糖尿病大鼠阴茎海绵体组织中一氧化氮合酶亚型表达的研究

目的:通过观察糖尿病性雄性大鼠阴茎海绵体组织中一氧化氮合酶亚型(nNOS、iNOS、eNOS)表达和勃起功能的变化,以及应用胰岛素、α-硫辛酸(LA)干预对其的影响,进一步探讨糖尿病性ED发病机制,并为临床治疗提供新的思路。方法:将50只成年雄性SD大鼠分为4组:A组为正常对照组(10只),B组为糖尿病无干预组(13只),C组为糖尿病胰岛素干预组(12只),D组为糖尿病胰岛素+LA干预组(15只),采用腹腔注射链脲佐菌素法制作糖尿病模型。8周后各组大鼠通过注射阿朴吗啡后评价勃起功能并取阴茎海绵体组织用免疫组化EnV i-sion法观察海绵体组织中nNOS、iNOS、eNOS表达的变化。结果:A组大鼠勃起功能正常,勃起率100%;与A组相比,B组、C组、D组勃起功能水平显著降低(P<0.05),勃起率:B组28.6%,C组62.5%,D组80.9%,其海绵体组织中nNOS、eNOS表达显著降低,A组大鼠海绵体组织每视野nNOS、eNOS阳性神经纤维数为86.7、9.6,B组为36.5、3.3,C组为52.7、5.7,D组为71.4、7.4,而iNOS表达显著增加(P<0.05),A组为6.9,B组为43.6,C组为36.2,D组为19.3;与B、C组相比,D组勃起功能显著上升,海绵体组织中nNOS、eNOS表达均显著上升,iNOS显著下降(P<0.05)。结论:糖尿病严重影响阴茎勃起功能和nNOS、iNOS、eNOS的表达,高血糖是其发病基础之一,而LA对其有显著疗效,推测可能与其抗氧化作用机制有关。     

伊木萨克片对糖尿病性ED大鼠阴茎组织中eNoS和nNoS的影响

目的 研究维药伊木萨克片对DM性ED大鼠阴茎海绵体组织中eNOS和nNOS蛋白表达水平的影响.方法 取雄性SD大鼠70只,从中随机取10只为正常对照组,余60只以链脲佐菌素诱导建立DM模型后,行阿朴吗啡阴茎勃起实验筛选DM性ED模型,发生ED者随机分为DM性ED组、伊木萨克片组、胰岛素组、伊木萨克片 胰岛素(联用)组,未成DM者为STZ组.各组给药6周后,采用免疫组化SP法检测各组大鼠阴茎组织中eNOS、nNOS含量.结果 DM性ED组大鼠阴茎组织中eNOS、nNOS蛋白表达水平显著低于正常对照组(P＜0.01);伊木萨克片组、胰岛素组及联用组大鼠阴茎组织中eNOS、nNOS蛋白表达水平均显著高于DM性ED组(P＜0.01),但伊木萨克片组与胰岛素组显著低于联用组(P＜0.01, P＜0.01;P＜0.05,P＜0.01);STZ组与正常对照组之间的差异则无统计学意义(P＞0.05).结论 维药伊木萨克片可以显著提高DM性ED大鼠阴茎组织中eNOS、nNOS蛋白表达水平,并提示在胰岛素控制血糖的基础上效果可能更佳.     

低氧分压对大鼠勃起功能的影响

目的探讨低氧分压对大鼠勃起功能障碍（ED）的影响。方法48只成年白色雄性SD大鼠随机分为对照组和实验组，每组按照实验时间（2周、6周、10周）分为3个亚组，每个亚组8只。实验组置于密闭低氧舱中饲养，对照组在正常环境中饲养，其他条件相同。分别观察其勃起功能，采用免疫组化SP法检测神经源性一氧化氮合成酶（nNOS）阳性神经纤维的数量、内皮源性一氧化氮合成酶（eNOS）的表达。结果实验组与对照组比较：（1）大鼠勃起次数明显降低（P〈0．001）；（2）nNOS阳性神经纤维数量、eNOS蛋白的表达均有显著性差异（P〈0．01）。实验组间比较：勃起次数6周组明显低于2周组，10周组稍高于6周组；nNOS阳性神经纤维数量有显著性差异（P〈0．01）；eNOS的表达6周组最低，10周组有所回升。结论低氧分压致大鼠勃起功能受损和nNOS、eNOS的表达下降。nNOS染色阳性神经纤维数量的减少、eNOS表达的下降，可能是低氧分压环境中大鼠ED发生的原因之一。     

髂内动脉结扎对大鼠阴茎海绵体组织nNOS神经纤维及eNOS表达的影响

目的 研究髂内动脉结扎对大鼠阴茎海绵体组织神经型一氧化氮合酶(nNOS)神经纤维及内皮型一氧化氮合酶(eNOS)表达的影响。方法 45只Wistar雄性成年大鼠随机分为假手术组(21只)和手术组(24只)。其中手术组采用双侧髂内动脉结扎的方法制备动脉性勃起功能障碍(ED)大鼠模型,假手术组作为对照组只切开腹腔不结扎髂内动脉。于术后1周、3周、6周末分别取各组1/3大鼠,观察其勃起功能,并采用免疫组化SP法检测大鼠阴茎组织中nNOS神经纤维的数量及eNOS的表达。结果 手术组与假手术组相比,1周末、3周末和6周末在阴茎勃起次数和阴茎组织nNOS神经纤维数量及eNOS表达上均有显著差异(P<0.01)。手术组大鼠阴茎勃起次数3周末高于1周末,6周末高于3周末;eNOS的表达3周末明显低于1周末,而6周末高于3周末,差异均有显著性意义(P<0.001),并呈增加趋势。手术组大鼠阴茎组织nNOS神经纤维数量三者之间差异均有显著性(P<0.001),呈进行性下降。结论 髂内动脉结扎影响阴茎勃起功能和nNOS、eNOS的表达。阴茎组织中nNOS阳性神经纤维数量的减少,可能是髂内动脉结扎术后阴茎勃起功能障碍发生的原因之一。     

杜仲对糖尿病大鼠扑捉行为和阴茎组织神经传导通路的影响

目的:探讨杜仲改善勃起功能的药效和病理学机制。方法:雄性糖尿病(DM)大鼠30只随机分为3组:A组(10只,DM大鼠赋形剂灌胃组)、B组(10只,DM大鼠西地那非灌胃组)、C组(10只,DM大鼠杜仲灌胃组)及10只正常对照组大鼠(赋形剂灌胃,D组);灌胃4周后观察4组大鼠扑捉行为,透射电镜检查阴茎组织有髓神经纤维超微特征;用免疫组化二步法显示阴茎组织中神经元型一氧化氮合酶(nNOS)的表达。结果:与A组比较,C组大鼠扑捉次数显著增多(P<0.05),阴茎组织中nNOS表达显著增强(P<0.001)。透射电镜显示:A组大鼠阴茎组织有髓神经纤维排布失序,部分变性、板层分离形成透明空泡或网络状,C组大鼠有髓神经纤维排列规整,板层结构清晰。结论:杜仲可通过减轻有髓神经的损伤、增强阴茎组织中nNOS表达改善ED。     

生长激素补充对老龄大鼠勃起功能及阴茎海绵体nNOS表达的影响

目的探讨生长激素(GH)补充对老龄大鼠勃起功能及阴茎海绵体神经型一氧化氮合酶(nNOS)表达的影响。方法20只18月龄SD大鼠随机均分成A、B两组,10只2月龄SD大鼠为C组。A组给予GH1U/(kg·d),B、C组给予相同剂量的生理盐水,均皮下注射8周。于8周末观察阿朴吗啡(APO)皮下注射与大鼠海绵体内注射罂粟碱诱导的阴茎勃起情况,并用免疫组化SP法检测阴茎海绵体组织中nNOS神经纤维的数目。结果8周末时,A、C组大鼠APO诱导的勃起次数、海绵体内注射罂粟碱诱导的最大海绵体内压以及阴茎海绵体组织中nNOS神经纤维数目均显著高于B组(P<0.05或P<0.01)。结论老龄大鼠勃起功能及阴茎海绵体nNOS表达较成年大鼠明显下降,GH补充可以部分改善老龄大鼠的勃起功能,其机制之一可能与GH补充增加了老龄大鼠阴茎海绵体组织中nNOS神经纤维数目有关。     

还原型谷胱甘肽对糖尿病大鼠阴茎海绵体氧化损伤的影响

目的探讨氧化损伤在糖尿病性ED发病机制中的作用,以及还原型谷胱甘肽（GSH）对氧化损伤的干预作用,为临床治疗提供新的手段。方法成年雄性SD大鼠50只,随机分为4组：A组为对照组（n=10）,B组为糖尿病无干预组（n=13）,C组为糖尿病胰岛素干预组（n=13）和D组为糖尿病胰岛素＋GSH干预组（n=14）。采用腹腔注射STZ法制作糖尿病模型并进行筛选。8周后取大鼠阴茎海绵体组织进行组织匀浆,采用黄嘌呤氧化法测定阴茎海绵体组织中超氧化物岐化酶（SOD）活性;用TBA法测定丙二醛（MDA）含量;RT—PCR法检测胞外型SOD基因（SODEX基因）的表达;Western-blot法检测CuZnSOD蛋白含量。结果A组大鼠海绵体组织基本无过氧化产物蓄积。与A组相比,B、C、D组的SOD活性显著下降（P〈0．05）,MDA含量均显著升高（P〈0．05）,B、C、D组SODEX基因的表达、CuZnSOD蛋白含量显著下降（P〈0．05）;与B组相比,C、D组的SOD活性显著升高（P〈0．05）,MDA含量显著下降（P〈0．05）,SODEx基因的表达及CuZnSOD蛋白含量显著升高（P〈0．05）。与C组相比,D组SODEX基因的表达及CuZnSOD蛋白含量显著升高（P〈0．05）。结论糖尿病性雄性大鼠阴茎海绵体组织自由基代谢明显障碍,脂质过氧化产物增加,同时氧自由基清除能力下降;而抗氧化剂GSH的应用能部分减弱氧化损伤。提示氧化损伤可能在糖尿病性ED的发生发展中起一定作用。本研究为糖尿病性ED的预防和治疗提供新的策略。     

糖尿病性勃起功能障碍大鼠阴茎海绵体组织中神经生长因子的表达及其治疗作用的研究

目的:本研究通过检测糖尿病性勃起功能障碍(ED)大鼠阴茎组织中神经生长因子(NGF)表达,并使用hNGF进行治疗,以探讨糖尿病性ED发病机制及NGF治疗作用的机制。方法:成年雄性SD大鼠60只,随机取50只大鼠用于制作糖尿病模型,饲养8周后,取正常组和糖尿病组大鼠阴茎海绵体组织,采用RT-PCR和W estern印迹法检测NGF的mRNA及蛋白水平。从造模成功的糖尿病大鼠中筛选出有ED大鼠,把所有大鼠分为5组:正常组、糖尿病性ED组、糖尿病性ED单用NGF组(NGF组)、糖尿病性ED单用胰岛素组(R I组)、糖尿病性ED联合应用NGF和胰岛素组(NGF+R I组,胰岛素通过颈部皮下注射给药,NGF通过腹腔内注射给药),8周后测海绵体内压(ICP),并取所有大鼠阴茎海绵体组织用免疫组化法观察nNOS神经纤维的变化。结果:与正常组相比,糖尿病性ED组大鼠阴茎海绵体组织中NGF的mRNA表达增加,蛋白含量增加。与糖尿病性ED组相比,NGF组、R I组、NGF+R I组ICP水平显著升高(P<0.05);NGF组、R I组、NGF+R I组阴茎组织中nNOS神经纤维水平显著升高(P<0.05)。结论:糖尿病晚期勃起神经出现损伤并发生ED,推测可能与NGF分泌增加的幅度小于高血糖状态对勃起神经的损伤程度有关,也可能与NGF与其相应受体结合转运能力损害有关,给予外源性NGF可能有助于糖尿病性ED局部神经病变减轻和勃起功能改善。提示NGF的异常在糖尿病性ED的发病及治疗中可能具有重要作用。     

银芷肛肠熏洗剂对大鼠痔相近模型iNOS、VEGF、CD68表达的影响

为观察银芷肛肠熏洗剂对大鼠痔相近模型诱导型一氧化氮合酶（iNOS）、血管内皮生长因子（VEGF）、CD68表达的影响,本实验采用肛门外周注射冰醋酸法建立大鼠痔相近模型,造模成功后将50只大鼠随机分为空白对照组（A组）、马应龙金玄痔科熏洗散对照组（B组）和银芷肛肠熏洗剂低剂量组（C组）、中剂量组（D组）、高剂量组（E组）,每组10只,连续给予相应药物7d,于第8天处死大鼠,通过免疫组化染色法评估大鼠痔相近模型创面组织中iNOS、VEGF、CD68水平。结果显示,（1）iNOS：B～E组大鼠创面组织中iNOS阳性细胞率评分明显低于A组,P〈0．01;D、E组大鼠创面组织中iNOS阳性细胞率评分明显低于B、C组,P〈0．01;而B、C组比较差异无统计学意义,P〉0．05。（2）VEGF：B～E组大鼠创面组织中VEGF阳性细胞率评分明显低于A组,P〈0．05或P〈0．01;D、E组大鼠创面组织中VEGF阳性细胞率评分均低于B、C组,P〈0．01;而B、C组比较差异无统计学意义,P〉0．05。（3）CD68：B～E组大鼠创面组织中CD68阳性细胞率评分明显低于A组,P〈0．05或P〈0．01;C～E组大鼠创面组织中CD68阳性细胞率评分均明显低于B组,P〈0．05或P〈0．01。结果表明,银芷肛肠熏洗剂能明显抑制大鼠痔相近模型iNOS、VEGF、CD68的表达,其对痔的治疗作用可能与抑制iNOS、VEGF、CD68表达有关。     

携带S1P3 siRNA慢病毒载体改善自发性高血压大鼠勃起功能

目的:探讨携带S1P3 siRNA慢病毒载体对自发性高血压大鼠(SHR)勃起功能的改善作用。方法:5只12周龄健康雄性WKY大鼠为A组,随机将12周龄健康雄性SHR大鼠分组(每组5只):B1组:携带S1P3 siRNA慢病毒转染的SHR;B2组:空慢病毒载体(GFP)转染的SHR;C组:SHR大鼠对照组。B1组阴茎海绵体中部注射20μl携带S1P3 siRNA慢病毒(2×10~8TU/ml),B2组注射GFP 20μl (2×10~8TU/ml),每侧10μl。1周后测各组大鼠ICPmax/MAP值,免疫组化、Western印迹、RT-qPCR检测S1P3、ROCK1、ROCK2、eNOS mRNA及蛋白在各组大鼠阴茎海绵体中的表达。结果:各组大鼠体重、血清T水平无明显差异。A、B1、B2、C组大鼠在0、3、5 V电压刺激下ICPmax/MAP分别为:0V:0.16±0.01、0.15±0.01、0.10±0.00、0.11±0.01,3 V:0.55±0.03、0.55±0.01、0.22±0.01、0.22±0.01,5 V:0.82±0.02、0.79±0.03、0.43±0.01、0.42±0.02,C、B2组ICPmax/MAP较A、B1组显著降低(P0.05)。免疫组化结果显示,S1P3、ROCK1、ROCK2在A、B1组大鼠阴茎海绵体组织中表达水平与B2、C组相比明显减少(P0.05),eNOS则明显增加(P0.05);Western印迹结果显示,与B2、C组相比,A、B1组大鼠阴茎海绵体组织中S1P3、ROCK1、ROCK2蛋白表达显著减低(P0.05),eNOS显著升高(P0.05);A、B1、B2、C组S1P3基因相对表达量分别为:0.72±0.04、0.71±0.07、1.00±0.06、1.00±0.10,ROCK1:0.99±0.05、1.08±0.16、1.85±0.44、2.02±0.38,ROCK2:1.00±0.03、1.08±0.16、2.16±0.78、2.46±0.69,eNOS:1.04±0.15、0.81±0.23、0.32±0.08、0.32±0.04,S1P3、ROCK1、ROCK2在A组和B1组中的表达水平与C组、B2组相比明显减少(P0.05),eNOS明显增加(P0.05)。结论:携带靶向S1P3 siRNA慢病毒载体抑制阴茎海绵体组织内S1P3基因的表达,并下调RhoA/Rho激酶信号通路,从而改善SHR大鼠勃起功能。     

伊木萨克片对半去势雄性大鼠勃起功能的影响

目的 研究伊木萨克片对半去势雄性大鼠勃起功能的影响.方法 从60只性功能正常的雄性SD大鼠中随机取出10只为正常对照组,余50只行右侧睾丸摘除后随机分为半去势空白组、男宝对照组、伊木萨克低、中、高剂量组,经药物干预6周后行阿朴吗啡(Apomorphine,APO)勃起试验,镜检阴茎海绵体的形态改变,采用免疫组化SP法检测各组大鼠阴茎组织中nNOS、eNOS蛋白表达.结果 (1)半去势空白组勃起潜伏期显著长于正常对照组(P<0.01),伊木萨克低、中、高剂量组勃起潜伏期显著短于半去势空白组和男宝对照组(P<0.05),但长于正常对照组(P<0.05),伊木萨克片低、中、高剂量组之间差异则无统计学意义(P>0.05):(2)半去势空白组阴茎组织中eNOS、nNOS蛋白表达低于正常对照组(P<0.05).伊木萨克低、中、高剂量组大鼠阴茎组织中eNOS、nNOS蛋白表达均显著高于半去势空白组和男宝对照组(P<0.05),其中eNOS在伊术萨克片中剂量组显著高于正常对照组(P<0.01),而伊木萨克片低、高剂量组与正常对照组之间的差异则无统计学意义(均为P>0.05);nNOS在伊木萨克片低、中、高剂量组与正常对照组及伊木萨克片低、中、高剂量组之间差异无统计学意义(P>0.05).结论 (1)半去势可显著影响雄性大鼠的阴茎勃起功能;(2)伊木萨克片干预后能够显著增强半去势大鼠的阴茎勃起功能,其机制可能和增加阴茎组织中eNOS、nNOS蛋白表达有关.     

Effects of icariin on erectile function and expression of nitric oxide synthase isoforms in castrated rats

Aim： To investigate the effect of icariin on erectile function and the expression of nitric oxide synthase （NOS） isoforms in castrated rats. Methods： Thirty-two adult male Wistar rats were randomly divided into one shamoperated group （A） and three castrated groups （B, C and D）. One week after surgery, rats were treated with normal saline （groups A and B） or oral icariin （1 mg/[kg·day] for group C and 5 mg/[kg·day] for group D） for 4 weeks. One week after treatment, the erectile function of the rats was assessed by measuring intracavernosal pressure （ICP） during electrostimulation of the cavernosal nerve. The serum testosterone （ST） levels, the percent of smooth muscle （PSM） in trabecular tissue, and the expression of mRNA and proteins of neuronal nitric oxide synthase （nNOS）, inducible nitric oxide synthase （iNOS）, endothelial nitric oxide synthase （eNOS） and phosphodiesterase V （PDES） in corpus cavernosum （CC） were also evaluated. Results： ICP, PSM, ST and the expression of nNOS, iNOS, eNOS and PDE5 were significantly decreased in group B compared with those in group A （P 〈 0.01）. However, ICE PSM and the expression of nNOS and iNOS were increased in groups C and D compared with those in group B （P 〈 0.05）. Changes in ST and the expression of eNOS and PDE5 were not significant （P 〉 0.05） in groups C and D compared with those in group B. Conclusion： Oral treatment with icariin （〉 98.6 % purity） for 4 weeks potentially improves erectile function. This effect is correlated with an increase in PSM and the expression of certain NOS in the CC of castrated rats. These results suggest that icariin may have a therapeutic effect on erectile dysfunction.     

糖尿病性勃起功能障碍大鼠阴茎组织内Cx43和eNOS表达的研究

目的:探讨糖尿病(DM)性勃起功能障碍(DMED)与阴茎组织中连接蛋白(Cx43)和eNOS水平变化的相关性。方法:25只SD大鼠腹腔内注射四氧嘧啶建立DM模型,8周后注射阿朴吗啡观察大鼠阴茎勃起情况,筛选出DMED大鼠,应用RT-PCR和免疫组化法分别检测阴茎组织中Cx43和eNOSmRNA表达水平。结果:DMED大鼠阴茎海绵体组织eNOSmRNA表达(0.155±0.157)与DM组(0.508±0.242)比较,显著降低(P<0.01),而Cx43mRNA则显著升高(0.993±0.157vs0.545±0.138,P<0.01),二者存在负相关性(r=-0.987)。免疫组化检测也显示DMED组大鼠阴茎组织平滑肌细胞中eNOS蛋白表达减少,而Cx43蛋白水平增多。结论:阴茎组织内eNOS水平下降可能是DM患者发生ED的主要机制之一;同时,Cx43的表达呈代偿性增加,其功能有待进一步探讨。     

Advanced glycation end products in human penis: elevation in diabetic tissue, site of deposition, and possible effect through inos or enos

Objectives

We hypothesized that advanced glycation end product (AGE) formation contributes to erectile dysfunction (ED) by quenching nitric oxide. Our first goal was to identify the specific AGE pentosidine in the diabetic human penis. Because AGE-mediated effects may involve inducible nitric oxide synthase (iNOS), we performed immunohistochemical and Western blot analysis of diabetic and nondiabetic human penile tissue for iNOS. Finally, because AGEs may act intracellularly to affect proteins, we set out to identify endothelial NOS (eNOS) in the human penis as an initial step in examining a possible intracellular interaction between eNOS and AGEs.

Methods

We performed high-performance liquid chromatographic analysis of diabetic human penile corpus cavernosum and serum for pentosidine and performed immunohistochemical, electron microscopic (EM), and Western blot analysis of the diabetic and nondiabetic penile corpus cavernosum and tunica for pyrraline, iNOS, and eNOS (and neural NOS [nNOS]for comparative purposes) via standard methods.

Results

We found a significant elevation of pentosidine in the penile tissue but not the serum of diabetic patients (average age 55.6 ± 2.3 years) compared with that of nondiabetic patients (average age 61.8 ± 3.6 years). Pentosidine was 117.06 ± 9.19 pmol/mg collagen in the diabetic tunica versus 77.58 ± 5.5 pmol/mg collagen in the nondiabetic tunica (P < 0.01) and 74.58 ± 8.49 pmol/mg collagen in the diabetic corpus cavernosum versus 46.59 ± 2.53 pmol/mg collagen in the nondiabetic corpus cavernosum (P < 0.01), suggesting a tissue-specific effect of the AGEs. We localized the site of deposition of the specific AGE pyrraline to the human penile tunica and the penile corpus cavernosum collagen. Immunohistochemical and EM analysis localized eNOS and iNOS to the cavernosal endothelium and smooth muscle. Western blot analysis in 6 patients revealed the following: iNOS, but no eNOS, in penile tissue from 1 insulin-dependent diabetic man; eNOS only in 1 man after radical prostatectomy; both eNOS and iNOS in 2 men with Peyronie's disease, as well as in 2 other men with impotence and hypertension. Finally, the specific iNOS inhibitor PNU-19451A significantly augmented relaxation of precontracted human cavernosal tissue, from 64.7% ± 5.58 to 80.03% ± 4.55 at 10 μM acetylcholine and 65.06% ± 2.84 to 86.16% ± 3.96 at 0.1 mM acetylcholine (n = 4, P < 0.002 and P < 0.02, respectively).

Conclusions

AGEs are elevated in diabetic human penile tissue, but not in serum, and are localized to the collagen of the penile tunica and corpus cavernosum. We identified eNOS and iNOS in the human penile cavernosal smooth muscle and endothelium. The augmentation of cavernosal relaxation with a specific iNOS inhibitor, combined with the identification of iNOS protein, but not eNOS, in a patient with severe diabetes and ED, allows for speculation of a pathophysiologic mechanism for AGE-mediated ED via upregulation of iNOS and downregulation of eNOS. These data provide further insight into the mechanisms of advanced glycation end product-mediated ED and provide a foundation for further study. UROLOGY 50: 1016-1026, 1997.     

Therapeutic effect of combination of alagebrium (ALT-711) and sildenafil on erectile function in diabetic rats

Recently, the relationship between advanced glycation end products (AGEs) and erectile dysfunction (ED) has been reported. The present study aimed to investigate whether a combination of an AGE cross-link breaker (alagebrium/ALT-711) and sildenafil could enhance the erectile capacity in streptozotocin (STZ) diabetic rats. Additionally, we assessed the effect of that treatment option on some molecules that have been suggested to have crucial roles in AGE-related ED pathways. Four groups of animals were utilized: (1) age-matched control rats, (2) STZ-induced diabetic rats (40 mg kg(-1) i.p.), (3) STZ rats+sildenafil (5 mg kg(-1) p.o.), (4) STZ rats treated with a combination of sildenafil (5 mg kg(-1) p.o)+alagebrium/ALT-711 (10 mg kg(-1) p.o.) for the final 1 month of the 2 months of diabetes period. At 2 months after i.p. injection of STZ, all animals underwent cavernosal nerve stimulation (CNS) to assess erectile function. Penile tissue AGEs, MDA (malondialdehyde), cyclic guanosine monophosphate (cGMP) (ELISA), endothelial nitric oxide (NO) synthase (eNOS), inducible NO synthase (iNOS) (western blot), nuclear factor (NF)-κB, mitogen-activated protein (MAP) kinase (immunohistochemistry) and apoptosis (terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling) analyses were performed in all groups of rats. STZ diabetic rats had a significant decrease in erectile function as determined by the peak intracavernosal pressure (ICP) and total ICP (area under the erectile curve) after CNS when compared with control rats (P<0.05). The increase in both ICP and area under the erectile curve of STZ diabetic rats treated with a combination of sildenafil+alagebrium/ALT-711 as well as in STZ diabetic rats treated with sildenafil alone was significantly greater than STZ diabetic rats. Additionally, combination treatment decreased AGE, MDA, iNOS, NF-κB, MAP kinase and apoptosis levels, whereas it preserved cGMP contents in diabetic penile tissue. Decreased AGE, MDA, iNOS, NF-κB, MAP kinase and increased cGMP levels at the combination (sildenafil+alagebrium/ALT-711) therapy group increased both the peak ICP and total ICP to CNS in the STZ diabetic rats, which was similar to the response observed in control rats. These results may explain the role of AGEs in diabetes-related ED and the effect of an AGE cross-link breaker alagebrium/ALT-711+sildenafil therapy on some critical molecules related to AGE-related ED pathways.     

Effects of hydrogen sulphide donor,sodium hydrosulphide treatment on the erectile dysfunction in L‐NAME‐induced hypertensive rats

Men with hypertension often develop erectile dysfunction (ED). The present study aimed to examine the effects of sodium hydrosulphide (NaHS), a hydrogen (H₂S) donor, treatment on ED in nitric oxide synthase (NOS) inhibitor (L‐NAME)‐induced hypertensive rats. Forty adult Sprague‐Dawley rats were divided into four groups: control, NaHS (0.037 mg kg day^?1)‐treated control, L‐NAME‐induced hypertension (40 mg kg day^?1) and NaHS‐treated L‐NAME‐induced hypertension. The ratio of intracavernosal pressure to mean arterial pressure and isometric tension of corpus cavernosum (CC) were measured. The penile expression of endothelial and neuronal NOS (eNOS and nNOS), inflammation markers [nuclear factor kappa B (NF‐κB) and inhibitor kappa B alpha (IκBα)], H₂S‐producing enzymes[cystathionine β‐synthase (CBS) and cystathionine γ‐lyase (CSE)], the smooth muscle/collagen ratio and H₂S concentrations were determined. The blood pressure was significantly increased in the hypertensive group, but not reversed by NaHS. The erectile response in hypertensive rats was partially prevented by NaHS. The relaxation response to electrical field stimulation was increased in CC from NaHS‐treated hypertensive rats. NaHS treatment restored decreased protein expression of eNOS, nNOS and CSE as well as smooth muscle/collagen ratio and H₂S levels and increased NF‐κB and IκBα protein expression in the penile tissue of hypertensive rats. NaHS promoted the recovery of erectile responses in hypertensive rats by improvement of neuronal function and downregulation of fibrosis and NF‐κB signalling.     

Effects of diabetes on nitric oxide synthase and growth factor genes and protein expression in an animal model.

Erectile dysfunction occurs frequently in humans with diabetes mellitus; the molecular basis of this phenomenon is not known. We investigated the effects of diabetes on penile erection, nitric oxide synthase and growth factors expression in an animal model. Forty male rats were divided into two groups: the experimental group (n = 30) received intraperitoneal injection of Streptozotocin (STZ) dissolved in citrate buffer to induce diabetes; ten age-matched control rats received injection of citrate buffer vehicle only. Before euthanization at eight weeks, erectile function was assessed by electrostimulation of the cavernous nerves. NADPH diaphorase staining was used to identify NOS and immunostaining technique was used to identify nNOS in the penile nerve fibers. RT-PCR was used to identify mRNA expression of nNOS, eNOS, iNOS, ER-beta, ER-alpha, NGF, IGF-I, TGF-beta 1, and AR. Western blot was used to identify nNOS, IGF-I, NGF, and TFG-beta protein expressions. In the diabetic group, there was: (1) a significant decrease in NOS containing nerve fibers in the dorsal and intracavernosal nerves; (2) a significant lower maximal intracavernosal pressure. RT-PCR showed down-regulation of nNOS (large form), iNOS and ER-beta mRNA expression, Immunoblot showed down-regulation of nNOS protein expression and nNOS immunostaining showed less positive staining in the dorsal and intracavernous nerves in the diabetic group. These molecular changes may provide the basis for further studies to explore the association between diabetes and impotence.     

Metformin restores the penile expression of nitric oxide synthase in high-fat-fed obese rats

Obesity is a well-known risk factor for erectile dysfunction, which is associated with reduced penile nitric oxide synthase (NOS) expression. Recently it was reported that metformin activates AMP-activated protein kinase (AMPK), which increases the expression of neuronal (n) NOS and endothelial (e) NOS. Thus, to evaluate whether metformin restores NOS expression in penile tissue, we measured penile expression of nNOS and eNOS after 4 weeks of metformin treatment (300 mg/kg/d) in 5-month-old high-fat-fed obese (HFO) rats. HFO rats have increased fat accumulation in visceral areas and marked suppression of nNOS and eNOS expression in penile tissue. However, metformin treatment decreased visceral fat deposition and restored nNOS and eNOS expression in penile tissue. The levels of AMPK and phosphorylated AMPK were also decreased in HFO rats but were subsequently elevated by metformin treatment. These results suggest that expression of NOS was suppressed by the high-fat diet but restored by metformin treatment. The effect of metformin on the expression of NOS may be associated with its activation of AMPK.