The expression and distribution of TACAN in human and rat bladders.

OBJECTIVES: A lot of ion channels participate in the regulation of bladder function. TACAN, a new mechanosensitive ion channel, was first discovered in 2020. TACAN has been found to be expressed in many tissues, such as the dorsal root ganglia (DRG) and adipose tissue. However, it is unclear whether or not TACAN is expressed in the bladder. In this work, we decided to study the expression and distribution of TACAN in human and rat bladders. Meanwhile, the expression of TACAN in the rat model of interstitial cystitis/bladder pain syndrome (IC/BPS) was studied. METHODS: Human bladder tissues were obtained from female patients. Cyclophosphamide (CYP) was used to build the rat model of IC/BPS. Real-time polymerase chain reaction, agarose gel electrophoresis, and western blotting were used to assess the expression of TACAN in human and rat bladders. Immunohistochemistry and immunofluorescence were used to observe the distribution of TACAN in human and rat bladders. Hematoxylin-eosin stain, withdrawal threshold, and micturition interval were used to evaluate animal models. RESULTS: The results of agarose gel electrophoresis and western blotting suggested that TACAN was expressed in human and rat bladders. Immunohistochemical results suggested that TACAN showed positive immunoreaction in the urothelial and detrusor layers. The immunofluorescence results indicated that TACAN was co-stained with UPKIII, α-SMA, and PGP9.5. The IC/BPS model was successfully established with CYP. The mRNA and protein expression of TACAN was upregulated in the CYP-induced rat model of IC/BPS. CONCLUSIONS: TACAN was found in human and rat bladders. TACAN was mainly distributed in the urothelial and detrusor layers and bladder nerves. The expression of TACAN was upregulated in the CYP-induced rat model of IC/BPS. This new discovery will provide a theoretical basis for future research on the function of TACAN in the bladder and a potential therapeutic target for IC/BPS.

Decreased autophagic activity of detrusor cells is involved in the inflammatory response of interstitial cystitis/bladder pain syndrome.

INTRODUCTION AND HYPOTHESIS: Genome-wide association studies suggest that autophagy plays an important regulatory role in inflammatory and autoimmune diseases. Inflammation and immune regulation disorders are involved in the occurrence and development of interstitial cystitis/bladder pain syndrome (IC/BPS). However, the changes and roles of autophagy in IC/BPS have not been reported. Therefore, this study aimed to investigate bladder autophagy and inflammation changes in patients with IC/BPS. METHODS: Bladder specimens (n = 5) from patients with cystectomy due to end-stage IC/BPS were collected. The bladder samples of the control group (n = 5) were derived from the normal area bladder tissue after radical cystectomy. H&E and toluidine blue staining were used for histological evaluation. The co-location of LC3, alpha-smooth muscle actin (α-SMA), and autophagosome was investigated with double-labeled immunofluorescence and transmission electron microscopy (TEM). The expression of IL-6, TNF-α, Bax, caspase-3, and BCL-2 in the detrusor layer was analyzed using immunohistochemistry (IHC) and Western blot (WB). RESULTS: Compared with the control group, bladder tissue from IC/BPS patients revealed thinner and edematous epithelium with many mast cells (P < 0.05) infiltrating into the muscle layer. By using TEM (P < 0.05), double-labeled immunofluorescence (P < 0.05), and Western blot (P < 0.05) in IC/BPS patients, autophagy was also found and was significantly increased in detrusor myocytes. IHC and WB indicate the expression of BCL-2 (P < 0.05) was decreased, while IL-6, TNF-α, Bax, and caspase-3 expression was elevated (P < 0.05). CONCLUSIONS: The number of autophagosomes in detrusor cells was increased in IC/BPS. However, autophagy of detrusor muscle cells may not have sufficient phagocytic activity to effectively remove damaged proteins and mitochondria, which may lead to the continued deterioration of IC/BPS inflammation and apoptosis.

P311 Promotes IL-4 Receptor‒Mediated M2 Polarization of Macrophages to Enhance Angiogenesis for Efficient Skin Wound Healing.

The transition from the proinflammatory phase to the prohealing phase in wound healing is essential for effective skin wound repair, which involves the balance of M1 and M2 polarization of wound-infiltrating macrophages. P311 plays an essential role in promoting wound closure by enhancing the biological function of epidermal stem cells, endothelial cells, and fibroblasts. Nevertheless, whether and how P311 regulates macrophage polarization remains unclear. In this study, we showed that P311 deficiency reduced the M2 polarization of macrophages, thereby attenuating the secretion of M2-like cytokines. The P311 deficiency prolonged the transition from the proinflammatory phase to the prohealing phase, accompanied by weakened angiogenesis and retarded granulation tissue formation, both of which coordinately hinder the healing of skin wounds. Mechanistically, P311 deficiency downregulated the expression of IL-4 receptor on macrophages, followed by less activation of the IL-4 receptor‒signal transducer and activator of transcription 6 signaling pathway, resulting in impaired M2 macrophage polarization. We further revealed that the mTOR signaling pathway was associated with the regulation of P311 on the expression of IL-4 receptor in macrophages. Thus, our study has highlighted the pivotal role of P311 in promoting the M2 polarization of macrophages for effective skin wound healing.

Inhibition of U-II/UT signaling ameliorates cystitis-associated bladder hyperactivity by targeting the RhoA/Rho-kinase pathway.

Urotensin II (U-II) and its receptor (UT) are involved in the pathogenesis of various diseases; however, their association with the development of cystitis has not been elucidated. The present study was designed to investigate the functional role of U-II/UT signaling in cyclophosphamide (CYP)-induced cystitis. A total of 60 female rats were randomly divided into the control and CYP-treated groups. Intraperitoneal injection of CYP successfully induced cystitis in rats of the CYP-treated group. The protein and mRNA expression levels of U-II and UT were significantly enhanced in rat bladder tissues of the CYP-treated group. Furthermore, the results of the immunofluorescence staining analysis demonstrated that CYP treatment apparently increased the expression levels of UT in the urothelium layer, detrusor smooth muscle, and bladder interstitial Cajal-like cells. The selective antagonist of UT, SB657510 (10 µm), significantly suppressed the CYP-induced increase in the spontaneous contractions of muscle strips and ameliorated the bladder hyperactivity of CYP-treated rats. Moreover, CYP treatment significantly increased the protein expression levels of Ras homolog family member (Rho) A and Rho-associated protein kinase 2 in rat bladder tissues. Following pretreatment with the Rho-kinase inhibitor Y-27632 (10 µm), the inhibitory effects of SB657510 (10 µm) on the spontaneous contractions of muscle strips were eliminated. In conclusion, the results of the present study suggested that activation of U-II/UT signaling promoted the development of cystitis-associated-bladder hyperactivity by targeting the RhoA/Rho-kinase pathway, indicating that the U-II/UT signaling could serve as a novel target for the treatment of interstitial cystitis/bladder pain syndrome.

Activation of CXCL13/CXCR5 axis aggravates experimental autoimmune cystitis and interstitial cystitis/bladder pain syndrome.

The abnormal CXCL13/CXCR5 axis is involved in many inflammatory diseases and its selective inhibitor, TAK-799 has exhibited strong anti-inflammatory potency. The sequencing of clinical specimens from interstitial cystitis/bladder pain syndrome (IC/BPS) has shown that CXCL13 and CXCR5 are highly expressed, but the role of CXCL13/CXCR5 axis in IC/BPS has not been rarely reported. Therefore, in this study, we analyzed the GSE11783 sequencing data of IC/BPS patients and investigate the role and mechanism of CXCL13/CXCR5 axis and TAK-779 in the mouse model of experimental autoimmune cystitis (EAC). We verified that CXCL13 and CXCR5 were significantly up-regulated in EAC model. EAC mice exhibited increased bladder inflammatory factors (IL-6, TNF-α, IL-1ß), apoptosis-related proteins (Bax, Caspase-3, Caspase-8), frequency of voiding. Using TAK779 to block CXCL13/CXCR5 axis significantly attenuated these inflammatory damages and efficiently improved bladder function (significant reduction in micturition frequency, significant prolongation of inter-contraction interval). Further investigation showed that inhibiton of JNK and NF-kappaB activation, the bioinformatics analysis-indicated downstream signaling of CXCL13/CXCR5 axis, is responsible for the protective effect of TAK779. Taken together, we demonstrate that activation of the CXCL13/CXCR5 axis is involved in the pathophysiology of IC/BPS and EAC. Blocking CXCL13/CXCR5 axis activation by TAK-779 reduces bladder inflammation and improves bladder function in EAC mice.

Inhibition of CXCR4 in Spinal Cord and DRG with AMD3100 Attenuates Colon-Bladder Cross-Organ Sensitization.

BACKGROUND: Cross-sensitization of pelvic organs is one theory for why symptoms of gut sickness and interstitial cystitis/bladder pain syndrome overlap. Experimental colitis has been shown to trigger bladder hyperactivity and hyperalgesia in rats. The chemokine receptor CXCR4 plays a key role in bladder function and central sensitization. We aim to study the role of CXCR4 and its inhibitor AMD3100 in colon-bladder cross-organ sensitization. METHODS: The colitis model was established by rectal infusion of trinitrobenzene sulfonic acid. Western blot and immunofluorescence were used to assess the expression and distribution of CXCR4. Intrathecal injection of AMD3100 (a CXCR4 inhibitor) and PD98059 (an ERK inhibitor) were used to inhibit CXCR4 and downstream extracellular signal-regulated kinase (ERK) in the spinal cord and dorsal root ganglion (DRG). Intravesical perfusion of resiniferatoxin was performed to measure the pain behavior counts of rats, and continuous cystometry was performed to evaluate bladder voiding function. RESULTS: Compared to the control group, CXCR4 was expressed more in bladder mucosa and colon mucosa, L6-S1 dorsal root ganglion (DRG), and the corresponding segment of the spinal dorsal horn (SDH) in rats with colitis. Moreover, intrathecal injection of the AMD3100 suppressed bladder overactivity, bladder hyperalgesia, and mastocytosis symptoms caused by colitis. Furthermore, AMD3100 effectively inhibited ERK activation in the spinal cord induced by experimental colitis. Finally, treatment with PD98059 alleviated bladder overactivity and hyperalgesia caused by colitis. CONCLUSION: Increased CXCR4 in the DRG and SDH contributes to colon inflammation-induced bladder overactivity and hyperalgesia partly via the phosphorylation of spinal ERK. Treatment targeting the CXCR4/ERK pathway might provide a potential new approach for the comorbidity between the digestive system and the urinary system.

Luteolin Improves Cyclophosphamide-Induced Cystitis through TXNIP/NLRP3 and NF-κB Pathways.

Hemorrhagic cystitis is an important complication of cyclophosphamide chemotherapy, and current therapies for the disease are limited. The natural flavonoid luteolin (LUT) has significant anti-inflammatory and antioxidant properties, but its protective effect on cyclophosphamide (CYP)-induced bladder toxicity has yet to be evaluated. This study aims to explore the protective effect of LUT on CYP-induced acute cystitis in rats. Female Sprague-Dawley rats were randomly assigned to the control (CON) group, CON + LUT group, CYP group, and CYP + LUT group. A single intraperitoneal injection of CYP was administered to establish an acute hemorrhagic cystitis model. HE staining was performed to detect the degree of bladder tissue damage, and TUNEL staining was performed to count apoptotic cells. Oxidative stress indicators were measured using commercial kits, and bladder surgery was performed to assess urinary function. The levels of inflammatory cytokines, apoptosis-related indicators, TXNIP/NLRP3 pathway, and NF-κB pathway were detected by western blot. We found that LUT treatment reduced bladder bleeding, congestion, and edema caused by CYP. Compared with the CYP + LUT group, the level of apoptosis was more highly expressed in the CYP group. We also found that caspase-3, caspase-8, and Bax were significantly upregulated and Bcl-2 was downregulated after LUT treatment. In addition, LUT inhibited the activation of NF-κB signal pathway in the rat bladder tissue after CYP exposure. LUT treatment can also reduce the NLRP3 inflammasome (NLRP3, ASC, and caspase-1) and TXNIP in the bladder. Finally, LUT can reduce the increase in the urination frequency and maximum urination pressure caused by cystitis. These results indicate that LUT displays effective anti-inflammatory, antioxidant, and antiapoptotic properties in CYP-induced acute hemorrhagic cystitis rats by inhibiting the TXNIP/NLRP3 and NF-κB pathways. LUT may be a potent therapeutic agent for the prevention and treatment of hemorrhagic cystitis.

Activation of GPR18 by Resolvin D2 Relieves Pain and Improves Bladder Function in Cyclophosphamide-Induced Cystitis Through Inhibiting TRPV1.

PURPOSE: Hyperalgesia and bladder overactivity are two main symptoms of interstitial cystitis/bladder pain syndrome (IC/BPS). Cannabinoid receptors participate in the modulation of pain and bladder function. GPR18, a member of the cannabinoid receptor family, also participates in the regulation of pain and bladder function, but its underlying mechanisms are unknown. In this work, we sought to study the role of GPR18 in IC/BPS. METHODS: A rat model of IC/BPS was established with cyclophosphamide (CYP). Paw withdrawal threshold (PWT) measurement and cystometry were used to evaluate pain and bladder function, respectively. RT-PCR, Western blotting and immunofluorescence were used to assess the expression and distribution of GPR18. The role of GPR18 in pain and bladder function was studied by intrathecal injection of resolvin D2 (RvD2, a GPR18 agonist) and O-1918 (a GPR18 antagonist). Calcium imaging was used to study the relationship between GPR18 and TRPV1. RESULTS: A rat model of IC/BPS, which exhibited a decreased PWT and micturition interval, was successfully established with CYP. The mRNA and protein expression of GPR18 was reduced in the bladder and dorsal root ganglia (DRG) in rats with CYP-induced cystitis. Intrathecal injection of RvD2 increased the PWT and micturition interval. However, O-1918 blocked the therapeutic effect of RvD2. GPR18 was present in bladder afferent nerves and colocalized with TRPV1 in DRG, and RvD2 decreased capsaicin-induced calcium influx in DRG. CONCLUSION: Activation of GPR18 by RvD2 alleviated hyperalgesia and improved bladder function, possibly by inhibiting TRPV1 in rats with CYP-induced cystitis.

Suppression of adenosine A2a receptors alleviates bladder overactivity and hyperalgesia in cyclophosphamide-induced cystitis by inhibiting TRPV1.

Interstitial cystitis/bladder pain syndrome (IC/BPS) is a type of chronic bladder inflammation characterized by increased voiding frequency, urgency and pelvic pain. The sensitization of bladder afferents is widely regarded as one of the pathophysiological changes in the development of IC/BPS. There is evidence that adenosine A2a receptors are involved in regulating the sensitization of sensory afferents. However, the effect of adenosine A2a receptors on cystitis remains unknown. In the present study, a rat model of chronic cystitis was established by intraperitoneal injection with cyclophosphamide (CYP). Cystometry and behavioral tests were performed to investigate bladder micturition function and nociceptive pain. The rats with chronic cystitis showed symptoms of bladder overactivity, characterized by an increase in bladder voiding frequency and voiding pressure. CYP treatment significantly increased the expression of the A2a receptor in bladder afferent fibers and dorsal root ganglion (DRG) neurons. The A2a receptor antagonist ZM241385 prevented bladder overactivity and hyperalgesia elicited by CYP-induced cystitis. In addition, the A2a receptor and TRPV1 were coexpressed on DRG neurons. The TRPV1 antagonist capsazepine blocked bladder overactivity induced by the A2a receptor agonist CGS21680. In contrast, ZM241385 significantly inhibited the capsaicin-induced increase in intracellular calcium concentration in DRG neurons. These results suggest that suppression of adenosine A2a receptors in bladder afferents alleviates bladder overactivity and hyperalgesia elicited by CYP-induced cystitis in rats by inhibiting TRPV1, indicating that the adenosine A2a receptor in bladder afferents is a potential therapeutic target for the treatment of IC/BPS.