去势大鼠阴茎海绵体胱硫醚γ裂解酶与硫化氢的表达

目的观察去势大鼠阴茎海绵体胱硫醚γ裂解酶（Cystathionine-γ-lyase,CSE）/硫化氢（Hydrogen sulphide,H2S）的变化,进一步探讨勃起功能障碍的发病机制。方法雄性SD大鼠72只分4组：对照组、假手术组,去势组和去势炔丙基甘氨酸（PAG,CSE阻断剂）组,检测基础条件下和阿扑吗啡（Apomorphine,APO）刺激后的海绵体内压（Intracavernous pressure,ICP）及勃起率;激光共聚焦显微镜检测CSE在大鼠勃起不同时期阴茎海绵体组织中的表达,敏感硫电极测定H2S在勃起不同时期的含量。结果与假手术组比较,去势组和去势PAG组ICP与勃起率下降（P〈0.01）;且去势PAG组较去势组ICP明显下降（P〈0.01）;阿朴吗啡刺激后,与假手术组比较,勃起前去势组和去势PAG组CSE蛋白表达降低（P〈0.01）,勃起中去势各组较假手术组CSE蛋白表达降低（P〈0.01）,且去势PAG组较去势组CSE蛋白表达明显降低（P〈0.01）;勃起后各组间CSE蛋白表达变化无差异。勃起前和勃起中去势各组较假手术组H2S含量下降（P〈0.05）,且勃起中去势PAG组较去势组H2S含量明显下降（P〈0.01）：勃起后去势组和去势PAG组较假手术组H2S含量下降（P〈0.01）。结论去势大鼠勃起功能障碍与CSE和H2S表达下降有关。     

去势大鼠阴茎海绵体内CSE/H2S与HO-2/CO的交叉调控作用

摘要：目的 观察去势大鼠阴茎海绵体胱硫醚γ裂解酶（cystathionine-γ-lyase,CSE）/硫化氢（Hydrogen sulphide,H2S）与血红素加氧酶2（heme oxygenase,HO-2）/一氧化碳（carbon monoxide,CO）的交叉调控作用,初步探讨勃起功能障碍的发病机制。方法 雄性SD大鼠制备去势大鼠模型,分别施加锌原卟啉（ZnPP,HO阻断剂）和炔丙基甘氨酸（PAG,CSE阻断剂）干预,检测基础条件下和阿扑吗啡（Apomorphine,APO）刺激后的海绵体内压（Intracavernous pressure,ICP）和勃起率;通过Western-blot、敏感硫电极和分光光度计分别检测大鼠勃起不同时期ZnPP（HO阻断剂)对CSE/H2S系统的影响,PAG（CSE阻断剂）对HO-2/CO系统的影响。结果 基础条件和APO刺激后去势组、去势PAG组和ZnPP组与假手术组比较,ICP和勃起率下降（P<0.01）,且去势PAG组和ZnPP组较去势组ICP明显下降（P<0.01）;阿朴吗啡刺激勃起前和勃起中,去势组和去势Znpp（HO阻断剂）组较假手术组CSE蛋白表达和H2S含量下降（P<0.05）,且勃起中去势Znpp组较去势组CSE蛋白表达和H2S含量明显降低（P<0.05）;勃起后去势组和去势Znpp（HO阻断剂）组较假手术组H2S含量下降（P<0.01）。勃起前和勃起中去势组和去势PAG（CSE阻断剂）组较假手术组HO-2蛋白表达和CO含量下降（P<0.05）,且勃起中去势PAG组较去势组HO-2蛋白表达和CO含量明显降低（P<0.05）;勃起后去势组和去势PAG（CSE阻断剂）组较假手术组CO含量下降（P<0.01）。勃起前、勃起中和勃起后,CSE和HO-2蛋白表达分别呈正相关（r=0.732,0.850和0.727）,H2S和CO水平分别呈正相关（r=0.765,0.912和0.929）。结论 CSE/H2S与HO-2/CO交叉调控作用与去势大鼠勃起功能障碍有关。     

缺血性阴茎异常勃起的诊断及阶梯治疗

阴茎异常勃起是泌尿男科一种罕见的疾病,需要急诊干预治疗。临床常分为缺血性,非缺血性二种类型,其中缺血性阴茎异常勃起占绝大多数。缺血性阴茎异常勃起会导致海绵体组织纤维化和勃起功能障碍,随着缺血时间的延长,海绵体会永久损伤。不同的阴茎持续勃起时间,不同的治疗方式影响患者的预后,会造成不同程度的勃起功能障碍。遂此病需尽早明确所属类型。通过询问患者病史,细致查体,结合实验室检查中阴茎海绵体血气分析及彩色多普勒检查,明确患者状态。并采取冷敷、镇静,海绵体抽吸灌注或海绵体注射拟交感类药物,外科分流手术等阶梯治疗的方式,最大限度保护患者勃起功能。本文对缺血性阴茎异常勃起的诊断及阶梯治疗进行综述。     

被中断化合物等的再评价提高制药效率的有利手段 药物再开发

作为提高新药开发效率的手段,对现有药物的再开发备受人们关注。采用现有药物再开发（drug re—profiling,DRP）的方法开发出了多发性骨髓肿瘤和勃起功能障碍（erectile dysfimction）等的治疗新药。美国辉瑞公司已经成立了DRP专门机构,而日本企业却还在观望。     

一氧化氮合酶与脓毒症的研究进展

脓毒症在外科临床工作中较常见,治疗相当困难;本文主要概述了一氧化氮合酶的基因定位、结构特点以及一氧化氮合酶与脓毒症的关系,进一步说明由一氧化氮合酶介导的一氧化氮生成与脓毒症关系密切,而选择性一氧化氮合酶抑制剂在脓毒症各阶段恰当的应用可能是有效治疗脓毒症、降低病死率的一个重要途径,也将成为今后研究的热点。     

一氧化氮合酶与脓毒症的研究进展

脓毒症在外科临床工作中较常见,治疗相当困难;本文主要概述了一氧化氮合酶的基因定位、结构特点以及一氧化氮合酶与脓毒症的关系,进一步说明由一氧化氮合酶介导的一氧化氮生成与脓毒症关系密切,而选择性一氧化氮合酶抑制剂在脓毒症各阶段恰当的应用可能是有效治疗脓毒症、降低病死率的一个重要途径,也将成为今后研究的热点。     

大鼠回肠肌间神经丛一氧化氮合酶和乙酰胆碱酯酶双重显示法研究

本文用一氧化氮合酶和乙酰胆碱酯酶双重显示法,对大鼠回肠肌间神经丛进行了组织化学观察,结果发现三种不同染色的神经元：（１）乙酰胆碱酯酶阳性神经元（占８２％）;（２）一氧化氮合酶阳性神经元（占１６％）;（３）一氧化氮合酶和乙酰胆碱酯酶阳性神经元（占２％）。以上结果提示,一氧化氮可以与乙酰胆碱共存于大鼠回肠肌间神经丛的少数神经元内。本文还对肠肌间神经丛内神经元的类型和一氧化氮的作用进行了讨论。     

马来酸麦角新碱联合米索前列醇对剖宫产产后出血患者凝血功能、血流动力学及血清NO、NOS水平的影响

摘要 目的：探讨马来酸麦角新碱联合米索前列醇对剖宫产产后出血患者凝血功能、血流动力学及血清一氧化氮（NO）、一氧化氮合酶（NOS）水平的影响。方法：选取2017年6月~2019年10月期间我院收治的剖宫产产后出血患者98例,根据信封抽签法分为对照组（n=49）和研究组（n=49）,对照组患者给予米索前列醇,研究组在对照组基础上联合马来酸麦角新碱治疗,比较两组患者凝血功能、血流动力学、临床指标、血清NO、NOS水平及不良反应。结果：研究组治疗后的产后恶露持续时间、住院时间短于对照组,产后2 h内出血、产后2～24 h出血少于对照组（P<0.05）。两组不良反应发生率比较无差异（P>0.05）。两组治疗后凝血酶原时间（PT）、凝血活酶时间（APTT）,纤维蛋白原（FIB）、D-二聚体（D-D）水平均下降,且研究组低于对照组（P<0.05）。两组治疗后收缩压（SBP）、舒张压（DBP）均下降,但研究组高于对照组（P<0.05）,两组治疗后心率（HR）升高,但研究组低于对照组（P<0.05）。两组治疗后血清NO、NOS水平均下降,且研究组低于对照组（P<0.05）。结论：剖宫产产后出血患者给予马来酸麦角新碱联合米索前列醇治疗,可有效维持血流动力学平稳,降低血清NO、NOS水平,改善患者凝血功能及临床指标。     

二甲基精氨酸二甲基氨基水解酶与内皮功能异常

非对称二甲基精氨酸（asymmetric dimethylarginine,ADMA）是一氧化氮合酶（NOS）的内源性竞争性抑制物,竞争性抑制NOS使NO含量减少,引起一系列血管异常效应。二甲基精氨酸Z-甲基氨基水解酶（dimethylarginine dimethylaminohydrolase,DDAH）是主导ADMA代谢的催化酶。DDAH减少或活性降低会使ADMA在体内积累,导致内皮功能异常。本文介绍了DDAH引起内皮功能异常的机制,并对有关影响DDAH活性或表达的药物研究作一概述。     

脑血管的一氧化氮合酶分布的组织化学研究

本研究用ＮＡＤＰＨ－ｄ组织化学方法观察了脑表面及脑实质（皮质、纹状体）血管壁的ＮＯＳ阳性细胞。结果发现,在正常脑大于５０μｍ的血管壁上可见有内皮型一氧化氮合酶（ｅＮＯＳ）阳性内皮细胞,并有神经元型一氧化氮合酶（ｎＮＯＳ）阳性细胞紧贴于管壁或发出突起布于管壁。在神经毒损伤的纹状体中,可见有诱导型一氧化氮阳性胶质细胞,终足附于脑血管壁上,而周围未受损伤的脑实质中未见有诱导型一氧化氮合酶阳性的胶质细胞     

Antagonism of Rho-kinase stimulates rat penile erection via a nitric oxide-independent pathway

Relaxation of the smooth muscle cells in the cavernosal arterioles and sinuses results in increased blood flow into the penis, raising corpus cavernosum pressure to culminate in penile erection. Nitric oxide, released from non-adrenergic/non-cholinergic nerves, is considered the principle stimulator of cavernosal smooth muscle relaxation, however, the inhibition of vasoconstrictors (that is, norepinephrine and endothelin-1, refs. 5-9) cannot be ignored as a potential regulator of penile erection. The calcium-sensitizing rho-A/Rho-kinase pathway may play a synergistic role in cavernosal vasoconstriction to maintain penile flaccidity. Rho-kinase is known to inhibit myosin light chain phosphatase, and to directly phosphorylate myosin light-chain (in solution), altogether resulting in a net increase in activated myosin and the promotion of cellular contraction. Although Rho-kinase protein and mRNA have been detected in cavernosal tissue, the role of Rho-kinase in the regulation of cavernosal tone is unknown. Using pharmacologic antagonism (Y-27632, ref. 13, 18), we examined the role of Rho-kinase in cavernosal tone, based on the hypothesis that antagonism of Rho-kinase results in increased corpus cavernosum pressure, initiating the erectile response independently of nitric oxide. Our finding, that Rho-kinase antagonism stimulates rat penile erection independently of nitric oxide, introduces a potential alternate avenue for the treatment of erectile dysfunction.     

Relaxation of canine corporal smooth muscle relaxation by ginsenoside saponin Rg3 is independent from eNOS activation

It has been reported that nitric oxide (NO) is involved in the relaxation mechanism of ginsenoside saponin in various smooth muscle in experimental animals. Although ginsenoside Rg(3) showed both endothelium-dependent and -independent component relaxation in vascular smooth muscle, the action mechanism of the relaxation of corporal muscle is not clear. We, thus, investigated the relaxation mechanism of ginsenoside Rg(3) using isolated canine corpus cavernosum. Ginsenoside Rg(3) concentration-dependently relaxed the canine corpus cavernosum that had been contracted by phenylephrine (PE), in which IC(50) was 1.68 x 10(-5) g/ml. Ginsenoside Rg(3) significantly (P < 0.05) potentiated acetylcholine (ACh)-induced relaxation in endothelium intact corpus cavernosum. Methylene blue (MB) but not N(omega)-nitro-L-arginine methylester (L-NAME) or ODQ (1H-[1,2,4]oxadiazol-[4,3-]quinoxsalin-1-one) modified the dose-response curve of ginsenoside Rg(3). Ginsenoside Rg(3) also significantly potentiated relaxation response to UV light in the presence of streptozotocin (STZ), which was almost completely (P < 0.01) blocked by ODQ. Ginsenoside Rg(3) concentration-dependently inhibited corporal phosphodiesterases (PDE), which resulted in increase of cyclic adenosine monophosphate (cAMP) as well as cyclic guanosine monophosphate (cGMP) contents in corporal smooth muscles. MB inhibited the accumulation of cGMP but not cAMP by ginsenoside Rg(3). These results indicate that mechanism responsible for the relaxation by ginsenoside Rg(3) is not by stimulating endothelial nitric oxide synthase (eNOS) of the canine corporal smooth muscle but by increasing cyclic nucleotide levels through PDE inhibition.     

Cardiovascular risk factors, erection disorders and endothelium dysfunction

Upon sexual stimulation, penile erection, occurring in response to the activation of pro-erectile autonomic pathways, is greatly dependent on adequate inflow of blood to the erectile tissue and requires coordinated arterial endothelium-dependent vasodilatation and sinusoidal endothelium-dependent corporal smooth muscle relaxation. Nitric oxide (NO) is the principal peripheral pro-erectile neurotransmitter which is released by both non-adrenergic, non-cholinergic neurons and the sinusoidal endothelium to relax corporal smooth muscle through the cGMP pathway. Any factors modifying the basal corporal tone, the arterial inflow of blood to the corpora, the synthesis/release of neurogenic or endothelial NO are prime suspects for being involved in the pathophysiology of erectile dysfunction (ED). In fact, conditions associated with altered endothelial function, such as ageing, hypertension, hypercholesterolemia and diabetes, may, by changing the balance between contractant and relaxant factors, cause circulatory and structural changes in penile tissues, resulting in arterial insufficiency and defect in smooth muscle relaxation and thus, ED. There is increasing evidence to suggest that ED is predominantly a vascular disease and may even be a marker for occult cardiovascular disease. Recent results illustrating the importance of endothelial dysfunction in the pathophysiology of different forms of experimental ED are discussed. These pathways may represent new potential treatment targets.     

Vasoactive intestinal polypeptide (VIP) as a putative neurotransmitter in penile erection

The localization of vasoactive intestinal polypeptide (VIP) in the male genitourinary tract was investigated in the rabbit and man by means of radioimmunoassay and immunohistochemistry. In addition, the in vitro effect of VIP upon penile smooth muscle from man, the Vervet monkey, and the rabbit was investigated. Significant concentrations of VIP immunoreactivity were found in the human penis and all the organs of the rabbit genital tract apart from the testis. VIP immunoreactive nerve fibres were observed in the erectile tissue of the human and rabbit penis and in the other organs of the rabbit genital tract apart from the testis. Fibres were most abundant in association with blood vessels, in smooth muscle tissue, and subepithelially in glandular tissue. Strips of smooth muscle taken from the corpus cavernosum of Vervet monkey and man showed a dose-dependent relaxation in response to VIP at concentrations of 6 X 10(-8) mol X L-1 and 6 X 10(-7) mol X L-1. The data indicate that VIP may be an inhibitory neurotransmitter involved in the nervous control of penile erection.     

Nitric oxide and cyclic GMP formation upon electrical field stimulation cause relaxation of corpus cavernosum smooth muscle

In the presence of functional adrenergic and cholinergic blockade, electrical field stimulation relaxes corpus cavernosum smooth muscle by unknown mechanisms. We report here that electrical field stimulation of isolated strips of rabbit corpus cavernosum promotes the endogenous formation and release of nitric oxide (NO), nitrite, and cyclic GMP. Corporal smooth muscle relaxation in response to electrical field stimulation, in the presence of guanethidine and atropine, was abolished by tetrodotoxin and potassium-induced depolarization, and was markedly inhibited by NG-nitro-L-arginine, NG-amino-L-arginine, oxyhemoglobin, and methylene blue, but was unaffected by indomethacin. The inhibitory effects of NG-substituted analogs of L-arginine were nearly completely reversed by addition of excess L-arginine but not D-arginine. Corporal smooth muscle relaxation elicited by electrical field stimulation was accompanied by rapid and marked increases in tissue levels of nitrite and cyclic GMP, and all responses were nearly abolished by NG-nitro-L-arginine. These observations indicate that penile erection may be mediated by NO generated in response to nonadrenergic-noncholinergic neurotransmission.     

Evidence of relaxant effect of omeprazole in rabbit corpus cavernosum in vitro

The present experiments were designed to investigate the effects of omeprazole, a H(+)-K+ ATPase inhibitor, on corporal smooth muscle tone in vitro. All spontaneous contractile activity in the corpus cavernosum was blocked following omeprazole (0.1 mM-1 mM) administration. However atropine (1 microM), Nw-nitro L-arginine methyl ester (L-NAME, 30 microM) or indomethacin (10 microM) did not affect the spontaneous contraction. Omeprazole (10 microM-1 mM) concentration-dependently induced relaxation in corporal smooth muscle precontracted with 10 microM phenylephrine or 80 mM KCl. Pretreatment of corporal tissue with L-NAME (30 microM), indomethacin (10 microM), ammonium chloride (7.5 mM), sodium acetate (7.5 mM), tetraethyl ammonium chloride (0.5 mM) or glibenclamide (1 microM) had no effect on the omeprazole induced relaxant responses. Nimodipine, an L-type Ca++ channel blocker, relaxed corporal strips precontracted with 80 mM KCl. Collectively, these results indicate that the inhibition of spontaneous contraction and the relaxation of precontracted corporal smooth muscle by omeprazole is probably mediated by the blockade of calcium channels. Further work is needed to determine the cellular mechanism(s) of action by which omeprazole acts on corpus cavernosum smooth muscle.     

Mesenchymal stem cell‐derived exosomes ameliorate erection by reducing oxidative stress damage of corpus cavernosum in a rat model of artery injury

Erectile dysfunction (ED) is a common ageing male's disease, and vascular ED accounts for the largest proportion of all types of ED. One of the mechanisms of vascular ED in the clinic is arterial insufficiency, which mainly caused by atherosclerosis, trauma and surgical. Moreover, oxidative stress damage after tissue ischemia usually aggravated the progress of ED. As a new way of acellular therapy, mesenchymal stem cell‐derived exosomes (MSC‐Exos) have great potential in ED treatment. In the current study, we have explored the mechanism of MSC‐Exos therapy in a rat model of internal iliac artery injury‐induced ED. Compared with intracavernous (IC) injection of phosphate‐buffered saline after artery injury, of note, we observed that both mesenchymal stem cells (MSCs) and MSC‐Exos through IC injection could improve the erectile function to varying degrees. More specifically, IC injection MSC‐Exos could promote cavernous sinus endothelial formation, reduce the organization oxidative stress damage, and improve the nitric oxide synthase and smooth muscle content in the corpus cavernosum. With similar potency compared with the stem cell therapy and other unique advantages, IC injection of MSC‐ Exos could be an effective treatment to ameliorate erectile function in a rat model of arterial injury.     

d-Galactose induces the senescence and phenotype switch of corpus cavernosum smooth muscle cells

Studies regarding age-related erectile dysfunction (ED) based on naturally aging models are limited by their high costs, especially for the acquisition of primary cells from the corpus cavernosum. Herein, d -galactose ( d -gal) was employed to accelerate cell senescence, and the underlying mechanism was explored. As predominant functional cells involved in the erectile response, corpus cavernosum smooth muscle cells (CCSMCs) were isolated from 2-month-old rats. Following this, d -gal was introduced to induce cell senescence, which was verified via β-galactosidase staining. The effects of d -gal on CCSMCs were evaluated by terminal deoxynucleoitidyl transferase dUTP nick-end labeling (TUNEL), immunofluorescence staining, flow cytometry, western blot, and quantitative real-time polymerase chain reaction (qRT-PCR). Furthermore, RNA interference (RNAi) was carried out for rescue experiments. Subsequently, the influence of senescence on the corpus cavernosum was determined via scanning electron microscopy, qRT-PCR, immunohistochemistry, TUNEL, and Masson stainings. The results revealed that the accelerated senescence of CCSMCs was promoted by d -gal. Simultaneously, smooth muscle alpha-actin (alpha-SMA) expression was inhibited, while that of osteopontin (OPN) and Krüppel-like factor 4 (KLF4), as well as fibrotic and apoptotic levels, were elevated. After knocking down KLF4 expression in d -gal-induced CCSMCs by RNAi, the expression level of cellular alpha-SMA increased. Contrastingly, the OPN expression, apoptotic and fibrotic levels declined. In addition, cellular senescence acquired partial remission. Accordingly, in the aged corpus cavernosum, the fibrotic and apoptotic rates were increased, followed by downregulation in the expression of alpha-SMA and the concurrent upregulation in the expression of OPN and KLF4. Overall, our results signaled that d -gal-induced accelerated senescence of CCSMCs could trigger fibrosis, apoptosis and phenotypic switch to the synthetic state, potentially attributed to the upregulation of KLF4 expression, which may be a multipotential therapeutic target of age-related ED.     

Ca2+-activated Cl- channels in corpus cavernosum smooth muscle: a novel mechanism for control of penile erection.

Little is known of the excitatory mechanisms that contribute to the tonic contraction of the corpus cavernosum smooth muscle in the flaccid state. We used patch-clamp electrophysiology to investigate a previously unidentified inward current in freshly isolated rat and human corporal myocytes. Phenylephrine (PE) contracted cells and activated whole cell currents. Outward current was identified as large-conductance Ca(2+)-activated K(+) current. The inward current elicited by PE was dependent on the Cl(-) gradient and was inhibited by niflumic acid, indicative of a Ca(2+)-activated Cl(-) (Cl(Ca)) current. Furthermore, spontaneous transient outward and inward currents (STOCs and STICs, respectively) were identified in both rat and human corporal myocytes and derived from large-conductance Ca(2+)-activated K(+) and Cl(Ca) channel activity. STICs and STOCs were inhibited by PE and A-23187, and combined 8-bromoadenosine cAMP and 8-bromoadenosine cGMP decreased their frequency. When studied in vivo, chloride channel blockers transiently increased intracavernosal pressure and prolonged nerve-evoked erections. This report reveals for the first time Cl(Ca) current in rat and human corpus cavernosum smooth muscle cells and demonstrates its key functional role in the regulation of penile erection.     

Downregulation of cGMP-dependent protein kinase-1 activity in the corpus cavernosum smooth muscle of diabetic rabbits

Increased guanosine 3',5'-cyclic monophosphate (cGMP), induced by nitric oxide release, is crucial for corpus cavernosum smooth muscle (CCSM) relaxation within the penis. This CCSM relaxation (necessary for penile erection) is impaired in men with erectile dysfunction (ED), especially those men with diabetes. One of the effector proteins for cGMP is cGMP-dependent protein kinase-1 (PKG-1). PKG-1 knockout mice exhibit detrusor overactivity (Am J Physiol Regul Integr Comp Physiol 279: R1112-R1120, 2000) and, more relevant to this study, ED (Proc Natl Acad Sci USA 97: 2349-2354, 2000), suggesting an in vivo role for PKG-1 in urogenital smooth muscle relaxation. In the current study, using normal rabbit CCSM, Western blot analysis revealed high expression of PKG-1 at levels almost equivalent to aorta (previously shown to have high PKG-1 expression) and that the two known alternatively spliced isoforms of PKG-1 (alpha and beta) are expressed in nearly equal amounts in the CCSM. However, in response to alloxan-induced diabetes, there was a decrease in expression of both PKG-1 isoforms at the mRNA and protein levels as determined by real-time RT-PCR and Western blotting, respectively, but with the PKG-1alpha isoform expression decreased to a greater extent. Moreover, diabetes was associated with significantly decreased PKG-1 activity of CCSM in vitro, correlating with decreased CCSM relaxation. Immunofluorescence microscopy revealed a diabetes-associated decrease in PKG-1 in the CCSM cells. In conclusion, our results demonstrate for the first time a significant downregulation of PKG-1 expression associated with decreased PKG-1 activity in the CCSM in response to diabetes. Furthermore, these results suggest a mechanistic basis for the decreased efficacy of phosphodiesterase V inhibitors in treating diabetic patients with ED.