一氧化氮和一氧化氮合酶在大鼠局灶性脑缺血中的表达特点

目的建立脑缺血SD大鼠模型,探讨一氧化氮(NO)和一氧化氮合酶(NOS)在脑缺血模型中的表达特点。方法应用线栓法制作大脑中动脉阻塞(MCAO)局灶性脑缺血模型,根据缺血不同时间分为8组,设立假手术组和正常对照组,每组各有6只大鼠。应用硝酸还原酶法测定脑组织NO的含量,流式细胞术(FCM)定量检测硝基酪氨酸(NT)表达,化学比色法测定脑组织NOS的活性,免疫组织化学方法定位检测eNOS、nNOS和iNOS表达位置,逆转录反应系统(RT-PCR)半定量分析eNOS、nNOS和iNOS的 mRNA在脑缺血区域的表达。结果神经功能缺失评分发现缺血时间越长,神经功能缺失越明显;脑组织中NO含量与缺血时间正相关;缺血1h后NT阳性细胞百分比开始明显升高(9.50%);缺血0.5h时NOS的活性开始升高,缺血3d达到高峰[0.94nmol/(g.min)];免疫组织化学提示eNOS在神经细胞和血管内皮细胞胞浆均有表达,nNOS和iNOS抗体主要在神经细胞胞浆中表达;RT-PCR半定量分析在缺血早期(0.5h~6h),随着缺血时间延长,eNOS和nNOS表达增加,iNOS未见表达或低表达;缺血中晚期(6h~5d),iNOS高表达,并与缺血时间呈正相关,eNOS和nNOS表达明显减少。结论脑缺血时间延长,NOS的活性升高,NO在体内特异性代谢产物NT量增加,神经功能缺失越明显;NOS在缺血早期以eNOS和nNOS为主,在缺血晚期以iNOS为主。     

内皮型一氧化氮合酶与缺血性脑卒中

缺血性脑血管病一向在国内外具有很高的致残率和病死率,随着对其研究的深入,其治疗方向逐渐分为改善缺血区域供血及保护缺血神经细胞两方面,其中血管内皮细胞产生的一氧化氮(NO)是一项重要的内在脑血流调节剂及保护剂.本文综述了内皮型一氧化氮合酶(eNOS)对于缺血性脑卒中保护及治疗意义.     

慢性间断性缺氧诱导一氧化氮合酶表达的研究

目的:建立大鼠缺氧模型,检测神经元型一氧化氮合酶(nNOS)及诱导型一氧化氮合酶(iNOS)的表达情况。方法:1.建立缺氧模型:将SD大鼠置于常压低氧舱中,充入氮气调节氧浓度至所需氧浓度。2.动物分组:(1)急性缺氧组:在低氧舱中缺氧1.5小时。(2)慢性间断性缺氧组:每日在低氧舱中6小时。每周缺氧6天,共缺氧28天。3.采用免疫组化法检测nNOS和iNOS的表达。4.统计学分析检验。结果:急性缺氧后,iNOS、nNOS阳性神经元增加;慢性缺氧后,iNOS、nNOS阳性神经元仍持续增多,慢性缺氧时增加iNOS-IR细胞远远多于nNOS-IR细胞。结论:我们的研究表明缺氧可引起iNOS、nNOS阳性神经元增加,NOS亚型表达时间的不同说明其脑损伤具有阶段性。     

一氧化氮合酶及血红素氧化酶在颈动脉粥样硬化斑块中的表达

目的 探讨内皮型一氧化氮合酶(eNOS)、诱导型一氧化氮合酶(iNOS)和血红素氧化酶-1(HO-1)在动脉粥样硬化过程中表达的变化及相互关系.方法 选取10例经颈动脉内膜剥脱术取下的粥样硬化斑块标本,以10例正常颈内动脉为对照,用免疫组化方法检测eNOS、iNOS及HO-1在两组颈动脉内的表达,应用统计学的方法对结果进行分析.结果 (1)eNOS在粥样硬化斑块中表达较正常颈动脉表达明显减弱(P<0.001);(2)iNOS在正常颈动脉中没有表达,但在粥样硬化斑块中的平滑肌细胞以及泡沫细胞中有明显表达;(3)HO-1仅在正常颈动脉内皮细胞中表达,而在粥样硬化斑块中,HO-1不仅在内皮细胞中表达,而且在平滑肌细胞以及泡沫细胞中也有较高表达,与正常组相比其表达明显增高(P<0.001);(4)统计分析显示:eNOS阳性表达与iNOS阳性表达呈负相关(r=-0.680,P<0.001),iNOS阳性表达与HO-1阳性表达呈正相关(r=0.898,P<0.001),eNOS阳性表达与HO-1阳性表达呈负相关(r=-0.713,P<0.001).结论 随着动脉粥样硬化的形成,eNOS产生减少而iNOS和HO-1产生增多,一氧化氮合酶及血红素氧化酶在动脉粥样硬化中显示互补及代偿性调节的作用.     

内皮型一氧化氮合酶基因第4内含子基因多态与福建汉族脑血管病的相关性

目的 研究福建汉族人内皮型一氧化氮合酶(endothelial nitric oxide synthase,eNOS)基因第4内含子27 bp可变串联重复序列(variable number of tandem repeat,VNTR)与脑血管病关系.方法 依据缺血性卒中TOAST(Trial of ORG 10172 in Acute Stroke Treatment)分型收集132例大动脉粥样硬化性卒中(large artery atherosclerosis,LAA),63例腔隙性脑梗死(lacunar infarction,LI)及62例高血压性脑出血患者为病例组,同时选择年龄、性别匹配的237名健康者为对照组,采用聚合酶链反应结合2%琼脂糖电泳技术检测eNOS基因第4号内含子27 bp VNTR的多态性.结果 正常福建汉族人eNOS基因第4内含子除bb,ab,aa基因型外,尚出现罕见的bc基因型,其基因型频率分别为0.789,0.203,0.004,0.004;b、a、c等位基因频率分别为.892,0.106及0.002.LAA及LI患者27 bp VNTR多态与对照组差别无统计学意义,LAA组bc基因型频率(0.030)虽明显高于对照组(0.004),但差异无统计学意义;缺血性卒中伴/不伴高血压病与该基因多态无相关性.高血压性脑出血患者的aa基因型频率为0.048.明显高于bb+ab基因型(OR=11.42,95% CI:1.209-117.892,P<0.05).结论 eNOS第4内含子aa基因型可能是福建汉族人高血压性脑出血的遗传危险因素,但该位点多态性与福建汉族LAA及LI无相关性.     

血管内皮生长因子表达载体的构建及在内皮祖细胞中的表达

背景：血管内皮生长因子(vascular endothelial growth factor, VEGF)具有很强的促进血管生成作用，但构建其真核表达质粒是否可转染血管内皮祖细胞并完整表达还不清楚。 目的：构建VEGF表达载体,并观察其在内皮祖细胞中的表达情况。 设计、时间及地点：观察性试验，于2007-12/2008-03在上海交通大学医学院附属新华医院科研中心实验室完成。 材料：质粒PDC315-VEGF165自备，质粒pIRES2-EGFP由美国国立卫生研究院Stanko Stojilkovic教授惠赠。 方法：运用DNA重组技术构建VEGF表达载体pIRES2-EGFP-VEGF，应用脂质体包裹的方法将载体转染猪外周血血管内皮祖细胞。 主要观察指标：采用荧光显微镜观察VEGF在内皮祖细胞内的表达，反转录-聚合酶链反应法检测VEGFmRNA表达水平， ELISA检测VEGF165蛋白表达情况。 结果：成功构建VEGF真核表达载体pIRES2-EGFP-VEGF。转染重组质粒pIRES2-EGFP-VEGF后,在内皮祖细胞内有VEGF的表达，VEGFmRNA和VEGF165 蛋白表达水平均明显增加。 结论：VEGF 表达载体转染猪外周血血管内皮祖细胞后能有效增加VEGF基因的表达，能够获得较高水平的VEGF蛋白。     

神经元型一氧化氮合酶在血管性痴呆大鼠海马中的表达

目的 探讨神经元型一氧化氮合酶(nNOS)在血管性痴呆(VD)大鼠海马中的表达。方法 将60只大鼠随机分为：对照组、VD12h组、VD1d组、VD3d组、VD7d组。采用反复夹闭双侧颈总动脉方法建立VD大鼠模型，用HE染色观察各组大鼠海马CA1区神经元的数目；应用免疫组化染色和Western印迹方法检测nNOS在大鼠海马中的表达。结果 VD12h组、VD1d组、VD3d组、VD7d组大鼠海马CA1区神经元数均明显下降。nNOS在对照组大鼠海马CA1区中弱表达．在VD12h组表达增强．VD1d组进一步增强，VD3d和7d组表达逐渐减弱。结论 nNOS可能参与缺血早期海马神经元的损害，是VD的发病机制之一。     

内皮型一氧化氮合酶与蛛网膜下腔出血的研究进展

内皮型一氧化氮合酶(e NOS)的产物一氧化氮,具有舒张血管和抑制炎症细胞的浸润等生理作用。蛛网膜下腔出血后,内皮型一氧化氮合酶表达下调、功能紊乱、一氧化氮(NO)消耗加剧,修复e NOS并促进其磷酸化有助于抑制细胞凋亡,促进血管新生,减轻血管痉挛,从而达到防治蛛网膜下腔出血后早期脑损伤和减轻脑血管痉挛的作用。     

内皮型一氧化氮合酶基因+894G/T多态性与偏头痛发病的meta分析

目的分析内皮型一氧化氮合酶(endothelial nitric oxide synthase,eNOS)基因+894G/T多态性与偏头痛发病风险之间的相关性。方法本研究检索时间从建库至2016年10月。纳入评估eNOS多态性(+894G/T)与偏头痛发病风险之间相关性的病例对照研究。初筛得到365篇全文,经筛选后最终5篇纳入meta分析。异质性分析采用I~2检验,文献质量评估采用英国牛津循证医学中心文献严格评价项目。结果与GG+GT基因型相比,TT基因型增加有先兆偏头痛(migraine with aura,MA)的发生风险(OR=1.56,95%CI 1.11~2.20;I~2=0%,P=0.01)。在非高加索人群中,GT+TT基因型可增加偏头痛发病风险(OR=1.53,95%CI 1.08~2.16;I~2=0%,P=0.02)。结论本研究提示内皮型一氧化氮合酶基因+894G/T多态性与偏头痛发病风险之间有一定的关系,并且可能因人群遗传背景的不同与偏头痛亚型有一定关联。     

一氧化氮合酶在实验性动脉瘤中的表达及作用

目的探讨一氧化氮合酶(NOS)在实验性动脉瘤中的表达及其作用。方法在13只家兔上成功复制了动脉瘤,并以正常的兔颈总动脉作为对照。用生物化学方法检测了动脉瘤组织和对照组中总一氧化氮合酶(T-NOS)和诱导型一氧化氮合酶(i-NOS)的活性,并以免疫组化的方法对i-NOS在血管壁中的表达进行了观察定位。结果动脉瘤组织与正常血管相比,T-NOS的活性无显著性差异(P>0.05),但i-NOS显著增加(P<0.01),且i-NOS与T-NOS的比例亦显著升高(P<0.01)。动脉瘤瘤壁肌层结构明显紊乱,可见炎症细胞浸润,细胞外基质明显破坏或消失。动脉瘤组织内i-NOS的表达呈局灶性分布在血管壁,主要集中在炎症细胞浸润的区域。结论提示主要由炎症细胞产生的i-NOS在动脉瘤发生发展的病理过程中可能起重要作用。     

Ethanol increases endothelial nitric oxide production through modulation of nitric oxide synthase expression

Moderate alcohol consumption has been shown to reduce the risk of ischemic heart disease potentially through its effect on specific endothelial-derived compounds. We tested the hypothesis that ethanol increases the expression of endothelial nitric oxide synthase (eNOS) and nitric oxide (NO) production in bovine aortic endothelial cells (BAEC). Primary cultures of BAEC grown to confluence under standard conditions were treated 3-6 h with 0.1% ethanol in the presence of indomethacin. Ethanol induced a significant increase in both basal and stimulated NO production as determined by chemiluminescence method. This effect was accompanied by a rapid increase of eNOS protein and mRNA expression levels. eNOS mRNA increased two-fold within 3 h and gradually declined, but the increased levels of mRNA persisted for >24 h. A similar increase of eNOS expression was observed in human umbilical endothelial cells exposed to ethanol. These results demonstrate that ethanol augments both basal and stimulated NO production and that this effect is associated with increased eNOS protein and mRNA expression levels. The data are consistent with the hypothesis that the reduced incidence of ischemic heart disease associated with alcohol may be related, at least in part, to the modulation of vascular endothelial cell production of NO.     

High glucose-mediated imbalance of nitric oxide synthase and dimethylarginine dimethylaminohydrolase expression in endothelial cells

The mechanisms involved in endothelial dysfunction are multifactorial. A correlation between oxidative stress and derangements of nitric oxide synthase (NOS) pathways in altered endothelial homeostasis has been most studied and demonstrated in different pathophysiological conditions. NOS activities are regulated by endogenous inhibitors such as asymmetric dimethyl-L-arginine (ADMA) that is metabolized by dimethylarginine dimethylaminohydrolase (DDAH). Since recent data demonstrated that some endothelial dysfunction may be related to reduced expression and/or activity of DDAH, the aim of the present research was to investigate the expression of DDAH-2 and NOS isoforms in high glucose-mediated oxidative stress. Endothelial cells were incubated with normal (7 mM) and high concentrations (33 mM) of D-glucose for 5 days; mannose (26 mM) plus D-glucose (7 mM) was used as osmotic control. Data obtained in the present study show that the exposure for 5 days to high glucose increases oxidative stress, reduces DDAH-2 and eNOS expression and increases iNOS expression. These results indicate that DDAH-2 and iNOS/eNOS dysregulation may play a key role in high glucose-mediated oxidative stress, suggesting that selective modulation of DDAH isoforms may result in selective inhibition/activation of NOS isoforms, thereby providing a novel strategy of approach in vascular complications of several pathologies.     

Expression of endothelial nitric oxide synthase in the ischemic penumbra: relationship to expression of neuronal nitric oxide synthase and vascular endothelial growth factor

Expressional patterns of the endothelial and neuronal forms of nitric oxide synthase (NOS) in cerebral ischemia were studied utilizing a permanent middle cerebral artery occlusion (PMCAO) model. Motor performance and infarct volumes were determined in the rats. Immunohistochemical staining for eNOS, nNOS and neurofilament were performed at 1, 2, 3, 5, 7 and 14 days after PMCAO. Vascular endothelial growth factor (VEGF) expression was determined by in-situ hybridization. PMCAO caused a reproducible cortical infarct with motor deficits in the rats. Double immunohistochemical stainings indicated that eNOS and nNOS were induced in ischemic neurons. Most stained neurons were positive for both NOS forms but some reacted with only one NOS antibody. nNOS expression peaked at 24-48 h after PMCAO, stained mainly the cytoplasm of core neurons, and disappeared after the 3rd day. eNOS expression increased until the 7th day, stained mainly the cytoplasm and membrane of penumbral cells and disappeared by the 14th day after PMCAO. VEGF expression was significantly induced in the penumbral zone in a similar distribution to eNOS. The anatomical and temporal pattern of VEGF and eNOS induction in the brain after permanent ischemia suggest that these mediators may play a role in protecting penumbral tissue from additional ischemic damage.     

Increased neuronal nitric oxide synthase expression in brain following portacaval anastomosis

It has previously been suggested that increases of l-arginine uptake into brain following portacaval shunting may result in increased activities of constitutive neuronal nitric oxide synthase (nNOS). In order to further address this issue, nNOS protein and gene expression were studied by Western blot analysis using a monoclonal nNOS antibody and RT-PCR respectively in the brains of rats following portacaval shunting or sham operation. Portacaval shunting resulted in a 2-fold increase (P<0.01) in nNOS protein and a concomitant 2.4-fold increase (P<0.01) in nNOS mRNA. Increased nNOS activity in brain and the resulting increase in nitric oxide production could contribute to the increased cerebral blood flow and to the pathogenesis of hepatic encephalopathy in chronic liver disease.     

Reduction expression of thrombomodulin and endothelial cell nitric oxide synthase in dermatomyositis

Dermatomyositis (DM) is a systemic microvasculitis predominantly involving the capillaries. We investigated the expression of thrombomodulin (TM) and endothelial cell nitric oxide synthase (eNOS) in microvessels of DM patients. Twelve patients with acute or subacute onset of proximal muscle weakness and erythematous rash over their faces and shoulders were included in this study. Serum creatine phosphokinase was elevated in almost all patients. Electromyograph showed a myopathic pattern in all patients. Muscle biopsies were performed in all patients and 10 non‐DM controls and studied with histological, enzyme histochemical and immunohistochemical staining. von Willebrand factor, TM and eNOS antibodies were used as the primary antibodies. Perifascicular degeneration and inflammatory cell infiltration in the perimysium were noted in almost all patients. Non‐special esterase staining was markedly positive in capillary and microvascular endothelium. Marked reduction in TM and eNOS staining was noted in DM patients in perimysium microvessels and perifascicular area capillaries. Vascular lesions in DM were not only limited to capillaries. The low expression of TM and eNOS in microvessels suggests the anticoagulation and vasodilation functions of vascular endothelium is reduced. DM is an inflammatory vascular endothelial disease.     

Change in endothelial nitric oxide synthase in the rat retina following transient ischemia

Patterns of endothelial nitric oxide synthase (eNOS) expression in retinal ischemia were studied utilizing a transient high intraocular pressure (HIOP) model. We investigated neuronal cell damage and changes in eNOS immunoreactive expression in the ischemic retina, and its relationship to the neuroprotection of betaxolol treatment after ischemic injury. Immunohistochemical staining for eNOS was performed at 3, 7, 14 and 28 days after ischemia/reperfusion. In controls, eNOS immunoreactivity was detected in retinal vessels, but was not detected in neurons. After ischemia/reperfusion, the intensity of eNOS immunoreactivity increased in both retinal vessels and the ganglion cell layer (GCL) compared with controls. eNOS-positive neurons were induced first in the inner nuclear layer (INL) 7 days after reperfusion. However, when experiments were carried out on animals that had been treated with betaxolol after ischemia/reperfusion, the intensity of eNOS immunoreactivity decreased compared to the untreated ischemic retinas. These results suggest that an increase in eNOS expression could be associated with the degenerative changes in the ischemic retina, and that betaxolol treatment appears to protect retinal tissue from ischemic damage.     

Agmatine inhibits matrix metalloproteinase-9 via endothelial nitric oxide synthase in cerebral endothelial cells

OBJECTIVES: Matrix metalloproteinases (MMPs) are up-regulated by ischemic injury and degrade the basement membrane of brain vessels to promote cell death and tissue injury. We previously showed that agmatine has a neuroprotective effect on neurons against ischemic injury. In the present study, we investigated the effect of agmatine on the expression of MMPs and nitric oxide (NO) production in cerebral endothelial cells (CECs) after oxygen-glucose deprivation (OGD)-reperfusion injury and its potential association with endothelial nitric oxide synthase (eNOS). METHODS: Primary cultured endothelial cells from murine brain and bEnd.3 cells were subjected to OGD-reperfusion injury. Protein and mRNA levels of both MMP-2 and MMP-9 were determined by immunocytochemical analysis, Western blot and RT-PCR. Protein levels of eNOS were evaluated by Western blot in the CECs. The production of NO was measured using the Griess reagent. RESULTS: Agmatine attenuated the expression of MMP-2 and MMP-9 induced by ischemic injury at the protein and mRNA level, while agmatine increased the expression of eNOS directly. NO production was decreased in CECs after similar insult and was increased by agmatine treatment. In the presence of a nitric oxide synthase (NOS) inhibitor, N(omega)-nitro-L-arginine methyl ester (L-NAME), the expression levels of MMP-2 were decreased, but the expression of MMP-9 was not decreased by agmatine administration. However, NO production was suppressed by a non-specific NOS inhibitor in the agmatine treatment group. CONCLUSION: Our study supports that the down-regulation of MMP-9 by agmatine runs parallel to the up-regulation of eNOS and the maintenance of functional NO release.