携带siRNA的慢病毒载体抑制自发性高血压大鼠阴茎海绵体平滑肌细胞S1PR3的表达

目的:筛选出能够高效特异性沉默自发性高血压大鼠(SHR)阴茎海绵体平滑肌细胞(CCSMC)内1-磷酸鞘氨醇受体3(S1PR3)基因表达的siRNA慢病毒载体,并观察其对SHR CCSMC ROCK1、ROCK2、e NOS表达的影响。方法:以大鼠S1PR3基因mRNA序列作为干扰靶点,设计并合成3对靶向S1PR3的siRNA序列(siRNA1、siRNA2、siRNA3)及1对阴性对照序列,构建并包装成慢病毒载体。体外培养SHR CCSMC及魏-凯二氏大鼠(WKY)CCSMC,随机分为A组(SHR对照组)、B组(携带阴性对照病毒的SHR CCSMC转染组)、C~E组(分别携带靶向S1PR3基因siRNA 1~3号靶点慢病毒的SHR CCSMC转染组),F组(WKY对照组),以感染复数(MOI)=60转染SHR CCSMC,转染后观察细胞绿色荧光蛋白(GFP)表达情况,并用RT-PCR和Western印迹检测转染后细胞中S1PR3、ROCK1、ROCK2、e NOS mRNA及蛋白的表达情况。结果:经基因测序证明慢病毒载体构建成功。荧光显微镜下观察B~E组细胞转染效率均80%。与A组相比,B组S1PR3、ROCK1、ROCK2 mRNA及蛋白的表达均无明显改变(P均0.05),C、D、E、F组的S1PR3、ROCK1、ROCK2 mRNA及蛋白的表达均较A组显著下降(P均0.05),其中E组抑制作用最为明显,使S1PR3 mRNA及蛋白表达的抑制效率分别为(34.2±2.9)%、(77.7±4.7)%;ROCK1 mRNA及蛋白表达的抑制效率分别为(33.3±1.4)%、(51.1±7.3)%;ROCK2 mRNA及蛋白表达的抑制效率分别为(30.8±3.6)%、(58.32±5.5)%。A组e NOS mRNA及蛋白表达分别与B、C、D、E比较无明显差异(P均0.05),但较F组显著降低(P0.05);与F组比较,E组S1PR3、ROCK1、ROCK2 mRNA及蛋白的表达均无明显改变(P均0.05),A、B、C、D组的S1PR3、ROCK1、ROCK2 mRNA及蛋白表达均显著高于F组(P均0.05),A、B、C、D、E组e NOS mRNA及蛋白表达均较F组显著降低(P均0.05)。结论:本研究构建的3条携带不同位点的S1PR3基因的siRNA慢病毒载体均能够显著抑制SHR CCSMC内S1PR3基因的表达,并能有效抑制SHR CCSMC中上调的Rho A/Rho激酶信号通路,其中携带siRNA3慢病毒载体的抑制效率最高。     

携带ROCK2基因siRNA有效片段的慢病毒载体的筛选

目的:筛选能够显著抑制自发性高血压大鼠(SHR)阴茎海绵体平滑肌细胞内ROCK2基因表达的携带ROCK2基因siRNA的慢病毒载体。方法:设计并合成4个靶向ROCK2的siRNA片段,构建并包装成慢病毒载体。随机将5只SHR阴茎海绵体平滑肌细胞分为6组,每组每个样本3×104个细胞,每组5个样本,分别为:A组(未转染对照组)、B组(携带慢病毒转染组)、C~F组(分别携带靶向ROCK2基因siRNA 1~4号靶点的慢病毒转染组),以感染复(MOI)=80转染SHR阴茎海绵体平滑肌细胞,转染后48 h荧光显微镜下观察细胞GFP表达情况,并用RT-PCR检测各组被转染细胞ROCK2 mRNA的表达。结果:荧光显微镜下观察各组细胞转染效率均50%。与A组相比,B组ROCK2 mRNA的表达无明显改变(P﹥0.05);C、D、F组SHR阴茎海绵体平滑肌细胞ROCK2基因mRNA的表达较A组极显著下降(P0.01),抑制效率分别达到(43.91±8.19)%、(47.15±6.64)%、(25.7±6.03)%;E组SHR阴茎海绵体平滑肌细胞ROCK2基因mRNA的表达较A组显著下降(P0.05),抑制效率为(16.81±5.94)%。结论:本研究构建的4种携带ROCK2基因的siRNA慢病毒载体均能够显著抑制SHR阴茎海绵体平滑肌细胞内ROCK2基因的表达,其中有1种慢病毒载体抑制作用最强。     

1-磷酸鞘氨醇受体1-3(S1P1-3)在去势雄性大鼠阴茎海绵体组织中的表达

目的:探讨1-磷酸鞘氨醇受体1-3(S1P1-3)在去势雄性大鼠阴茎海绵体内的表达,及其与NOS/NO/c GMP、Rho A/Rho激酶等信号通路的关系。方法:18只8周龄健康雄性SD大鼠,随机分为去势组、对照组及去势后睾酮替代组(替代组)各6只,去势组和替代组大鼠切除双侧睾丸、附睾,替代组大鼠去势术后给予生理剂量丙酸睾酮3 mg/(kg·d)皮下注射4周,对照组为假手术组,去势组及对照组大鼠术后给予等量植物油皮下注射4周,12周龄时,测定各组大鼠阴茎海绵体内压/平均动脉压(ICPmax/MAP)、采用免疫组化和Western印迹分析S1P1-3、e NOS、P-e NOS、ROCK1、ROCK2在阴茎海绵体内的表达变化。结果:去势组大鼠血清睾酮水平[(0.41±0.04)nmol/L]显著低于对照组[(16.01±1.02)nmol/L]及替代组[(15.84±1.32)nmol/L](P0.01),而替代组与对照组睾酮水平无显著差异。去势组ICPmax/MAP比值在0 V、3 V和5 V电刺激盆神经节时(0.088±0.014、0.323±0.014、0.432±0.012)均显著低于对照组(0.155±0.011、0.711±0.010、0.819±0.024)及替代组(0.153±0.012、0.696±0.017、0.763±0.027)(P0.01),而对照组与替代组无显著差异。去势组S1P1、e NOS、P-e NOS的蛋白表达量[以目的蛋白占内参GAPDH的百分率表示:S1P1(49.99±3.39)%,e NOS(46.82±3.81)%,P-e NOS(45.42±4.35)%]显著低于对照组[S1P1(72.57±3.06)%,e NOS(89.76±3.98)%,P-e NOS(82.53±8.92)%]和替代组[S1P1(71.77±4.43)%,e NOS(87.19±4.23)%,P-e NOS(79.82±7.38)%](P0.01),去势组S1P2、S1P3、ROCK1、ROCK2蛋白表达量[以目的蛋白占内参GAPDH的百分率表示:S1P2(82.35±4.13)%,S1P3(61.03±5.14)%,ROCK1(74.50±4.02)%,ROCK2(69.83±5.75)%]显著高于对照组[S1P2(41.67±1.68)%,S1P3(31.66±2.67)%,ROCK1(35.69±5.56)%),ROCK2(39.85±7.17)%]和替代组[S1P2(42.80±3.87)%,S1P3(32.25±4.22)%,ROCK1(38.06±5.21)%,ROCK2(42.36±4.44)%](P0.01)。结论:雄激素缺乏导致大鼠ICPmax/MAP显著降低,可能与阴茎海绵体内S1P1表达下调、抑制e NOS/NO/c GMP信号通路,S1P2、S1P3表达升高、激活Rho A/Rho激酶信号通路有关。     

SphK1/S1P信号通路在肾脏疾病中的作用

鞘氨醇-1-磷酸（sphingosine 1-phosphate,S1P）具有很高的生物学活性,它参与细胞的增殖、凋亡、代谢等多个生理过程,是治疗多种疾病的一个潜在靶点。鞘氨醇激酶1(sphingosine kinase-1,SphK1)是一种广泛存在的脂类激酶,是维持体内S1P水平的关键调节酶,其在生物体内参与癌症、感染和炎症等诸多疾病的发生发展。目前已证实SphK1和S1P受体在肾脏中表达,并且多种肾脏疾病的发生发展与之相关,因此SphK1和S1P在肾脏疾病中作用受到人们越来越多的关注。本文就SphK1/S1P信号通路在肾脏疾病中的作用的研究现状进行综述,重点介绍SphK1/S1P信号通路及其在多种肾脏疾病进展中的作用。     

盐酸芬戈莫德对大鼠颈动脉球囊损伤后1型和3型1-磷酸鞘氨醇受体表达的影响

目的探讨新型免疫抑制剂盐酸芬戈莫德对大鼠颈动脉球囊损伤后1型和3型1-磷酸鞘氨醇受体(S1P1,S1P3)表达的影响。方法 60只SD大鼠随机分为假手术组(n=15)、阴性对照组(n=15)、模型组(n=15)和药物处理组(n=15),采用球囊损伤的方法制备大鼠颈动脉球囊损伤模型,于术后3天、7天和21天取材,行HE染色观察其组织学变化,采用Real-time PCR(RT-PCR)检测大鼠血管中丝氨酸/苏氨酸蛋白激酶2(AKT2)的表达,Western Blot检测大鼠血管中S1P1和S1P3的表达水平。结果 HE染色显示模型组与其他组相比血管增殖明显;RT-PCR显示AKT2在药物处理组的表达低于模型组,但只在7天时差异有统计学意义(P0.05),在同一时间点模型组和药物处理组的表达量均高于假手术组和阴性对照组(P均0.05),假手术组和阴性对照组在各时间点的表达量差异均无统计学意义(P均0.05);Western Blot在球囊损伤初期S1P1和S1P3表达增加,随着时间推移特别是药物干预作用后,到21天时的表达与正常组织相比无明显差异。结论S1P1和S1P3参与了球囊损伤后平滑肌细胞的迁移与增殖,新型免疫抑制剂盐酸芬戈莫德可以抑制AKT2及S1P1和S1P3的表达,减轻球囊损伤后的再狭窄。     

自发性高血压大鼠阴茎组织结构和勃起功能的改变

目的:探讨自发性高血压大鼠(SHR)勃起功能的改变及其发病机制。方法:20周龄雄性SHR大鼠及同系WKY大鼠各15只,夹尾法测量大鼠收缩压(SBP),皮下注射阿朴吗啡(APO)检测阴茎勃起功能,免疫组化染色观察阴茎海绵体α-平滑肌肌动蛋白(α-SMA)及Ⅲ型胶原(COLⅢ)的表达。结果:SHR组及WKY组收缩压分别为(205.7±11.9)、(114.3±10.2)mmHg(1 mmHg=0.133 kPa),阴茎勃起次数分别为(0.6±0.5)、(2.4±0.6)次,差异均有极显著性(P<0.01)。SHR大鼠阴茎海绵体组织平滑肌及COLⅢ的表达显著高于WKY大鼠(P<0.01)。结论:高血压严重影响大鼠阴茎勃起功能,海绵体组织结构的病理改变可能是自发性高血压大鼠勃起功能下降的机制之一。     

Rho激酶及血红素氧合酶在自发性高血压大鼠阴茎海绵体中的表达

目的:探讨NOS/NO、HO/CO、RhoA/Rho激酶等信号通路在自发性高血压大鼠(SHR)阴茎海绵体中的表达及相互关系。方法:健康成年雄性SPF级SHR与对照组WKY大鼠各7只,16周龄,体重250~300g。麻醉后颈动脉和海绵体内插管连续监测平均动脉压(MAP)和海绵体内压(ICP)。利用电刺激海绵体神经,记录ICP/MAP比值变化。利用免疫组化和Western印迹方法分析ROCK2、HO-2、eNOS在阴茎海绵体中的表达变化。结果:SHR组在利用电刺激海绵体神经后ICP/MAP比值升高不明显(P>0.05),海绵体组织中ROCK2蛋白表达水平升高显著(P=0.017),HO-2表达水平则降低显著(P=0.006)。HO-2主要位于阴茎海绵体的平滑肌细胞及神经细胞内,eNOS则主要位于阴茎海绵体血管内皮细胞,两者在SHR组表达明显降低。结论:NOS/NO、HO/CO、RhoA/Rho激酶与SHRED有关,并且可能互相影响。     

P-Erk1/2和P-Akt1在高血压大鼠阴茎海绵体中的表达

目的:了解磷酸化Erk1/2(P-Erk1/2)和磷酸化Akt1(P-Akt1)在自发性高血压大鼠(SHR)和正常血压大鼠阴茎海绵体中的表达及与勃起功能的关系。方法:健康成年雄性SPF级SHR与对照组WKY大鼠各8只,14周龄,体重250～300g。麻醉后颈动脉和海绵体内插管连续监测平均动脉压(MAP)和海绵体内压(ICP),利用电刺激海绵体神经,记录ICP/MAP比值变化;免疫组织化学及RT-PCR技术检测P-Erk1/2和P-Akt1在大鼠阴茎海绵体的表达。结果:3V和5V电刺激海绵体神经后SHR组ICP/MAP比值(0.26±0.06、0.28±0.04)均较WKY组(0.46±0.12、0.76±0.13)显著降低(P均<0.05),P-Erk1、P-Erk2mRNA和P-Erk1/2蛋白的相对表达量在SHR组(0.81±0.05、0.91±0.06、54.22±10.05)较WKY组(0.42±0.04、0.68±0.14、7.05±1.45)显著升高(P均<0.05);P-Akt1mRNA和P-Akt1蛋白的相对表达量在SHR组(0.90±0.05、11.17±2.21)与WKY组(0.92±0.06、10.91±1.86)无显著差异(P均>0.05)。结论:高血压性勃起功能障碍的发生与阴茎海绵体P-Erk1/2的过度表达有关,而与P-Akt1的表达水平无明显相关。     

抗高血压药物对自发性高血压大鼠勃起功能和nNOS神经纤维的影响

目的 :研究抗高血压药物对自发性高血压大鼠 (SHR)勃起功能和神经元型一氧化氮合酶 (nNOS)神经纤维的影响。　方法 :18只 6周龄雄性SHR随机均分为 3组 :缬沙坦 [30mg (kg·d) ]干预组、螺内酯 [2 0mg (kg·d) ]干预组和对照组。 12周后 ,观察大鼠阴茎勃起功能 ,并用免疫组化SP法检测阴茎组织中nNOS神经纤维的数目。　结果 :缬沙坦干预组勃起次数与另外两组相比差异有显著性 (P <0 .0 5 ) ,后两组之间差异无显著性 (P >0 .0 5 ) ,勃起率 3组之间差异不明显 (P >0 .0 5 ) ,阴茎组织中nNOS神经纤维数目 ,两干预组与对照组之间差异有显著性 (P <0 .0 1) ,而两干预组之间差异无显著性 (P >0 .0 5 )。　结论 :缬沙坦能改善SHR的勃起功能 ,而螺内酯无改善作用 ,这种差异与nNOS神经纤维数目无关 ,可能与血管紧张素Ⅱ(AngⅡ)受体阻滞剂引起血管重构等其他作用有关     

血清S1P与绝经后2型糖尿病患者骨密度和骨代谢指标之间的相关性研究

目的探讨血清1-磷酸鞘氨醇(S1P)与绝经后2型糖尿病患者骨密度(bone mineral density,BMD)和骨代谢指标之间的相关性。方法选取2018年2月至2019年12月期间在海口市妇幼保健院就诊的绝经后2型糖尿病女性,收集患者一般临床资料和获取其血液标本,检测生化指标、S1P和髋部、腰椎骨密度。结果最终选取130名血糖控制较好的绝经后2型糖尿病女性参与本研究,年龄为(59.3±8.9)岁,血糖为(8.75±1.5)mmol/L;S1P平均浓度为(6.46±0.78)μmol/L。相关分析表明S1P与腰椎(L1~4)、全髋和股骨颈BMD呈显著负相关(P均<0.05)。多步逐步回归分析表明,血清S1P和Ⅰ型胶原交联C末端肽(β-CTX)与腰椎(L1~4)、全髋和股骨颈BMD密切相关;而血清S1P和β-CTX是各部位BMD独立危险因素。结论1-磷酸鞘氨醇与绝经后2型糖尿病女性骨密度和β-CTX水平密切相关。     

法舒地尔对高血压大鼠勃起功能的影响

目的:探讨Rho激酶抑制剂法舒地尔对高血压大鼠勃起功能的影响及其机制。方法:12周龄雄性SD大鼠随机分成对照组(A组)、高血压组(B组)、法舒地尔治疗组(C组),建立高血压大鼠模型后,C组给予法舒地尔[30 mg/(kg.d)]腹腔注射,A组、B组给予等量生理盐水腹腔注射,术后10周测量大鼠阴茎海绵体内压/平均颈动脉压(ICPmax/MAP),Western印迹法测定ROCK1、ROCK2蛋白在阴茎海绵体的表达水平。结果:B组收缩压(mmHg)、ROCK1、ROCK2蛋白表达(190.39±5.07、0.048±0.002、0.143±0.011)较A组(124.81±4.01、0.036±0.001、0.101±0.011)显著增加(P<0.05),C组收缩压(mmHg)、ROCK1、ROCK2蛋白表达(182.03±4.32、0.044±0.001、0.126±0.007)较B组显著降低(P<0.05),B组ICPmax/MAP(36.82±5.47)较A组(59.99±5.69)显著降低(P<0.05),C组(51.1±5.63)较B组显著提高(P<0.05)。结论:法舒地尔可通过抑制RhoA/Rho激酶信号高表达及可能的降血压作用而改善高血压大鼠勃起功能。     

Rho-kinase inhibition improves erectile function in aging male Brown-Norway rats

Physiological aging is a significant risk factor in the on-set of male erectile dysfunction (ED) and an imbalance in factors that modulate cavernosal smooth-muscle tone may play a role in these altered penile hemodynamic mechanisms. To evaluate the association between aging and male erectile function, we monitored neurogenic erectile response and its correlation to systemic arterial pressure changes in old (21-23 months of age) vs young (6-9 months of age) Brown-Norway (BN) rats. We tested the hypothesis that age-associated ED is due to unregulated vasoconstrictive tone, contributed in part by an increased Rho-kinase activity, and that antagonism of Rho-kinase activity attenuates the age-related decline in male erectile function. We also examined the hypothesis that a combination of Rho-kinase antagonism and phosphodiesterase-5 (PDE-5) inhibition has a synergistic effect in improving the erectile response in these aging animals. Erectile function in old BN rats was evaluated before and after intracavernosal injection of a specific inhibitor of Rho-kinase (Y-27632) alone or in combination with zaprinast, a PDE-5 inhibitor. Erectile capabilities of the young and old BN rat groups were significantly different in corpus cavernosum pressure response after electrical-field stimulation of the major pelvic ganglion. Y-27632 administration attenuated the aging-related changes in male erectile function seen in BN rats. Rho-kinase antagonism and PDE-5 inhibition had a synergistic effect in improving erectile function in old rats. Our data indicate that aging leads to impairment in the neurogenic erectile response in BN rats involving a possible derangement in penile hemodynamic mechanisms of the erectile tissue. Rho-kinase inhibition may be of value in treating age-related ED.     

淫羊藿苷上调NRF2改善SHR大鼠勃起功能

目的:研究淫羊藿苷是否通过调节核因子类红细胞2-相关因子2(NRF2)通路改善自发性高血压大鼠(SHR)的勃起功能.方法:随机将10周龄健康雄性WKY大鼠(WKR)与雄性SHR大鼠分为4组(每组6只,共24只):WKY对照组、WKR+淫羊藿苷组[10 mg/(kg·d)灌胃]、SHR对照组,SHR+淫羊藿苷组[10 m...     

Intracavernosal injection of vascular endothelial growth factor improves erectile function in aged rats

OBJECTIVES: To investigate whether intracavernosal injection of vascular endothelial growth factor (VEGF) can restore erectile function in the aging rat. MATERIALS AND METHODS: Ten young (4-5 months) and 30 old (24 months) Sprague-Dawley male rats were used. The old rats were divided into 3 groups: vehicle-only (phosphate buffered saline plus 0.1% bovine serum albumin; n = 10), VEGF 1 microg/kg (n = 10), and VEGF 10 microg/kg (n = 10). At 2 and 4 weeks after treatment, erectile function and histology were evaluated by hemodynamic study, histomorphometric analysis, and immunohistochemistry. RESULTS: After 4 weeks of treatment, the ratio of peak intracavernosal pressure to systemic arterial blood pressure in response to neurostimulation was significantly higher in both the VEGF 1 microg/kg (79.9 +/- 7.7%) and the VEGF 10 microg/kg group (76.8 +/- 5.8%) compared to the vehicle-only group (63.1 +/- 8.5%; p < 0.05). The percentage of cavernosal smooth muscle was significantly higher in the VEGF 10 microg/kg group (16.1 +/- 1.4%) compared to the vehicle-only group (12.8 +/- 2.2%; p = 0.047). VEGF treatment in old rats increased e-NOS and VEGF expression in both treatment groups. CONCLUSION: Intracavernosal injection of VEGF appears to restore smooth muscle integrity and improve erectile function in aged rats.     

Vasoactive intestinal polypeptide, an erectile neurotransmitter, improves erectile function more significantly in castrated rats than in normal rats

What’s known on the subject? and What does the study add? Vasoactive intestinal polypeptide (VIP) is an important erectile neurotransmitter, and our previous study found that the mRNA expression of VIP was independent of androgens. The present study further investigated the vivo effect of VIP on erection. We found that not only the expression of VIP was independent of androgens, but also the effect of VIP on erection was independent of androgens. In fact, we found that VIP played a more significant role on erection in castrated rats than in normal rats.

OBJECTIVE

• ? To investigate the regulatory role of androgen in VIP‐mediated erectile effect. Androgen is essential for physiological erection. Vasoactive intestinal polypeptide (VIP) is an important erectile neurotransmitter. While previous studies demonstrated that VIP expression in the penis was androgen‐independent, it remains controversial whether androgen has any effect on VIP‐mediated erection.

MATERIALS AND METHODS

• ? Male SD rats were divided into a control group, a castration group, and a castration‐with‐testosterone‐replacement group. Four weeks later, each group was subdivided into low and high‐dose VIP subgroups and subjected to intracavernous injection of 0.5 and 2 µg VIP, respectively.
• ? Erectile function was tested by recording intracavernosal pressure (ICP) and mean arterial blood pressure (MAP) before and after VIP injection.
• ? The expressions of the VIP‐receptor (VPAC2), G‐protein stimulatory and inhibitory alpha subunits (Gs‐α, Gi‐α), and PDE3A in rat corpus cavernosum (CC) was qualified by real‐time PCR and Western blot analysis.

RESULTS

• ? Castration reduced erectile function while testosterone restored it. VIP improved erectile function in a dose‐dependent manner.
• ? High‐dose VIP significantly enhanced erectile function in castrated rats and there was no difference of ICP/MAP among three groups after injection of high‐dose VIP.
• ? Low‐dose VIP also resulted in a higher improvement of erectile function in castrated rats, although the ICP/MAP was lower in these rats than in the other two groups. VPAC2 and Gs‐α were up‐regulated while Gi‐α and PDE3A were down‐regulated in CC of castrated rats.

CONCLUSION

• ? VIP improves erectile function much more significantly in hypogonadal condition, mainly due to the higher expression of VPAC2, Gs‐α, and lower expression of Gi‐α and PDE3A in CC of castrated rats. Androgen may negatively regulate the erectile effect of VIP.

     

Probucol improves erectile function by regulating endoplasmic reticulum stress in rats with streptozotocin-induced diabetes

This study was to explore the effect and mechanism of Probucol on STZ-induced erectile dysfunction in diabetic rats. Thirty SD male rats aged 12 weeks were given intraperitoneal injection of STZ after fasting for 12 hr. Diabetic rats were haphazardly partitioned under two assemblies and administered 0 or 500 mg/kg probucol by oral gavage to 12 weeks. Control group was intraperitoneally injected with physiological saline, and saline was administered by oral gavage daily. Intracorporeal pressure was used to evaluate erectile function. Levels of proteins were detected using immunohistochemistry and Western blotting. α-SMA and vWF were detected using immunofluorescence staining. After treatment, erectile function in probucol group was significantly improved. Endoplasmic reticulum stress-related proteins were expressed higher in DM group than in sham group, while expression of these proteins decreased significantly in probucol group. However, α-SMA and vWF were expressed at lower levels in DM group than in sham group, and probucol treatment reversed this phenomenon. Finally, Bax and Caspase3 were expressed at higher levels and Bcl-2 was expressed at lower levels in DM group, while the opposite result was obtained in probucol group. In conclusions, probucol improves erectile function by reducing endothelial dysfunction and inhibiting PERK/ATF4/CHOP pathway in STZ-induced diabetic rats.     

Chronic sildenafil improves erectile function and endothelium-dependent cavernosal relaxations in rats: lack of tachyphylaxis

OBJECTIVES: Sildenafil is a widely-prescribed effective on-demand treatment of erectile dysfunction (ED). Chronic treatment with sildenafil could help patients with ED. METHODS: The effects of an 8-week long treatment with sildenafil (60 mg/kg/d sc) in male Sprague Dawley rats were evaluated on electrically-elicited erectile responses in vivo before and after an acute injection of sildenafil (0.3mg/kg iv). In addition, endothelium-dependent and -independent relaxations of strips of corpus cavernosum in vitro were examined. All experiments were performed 36 hours after the last injection of sildenafil. RESULTS: Endothelium-dependent relaxations of cavernosal strips to acetylcholine were enhanced after chronic treatment with sildenafil while relaxations to A23187 or sodium nitroprusside were unchanged. Frequency-dependent erectile responses elicited by cavernous nerve stimulation were significantly improved. Moreover, the erectile responses to acute sildenafil were greater in chronically-treated rats with sildenafil. CONCLUSIONS: This is the first report providing experimental support for chronic dosing with sildenafil which could be of use for patients that are poor responders to on-demand treatment. Chronic sildenafil may regulate the transduction pathway leading to the activation of eNOS but has no effect on NO bioavailability or on the cGMP pathway, thereby eliminating a possible concern for tachyphylaxis.     

Injection of skeletal muscle-derived cells into the penis improves erectile function

We investigated the effect of intrapenile injection of muscle-derived cells (MDC) on the erectile function in rats with bilateral cavernous nerve injury. Rat MDC were harvested and transduced with a retrovirus expressing the lacZ gene. Hanks' balanced salt solution (HBSS) (20 microl) or MDC (1 x 10(6) cells/side) were injected in each corpora cavernosa immediately before bilateral cavernous nerve transection. Intracavernous pressures (ICP) were measured 2 or 4 weeks after surgery with electrical stimulation of the pelvic nerves. Mean maximal ICP of sham group was significantly lower than that of control group both at 2 and 4 weeks after surgery. When MDC were injected into the penis, ICP improved over the sham-injected group at both 2 and 4 weeks after surgery. Percent area of PGP 9.5 staining was significantly greater in MDC-injected penis than in sham-injected at 2 and 4 weeks. Penile MDC injection can facilitate recovery of injured penile innervation and improve erectile function.