RESUMO
Enhanced afferent excitability is considered to be an important pathophysiological basis of interstitial cystitis/bladder pain syndrome (IC/BPS). In addition, transient receptor potential vanilloid-1 (TRPV1) receptors are known to be involved in afferent sensitization. Animals with hydrogen peroxide (HP)-induced cystitis have been used as a model exhibiting pathologic characteristics of chronic inflammatory condition of the bladder. This study investigated the effect of gene therapy with replication-defective herpes simplex virus (HSV) vectors encoding poreless TRPV1 (PL) or protein phosphatase 1 α (PP1α), a negative regulator of TRPV1, using a HP-induced rat model of cystitis. HSV vectors encoding green fluorescent protein, PL or PP1α were inoculated into the bladder wall of female rats. After 1 week, 1% HP or normal saline was administered into the bladder, and the evaluations were performed 2 weeks after viral inoculation. In HP-induced cystitis rats, gene delivery of PL or PP1α decreased pain behavior as well as a reduction in the intercontraction interval. Also, both treatments reduced nerve growth factor expression in the bladder mucosa, reduced bladder inflammation characterized by infiltration of inflammatory cells and increased bladder weight. Taken together, HSV-mediated gene therapy targeting TRPV1 receptors could be effective for the treatment of IC/BPS.
Assuntos
Cistite/induzido quimicamente , Cistite/terapia , Terapia Genética/métodos , Vetores Genéticos , Peróxido de Hidrogênio/toxicidade , Proteína Fosfatase 1/genética , Simplexvirus/genética , Canais de Cátion TRPV/genética , Animais , Cistite/enzimologia , Cistite/metabolismo , Vírus Defeituosos/genética , Modelos Animais de Doenças , Feminino , Expressão Gênica , Proteínas de Fluorescência Verde/genética , Tamanho do Órgão , Ratos , Bexiga Urinária/efeitos dos fármacos , Bexiga Urinária/patologiaRESUMO
Glandular lesions of the urinary bladder include a broad spectrum of entities ranging from completely benign to primary and secondary malignancies. The accurate diagnosis of these lesions is both important and challenging. Recently, studies suggest that telomerase reverse transcriptase (TERT) promoter mutations could be a biomarker for urothelial carcinoma (UC). We hypothesized that these mutations can distinguish UC with glandular differentiation from nephrogenic adenoma, primary adenocarcinoma of the urinary bladder (PAUB), or secondary malignancies. Twenty-five cases of benign glandular lesions (including nephrogenic adenoma); 29 cases of UC with glandular differentiation; 10 cases of PAUB; and 10 cases each of metastatic colon cancer, prostatic carcinoma, and carcinoma from Mullerian origin were collected. Slides were reviewed and selected to make sure the lesion was at least 10% to 20% of all tissue. Macrodissection was performed in some of cases, and genomic DNA was extracted from the tissue. Telomerase reverse transcriptase promoter mutations were determined by standard polymerase chain reaction sequencing. Twenty-one cases (72%) of UC with glandular differentiation were positive for TERT promoter mutations. However, none of the remaining cases (total 65 cases of benign lesions, PAUB, and metastatic carcinomas) was positive for TERT promoter mutation. Telomerase reverse transcriptase promoter mutations were highly associated with UC including UC with glandular differentiation but not other glandular lesions of bladder. Therefore, in conjunction with morphologic features, Immunohistochemistry stain profile, and clinical information, TERT promoter mutations could distinguish UC with glandular differentiation from other bladder glandular lesions. In addition, lack of TERT promoter mutations in primary adenocarcinoma of bladder suggests that this entity may have different origin or carcinogenesis from those of UC.
Assuntos
Mutação , Neoplasias Epiteliais e Glandulares/genética , Telomerase/genética , Doenças da Bexiga Urinária/genética , Neoplasias da Bexiga Urinária/genética , Adenoma/enzimologia , Adenoma/genética , Adenoma/patologia , Biomarcadores Tumorais/genética , Cistite/enzimologia , Cistite/genética , Cistite/patologia , Feminino , Humanos , Masculino , Neoplasias Epiteliais e Glandulares/enzimologia , Neoplasias Epiteliais e Glandulares/patologia , Reação em Cadeia da Polimerase , Regiões Promotoras Genéticas , Doenças da Bexiga Urinária/enzimologia , Doenças da Bexiga Urinária/patologia , Neoplasias da Bexiga Urinária/enzimologia , Neoplasias da Bexiga Urinária/patologia , Neoplasias da Bexiga Urinária/secundárioRESUMO
OBJECTIVE: To investigate the effect of intravesical hyaluronic acid on Escherichia coli-induced cystitis and cystitis-induced hypercontractility in rats. METHODS: Bacterial cystitis was induced in Wistar female rats by intravesical inoculation of E. coli. Isotonic saline was instilled in the control group (n = 6). The rats were either non-treated, treated with gentamycin (4 mg/kg, 5 days) or treated intravesically with hyaluronic acid (0.5 mL, 0.5%). On the eighth day, the bladder tissues were excised for histological examination, and the measurements of myeloperoxidase, superoxide dismutase and catalase activities. Contraction/relaxation responses to carbachol, isoprotrenol and papaverine were studied. RESULTS: Tissue myeloperoxidase activity was increased, but superoxide dismutase and catalase activities were decreased in bacterial cystitis, while hyaluronic acid treatment reversed these changes. In the hyaluronic acid-treated group, healing of the uroepithelium was observed, while decreased inflammatory cell infiltration was obvious in gentamycin-treated group. E. coli-induced cystitis in all rats resulted in increased contraction responses to carbachol compared with controls (P < 0.01). Treatment with hyaluronic acid, but not gentamycin, significantly (P < 0.05) depressed hypercontractility at maximum carbachol concentrations. In all rats with cystitis, papaverine-induced relaxation was increased, whereas isoproterenol-induced relaxation curves were not different between the studied groups. CONCLUSION: Gentamycin treatment, despite its ameliorative effect on inflammation, had no impact on the contractile dysfunction of the injured bladder. Intravesical hyaluronic acid, in addition to its supportive role in the healing of the epithelium, seems to lower the increased threshold for contraction and to reduce oxidative stress. These findings support a potential role for hyaluronic acid in the treatment of bacterial cystitis.
Assuntos
Adjuvantes Imunológicos/administração & dosagem , Cistite/tratamento farmacológico , Infecções por Escherichia coli/tratamento farmacológico , Ácido Hialurônico/administração & dosagem , Contração Muscular/efeitos dos fármacos , Relaxamento Muscular/efeitos dos fármacos , Bexiga Urinária/efeitos dos fármacos , Administração Intravesical , Animais , Antibacterianos/uso terapêutico , Catalase/metabolismo , Cistite/enzimologia , Cistite/microbiologia , Cistite/patologia , Escherichia coli , Infecções por Escherichia coli/complicações , Feminino , Peroxidase/metabolismo , Ratos , Ratos Wistar , Superóxido Dismutase/metabolismo , Bexiga Urinária/enzimologia , Agentes Urológicos/farmacologiaRESUMO
AIMS: The aims of the present study were to investigate voiding patterns, tissue constituents and the expressions of cyclooxygenase-2 (COX-2) and nitric oxide synthase (NOS) involved in ketamine-induced ulcerative cystitis in rat urinary bladder. METHODS: Thirty Sprague-Dawley rats were distributed into three groups which received saline or ketamine (25 mg/kg/day) for a period of 14 and 28 days. In each group, cystometry was performed weekly and the concentration of ketamine and its metabolites (norketamine) was assayed. Paraffin-embedded sections were stained with Masson's trichrome stain, and ketamine-induced morphological changes were examined. Western blot analyses were carried out to examine the expressions of COX-2 and different NOS isoforms in bladder tissues. Immunofluorescence study was done to evaluate the expressions of COX-2 and macrophage infiltration (stained with ED-1 macrophage cell surface antigen) within the bladder. RESULTS: Ketamine treatment resulted in bladder hyperactivity and the non-voiding contractions were significantly increased. The urine concentrations of ketamine and norketamine were much higher in ketamine-treated group. Moreover, ulcerated urothelium and mononuclear cell infiltration were noted in ketamine-treated group. These alterations in urodynamic functions and tissue constituents were accompanied by increases in the expression of COX-2. Two NOS isoforms (iNOS and eNOS) were also overexpressed, but no significant change was observed for nNOS. COX-2 positive stained cells were significantly increased. Meanwhile, increased amounts of ED-1 positive stained macrophages were present and most of COX-2 expressed cells were co-stained with ED-1 in the early stage of ketamine treatment. CONCLUSIONS: Ketamine treatment affected bladder tissues by enhancing interstitial fibrosis and accelerating macrophages infiltration. Ketamine also initiated the up-regulations of COX-2 and iNOS and eNOS expressions. These up-regulated enzymes might play an important role in contributing to ketamine-induced alterations in micturition patterns and ulcerative cystitis.
Assuntos
Cistite/enzimologia , Óxido Nítrico Sintase/metabolismo , Prostaglandina-Endoperóxido Sintases/metabolismo , Bexiga Urinária/enzimologia , Animais , Cistite/induzido quimicamente , Cistite/fisiopatologia , Ketamina , Ratos , Ratos Sprague-Dawley , Regulação para Cima , Bexiga Urinária/fisiopatologia , Micção/fisiologia , Urodinâmica/fisiologiaRESUMO
Hemorrhagic cystitis is a common complication in children receiving cyclophosphamide, a chemotherapeutic alkylating agent. Acrolein is a urinary metabolite from cyclophosphamide and can induce hemorrhagic cystitis. Here, we investigated the effects and mechanisms of acrolein by intravesical instillation on urinary bladder muscle contractions and pathological alterations in rats. Acrolein instillation significantly increased the muscle contractions of rat bladder detrusor after 1 and 6 h but markedly decreased detrusor contractions after 24 h. Acrolein increased phosphorylated protein kinase C (pan-PKC) expressions in bladders after 1 and 6 h but inhibited it after 24 h. Inducible nitric oxide (NO) synthase (iNOS) protein expressions were markedly induced in bladders 24 h after acrolein treatment. Twenty-four-hour acrolein instillation increased the levels of nitrite/nitrate and interleukin-6 (IL-6) in the urinary bladder. The iNOS inhibitors significantly inhibited the acrolein-increased nitrite/nitrate levels, but not IL-6 levels. IL-6-neutralizing antibodies effectively inhibited the acrolein-increased NOx levels. The increased detrusor contractions by 1-h acrolein treatment were significantly reversed by the PKC inhibitor RO32-0432, and the decreased detrusor contractions by 24-h acrolein treatment were significantly reversed by the iNOS inhibitor and IL-6-neutralizing antibody. Both the iNOS inhibitor and IL-6-neutralizing antibody effectively reversed the increased iNOS expression, decreased PKC phosphorylation, increased bladder weight, and hemorrhagic cystitis in rats 24 h after acrolein treatment. Taken together, these results suggest that an IL-6-regulated iNOS/NO signaling pathway participates in the acrolein-triggered detrusor contraction inhibition and hemorrhagic cystitis. These findings may help us to find a new strategy to treat cyclophosphamide-induced hemorrhagic cystitis.
Assuntos
Acroleína/toxicidade , Ciclofosfamida/urina , Cistite/induzido quimicamente , Hemorragia/induzido quimicamente , Interleucina-6/metabolismo , Contração Muscular/efeitos dos fármacos , Óxido Nítrico Sintase Tipo II/metabolismo , Bexiga Urinária/efeitos dos fármacos , Acroleína/urina , Animais , Western Blotting , Cistite/enzimologia , Cistite/fisiopatologia , Feminino , Hemorragia/enzimologia , Hemorragia/fisiopatologia , Imuno-Histoquímica , Interleucina-6/urina , Músculo Liso/efeitos dos fármacos , Músculo Liso/enzimologia , Músculo Liso/imunologia , Músculo Liso/fisiopatologia , Óxido Nítrico Sintase Tipo II/urina , Ratos , Ratos Wistar , Bexiga Urinária/enzimologia , Bexiga Urinária/imunologia , Bexiga Urinária/fisiopatologiaRESUMO
Interstitial cystitis/painful bladder syndrome is a chronic bladder inflammatory disease of unknown etiology that is often regarded as a neurogenic cystitis. Interstitial cystitis is associated with urothelial lesions, voiding dysfunction, and pain in the pelvic/perineal area. In this study, we used a murine neurogenic cystitis model to identify genes participating in the development of pelvic pain. Neurogenic cystitis was induced by the injection of Bartha's strain of pseudorabies virus (PRV) into the abductor caudalis dorsalis (tail base) muscle of female C57BL/6J mice. Mice infected with PRV developed progressive pelvic pain. The sacral spinal cord was harvested on postinfection days (PID) 2 and 4, and gene expression was analyzed by microarrays and confirmed by quantitative RT-PCR. On PID 2, the overall expression profile was similar to that of uninfected sacral spinal cord; by PID 4, there were substantial differences in expression of multiple functional classes of genes, especially inflammation. Analysis of pain-signaling pathways at the dorsal horn suggested that Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) contributes to neurogenic cystitis pelvic pain. Consistent with this, CaMKIIδ expression exhibited a mast cell-dependent increase in the sacral spinal cord at the mRNA level, and phospho-CaMKII immunoreactivity in the dorsal horn was increased on postinfection day (PID) 4 during PRV infection. Finally, intrathecal injection of the CaMKII inhibitor KN-93 attenuated the PRV pain response. These data suggest that CaMKII plays a functional role in pelvic pain due to neurogenic cystitis.
Assuntos
Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/metabolismo , Cistite/complicações , Cistite/enzimologia , Dor Pélvica/enzimologia , Dor Pélvica/etiologia , Animais , Comportamento Animal/fisiologia , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/antagonistas & inibidores , Cistite/virologia , Relação Dose-Resposta a Droga , Feminino , Herpesvirus Suídeo 1 , Hiperalgesia/etiologia , Processamento de Imagem Assistida por Computador , Imuno-Histoquímica , Injeções Espinhais , Isoenzimas/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Análise em Microsséries , Dor Pélvica/psicologia , Fosforilação , Células do Corno Posterior/enzimologia , RNA/biossíntese , RNA/isolamento & purificação , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Medula Espinal/metabolismo , Transcrição GênicaRESUMO
BACKGROUND: Cystitis causes considerable neuronal plasticity in the primary afferent pathways. The molecular mechanism and signal transduction underlying cross talk between the inflamed urinary bladder and sensory sensitization has not been investigated. RESULTS: In a rat cystitis model induced by cyclophosphamide (CYP) for 48 h, the mRNA and protein levels of the excitatory neurotransmitter calcitonin gene-related peptide (CGRP) are increased in the L6 dorsal root ganglia (DRG) in response to bladder inflammation. Cystitis-induced CGRP expression in L6 DRG is triggered by endogenous nerve growth factor (NGF) because neutralization of NGF with a specific NGF antibody reverses CGRP up-regulation during cystitis. CGRP expression in the L6 DRG neurons is also enhanced by retrograde NGF signaling when NGF is applied to the nerve terminals of the ganglion-nerve two-compartmented preparation. Characterization of the signaling pathways in cystitis- or NGF-induced CGRP expression reveals that the activation (phosphorylation) of extracellular signal-regulated protein kinase (ERK)5 but not Akt is involved. In L6 DRG during cystitis, CGRP is co-localized with phospho-ERK5 but not phospho-Akt. NGF-evoked CGRP up-regulation is also blocked by inhibition of the MEK/ERK pathway with specific MEK inhibitors U0126 and PD98059, but not by inhibition of the PI3K/Akt pathway with inhibitor LY294002. Further examination shows that cystitis-induced cAMP-responsive element binding protein (CREB) activity is expressed in CGRP bladder afferent neurons and is co-localized with phospho-ERK5 but not phospho-Akt. Blockade of NGF action in vivo reduces the number of DRG neurons co-expressing CGRP and phospho-CREB, and reverses cystitis-induced increases in micturition frequency. CONCLUSIONS: A specific pathway involving NGF-ERK5-CREB axis plays an essential role in cystitis-induced sensory activation.
Assuntos
Peptídeo Relacionado com Gene de Calcitonina/genética , Cistite/enzimologia , Cistite/patologia , Proteína Quinase 7 Ativada por Mitógeno/metabolismo , Fator de Crescimento Neural/metabolismo , Células Receptoras Sensoriais/enzimologia , Animais , Anticorpos Neutralizantes/farmacologia , Peptídeo Relacionado com Gene de Calcitonina/metabolismo , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Ativação Enzimática/efeitos dos fármacos , Gânglios Espinais/efeitos dos fármacos , Gânglios Espinais/metabolismo , Gânglios Espinais/patologia , Regulação da Expressão Gênica/efeitos dos fármacos , Vértebras Lombares/efeitos dos fármacos , Vértebras Lombares/enzimologia , Vértebras Lombares/patologia , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Masculino , Fator de Crescimento Neural/antagonistas & inibidores , Fosforilação/efeitos dos fármacos , Transporte Proteico/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-akt/metabolismo , Ratos , Células Receptoras Sensoriais/efeitos dos fármacos , Células Receptoras Sensoriais/patologia , Bexiga Urinária/efeitos dos fármacos , Bexiga Urinária/inervação , Bexiga Urinária/patologiaRESUMO
AKT phosphorylation following peripheral nerve injury or inflammation may play a role in somatic pain processes and visceral inflammation. To examine such a role in micturition reflexes with bladder inflammation, we induced bladder inflammation in adult female Wistar rats (200-300 g) by injecting cyclophosphamide (CYP) intraperitoneally at acute (150 mg/kg; 4 h), intermediate (150 mg/kg; 48 h), and chronic (75 mg/kg; every third day for 10 days) time points. Western blot analyses of whole urinary bladders showed significant increases (P ≤ 0.01) in phosphorylated (p) AKT at all time points; however, the magnitude of AKT phosphorylation varied with duration of CYP treatment. Immunohistochemical analyses of pAKT immunoreactivity (pAKT-IR) in cryostat bladder sections demonstrated duration-dependent, significant (P ≤ 0.01) increases in pAKT-IR in both the urothelium and detrusor smooth muscle of CYP-inflamed bladders. Additionally, a suburothelial population of pAKT-IR macrophages (CD68-, MAC2-, and F4/80-positive) was present in chronic CYP-treated bladders. The functional role of pAKT in micturition was evaluated using open, conscious cystometry with continuous instillation of saline in conjunction with administration of an inhibitor of AKT phosphorylation, deguelin (1.0 µg/10 µl), or vehicle (1% DMSO in saline) in control (no inflammation) and CYP (48 h)-treated rats. Bladder capacity, void volume, and intercontraction void interval increased significantly (P ≤ 0.05) following intravesical instillation of deguelin in CYP (48 h)-treated rats. These results demonstrate increased AKT phosphorylation in the urinary bladder with urinary bladder inflammation and that blockade of AKT phosphorylation in the urothelium improves overall bladder function.
Assuntos
Cistite/enzimologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Bexiga Urinária/enzimologia , Animais , Western Blotting , Ciclofosfamida , Cistite/induzido quimicamente , Cistite/tratamento farmacológico , Avaliação Pré-Clínica de Medicamentos , Ativação Enzimática , Feminino , Imuno-Histoquímica , Fosforilação , Proteínas Proto-Oncogênicas c-akt/antagonistas & inibidores , Ratos , Ratos Wistar , Rotenona/análogos & derivados , Rotenona/farmacologia , Rotenona/uso terapêutico , Bexiga Urinária/efeitos dos fármacos , Urotélio/enzimologiaRESUMO
AIMS: Macrophage migration inhibitory factor (MIF) is a pro-inflammatory cytokine found pre-formed in the urothelium. During inflammation, MIF is released into the bladder lumen and bladder MIF mRNA is upregulated. Since MIF also has tautomerase activity and blocking tautomerase activity also blocks MIF's biological activity, we hypothesized that blocking MIF's tautomerase activity would prevent bladder inflammation. Therefore, we examined the effects of a MIF tautomerase inhibitor (ISO-1; also blocks biological activity) on cyclophosphamide (CYP)-induced cystitis in mice. METHODS: Mice receiving CYP (300 mg/kg; i.p.) to induce cystitis or saline (control) were treated either with ISO-1 (20 mg/kg; i.p.; daily) or vehicle (20% DMSO; i.p.; daily) for 2 days. After 2 days, micturition volume and frequency in awake mice were recorded and also mechanical sensitivity to abdominal stimulation using von Frey monofilaments. Bladders were collected under anesthesia and examined histologically, nerve growth factor levels were assayed in bladder homogenates, and production of inflammatory cytokines in the bladder was determined using a targeted array. RESULTS: CYP treatment resulted in decreased micturition volume, increased frequency, decreased threshold, increased histological signs of cystitis, increased bladder NGF levels and production of inflammatory cytokines when compared to the control group. Treatment with ISO-1 prevented or greatly decreased all these changes. CONCLUSION: Antagonizing MIF's activity with a systemic MIF tautomerase inhibitor was able to prevent or greatly reduced chemical cystitis in mice, thus indicating the MIF mediates bladder inflammation in this model. MIF represents a novel and important modulator of cystitis.
Assuntos
Cistite/prevenção & controle , Inibidores Enzimáticos/farmacologia , Oxirredutases Intramoleculares/antagonistas & inibidores , Isoxazóis/farmacologia , Fatores Inibidores da Migração de Macrófagos/antagonistas & inibidores , Bexiga Urinária/efeitos dos fármacos , Animais , Ciclofosfamida , Cistite/induzido quimicamente , Cistite/enzimologia , Cistite/imunologia , Cistite/fisiopatologia , Citocinas/metabolismo , Modelos Animais de Doenças , Feminino , Hiperalgesia/induzido quimicamente , Hiperalgesia/prevenção & controle , Mediadores da Inflamação/metabolismo , Oxirredutases Intramoleculares/metabolismo , Fatores Inibidores da Migração de Macrófagos/metabolismo , Masculino , Mecanotransdução Celular/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , Fator de Crescimento Neural/metabolismo , Limiar Sensorial , Bexiga Urinária/enzimologia , Bexiga Urinária/imunologia , Bexiga Urinária/fisiopatologia , Micção/efeitos dos fármacosRESUMO
PURPOSE: Cyclophosphamide induced cystitis is an established model for the study of bladder injury and wound healing. Glycosylation is an important modification mechanism that regulates the structure and function of secreted proteins and growth factors from inflammation sites. We determined the effect of cyclophosphamide induced cystitis on O-GlcNAc mediated glycosylation in the bladder. MATERIALS AND METHODS: Cystitis in WT C57BL6 mice was induced with intraperitoneal cyclophosphamide. Retrieved bladders were analyzed using histology, immunohistochemistry, reverse transcriptase-polymerase chain reaction and Western blot for glycosylation associated factors. RESULTS: Acute bladder injury was seen up to 168 hours (7 days) after injection. Reverse transcriptase-polymerase chain reaction revealed down-regulation of O-GlcNAc transferase, a key enzyme in O-GlcNAc mediated glycosylation, at the 8, 48 and 168-hour time points. Also, the glycosidase menangioma expressed antigen 5 was up-regulated at similar time points. Western blot analysis revealed decreased glycosylated protein during cyclophosphamide induced inflammation. CONCLUSIONS: To our knowledge we report the first study of alterations in O-GlcNAc mediated glycosylation activity in bladders with cyclophosphamide induced cystitis. Glycosylation may have a significant role in the bladder wound healing process. Future studies of the glycosylation signaling pathways in the bladder would assist in future potential therapy for bladder inflammatory disease and cancer by elucidating pathways that guide bladder development and wound healing.
Assuntos
Cistite/enzimologia , Regulação para Baixo/fisiologia , Glicosilação , N-Acetilglucosaminiltransferases/fisiologia , Animais , Ciclofosfamida/administração & dosagem , Cistite/induzido quimicamente , CamundongosRESUMO
BACKGROUND: Interstitial cystitis is a chronic condition associated with bladder inflammation and, like a number of other chronic pain states, symptoms associated with interstitial cystitis are more common in females and fluctuate during the menstrual cycle. The aim of this study was to determine if estrogens could directly modulate signalling pathways within bladder sensory neurons, such as extracellular signal-related kinase (ERK) and p38 mitogen-activated protein (MAP) kinases. These signalling pathways have been implicated in neuronal plasticity underlying development of inflammatory somatic pain but have not been as extensively investigated in visceral nociceptors. We have focused on lumbosacral dorsal root ganglion (DRG) neurons projecting to pelvic viscera (L1, L2, L6, S1) of adult female Sprague-Dawley rats and performed both in vitro and in vivo manipulations to compare the effects of short- and long-term changes in estrogen levels on MAPK expression and activation. We have also investigated if prolonged estrogen deprivation influences the effects of lower urinary tract inflammation on MAPK signalling. RESULTS: In studies of isolated DRG neurons in short-term (overnight) culture, we found that estradiol and estrogen receptor (ER) agonists rapidly stimulated ER-dependent p38 phosphorylation relative to total p38. Examination of DRGs following chronic estrogen deprivation in vivo (ovariectomy) showed a parallel increase in total and phosphorylated p38 (relative to beta-tubulin). We also observed an increase in ERK1 phosphorylation (relative to total ERK1), but no change in ERK1 expression (relative to beta-tubulin). We observed no change in ERK2 expression or phosphorylation. Although ovariectomy increased the level of phosphorylated ERK1 (vs. total ERK1), cyclophosphamide-induced lower urinary tract inflammation did not cause a net increase of either ERK1 or ERK2, or their phosphorylation. Inflammation did, however, cause an increase in p38 protein levels, relative to beta-tubulin. Prior ovariectomy did not alter the response to inflammation. CONCLUSIONS: These results provide new insights into the complex effects of estrogens on bladder nociceptor signalling. The diversity of estrogen actions in these ganglia raises the possibility of developing new ways to modulate their function in pelvic hyperactivity or pain states.
Assuntos
Cistite/enzimologia , Estrogênios/metabolismo , Gânglios Espinais/enzimologia , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Animais , Células Cultivadas , Doença Crônica , Ativação Enzimática/fisiologia , Estradiol/farmacologia , Feminino , Região Lombossacral , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Plasticidade Neuronal , Nociceptores/metabolismo , Ovariectomia , Fosforilação , Ratos , Ratos Sprague-DawleyRESUMO
Mast cells have been shown to play a role in development and persistence of various inflammatory bladder disorders. Mast cell-derived tryptase specifically activates protease-activated receptor-2 (PAR-2), and PAR-2 is known to be involved in inflammation. We investigated whether mast cells participate in increase of cyclooxygenase-2 (COX-2) protein abundance in urothelium/suburothelium of bladders of mice subsequent to cyclophosphamide (CYP)-induced bladder inflammation. We also used primary cultures of human urothelial cells to investigate cellular mechanisms underlying activation of PAR-2 resulting in increased COX-2 expression. We found that treatment of mice with CYP (150 mg/kg ip) increased COX-2 protein abundance in bladder urothelium/suburothelium 3, 6, and 24 h after CYP (P < 0.01), and increased COX-2 protein abundance was prevented by treatment of mice with the mast cell stabilizer sodium cromolyn (10 mg/kg ip) for 4 consecutive days before CYP treatment. Incubation of freshly isolated mouse urothelium/suburothelium with a selective PAR-2 agonist, 2-furoyl-LIGRLO-amide (3 microM), also increased COX-2 protein abundance (P < 0.05). We further demonstrated that 2-furoyl-LIGRLO-amide (3 microM) increased COX-2 mRNA expression and protein abundance in primary cultures of human urothelial cells (P < 0.01), and the effects of PAR-2 activation were mediated primarily by the ERK1/2 MAP kinase pathway. These data indicate that there are functional interactions among mast cells, PAR-2 activation, and increased expression of COX-2 in bladder inflammation.
Assuntos
Comunicação Celular , Ciclo-Oxigenase 2/metabolismo , Cistite/enzimologia , Mastócitos/metabolismo , Receptor PAR-2/metabolismo , Bexiga Urinária/enzimologia , Animais , Células Cultivadas , Cromolina Sódica/farmacologia , Ciclo-Oxigenase 2/genética , Ciclofosfamida , Cistite/induzido quimicamente , Cistite/imunologia , Modelos Animais de Doenças , Humanos , Masculino , Mastócitos/efeitos dos fármacos , Mastócitos/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Oligopeptídeos/farmacologia , RNA Mensageiro/metabolismo , Receptor PAR-2/agonistas , Transdução de Sinais , Fatores de Tempo , Regulação para Cima , Bexiga Urinária/imunologia , Urotélio/enzimologia , Urotélio/imunologiaRESUMO
The possible uroprotective effects of curcumin have been addressed in the current study. Haemorrhagic cystitis was induced by challenging male Swiss albino rats with a single dose of cyclophosphamide (150 mg/kg, i.p.). Curcumin (200 mg/kg, i.p.) was administered for 10 consecutive days followed by a single dose of cyclophosphamide. Haemorrhagic cystitis was well characterized morphologically and biochemically. The hallmark of this toxicity was marked congestion, oedema and extravasation in rat urinary bladder, as well as a marked desquamative damage to the urothelium and severe inflammation in the lamina propria. Leucocytic infiltration was also observed and determined by histopathological examination. Serum level of tumour necrosis factor-alpha was notably elevated associated with apparent hypokalaemia and hyponatraemia. Bladder contents of adenosine triphosphate, reduced glutathione and glutathione-S-transferase activity were markedly reduced. Malondialdehyde level, myeloperoxidase activity and urinary nitrite-nitrate levels, expressed as nitric oxide, were dramatically increased. Prior administration of curcumin ahead of cyclophosphamide challenge improved all the biochemical and histologic alterations induced by the cytotoxic drug. Based on these broad findings, it could be concluded that curcumin has proven uroprotective efficacy in this cyclophosphamide haemorrhagic cystitis model, possibly through modulating the release of inflammatory endocoids, namely tumour necrosis factor-alpha and nitric oxide, improving the energy status and restoring the oxidant/antioxidant balance.
Assuntos
Anti-Inflamatórios não Esteroides/uso terapêutico , Curcumina/uso terapêutico , Ciclofosfamida/efeitos adversos , Cistite/prevenção & controle , Hemorragia/prevenção & controle , Urotélio/efeitos dos fármacos , Animais , Anti-Inflamatórios não Esteroides/administração & dosagem , Curcumina/administração & dosagem , Cistite/induzido quimicamente , Cistite/enzimologia , Cistite/patologia , Hemorragia/induzido quimicamente , Hemorragia/enzimologia , Hemorragia/patologia , Masculino , Camundongos , Óxido Nítrico/urina , Potássio/sangue , Sódio/sangue , Fator de Necrose Tumoral alfa/sangue , Bexiga Urinária/efeitos dos fármacos , Bexiga Urinária/enzimologia , Bexiga Urinária/patologia , Urotélio/enzimologia , Urotélio/patologiaRESUMO
AIM: Acrolein (ACR) is a urinary metabolite of cyclophosphamide (CPS) and ifosfamide (IFS), which has been demonstrated to be the causative agent of hemorrhagic cystitis (HC), induced by these compounds. In this study, we investigate the participation of cyclooxygenase-2 (COX-2) on ACR-induced HC. METHODS: Male Wistar rats (150-200g; six rats per group) were treated with distilled water or intravesical ACR and analyzed by changes in bladder wet weight, macroscopic and microscopic parameters and COX-2 expression. RESULTS: COX-2 immunohistochemical expression was significant 12h after ACR administration mainly in subepithelial cells. ACR injection also alters some macroscopic and microscopic parameters in bladder of rats analyzed by Gray's criteria. CONCLUSIONS: COX-2 participates in the pathogenesis of ACR-induced HC first seen 12h after initial contact between ACR and urothelium.
Assuntos
Acroleína/toxicidade , Ciclo-Oxigenase 2/biossíntese , Cistite/induzido quimicamente , Hemorragia/induzido quimicamente , Acroleína/metabolismo , Administração Intravesical , Animais , Cistite/complicações , Cistite/enzimologia , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Indução Enzimática , Hemorragia/complicações , Hemorragia/enzimologia , Masculino , Tamanho do Órgão/efeitos dos fármacos , Ratos , Ratos Wistar , Fatores de Tempo , Bexiga Urinária/efeitos dos fármacos , Bexiga Urinária/enzimologia , Bexiga Urinária/patologiaRESUMO
PURPOSE: Hemorrhagic cystitis (HC) is a limiting side effect of chemotherapy with ifosfamide (IFS). In this study, we investigated the participation of cyclooxygenase-2 (COX-2) upon ifosfamide-induced HC. METHODS: Male Wistar rats (150-200 g; six rats per group) were treated with saline, IFS (400 mg/kg, i.p.) and analyzed by changes in bladder wet weight, macroscopic and microscopic parameters, and COX-2 expression. In other groups etoricoxib (selective COX-2 inhibitor), indomethacin (non-selective COX inhibitor), thalidomide (selective TNF-alpha inhibitor), pentoxifyllin (non-selective TNF-alpha inhibitor) were added 1 h before IFS administration. The classical protocol using three doses of Mesna was also evaluated and compared with two extra doses of etoricoxib or indomethacin. RESULTS: COX-2 was expressed significantly 24 h after IFS administration mainly in myofibroblasts and mast cells evaluated by immunohistochemistry. Treatment 1 h before IFS injection with etoricoxib, indomethacin, thalidomide, and pentoxifylline reduced COX-2 expression and some macroscopic and microscopic parameters in IFS-induced HC. Moreover, addition of etoricoxib or indomethacin with the last two doses of Mesna was more efficient than three doses of Mesna alone when evaluated microscopically. CONCLUSIONS: COX-2 participates in the pathogenesis of IFS-induced HC and the treatment with COX and TNF-alpha inhibitors reduced COX-2 expression. The addition of COX-inhibitors to the last two doses of Mesna represents a new therapeutic strategy of preventing HC.
Assuntos
Inibidores de Ciclo-Oxigenase 2/uso terapêutico , Ciclo-Oxigenase 2/metabolismo , Cistite/enzimologia , Hemorragia/enzimologia , Ifosfamida/efeitos adversos , Animais , Cistite/induzido quimicamente , Cistite/patologia , Quimioterapia Combinada , Etoricoxib , Hemorragia/induzido quimicamente , Hemorragia/patologia , Técnicas Imunoenzimáticas , Indometacina/uso terapêutico , Masculino , Mesna/uso terapêutico , Pentoxifilina/uso terapêutico , Substâncias Protetoras/uso terapêutico , Piridinas/uso terapêutico , Ratos , Ratos Wistar , Sulfonas/uso terapêutico , Talidomida/uso terapêuticoRESUMO
Phosphorylated ERK expression has been demonstrated in the central and peripheral nervous system after various stimuli, including visceral stimulation. Changes in the activation (i.e., phosphorylation) of extracellular signal-regulated kinases (pERK) were examined in the urinary bladder after 4 h (acute), 48 h (intermediate), or chronic (10 day) cyclophosphamide (CYP) treatment. CYP-induced cystitis significantly (P < or = 0.01) increased pERK expression in the urinary bladder with intermediate (48 h) and chronic CYP treatment. Immunohistochemistry for pERK immunoreactivity revealed little pERK-IR in control or acute (4 h) CYP-treated rat urinary bladders. However, pERK expression was significantly (P < or = 0.01) upregulated in the urothelium after 48 h or chronic CYP treatment. Whole mount preparations of urothelium/lamina propria or detrusor smooth muscle from control (noninflamed) rats showed no pERK-IR in PGP9.5-labeled nerve fibers in the suburothelial plexus. However, with CYP-treatment (48 h, chronic), a few pERK-IR nerve fibers in the suburothelial plexus of whole mount preparations of bladder and at the serosal edge of urinary bladder sections were observed. pERK-IR cells expressing the CD86 antigen were also observed in urinary bladder from CYP-treated rats (48 h, chronic). Treatment with the upstream inhibitor of ERK phosphorylation, U0126, significantly (P < or= 0.01) increased bladder capacity in CYP-treated rats (48 h). These studies suggest that therapies targeted at pERK pathways may improve urinary bladder function in CYP-treated rats.
Assuntos
Antineoplásicos Alquilantes , Ciclofosfamida , Cistite/enzimologia , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Fosforilação , Bexiga Urinária/enzimologia , Animais , Western Blotting , Butadienos/farmacologia , Cistite/induzido quimicamente , Inibidores Enzimáticos/farmacologia , MAP Quinases Reguladas por Sinal Extracelular/antagonistas & inibidores , Feminino , Imuno-Histoquímica , Técnicas In Vitro , Fibras Nervosas/enzimologia , Nitrilas/farmacologia , Ratos , Ratos Wistar , Regulação para Cima/fisiologia , Bexiga Urinária/inervação , Cateterismo Urinário , Urotélio/enzimologiaRESUMO
PURPOSE: The role of substance P, inducible nitric oxide synthase, and cyclooxygenase-1 and 2 on the pathogenesis of cyclophosphamide induced cystitis was investigated in rats. MATERIALS AND METHODS: Sprague-Dawley male rats received 1 of certain treatments, including 1) 0.9 weight per volume saline (0.10 ml/100 gm intraperitoneally), 2) cyclophosphamide (75 mg/kg intraperitoneally), 3) cyclophosphamide plus the NK(1) receptor antagonist Win-51.708 (20 mg/kg intraperitoneally), 4) cyclophosphamide plus the inducible nitric oxide synthase inhibitor S-methylthiourea (20 mg/kg intraperitoneally), 5) cyclophosphamide plus the highly selective cyclooxygenase-2 inhibitor rofecoxib (15 mg/kg intraperitoneally), 6) cyclophosphamide plus the selective cyclooxygenase-2 inhibitor meloxicam (15 mg/kg intraperitoneally), 7) cyclophosphamide plus the nonselective cyclooxygenase inhibitor ketoprofen (20 mg/kg intraperitoneally) or 8) cyclophosphamide plus methylthiourea plus meloxicam. Parameters were evaluated 6 hours after cyclophosphamide administration, including plasma protein extravasation, histological changes, myeloperoxidase and inducible nitric oxide synthase activities in the bladder, plasmatic nitric oxide metabolites and urinary nitric oxide metabolites, and prostaglandin E(2) levels. RESULTS: Cyclophosphamide produced inflammatory and cytotoxic changes in the bladder, accompanied by increased nitric oxide metabolites, urinary prostaglandins, myeloperoxidase and inducible nitric oxide synthase activity. Pretreatment with Win-51.708 and with methylthiourea prevented all of these effects except myeloperoxidase activity, which was only prevented by Win-51.708. All inducible cyclooxygenases were able to prevent prostaglandin synthesis and increases in myeloperoxidase activity. Combined inhibition of inducible nitric oxide synthase and cyclooxygenase-2/cyclooxygenase-1 (methylthiourea plus meloxicam) did not provide any additional protection against bladder damage, increased inducible nitric oxide synthase activity or prostaglandin E(2) synthesis. Additionally, this combination was unable to prevent increased myeloperoxidase activity. CONCLUSIONS: The results of this study suggest that there is crosstalk between nitric oxide and the cyclooxygenase enzyme with cyclooxygenase-1/cyclooxygenase-2 isoforms having an important role in this relationship. Augmented myeloperoxidase activity seems to be associated with NK(1) receptor activation and low levels of nitric oxide with cyclooxygenase-1 having an important role.
Assuntos
Ciclo-Oxigenase 1/fisiologia , Ciclo-Oxigenase 2/fisiologia , Ciclofosfamida/administração & dosagem , Cistite/induzido quimicamente , Cistite/enzimologia , Óxido Nítrico Sintase/fisiologia , Receptores da Neurocinina-1/fisiologia , Substância P/fisiologia , Animais , Inibidores de Ciclo-Oxigenase 2/farmacologia , Inibidores de Ciclo-Oxigenase/farmacologia , Masculino , Antagonistas dos Receptores de Neurocinina-1 , Óxido Nítrico Sintase/antagonistas & inibidores , Ratos , Ratos Sprague-Dawley , Substância P/antagonistas & inibidoresRESUMO
PURPOSE: We assessed the effect of irradiation on COX-2 expression in blood vessels of the mouse bladder wall during the early and late radiation response phases. Vasodilatation was quantified as an additional marker of inflammation related to COX-2 activity. MATERIALS AND METHODS: Female C3H/Neu mice were irradiated with a single dose of 20 Gy. The intensity of the COX-2 immunohistochemical staining signal was assessed using an arbitrary semiquantitative score of 0 to 3. To evaluate vasodilatation the percent of the bladder wall covered by the lumen of the blood vessels, depicted as vascular area, was determined. RESULTS: Constitutive COX-2 expression was found in the tunica intima and media of bladder blood vessels. During the early response significant biphasic changes were detected in the COX-2 staining signal as well as in the vascular area with peak values on days 4 and 16 after irradiation. A significant association was found between vascular area and COX-2 expression in blood vessels during the early radiation response (p <0.0001). No changes in COX-2 expression were observed during the late phase, that is between days 90 and 360 after irradiation. Minor vasodilatation seen during the late phase did not correlate with COX-2 activity. CONCLUSIONS: Irradiation resulted in pronounced COX-2 dependent inflammatory changes in the bladder wall during the early but not during the late radiation reaction. Therefore, a potential effect of COX-2 inhibition on early radiation side effects in the bladder can be proposed.
Assuntos
Ciclo-Oxigenase 2/biossíntese , Cistite/enzimologia , Cistite/etiologia , Lesões por Radiação/complicações , Animais , Vasos Sanguíneos/metabolismo , Ciclo-Oxigenase 2/fisiologia , Ciclo-Oxigenase 2/efeitos da radiação , Feminino , Camundongos , Camundongos Endogâmicos C3H , Bexiga Urinária/irrigação sanguíneaRESUMO
Spinal processing of somatosensory and viscerosensory information is greatly facilitated in some persistent pain states. Growing evidence suggests that the so-called central sensitization depends in part on intracellular activation and signalling via specific MAP kinases. Here we studied the expression of phosphorylated extracellular signal-regulated kinases 1 and 2 (phosphoERK), the active form of these kinases, in spinal neurons following innocuous and noxious distension of non-inflamed and cyclophosphamide (CYP)-inflamed rat urinary bladders. Additionally, we investigated the nature of bladder primary afferents responsible for spinal ERK activation. Finally, we used a specific inhibitor of ERK phosphorylation to study the influence of these kinases on the bladder reflex activity of normal and inflamed bladders. Results indicated that, in non-inflamed rats, noxious but not innocuous bladder distension significantly increased spinal phosphoERK immunoreactivity from its normal very low level. However, in CYP-inflamed rats, innocuous and noxious bladder distension significantly increased the number of spinal neurons immunoreactive to phosphoERK. ERK activation was rapid (within minutes) and transient. Desensitization of vanilloid-sensitive afferents by intravesical resiniferatoxin, a capsaicin analogue, did not decrease phosphoERK immunoreactivity in normal or CYP-inflamed rats. ERK inhibition by intrathecal PD 98059 had no effect on bladder reflex contractions of non-inflamed bladders but significantly decreased its frequency in inflamed animals. Our results suggest that spinal ERK intervene in acute and chronic inflammatory pain perception and mediate bladder reflex overactivity accompanying chronic bladder inflammation. In addition, bladder noxious input conveyed in vanilloid-resistant primary afferents is important to spinal ERK phosphorylation in both noninflamed and CYP-inflamed animals.