TGF-β1Mediates Androgen Deprivation Induced Bladder Histological Abnormalities And Dysfunction In Orchiectomized Mature Male Rats

Part One:Androgen Deprivation Induces Bladder Histological Abnormalities via TGF-β in Orchiectomized Mature Male RatsObjectiveTo determine the pathologic consequences of androgen deprivation in bladder histology and address the underlying mechanism.Animal and Methods36male Sprague-Dawley120-day-old rats, weighing from210g to245g, were involved in this study. They were divided into4experimental groups:1. Emasculated rats group (EMR):After bilateral orchiectomy,9rats received intramuscular saline injection for2months;2. Emasculated rats treated with testosterone group (EMR+T): After bilateral orchiectomy,9rats were treated with intramuscular injection of testosterone propionate (10mg/kg.2injections every-week) for2months:3. Emasculated rats treated with anti-TGF-β neutralizing antibody group (EMR+A): After bilateral orchiectomy,9rats were treated with anti-TGF-P neutralizing antibody by intraperitoneal injection (0.5mg/kg,2injections every week) for2months, which is a neutralizing monoclonal anti-TGF-β antibody (aT) against TGF-β1.-β2, and-β3;4. Sham surgery rats group (Sham):9rats were received sham surgery procedure and served as control.The serum testosterone level in deffirent groups of rats was determined following by the protocol of Rat testosterone (T)ELISA Kit. Masson's trichrome coupled with biological electron microscope technique were used to evaluate the bladder smooth musle damage and bladder fibrosis. Realtime PCR was employed to determine the expression level of pro-collagen I gene.ResultsAt the end of experiment-The serum testosterone level in EMR group rats is0.002±0.0005ng/ml. significantly lower than sham group. Remarkably increased bladder fibrosis was seen in EMR group rats compared to sham group rats, which was completely reversed by testosterone propionate replacement therapy (EMR-T group). Quantitative image analysis showed a significantly increased ratio of collagen to smooth muscle in EMR group rats compared to sham group rat. indicating smooth muscle damage and increased bladder fibrosis. However, after testosterone propionate replacement therapy, the increased ratio of collagen to smooth muscle in orchiectomized rats was completely corrected, presenting no significant difference with sham group rats. To confirm our histological finding that androgen deprivation in orchiectomized rats lead to bladder fibrosis. pro-collagen I (fibrotic marker gene) gene expression level was determined by Real-time PCR in each group of rats. The results showed that the mRNA expression level of pro-collagen I was dramatically elevated in EMR group rats compared with sham group rats. After testosterone propionate replacement therapy (EMR+T group), the elevated pro-collagen I expression level was significant decreased compared to EMR group rats, revealing no significant difference with sham group rats. TGF-β mRNA levels were measured for each groups of rats by real-time PCR. the results showed that TGF-β1mRNA level increased dramatically in EMR group rats compared to sham groups rats and androgen replacement treatment totally blocked the induction of TGF-β1mRNA level in orchiectomized rats. These data suggest that androgen deprivation lead to increase of TGF-β1mRNA level and TGF-β1may be a signaling molecule response for androgen deprivation-induced bladder fibrosis. To test this possibility, we treated one group of orchiectomized rats with anti-TGF-β neutralizing antibody for2months. We found that the androgen deprivation-induced increased ratio of bladder collagen to smooth muscle and induction of pro-collagen I mRNA expression were significantly attenuated by anti-TGF-β neutralizing antibody treatment.ConclusionOur studies identify an essential role of TGF-β in the pathogenesis of androgen deprivation-induced bladder fibrosis and offer a potential target for prevention and treatment of bladder fibosis associated with androgen deficiency. ObjectiveTo determine the pathologic consequences of androgen deprivation in bladder function and address the underlying mechanism.Animal and MethodsMale rats were divided into4groups as described above:emasculated rats (EMR). emasculated rats treated with testosterone, emasculated rats treated with anti-transforming growth factor-β (TGF-β) neutralizing antibody, and sham surgery rats. Urodynamic evaluations were employed to determine the bladder function in vivoResultsAt the end of experiment. To determine the functional changes following by bladder fibrosis in orchiectomized rats, urodynamic evaluations were performed at the end of treatment for each group of rats. The maximal bladder volume and bladder compliance were determined. The maximal bladder volume after experiment in EMR group rats was0.51±0.16ml. significantly lower than sham group (1.25±0.20ml). After androgen replacement treatment, the maximal bladder volume in EMR+T group rats was1.37±0.22ml (p<0.05vs. EMR group rats). The mean bladder compliance was0.015±0.004ml/cm H2O in EMR group rats, which was dramatically decreased compare to sham group (0.029±0.007ml/cm H2O). However, the decreased bladder compliance was totally corrected by androgen replacement treatment in EMR+T group rats (0.028±0.007ml/cm H2O. p>0.05vs. sham group). Overall, our data demonstrated that androgen deprivation in rats decreases the maximal bladder volume and compliance and leads to bladder dvsfunction and these functional abnormalities could be corrected by androgen replacement therapy.Then we want to figure out whether anti-TGF-P treatment is effective to abolish androgen deprivation-induced bladder dysfunction by blocking the bladder fibrosis. The results showed that after anti-TGF-β neutralizing antibody treatment, the maximal bladder volume of orchiectomized rats was0.96±0.27ml and the bladder compliance was0.023±0.004ml/cm H2O. Both of them were significantly lower than orchiectomized rats without treatment group (EMR group). These data demonstrated that the androgen deprivation-induced bladder dysfunction is also mediated by TGF-β1and anti-TGF-β treatment abolishes androgen deprivation-induced bladder dysfunction.ConclusionOur studies identify an essential role of TGF-β in the pathogenesis of androgen deprivation-induced bladder dysfunction and offer a potential target for prevention and treatment of bladder dysfunction associated with androgen deficiency.