST and FST, increased SOD level in serum and hippocampus, and decreased MDA level and cytokine level. BMM reversed the changes of neurotransmitters. At the same time, BMM significantly decreased autophagy protein expression in hippocampus of rats. The above results show that BMM significantly relieve anxiety induced by chronic stress in rats.

Molecular and Morphological Characteristics of the De-Obstructed Rat Urinary Bladder-An Update.

Many patients with outlet obstruction secondary to prostatic enlargement have lower urinary tract symptoms (LUTSs) and an increased frequency of micturition. The standard treatment is transurethral resection of the prostate (TURP), which alleviates obstruction and symptoms. However, after TURP, 20-40 percent of patients continue to experience LUTSs. The aim of the present study in rats was to identify the mechanisms that do not normalize after the removal of the obstruction and that could explain the persisting symptoms. We had microarray data from control, obstructed, and de-obstructed female rat bladders, which made it possible to study 14,553 mRNA expressions. We also had a bank of electron micrographs from similar detrusors. Microarrays: There were significant differences between the control and obstructed bladders for 1111 mRNAs. The obstructed and de-obstructed bladders differed significantly for 1059 mRNAs. The controls and the de-obstructed bladders differed significantly for 798 mRNAs. We observed many mRNAs that were increased in the obstructed bladder and then decreased to control levels after de-obstruction, and many mRNAs that were decreased in the obstructed bladder and then increased following de-obstruction. mRNAs that were significantly higher or lower in the de-obstructed bladder than in the control bladder were also found. Ultrastructure: The detrusor cells in the obstructed bladders had cross-sectional areas that were much larger than those in the controls. The control cells had smooth outlines and similar cross-sectional areas. The de-obstructed detrusor cells had larger cross-sectional areas than the controls, as well as corrugated surfaces. The cell areas varied, suggesting that the shrinkage of the de-obstructed cells was not even. We did not find any points of contact of the gap junction plaque type between the detrusor cells. There were abundant finger-like processes between the detrusor cells in the obstructed and in de-obstructed bladders, which were only occasionally found in the control detrusors. They are the only possible localization for gap junction channels. The de-obstructed rat bladder is not an organ with properties intermediate between those of the control and obstructed bladders. Instead, de-obstructed bladders have gene expressions, morphologies, and functional properties of the individual cells and their organization, which make them distinctly different from both control and obstructed bladders.

Permixon®, hexane-extracted Serenoa repens, inhibits human prostate and bladder smooth muscle contraction and exerts growth-related functions in human prostate stromal cells.

AIMS: Recently, the European Association of Urology recommended hexane-extracted fruit of Serenoa repens (HESr) in their guidelines on management of non-neurogenic male lower urinary tracts symptoms (LUTS). Despite previously lacking recommendations, Permixon® is the most investigated HESr in clinical trials, where it proved effective for male LUTS. In contrast, underlying mechanisms were rarely addressed and are only marginally understood. We therefore investigated effects of Permixon® on human prostate and detrusor smooth muscle contraction and on growth-related functions in prostate stromal cells. MAIN METHODS: Permixon® capsules were dissolved using n-hexane. Contractions of human prostate and detrusor tissues were induced in organ bath. Proliferation (EdU assay), growth (colony formation), apoptosis and cell death (flow cytometry), viability (CCK-8) and actin organization (phalloidin staining) were studied in cultured human prostate stromal cells (WPMY-1). KEY FINDINGS: Permixon® inhibited α1-adrenergic and thromboxane-induced contractions in prostate tissues, and methacholine-and thromboxane-induced contractions in detrusor tissues. Endothelin-1-induced contractions were not inhibited. Neurogenic contractions were inhibited in both tissues in a concentration-dependent manner. In WPMY-1 cells, Permixon® caused concentration-dependent breakdown of actin polymerization, inhibited colony formation, reduced cell viability, and proliferation, without showing cytotoxic or pro-apoptotic effects. SIGNIFICANCE: Our results provide a novel basis that allows, for the first time, to fully explain the ubiquitous beneficial effects of HESr in clinical trials. HESr may inhibit at least neurogenic, α1-adrenergic and thromboxane-induced smooth muscle contraction in the prostate and detrusor, and in parallel, prostate stromal cell growth. Together, this may explain symptom improvements by Permixon® in previous clinical trials.

Therapeutic effects of a soluble guanylate cyclase activator, BAY 60-2770, on lower urinary tract dysfunction in mice with spinal cord injury.

We aimed to evaluate the effects of a soluble guanylate cyclase (sGC) activator, BAY 60-2770, on neurogenic lower urinary tract dysfunction in mice with spinal cord injury (SCI). Mice were divided into the following three groups: spinal cord intact (group A), SCI + vehicle (group B), and SCI + BAY 60-2770 (group C). SCI mice underwent Th8-Th9 spinal cord transection and treatment with BAY 60-2770 (10 mg/kg/day) once daily for 2-4 wk after SCI. We evaluated urodynamic parameters using awake cystometry and external urethral sphincter electromyograms (EMG); mRNA levels of mechanosensory channels, nitric oxide (NO)-, ischemia-, and inflammation-related markers in L6-S1 dorsal root ganglia, the urethra, and bladder tissues; and protein levels of cGMP in the urethra at 4 wk after SCI. With awake cystometry, nonvoiding contractions, postvoid residual, and bladder capacity were significantly larger in group B than in group C. Voiding efficiency (VE) was significantly higher in group C than in group B. In external urethral sphincter EMGs, the duration of notch-like reductions in intravesical pressure and reduced EMG activity time were significantly longer in group C than in group B. mRNA expression levels of transient receptor potential ankyrin 1, transient receptor potential vanilloid 1, acid-sensing ion channel (ASIC)1, ASIC2, ASIC3, and Piezo2 in the dorsal root ganglia, and hypoxia-inducible factor-1α, VEGF, and transforming growth factor-ß1 in the bladder were significantly higher in group B than in groups A and C. mRNA levels of neuronal NO synthase, endothelial NO synthase, and sGCα1 and protein levels of cGMP in the urethra were significantly lower in group B than in groups A and C. sGC modulation might be useful for the treatment of SCI-related neurogenic lower urinary tract dysfunction.NEW & NOTEWORTHY This is the first report to evaluate the effects of a soluble guanylate cyclase activator, BAY 60-2770, on neurogenic lower urinary tract dysfunction in mice with spinal cord injury.

Electroacupuncture protects against bladder dysfunction induced by acute urinary retention via TRPV1/ATP signaling pathway: An animal and human research study.

Electroacupuncture (EA) can protect against acute urinary retention (AUR); however, the underlying mechanism remains unclear. Non-vesicular ATP release mediated by transient receptor potential (TRP) channels were identified as a key contributor to signaling in urothelial cells. In this study, the AUR model was established by urethral outlet obstruction in female Sprague-Dawley rats. EA was performed at SP6 and BL32 for 0.5 h prior to induction of AUR. EA reduced TRPV1 expression and urinary ATP concentrations in rat bladder, decreased the peak intravesical pressure during AUR, and attenuated abnormal voiding patterns and bladder pathological injury induced by AUR. Besides, 179 patients who experienced postoperative urinary retention were recruited and found that EA reduced urinary ATP concentrations and accelerated the recovery of spontaneous voiding. These observations indicate that EA exerts protection against AUR-induced bladder dysfunction by reducing urinary ATP concentrations through the regulation of TRPV1.

Echinacoside Ameliorates Cyclophosphamide-Induced Bladder Damage in Mice.

Interstitial cystitis (IC) is featured by apoptosis and chronic inflammation in bladder tissue. We aimed to evaluate the effect of echinacoside (ECH), which is known to modulate inflammation and apoptosis on IC using relevant models. We established a mouse model of cystitis using cyclophosphamide (CYP) and treated human urothelium cells (SV-HUC-1) with lipopolysaccharide (LPS) + ATP as in vitro model. The bladder function was tested by urodynamics. Apoptosis of bladder cells was assessed by terminal deoxynucleotidyl transferase dUTP nick-end labeling assay. Expressions of apoptosis-associated and inflammation-related proteins were assessed using western blotting. Treatment with ECH significantly improved bladder function, reduced inflammatory damage, and decreased apoptosis in the models. Furthermore, ECH decreased the phosphorylation levels of IκB and NF-κB(p65), and upregulated the expression of peroxisome proliferator-activated receptor gamma (PPARγ), which are related to apoptosis and inflammation in CYP-induced mouse cystitis. Moreover, ECH did not reduce apoptosis of urothelial cells after treatment with PPARγ antagonist GW9662. Our findings suggest that ECH might have protective effect against IC in bladder and be mediated through modulation of the PPARγ/NF-κB pathway.

Therapeutic effect of modulating the NLRP3-regulated transforming growth factor-ß signaling pathway on interstitial cystitis/bladder pain syndrome.

Interstitial cystitis/bladder pain syndrome (IC/BPS) is a disorder with complex pathogenesis and lacks effective treatment. Chronic inflammation is the main pathogenesis of Hunner-type IC/BPS. The NLR family pyrin domain-containing 3 (NLRP3) inflammasome-related transforming growth factor-ß (TGF-ß)/Smad signaling pathway plays a crucial role in inflammation-related tissue fibrosis. Lipopolysaccharide (LPS) and protamine sulfate (LPS/PS) were instilled into the mouse bladder twice a week for 5 consecutive weeks to establish a chronic inflammation-induced IC/BPS model (LPS/PS model). Following LPS/PS treatment, curcumin (oral, 100 mg/kg; a potent NLRP3 modulator) was administered for 2 weeks in the curcumin treatment group, and normal saline was used for the sham group. Bladder function was evaluated by performing the voiding spot assay and examining the status of urothelial denudation and fibrosis in bladder tissues. The expression of NLRP3 inflammasome, interleukin-1ß, TGF-ß, Smad, vimentin, and E-cadherin in bladder tissues was evaluated through immunohistochemistry staining. Results revealed that the repeated instillation of LPS/PS leads to voiding dysfunction, bladder urothelium denudation, and detrusor muscle fibrosis through the upregulation of the NLRP3 inflammasome/IL-1ß-related TGF-ß/Smad pathway and the increased epithelial-mesenchymal transition process in bladder tissues. The downregulation of the NLRP3 inflammasome/IL-1ß-related TGF-ß/Smad pathway in bladder tissues through curcumin effectively mitigated bladder injury in the LPS/PS model. In conclusion, the NLRP3 inflammasome/IL-1ß-related TGF-ß/Smad pathway plays a crucial role in bladder injury in the LPS/PS model, and modulation of this pathway, such as by using curcumin, can effectively mitigate the sequelae of chronic inflammation-induced IC/BPS.

4,6,4'-Trimethylangelicin Photoactivated by Blue Light Might Represent an Interesting Option for Photochemotherapy of Non-Invasive Bladder Carcinoma: An In Vitro Study on T24 Cells.

Photodynamic therapy (PDT) is frequently used to treat non-muscle invasive bladder cancer due its low toxicity and high selectivity. Since recurrence often occurs, alternative approaches and/or designs of combined therapies to improve PDT effectiveness are needed. This work aimed to evaluate the cytotoxicity of 4,6,4'-trimethylangelicin (TMA) photoactivated by blue light (BL) on human bladder cancer T24 cells and investigate the mechanisms underlying its biological effects. TMA/BL exerted antiproliferative activity through the induction of apoptosis without genotoxicity, as demonstrated by the expression levels of phospho-H2AX, an indicator of DNA double-stranded breaks. It also modulated the Wnt canonical signal pathway by increasing the phospho-ß-catenin and decreasing the nuclear levels of ß-catenin. The inhibition of this pathway was due to the modulation of the GSK3ß phosphorylation state (Tyr 216) that induces a proteasomal degradation of ß-catenin. Indeed, a partial recovery of nuclear ß-catenin expression and reduction of its phosphorylated form after treatment with LiCl were detected. As demonstrated by RT-PCR and cytofluorimetric analysis, TMA/BL also decreased the expression of CD44v6, a marker of cancer stem cells. Taken together, our data suggest that TMA photoactivated by BL may represent an interesting option for the photochemotherapy of noninvasive bladder carcinomas, since this treatment is able to inhibit key pathways for tumour growth and progression in the absence of genotoxic effects.

Identification and Characterization of a Novel Large-Conductance Calcium-Activated Potassium Channel Activator, CTIBD, and Its Relaxation Effect on Urinary Bladder Smooth Muscle.

The large-conductance calcium-activated potassium channel (BKCa channel) is expressed on various tissues and is involved in smooth muscle relaxation. The channel is highly expressed on urinary bladder smooth muscle cells and regulates the repolarization phase of the spontaneous action potentials that control muscle contraction. To discover novel chemical activators of the BKCa channel, we screened a chemical library containing 8364 chemical compounds using a cell-based fluorescence assay. A chemical compound containing an isoxazolyl benzene skeleton (compound 1) was identified as a potent activator of the BKCa channel and was structurally optimized through a structure-activity relationship study to obtain 4-(4-(4-chlorophenyl)-3-(trifluoromethyl)isoxazol-5-yl)benzene-1,3-diol (CTIBD). When CTIBD was applied to the treated extracellular side of the channel, the conductance-voltage relationship of the channel shifted toward a negative value, and the maximum conductance increased in a concentration-dependent manner. CTIBD altered the gating kinetics of the channel by dramatically slowing channel closing without effecting channel opening. The effects of CTIBD on bladder muscle relaxation and micturition function were tested in rat tissue and in vivo. CTIBD concentration-dependently reduced acetylcholine-induced contraction of urinary bladder smooth muscle strips. In an acetic acid-induced overactive bladder (OAB) model, intraperitoneal injection of 20 mg/kg CTIBD effectively restored frequent voiding contraction and lowered voiding volume without affecting other bladder function parameters. Thus, our results indicate that CTIBD and its derivatives are novel chemical activators of the bladder BKCa channel and potential candidates for OAB therapeutics. SIGNIFICANCE STATEMENT: The novel BKCa channel activator CTIBD was identified and characterized in this study. CTIBD directly activates the BKCa channel and relaxes urinary bladder smooth muscle of rat, so CTIBD can be a potential candidate for overactive bladder therapeutics.

Shionone alleviates NLRP3 inflammasome mediated pyroptosis in interstitial cystitis injury.

Shionone is a triterpenoid component derived from the herbal medicine Aster tataricus, and it has been reported to possess marked anti-inflammatory properties. The activation of NLRP3 inflammasome plays an important role in cystitis, and the effect of Shionone on NLRP3 inflammasome-dependent pyroptosis remains unclear. In this study, we established an interstitial cystitis (IC) rat model and SV-HUC-1 cell model with CYP or LPS + ATP treatment to mimic inflammation response and induce NLRP3 inflammasome activation. Shionone treatment significantly attenuated the bladder wet weight, score of edema and hemorrhage, enhanced the viability of SV-HUC-1 cell, decreased the rate of pyroptosis. Moreover, Shionone reduced the expression of NF-κB, NLRP3, ASC, Pro-caspase-1, Caspase-1, GSDMD, GSDMD-N at the mRNA and protein levels both in rat and SV-HUC-1 cell model, demonstrating NLRP3 inflammasome pathway was blocked and pyroptosis degree was reduced. These results indicated that Shionone could alleviate interstitial cystitis in Rat model and enhancing the viability of SV-HUC-1 cells via NF-κB/NLRP3/GSDMD-N pathway, which illustrated that Shionone could be used as a drug candidate for the treatment of interstitial cystitis.

The intracellular Ca2+ release channel TRPML1 regulates lower urinary tract smooth muscle contractility.

TRPML1 (transient receptor potential mucolipin 1) is a Ca2+-permeable, nonselective cation channel that is predominantly localized to the membranes of late endosomes and lysosomes (LELs). Intracellular release of Ca2+ through TRPML1 is thought to be pivotal for maintenance of intravesicular acidic pH as well as the maturation, fusion, and trafficking of LELs. Interestingly, genetic ablation of TRPML1 in mice (Mcoln1-/- ) induces a hyperdistended/hypertrophic bladder phenotype. Here, we investigated this phenomenon further by exploring an unconventional role for TRPML1 channels in the regulation of Ca2+-signaling activity and contractility in bladder and urethral smooth muscle cells (SMCs). Four-dimensional (4D) lattice light-sheet live-cell imaging showed that the majority of LELs in freshly isolated bladder SMCs were essentially immobile. Superresolution microscopy revealed distinct nanoscale colocalization of LEL-expressing TRPML1 channels with ryanodine type 2 receptors (RyR2) in bladder SMCs. Spontaneous intracellular release of Ca2+ from the sarcoplasmic reticulum (SR) through RyR2 generates localized elevations of Ca2+ ("Ca2+ sparks") that activate plasmalemmal large-conductance Ca2+-activated K+ (BK) channels, a critical negative feedback mechanism that regulates smooth muscle contractility. This mechanism was impaired in Mcoln1-/- mice, which showed diminished spontaneous Ca2+ sparks and BK channel activity in bladder and urethra SMCs. Additionally, ex vivo contractility experiments showed that loss of Ca2+ spark-BK channel signaling in Mcoln1-/- mice rendered both bladder and urethra smooth muscle hypercontractile. Voiding activity analyses revealed bladder overactivity in Mcoln1-/- mice. We conclude that TRPML1 is critically important for Ca2+ spark signaling, and thus regulation of contractility and function, in lower urinary tract SMCs.

Hyperbaric oxygen therapy prevents subarachnoid hemorrhage-induced apoptosis and impaired contractility of the rabbit bladder.

AIM: To explore the effects of experimental subarachnoid hemorrhage (SAH) on rabbit urinary bladder and to assess the potential protective effects of hyperbaric oxygen therapy (HBOT). METHODS: A total of 15 male New Zealand white rabbits were divided randomly to one of three groups: group I was spared as the control group (n = 5), group II was exposed to SAH, received no treatment, and acted as the SAH group (n = 5) and group III was exposed to SAH and received five sessions of HBOT (started 12 hours after SAH induction and was given twice daily for the first 2 days and once on the third day) and acted as the treatment group (n = 5). At 72 hours after the SAH induction, bladders from all animals were removed for in vitro organ bath experiments and biochemical analyses. RESULTS: Isometric tension studies revealed that compared to group I, the contractile responses of the strips to carbachol in group II were significantly decreased whereas HBOT restored the contractile responses (P < .05). Caspase-3 and nitric oxide synthase (NOS) activities of bladder tissues were significantly increased in group II when compared with group I, whereas caspase-3 and NOS activities were significantly decreased in the tissues of group III (P < .01). CONCLUSIONS: Subarachnoid hemorrhage stimulates apoptosis of the rabbit bladder and impairs the contractile response of the rabbit bladder to carbachol. HBOT creates a protective effect in rabbit bladder tissues and restores SAH-induced changes.

Blueberry Prevents the Bladder Dysfunction in Bladder Outlet Obstruction Rats by Attenuating Oxidative Stress and Suppressing Bladder Remodeling.

Various berries demonstrate antioxidant activity, and this effect is expected to prevent chronic diseases. We examined whether a diet containing blueberry powder could prevent the development of bladder dysfunction secondary to bladder outlet obstruction (BOO). Eighteen 8-week-old male Sprague-Dawley rats were randomly divided into three groups: Sham (sham operated + normal diet), N-BOO (BOO operated + normal diet) and B-BOO (BOO operated + blueberry diet). Four weeks after BOO surgery, the N-BOO group developed bladder dysfunction with detrusor overactivity. The B-BOO group showed significantly improved micturition volume and micturition interval. The urinary levels of 8-hydroxy-2'-deoxyguanosine (8-OHdG) and malondialdehyde (MDA) were measured as oxidative stress markers. In the N-BOO group, 8-OHdG increased 1.6-fold and MDA increased 1.3-fold at 4 weeks after surgery, whereas the increase in 8-OHdG was significantly reduced by 1.1-fold, despite a similar increase in MDA, in the B-BOO group. Bladder remodeling was confirmed due to bladder hypertrophy, fibrosis and increased connexin43 expression in the N-BOO group, but these histological changes were reduced in the B-BOO group. The intake of blueberries prevented the development of bladder dysfunction secondary to BOO. This effect seems to be related to antioxidation and the inhibition of bladder remodeling.

Assessment and Management of Urinary Dysfunction in 187 Patients with Parkinson's Disease.

BACKGROUND: Urinary dysfunction is common in Parkinson's disease (PD) patients and management options are limited. OBJECTIVE: This study aimed to explore the management of urinary dysfunction by researching the special needs of PD patients. METHODS: PD patients with urinary dysfunction who underwent urodynamic testing were recruited from a single center from October 2013 to February 2019. The urinary symptoms, International Prostate Symptom Score and Hoehn-Yahr scale were evaluated. Management was made at the urologists' discretion with follow-up after three weeks. Urinary symptoms, urodynamics and the management of urinary dysfunction were analyzed. RESULTS: A total of 187 patients with a median age of 66.2 and Hoehn-Yahr scale soccer of 2 were enrolled. Irritative symptoms were more common than obstructive symptoms, while obstructive symptoms were more common in male than female patients, except for incomplete voiding. There were 51% cases of detrusor overactivity, followed by 33% with bladder outlet obstruction, 13% had normal function, 12% had detrusor underactivity, 9% had stress incontinence, 7% had increased bladder sensation and 4% had an acontractile bladder. Tolterodine and tamsulosin were the most common therapeutic agents, respectively prescribed to 38.5% and 27.3% of the patients. Other treatments included catheterization, botulinum toxin A bladder wall injection, transurethral resection of the prostate and urethral dilatation. Urinary symptoms were improved significantly in 74.5% of the patients (p < 0.001), including 27 patients treated with tamsulosin only and 54 patients with tolterodine only. CONCLUSIONS: Urinary symptoms and urodynamics were highly variable in PD patients, indicating that most patients may benefit from personalized management.