The expression and activity of renal nitric oxide synthase and circulating nitric oxide in polycystic kidney disease rats

Nitric oxide (NO) influences tubular fluid and electrolyte transport, and hence possibly also fluid accumulation in renal cysts. The expression and activity of intrarenal constitutive NO synthase (cNOS) [neuronal NOS, nNOS and endothelial NOS, eNOS] and inducible NOS (iNOS) and plasma nitrite/nitrate (PNOx) concentration were assessed in homozygous Han:SPRD polycystic kidney disease (PKD) rats (cy/cy), heterozygous Han:SPRD PKD rats (cy/+), homozygous normal Han:SPRD littermates (+/+) and Sprague Dawley rats (sd). The results showed: 1) nNOS expression was decreased in proximal tubules and thick ascending limbs of the loop of Henle in cy/cy and cy/+ rats compared to +/+ and sd rats (p<0.05). nNOS was weakly expressed in the epithelium of small cysts and unexpressed in epithelium of large cysts. 2) iNOS expression was increased in proximal tubular epithelial cells in cy/+ rats compared to +/+ rats and sd rats (p<0.01). iNOS expression in cyst epithelium was decreased in cy/+ rats (p<0.05) and absent in cy/cy rats. 3) eNOS expression was similar in the endothelium of intrarenal arteries in all groups. 4) The activity of renal cNOS was decreased in cy/cy and cy/+ rats; the activity of iNOS was decreased only in cy/cy rats, with no significant difference among the other three groups. 5) PNOx concentration was higher in cy/cy rats than in the other three groups, and correlated positively with plasma creatinine and urea. In conclusion, NOS expression and activity decreased as cysts developed, suggesting that NO downregulation is involved in the pathogenesis of PKD.     

Effect of focal cerebral ischemia on nitric oxide synthase expression in rats

Time- and cell-type-dependent immunohistochemical activity of nitric oxide synthase (NOS) was investigated in rat cerebral cortex following focal ischemia and the local concentration of nitric oxide (NO) was measured. NO concentration increased 2 min after the ischemia. Brain NOS-immunoreactive neurons increased in number 5 min after the ischemia. Endothelial cell NOS immunoreactivity was first detected in vascular endothelial cells and astrocytes 5 min after the ischemia, and it increased again during 60 min to 4 days after the ischemia in reactive astrocytes. Inducible NOS immunoreactivity was detected in astrocytes, vascular endothelium, and microglia/macrophages at the periphery of the ischemic core during 2–4 days after the ischemia.This study was presented at the 28th Annual Meeting of the Clinical Electron Microscopy Society of Japan, Osaka, October 17–19, 1996.     

miRNA-24对内皮型一氧化氮合酶表达调节及血管内皮细胞增殖的影响

 目的：为进一步探讨miRNA-24是否参与内皮型一氧化氮合酶（eNOS）表达的调控及其对血管内皮细胞增殖的影响,本研究构建miRNA-24高表达质粒,并用脂质体将其导入人脐静脉内皮细胞（HUVECs）,观察miRNA-24对eNOS表达及HUVECs增殖的影响。方法：用四甲基偶氮唑蓝(MTT)法检测细胞增殖情况,RT-PCR、免疫组织化学和Western blotting检测eNOS与Sp1转录因子mRNA和蛋白表达水平。结果：与对照组比较, miRNA-24高表达组细胞增殖减慢41.97 %（0.47±0.04 vs 0.81±0.03, P<0.01）,eNOS mRNA降低44.8%（0.48±0.01 vs 0.87±0.03,P<0.05）,蛋白表达减少71.92%（0.16±0.06 vs 0.57±0.08,P<0.05）;同时Sp1 mRNA降低53.00%（0.45±0.02 vs 0.93±0.01,P<0.05）,其蛋白质表达量也相应减少62.31%（0.13±0.07 vs 0.31±0.09,P<0.05）。miRNA-24抑制组中,上述指标比对照组降低,但比miRNA-24高表达组明显升高。结论：miRNA-24高表达明显抑制HUVECs的增殖及eNOS的表达;Sp1可能是参与miRNA-24调控eNOS表达的重要因素之一。     

一氧化氮/诱导型一氧化氮合酶在动脉粥样硬化过程中的作用

 目的：探讨一氧化氮（NO）/诱导型一氧化氮合酶（iNOS）在动脉粥样硬化（atherosclerosis,AS）过程中的动态变化,分析其对动脉粥样硬化形成过程的影响。方法：将60只SD大鼠随机分成2组：对照组及AS组,每组30只。AS组采用维生素D3腹腔注射联合高脂饲料饲养的方法构建动脉粥样硬化模型。用相关生化方法检测血清各项生化指标：总胆固醇、甘油三酯、高密度脂蛋白胆固醇、低密度脂蛋白胆固醇、空腹血糖和钙离子,比色法检测血清NO浓度,并对主动脉行HE染色,免疫组化技术检测iNOS蛋白表达,将所得数据进行统计分析,用简单线性相关分析NO与钙离子及动脉粥样硬化指数的相关性。结果：90 d后成功构建了主动脉中膜钙化型动脉粥样硬化模型。血清NO浓度在动脉粥样硬化过程中逐步下降,各组间差异均有统计学意义（均P<0.05）。动脉粥样硬化过程中动脉粥样硬化指数与钙离子呈正相关,与NO呈负相关。在90 d的AS组粥样斑块区免疫组化技术检测到iNOS蛋白表达。结论:在动脉粥样硬化形成过程中,主动脉粥样斑块区iNOS蛋白高表达,但血清NO浓度逐渐降低,NO抗动脉粥样硬化作用减弱。     

Endothelial nitric oxide synthase expression in pulmonary capillary hemangiomatosis

Pulmonary capillary hemangiomatosis (PCH) is an unusual disorder characterized by the proliferation of capillaries in the alveolar septa and pulmonary interstitium. Originally conceived as a primary idiopathic disorder of the pulmonary microcirculation, recent studies have demonstrated that PCH may be associated with other pathologies. Nitric oxide (NO) is a gaseous free radical with protean biological effects that is released during the intracellular conversion of arginine to citrulline. Nitric oxide synthases (NOS) mediate the production of NO and the release of NO in the microvasculature is specifically catalyzed by endothelial NOS (NOS-III). As NOS contributes to angiogenesis and is reduced in the hypertensive pulmonary microcirculation, we examined the expression of NOS-III protein in situ in the lungs of patients with PCH. Reduced microvascular expression of NOS-III protein by endothelial cells was observed in 4/6 (67%) cases of PCH, and all of these showed concomitant pulmonary vascular hypertensive remodeling. In 2/6 (33%) cases of PCH with no morphologic evidence of pulmonary hypertensive arteriopathy, endothelial expression of NOS-III protein was judged to be either minimally reduced or normal. These findings suggest that NOS-III is specifically reduced in PCH when pulmonary arterial hypertensive remodeling is concomitantly present.     

脑挫伤后一氧化氮合酶阳性细胞及一氧化氮含量的变化

目的　探讨脑挫伤后一氧化氮合酶 (NOS)阳性细胞和一氧化氮 (NO)的变化和意义。方法　采用自由落体法致Wistar大鼠顶叶皮质挫裂伤动物模型。伤后 2 4h、72h和 7d取脑 ,制作冰冻切片 ,采用NADPH组织化学染色 ,显示脑挫伤区NOS阳性细胞。用硝酸还原酶法测定血液和脑组织中NO含量。结果　脑挫伤后 72h ,NOS阳性细胞数密度 (Nv)和面密度(Sv)明显增高 (P <0 0 5 ) ,而且 7d时仍无明显下降。血液和脑中NO含量也增高 ,并与NOS细胞呈平行关系。结论　脑挫伤后不同时间NOS细胞数目和NO含量有明显改变 ,提示NOS和NO参与了脑挫伤的病理过程     

地尔硫(艹卓)对肺动脉高压大鼠血红素氧合酶1和一氧化氮合酶的影响

目的 :　探讨钙离子拮抗剂地尔硫 艹卓(商品名合心爽 )对大鼠缺氧性肺动脉高压模型肺小动脉 (SPA)血红素氧合酶 (HO) - 1、一氧化氮合酶 (NOS)表达的影响。方法 :常压缺氧 [(10 %± 1% )O2 ]6周复制大鼠肺动脉高压模型。缺氧 2周后随机分为模型组和地尔硫 艹卓 治疗组。检测右室收缩压 (RVSP)、右心肥大指数 (RVHI) ,用光镜和透射电镜观察肺的病理改变 ,并进行形态学定量。用免疫组化和Westernblot法观察肺内HO - 1、内皮型和诱生型NOS(eNOS和iNOS)的表达和分布。放免法检测肺组织环 -磷酸鸟苷 (cGMP)含量。结果 :地尔硫 艹卓 明显降低缺氧大鼠的RVSP和RVHI,减轻肺小动脉中膜肥厚 ,促进eNOS及抑制iNOS表达 ,但对缺氧引起的HO - 1表达增高和内皮超微结构损伤无显著影响。结论 :地尔硫 艹卓 能明显减轻肺动脉高压性结构重塑 ,这种作用可能部分通过抑制iNOS、促进eNOS ,而不减少HO - 1的表达等环节来实现。     

叶酸对去卵巢大鼠抗氧化酶、一氧化氮合酶和一氧化氮的影响

 目的: 观察叶酸对去卵巢大鼠抗氧化酶、一氧化氮合酶和一氧化氮的影响。方法: 40只3月龄健康雌性SD大鼠,随机分成5组:假手术组、去卵巢组、二乙基己烯雌酚(0.03 mg·kg^-1·d^-1)组、低剂量(5 mg·kg^-1·d^-1)叶酸组和高剂量(20 mg·kg^-1·d^-1)叶酸组。各组大鼠于术后1周开始灌胃给药,假手术组和去卵巢组给予蒸馏水,10周后,取L₅椎体和右股骨行骨密度(BMD)检测;测定血浆和骨匀浆总抗氧化能力(TAC)、谷胱甘肽过氧化物酶(GSH-Px)、丙二醛(MDA)、一氧化氮合酶(NOS)和一氧化氮(NO)水平。结果: 与假手术组比较,去卵巢组大鼠L₅椎体和股骨BMD显著降低(P < 0.01),血浆GSH-Px、NO和骨匀浆TAC、GSH-Px、NOS及NO水平明显降低(P < 0.01),MDA浓度升高显著(P < 0.01);与去卵巢组大鼠比较,高剂量叶酸组大鼠L₅椎体和股骨BMD增加(P < 0.01),骨匀浆TAC、GSH-Px、NOS和NO水平升高(P < 0.01),MDA浓度降低(P < 0.01),血浆GSH-Px和NO水平升高。结论: 去卵巢大鼠体内抗氧化酶、NOS和NO水平降低,氧化应激参与了去卵巢大鼠骨质疏松的发生;高剂量叶酸能提升去卵巢大鼠腰椎和股骨BMD,提高其体内抗氧化酶、NOS和NO水平,改善氧化应激,这可能是高剂量叶酸防治去卵巢大鼠骨质疏松的机制之一。     

老年大鼠空间记忆减退与cNOS基因表达变化

目的：从基因表达角度研究原生型NOS（cNOS,包括神经元型nNOS和内应型eNOS）与老年记忆减退发生的关系。方法：用Morris水迷宫将老年大鼠筛选为记忆正常和记忆减退两组,以老年记忆减退大鼠作为老年记忆减退模型;用半定量反转录一聚合酶链反应（RT－PCR）方法测定nNOS和eNOS的mRNA含量。结果：（1）海马组织中老年记忆正常组和老年记忆减退组较青年组nNOS和eNOSmRNA含量均下降,nNOS以记忆减退组下降更为显著;（2）小脑组织中老年记忆减退组nNOS和eNOSmRNA含量的下降与老年记忆正常组和青年组均有显著差异。（3）额叶组织nNOSmRNA含量三组之间均无显著差异;eNOSmRNA含量老年记忆正常组和老年记忆减退组技青年组均下降,以记忆减退组下降更为显著。结论：老年记忆减退的发生可能与有关脑区cNOS基因表达下降有关。     

Age-related alteration of activity and gene expression of endothelial nitric oxide synthase in different parts of the brain in rats

Nitric oxide (NO) plays important roles in aging and neurodegeneration. Our previous results indicated that aging differently affects NOS isoforms. Expression of nNOS mRNA was lower while iNOS was absent at any age. However, total NO synthesis increased in aged cerebral cortex and cerebellum as a consequence of changes of nNOS phosphorylation state. The question arise how aging influences activity and expression of eNOS in different parts of adult and aged brain. The levels of eNOS mRNA, protein and activity were measured using RT-PCR, immuno- and radiochemical methods, respectively. Our studies indicated that after inhibition of nNOS with 7-nitroindazole (7-NI) NO synthesis is lower in all parts of aged brain comparing to adults. However, eNOS activity significantly decreases only in cerebellum. The expression of eNOS determined on mRNA level was enhanced in all investigated aged brain parts to 140–190% of adult value and the data were statistically significant for cerebral cortex and cerebellum. The higher level of mRNA is probably the adaptive response to lower NOS activity. However, the Western-blot signal of eNOS protein was unchanged in aged brain parts comparing to adults suggesting age-related disturbances of protein synthesis and its function. It is also possible that a post-translational modification of the enzyme occurs in the aged rat brain. The lower eNOS activity in aged brain may significantly affects the signal transduction processes on the pathway NO/cGMP/PKG.     

阿司匹林对炎症条件下人血管内皮细胞一氧化氮的产生及诱导型一氧化氮合酶mRNA表达的影响

目的:探讨炎症时阿司匹林(AS)对内皮细胞一氧化氮(NO)的产生及诱导型一氧化氮合酶(iNOS)基因表达的抑制作用。方法:Griess法测上清液NO^-₂/NO^-₃水平、黄递酶法测NOS活性、常规生化法测乳酸脱氢酶(LDH)、丙二醛(MDA)浓度,染料排除法测细胞活力,RT-PCR技术分析iNOSmRNA水平。结果:白介素(IL)-1β、肿瘤坏死因子(TNF)-α、γ-干扰素(INF)联用脂多糖(LPS)诱导后上清液中NO^-₂/NO^-₃由(4.27±0.75)μmol/L增加到(9.35±1.25)μmol/L,对内皮细胞造成明显的损伤。但3mmol/LAS组NO生成及NOS活性明显降低,LDH释放率及MDA浓度下降,细胞存活率上升,与NO诱导组相比差异显著。并随AS剂量的增加对NO的抑制及对细胞的保护作用更加明显,但AS对生理水平的NO没有抑制作用。同时发现10mmol/L浓度以下AS对iNOSmRNA表达水平没有影响;但10-20mmol/L的AS则可在转录水平上抑制iNOSmRNA的表达。并观察到水杨酸钠及消炎痛不具有抑制NO产生的作用。结论:AS具有明显抑制IL-1β、TNF-α、γ-INF及LPS诱导NO生成的作用,从而保护血管内皮细胞避免炎症时高浓度NO的损伤。     

肝细胞癌一氧化氮合酶的表达及其临床意义

目的：研究肝细胞癌（HCC）组织中一氧化氮合酶（NOS）的表达及其临床意义。方法：通过免疫组化的方法检测51例HCC组织和46例癌旁肝组织（LTBC）中NOS1、NOS2、NOS3的表达,探讨3指标与HCC单发还是多发、肿瘤大小、有否合并肝硬化、肿瘤坏死、组织分化程度、门静脉癌栓形成、肝外转移和预后等的关系。结果：NOS1、NOS2、NOS3在46例LTBC的阳性率均明显高于51例HCC组织的阳性率（P<0.01）;NOS1两年内复发组的阳性率明显高于无复发组（P<0.01）;NOS2在无癌栓形成组的阳性率明显高于伴有癌栓形成组（P<0.05）;NOS3在复发组的阳性率明显高于无复发组（P<0.01）。结论：NOS的表达与HCC的组织分化程度、门静脉癌栓形成和预后等生物学行为有密切关系     

缺氧新生鼠胃壁内一氧化氮合成酶定量和定位研究

为探讨缺氧新生鼠胃壁局部一氧化氮（ＮＯ）的改变,本文直接测定其胃壁一氧化氮合成酶（ＮＯＳ）活性,并采用ＮＡＤＰＨ二氢硫辛酰胺脱氢酶组织化学方法（ＮＤ法）观察胃壁各层ＮＯＳ分布的变化,结果发现：急性缺氧组与正常组相比,差异无显著性（Ｐ＞０．０５）。但在缺氧缺血性脑病（ＨＩＥ）组,胃壁ＮＯＳ活性明显增高（Ｐ＜０．０１）,ＮＤ法定位显示ＮＯＳ阳性纤维及胞体明显增多表现在肌层,而粘膜及粘膜下层变化不明显。说明窒息时胃动力降低及胃粘膜病变与ＮＯ在胃壁内的改变有关。     

一氧化氮合酶(NOS)与子宫内膜关系的研究进展

女性子宫内膜在女性生殖生理功能方面担负着月经形成、胚胎着床、妊娠维持、激素分泌等重要作用。自从1995年Telfer首次发现人子宫内膜中NOS(N itric Oxide Synthase)mRNA和其蛋白的存在后,近年来越来越多的研究表明NO(N itric Oxide)在女性生殖过程中扮演着重要的角色。本文将就NOS在子宫内膜的活性表达,及其与子宫内膜关系的研究作一综述。     

低水平铅暴露人胎盘组织诱导型和内皮型一氧化氮合酶的表达及意义

目的探讨孕期低水平铅暴露对胎盘组织诱导型和内皮型一氧化氮合酶的表达及其与铅水平的关系。方法2005年2月至2006年12月孕期外周血铅水平大于30μg/l的67例孕妇,根据铅水平分组,血铅水平30μg/l-60μg/l的孕妇35例为A组;血铅水平在60μg/l-100μg/l的孕妇32例为B组。检测胎盘中一氧化氮合酶系统表达。结果低水平铅暴露下诱导型和内皮型一氧化氮合酶在各组胎盘中的表达均分布在合体滋养细胞、细胞滋养细胞、微小血管内皮细胞、蜕膜细胞、绒毛间纤维细胞(villus fibrocyte)的胞浆,定位显示无显著差异;高血铅组的表达水平与强度显著高于低铅水平组(P<0.05)。结论金属铅可诱导发育中胎盘组织诱导型和内皮型一氧化氮合酶产生,胎盘NOS酶活性上升,能够改善子宫-胎盘血循环障碍,抵御铅毒性,维持正常妊娠的进行。     

大鼠脑干神经元型一氧化氮合酶免疫阳性神经元的分布

目的　观察大鼠脑干神经元型一氧化氮合酶 (nNOS)免疫阳性神经元的分布 ,为探讨nNOS的作用提供形态学资料。方法　用ABC免疫细胞化学方法显示脑干nNOS免疫阳性神经元。结果　大鼠脑干nNOS免疫阳性神经元以中脑和脑桥分布丰富 ,延髓较稀少 ;在中脑 ,nNOS免疫阳性神经元主要分布于中脑水管周围灰质的背侧部、被盖背外侧核、中缝背核、上下丘灰质等部位 ;在脑桥 ,主要分布于被盖背外侧核、脑桥中缝核、被盖脚桥核、蓝斑、臂旁核、斜方体核 ,以及脑桥网状结构 ;与中脑和脑桥相比 ,延髓nNOS免疫阳性神经元较少 ,主要分布于延髓网状结构、三叉神经脊束核和孤束核等核团。结论　分布于脑干内丰富的nNOS免疫阳性神经元可能通过其生成的NO调节其他神经递质的分泌 ,共同参与内脏活动、感觉和运动的传导 ,以及睡眠和觉醒等脑的高级整合功能的调节。     

Repeated induction of nitric oxide synthase and leishmanicidal activity in murine macrophages

Murine macrophages express high levels of nitric oxide (NO) synthase and produce large amounts of NO when stimulated with interferon-y plus lipopolysaccharide in vitro. The expression of NO synthase peaks at 12 h after stimulation and declines rapidly to the background level by 72 h. These macrophages can be repeatedly reactivated to express similar levels of NO synthase. The reactivation is not due to newly divided cells since peritoneal macrophages which do not divide in vitro and J774 cells cultured in the presence of colchicine can also be restimulated to express NO synthase. The reactivation is accompanied by re-expression of NO synthase mRNA, as assessed by polymerase chain reaction analysis. Furthermore, the reactivated macrophages are fully capable of killing the intracellular protozoan parasite Leishmania major.     

肝移植围术期血浆一氧化氮和一氧化氮合酶活性的变化及意义

目的：动态观察肝移植围术期血浆一氧化氮（NO）水平和一氧化氮合酶（NOS）活性的变化，并探讨其意义。 方法： 30例终末期肝病患者接受原位肝移植术。用放免法、比色法分别测定肝移植围术期5个时点血浆NO2-/NO3-水平和NOS活性，观察其动态变化。同步抽取桡动脉和肺动脉血做血气分析，记录不同时期的PO2、PCO2、SO2、Hb，根据肺内分流标准模型公式计算(Qs/Qt)。并监测围术期心输出量（CO）、心率（HR）、中心静脉压（CVP）、平均动脉压（MABP）、体循环阻力（SVR）。 结果： (1) 无肝前10 min NO2-/NO3-水平明显高于麻醉后术前。无肝期30 min NO2-/NO3-显著低于无肝前10 min。新肝期30 min NO2-/NO3-显著高于麻醉后术前、无肝期30 min。（2）TNOS活性各时点无显著差异。无肝前10 min、新肝30 min时iNOS活性明显高于麻醉后术前。与无肝30 min值比较，新肝期30 min iNOS活性显著升高。（3）MABP在开放下腔静脉后1 min明显下降，CO和CVP在无肝期下降，新肝期增高。SVR在无肝期增高，新肝期明显下降。（4）Qs/Qt在无肝期下降，新肝期30 min升高。 结论： 在肝移植围术期各个时段，NO水平及iNOS活性各不相同。高NO水平可能是新肝期低阻力、肺内分流增加的原因。     

小凹蛋白与内皮型一氧化氮合成酶在人肝硬化组织中的表达及意义

目的 检测人肝硬化组织中小凹蛋白(caveolin-1)、内皮型一氧化氮合成酶(eNOS)的细胞定位及蛋白表达水平的变化,探讨caveolin-1的表达对eNOS的影响.方法 免疫组织化学染色检测30例肝硬化患者活检后肝组织和30例肝外伤、肝血管瘤患者正常肝组织中caveolin-1和eNOS的细胞定位.Western印迹检测caveolin-1和eNOS的蛋白表达水平变化.结果 caveolin-1和eNOS均主要分布于肝窦内皮细胞中,caveolin-1在肝硬化组和对照组表达阳性率有差异,分别为87%和40%(P＜0.05).eNOS在肝硬化组和对照组表达阳性率有差异,分别为33%和66%(P＜0.05).caveolin-1在肝硬化组织中表达较正常肝组织中明显增强.eNOS在肝硬化组织中呈低水平表达,较正常肝组织中表达明显减少.结论 肝硬化肝窦内皮细胞的损伤降低了eNOS的表达.caveolin-1的过表达促进eNOS-caveolin-1复合物的形成,加剧了门静脉高压症的发生.