诱导型一氧化氮合酶与疾病

炎症是众多疾病如自体免疫紊乱、神经退行性病变、心血管疾病和癌症发展的病理机制,诱导型一氧化氮合酶在炎症过程中被诱导表达,产生过量的一氧化氮,引发炎症级联反应,进而导致以上多种疾病发生。抑制诱导型一氧化氮合酶表达在体内体外实验及临床使用中均体现抗炎效果和症状改善。本文综述了诱导型一氧化氮合酶在炎症过程中诱导表达及与各类重大疾病联系的最新进展,并展望了诱导型一氧化氮合酶抑制剂作为抗炎治疗策略的前景。     

诱导型一氧化氮合酶的激活与血压的关系

本实验旨在探讨诱导型一氧化氮合酶（iNOS)的激活与血压之间的关系,三组SD大鼠分别静脉输注不同浓度（0．3％,4％及8％）NaCl溶液以使其处于不同的血压水平,运用同位素标记的L－精氨酸转换成L－Citrulline 的转换率变化及Greiss反应,分别测定不同血压时iNOS的活性及NO的生成量,另四组大鼠包括正常Wistar,正常SD,高盐诱导的高血压（NaHR)及自发性高血压大鼠（SHR）,经测定血压后,取主动脉血管并以Western印迹印交法测定其iNOS蛋白水平,结果表明,血压较低时,SD大鼠iNOS活基本没有改变,而在输入4％和8％NaCl并处于较高血压水平的SD大鼠,其iNOS活性及NO生存均明显升高,。此外Western 印迹表明,两种高血压大鼠主动脉组织iNOS蛋白水平均较正常Wistar及正常SD大鼠高,密度扫描表明,NaHR及SHR主动脉组织iNOS蛋白分别较正常SD大鼠及正常Wistar大鼠升高149％及261％,这一结果提示,诱导型一氧化氮合酶是血液动力学调控的重要组成部分,尤其是在血压处于较高水平时,iNOS具有重要的代偿调节作用,除细胞因子,细菌产物等之外,血压也是调节iNOS表达及活性的重要因素之一。     

中国人诱导型一氧化氮合酶基因STR多态性研究

一氧化氮（nitric oxide,NO）作为一种可在细胞间自由扩散的信使分子在神经递质传递和血管舒张调节等生理与病理过程中起着重要作用。NO通过一氧化氮合酶（nitric oxide synthase,NOS）催化L-精氨酸的氧化反应而生成。目前在哺乳动物中已发现细胞来源、表达方式和活性调节不同的3种NOS同工酶,分别为神经原型NOS(meuronal NOS,nNOS）、诱导型NOS(inducible NOS,iNOS）和内皮细胞型NOS（endothelial NOS,eNOS）。3种NOS由位于不同染色体上的基因所编码。人iNOS基因位于第17号染色体长臂(17q11.2～17q12）,全长约37kb,含有26个外显子,iNOS可在多种类型的细胞中通过IL-1、IFN-a、INF-γ等细胞因子和其他介质的刺激作用而诱导表达。iNOS基因5‘-端调控区内存在一个CCTTT串联重复的多态性位点,这一多态性基因座已居北爱尔兰的糖尿病患者中证实与微血管病变有关。另有实验表明,CCTTT串联重复序列的变化对iNOS基因的转录将产生不同影响。目前在东方种族中有关iNOS基因CCTTT串联重复多态性尚未见报道。将303名中国汉族人基因组DNA用于iNOS基因CCTTT串联重复多态性分析,鉴定出了12种等位基因和49种基因型,其中重复17次、18次和19次的等位基因是在人类中首次发现的新等位基因。统计学检验和比较表明,中国汉族人iNOS基因CCTTT串联重复序列的6个等位基因频率与来自英格兰的高加索人差异显著,说明这一多态性位点的等位基因频率分布存在种族差异。在中国正常人群中获得的有关iNOS基因STR多态性的统计资料,对于进一步研究这一多态性基因座与心脑血管疾病的相关性将有重要应用价值。     

诱导型一氧化氮合酶(iNOS)基因表达的调控

一氧化氮（ＮＯ）自由基有多方面的生物学功能。随着研究的深入,发现ＮＯ能与超氧阴离子（Ｏ－２·）反应生成激发态亚硝酸（ＯＮＯＯＨ＊）,它与靶分子能产生羟自由基（·ＯＨ）和二氧化氮（ＮＯ２）样反应,在体内原先认为的一些ＮＯ效应,现在知道主要是由于ＯＮＯＯ...     

双歧杆菌对实验性大肠癌诱导型一氧化氮合酶表达的影响

目的:用免疫组化法观察大肠癌移植瘤诱导型一氧化氮合酶(iNOS)的表达。方法:以大肠癌裸鼠移植瘤为动物模型,将青春型双歧杆菌注射于裸鼠腹腔。结果:显示双歧杆菌注射组大肠癌移植瘤iNOS的表达率、表达强度和阳性细胞数量均显著高于肿瘤对照组(P<0.01)。结论:青春型双歧杆菌能增强大肠癌移植瘤iNOS的蛋白表达水平。它的表达可能介导了双歧杆菌诱导大肠癌移植瘤细胞的凋亡。     

大鼠烧伤复合内毒素脏器损伤中诱导型一氧化氮合酶的免疫组织化学定位及意义初探

选用烧伤复合内毒素血症大鼠模型,应用免疫组化ＬＳＡＢ法观察肝、肾组织中诱导型一氧化氮合酶（ｉＮＯＳ）的定位分布与含量变化。结果表明：在肝损伤中,ｉＮＯＳ主要定位于肝窦内皮细胞、枯否细胞和肝细胞胞浆,在损伤严重时,定位于多发性、溶解性坏死灶内残留的窦内皮细胞、巨噬细胞、中性粒细胞以及坏死灶基质内。在肾损伤中,ｉＮＯＳ主要定位于肾小管上皮细胞胞浆。并且ｉＮＯＳ与ＴＮＦ－α、ＩＬ－６的蛋白和基因表达密切相关。提示：ｉＮＯＳ及合成产物ＮＯ在烧伤复合内毒素性脏器损伤中起着重要作用,并推测ＮＯ可能是单核／巨噬细胞激活反应的信号传导分子之一     

大鼠视网膜缺血后一氧化氮合酶阳性神经元的变化

本文用前房加压灌注视网膜缺血模型、β－ＮＡＤＰＨ脱氢酶组化方法研究了ＳＤ大鼠视网膜内含一氧化氮合酶（ＮＯＳ）神经元的分布及其变化。实验动物依缺血时间分四组,分别为缺血１０ｍｉｎ、１５ｍｉｎ、３０ｍｉｎ及６０ｍｉｎ组。将ＮＯＳ阳性细胞进行计数统计,做自身配对ｔ检验及方差分析。实验结果表明正常及缺血视网膜ＮＯＳ阳性神经元。大多数位于内核层,少数位于节细胞层;ＮＯＳ阳性细胞在视网膜中央区密度高于周边区,而各象限的平均密度分布无明显差别。缺血１５ｍｉｎ后内核层的ＮＯＳ阳性细胞开始减少,随缺血时间的延长细胞减少更为明显。各组均以视网膜中央区变化较为显著,提示视网膜缺血１５ｍｉｎ后即可出现神经生物学变化,视网膜中央区对缺血比周围区更为敏感。     

大鼠诱导型一氧化氮合酶基因转录调控区的克隆与鉴定

采用单特异引物ＰＣＲ克隆法,得到大鼠诱导型一氧化氮合酶（ｉＮＯＳ）基因转录调控区ＤＮＡ片段。核酸序列分析证实,大鼠ｉＮＯＳ基因的５＇－侧翼区含有ＩＦＮ－γ和ＴＮＦ－α应答元件及ＮＦ－ｋＢ结合位点的保守序列。这些保守序列的位置及排列显区别于人和小鼠的ｉＮＯＳ基因。电泳迁移率改变分析（ＥＭＳＡ）表达,ＶＳＭＣ受ＩＬ－１和ＩＦＮ－γ刺激后,细胞核内产生某种可与ｉＮＯＳ基因５＇－侧翼区特异结合的核蛋白因     

诱导型一氧化氮合酶基因多态性与2型糖尿病关系研究

目的：观察诱导型一氧化氮合酶（iNOs）基因Ser608Leu位点基因多态性与2型糖尿病的相关性。方法：采用测序法测定261例2型糖尿病患者和128例正常对照者iNOS基因Ser608Leu位点基因多态性。结果：两组间iNOS基因Ser608Leu位点基因型及等位基因频率构成存在差异。结论：iNOS基因Ser608Leu位点多态性与2型糖尿病有关。     

高原鼠兔诱导型一氧化氮合酶cDNA克隆及序列分析

高原鼠兔Ochotona curzoniae,世居在青藏高原海拔3000~5000 m的地区,是一种典型的低氧耐受哺乳动物。一氧化氮(NO)作为一种有效的血管舒张因子,在预防低氧诱导的低氧性肺血管收缩反应和肺动脉高压中发挥着重要功能。诱导型一氧化氮合酶(iNOS)是一种催化L-精氨酸合成NO的重要酶,受低氧调控。本研究经RTPCR和cDNA 3’末端快速扩增(3’RACE)方法成功克隆了高原鼠兔iNOS基因cDNA序列,并对其分子特征进行了分析。结果显示:高原鼠兔iNOS cDNA全长为3981 bp,开放阅读框(ORF)为3450 bp,共编码1149个氨基酸残基;预测的蛋白序列与北美鼠兔、兔、人、大鼠、小鼠、狗以及猪的同源性分别为98%、87%、82%、78%、78%、82%和83%;蛋白结构预测结果显示高原鼠兔iNOS具有氧化域、还原域及黄素腺苷酸结合区域等iNOS所具有的典型结构域;基于iNOS的最大似然树和贝叶斯树均支持鼠兔与兔有最近的亲缘关系,与形态或其他分子标记构建的进化关系相符;分子进化分析检测到高原鼠兔iNOS中存在3个正选择位点——32T、33Y和46R,但不同模型的结果表明哺乳动物iNOS基因所受选择以净化选择为主,不支持iNOS在高原鼠兔支系发生适应性进化。该研究为揭示高原鼠兔iNOS的表达特征及其在低氧适应中的作用与调控机制研究奠定了初步基础。     

Cobalt-60 gamma radiation increased the nitric oxide generation in cultured rat vascular smooth muscle cells

Radiation is a promising and new treatment for restenosis following angioplasty. Nitric oxide has been proposed as a potential "anti-restenotic" molecule. We radiated the cultured rat vascular smooth muscle cells with Cobalt-60 gamma radiation at doses of 14 and 25Gy and observed nitrite production, cGMP content, L-arginine uptake, inducible nitric oxide synthase (iNOS) activity, and the gene expression of iNOS. Results showed that radiation at doses of 14 and 25Gy increased cGMP content by 92.4% and 86.4%, respectively. Radiation at the dose of 25Gy increased the iNOS activity and nitrite content, but radiation at the dose of 14Gy had no significant effect on iNOS activity and NO production. Both doses of radiation significantly decreased the L-arginine transport. Radiation at the doses of 14 and 25Gy increased iNOS gene expression significantly, which was consistent with the effect of radiation on iNOS activity. In conclusion, radiation induces the NO generation by up-regulating the iNOS activity.     

Nitric oxide produced by iNOS is associated with collagen synthesis in keloid scar formation

Nitric oxide (NO) has emerged as an important mediator of many physiological functions. Recent reports have shown that NO participates in the wound healing process, however, its role in keloid formation remains unclear. This study aimed to investigate the effect of NO on keloid fibroblasts (KF) and to determine the levels of inducible nitric oxide synthase (iNOS) expression in clinical specimens of keloid. Scar tissue from seven keloid patients with matched perilesion skin tissue controls was studied for inducible nitric oxide synthase expression and location. In addition, primary keloid and normal scar skin fibroblast cultures were set up to investigate the effects of NO in inducing collagen type I expression. Inducible nitric oxide synthase expression, and NO production were elevated in keloid scar tissues but not in matched perilesion skin tissues. Furthermore, exposure of KF to exogenous NO resulted in increased expression of collagen type I in a dose-dependent manner. NO exposure also induced time-course dependent collagen I expression that peaked at 24h in KF. Taken together, these results indicate that excess collagen formations in keloid lesion may be attributed to iNOS overexpression.     

Pharmacological inhibition of inducible nitric oxide synthase attenuates the development of seizures in mice

The present study has been designed to pharmacologically expound the significance of inducible nitric oxide synthase in the pathophysiological progression of seizures using mouse models of chemically induced kindled epilepsy and status epilepticus induced spontaneous recurrent seizures. Pentylenetetrazole (40 mg kg⁻¹) (PTZ) administration every second day for a period of 15 days was used to elicit kindled seizure activity in mice. Severity of kindled seizures was assessed in terms of a composite kindled seizure severity score (KSSS). Pilocarpine (100 mg kg⁻¹) was injected every 20 min until the onset of status epilepticus. A spontaneous recurrent seizure severity score (SRSSS) was recorded as a measure of quantitative assessment of the progressive development of spontaneous recurrent seizures induced after pilocarpine status epilepticus. Sub-acute PTZ administration induced the development of severe form of kindled seizures in mice. Further, pharmacological status epilepticus elicited a progressive evolution of spontaneous recurrent seizures in the animals. However, treatment of aminoguanidine, a relatively selective inhibitor of inducible nitric oxide synthase, markedly and dose dependently suppressed the development of both PTZ induced kindled seizures as well as pilocarpine induced spontaneous recurrent seizures. Therefore inducible nitric oxide synthase may be implicated in the development of seizures.     

Synthesis and biological evaluation of 4,5-dihydro-1H-pyrazole derivatives as potential nNOS/iNOS selective inhibitors. Part 2: Influence of diverse substituents in both the phenyl moiety and the acyl group

In a preliminary article, we reported a series of 4,5-dihydro-1H-pyrazole derivatives as neuronal nitric oxide synthase (nNOS) inhibitors. Here we present the data about the inhibition of inducible nitric oxide synthase (iNOS) of these compounds. In general, we can confirm that these pyrazoles are nNOS selective inhibitors. In addition, taking these compounds as a reference, we have designed and synthesized a series of new derivatives by modification of the heterocycle in 1-position, and by introduction of electron-donating or electron-withdrawing substituents in the aromatic ring. These derivatives have been evaluated as nNOS and iNOS inhibitors in order to identify new compounds with improved activity and selectivity. Compound 3r, with three methoxy electron-donating groups in the phenyl moiety, is the most potent nNOS inhibitor, showing good selectivity nNOS/iNOS.     

Effects of moderate hypothermia on constitutive and inducible nitric oxide synthase activities after traumatic brain injury in the rat

We investigated the effects of therapeutic hypothermia (30 degrees C) on alterations in constitutive (cNOS) and inducible (iNOS) nitric oxide synthase activities following traumatic brain injury (TBI). Male Sprague-Dawley rats were anesthetized with 0.5% halothane and underwent moderate (1.8-2.2 atm) parasagittal fluid-percussion (F-P) brain injury. In normothermic rats (37 degrees C) the enzymatic activity of cNOS was significantly increased at 5 min within the injured cerebral cortex compared with contralateral values (286.5+/-68.9% of contralateral value; mean+/-SEM). This rise in nitric oxide synthase activity was significantly reduced with pretraumatic hypothermia (138.8+/-17% of contralateral value; p < 0.05). At 3 and 7 days after normothermic TBI the enzymatic activity of cNOS was decreased significantly (30+/-8.4 and 28.6+/-20.9% of contralateral value, respectively; p < 0.05). However, immediate posttraumatic hypothermia (3 h at 30 degrees C) preserved cNOS activity at 3 and 7 days (69.5+/-23.3 and 78.6+/-7.6% of contralateral value, respectively; mean+/-SEM; p < 0.05). Posttraumatic hypothermia also significantly reduced iNOS activity at 7 days compared with normothermic rats (0.021+/-0.06 and 0.23+/-0.06 pmol/mg of protein/min, respectively; p < 0.05). The present results indicate that hypothermia (a) decreases early cNOS activation after TBI, (b) preserves cNOS activity at later periods, and (c) prevents the delayed induction of iNOS. Temperature-dependent alterations in cNOS and iNOS enzymatic activities may participate in the neuroprotective effect of hypothermia in this TBI model.     

Inhibition of inducible nitric oxide synthesis by azathioprine in a macrophage cell line

Azathioprine is used as an anti-inflammatory agent. Although there are numerous data demonstrating cytotoxic and immunosuppressive properties of azathioprine and its metabolite 6-mercaptopurine, the mechanism of the anti-inflammatory action of azathioprine has not yet been fully clarified. During our study, we investigated the effects of azathioprine on the inducible nitric oxide synthase (iNOS) in lipopolysaccharide stimulated murine macrophages (RAW 264.7) by measurement of iNOS protein (immunoblotting), iNOS mRNA (semiquantitative competitive RT-PCR), and NO production (nitrite levels). Azathioprine (0-210 muM) induces a concentration dependent inhibition of inducible nitric oxide synthesis (IC50: 33.5 muM). iNOS protein expression showed a concentration dependent reduction as revealed by immunoblotting when cells were incubated with increasing amounts of azathioprine. Azathioprine decreases iNOS mRNA levels as shown by semiquantitative competitive RT-PCR. In contrast, 6-mercaptopurine showed no inhibition of inducible nitric oxide synthesis. Azathioprine did not reduce iNOS mRNA stability after the addition of actinomycin D. Enzymatic activity assays with increasing concentrations of azathioprine (0-210 muM) showed no statistically significant inhibition of iNOS enzyme activity compared to cell lysates without azathioprine. Nuclear translocation of NF-kappaB p65 subunit and binding of NF-kappaB p50 subunit from nuclear extracts to a biotinylated-consensus sequence was unaffected by azathioprine treatment. iNOS inhibition by azathioprine was associated with a decreased expression of IRF-1 (interferon regulatory factor 1) and IFN-beta (beta-interferon) mRNA. Azathioprine induced iNOS inhibition seems to be associated with an action of the methylnitroimidazolyl substituent. This suggests a route to the rational design of nontoxic anti-inflammatory agents by replacing the 6-mercaptopurine component of azathioprine with other substituents. The inhibition of inducible nitric oxide synthesis might contribute to the anti-inflammatory activities of azathioprine.     

Inducible nitric oxide synthase expression in the ischemic core and penumbra after transient focal cerebral ischemia in mice

The present observations examined the hypothesis that the iNOS expression in the ischemic penumbra after a transient focal ischemic insult is involved in the recruitment of penumbra into infarction. The middle cerebral artery in mice was occluded for 2 h by an intraluminal filament and then recirculated. The measurement of iNOS activity, iNOS protein formation and NO concentration in the ischemic core and penumbra, and the determination of infarct volume were performed at 6, 12, 24 and 48 h after reperfusion. iNOS protein and iNOS enzymatic activity appeared at 6 h, peaked at 24 h, and declined at 48 h in the penumbra after reperfusion. iNOS protein was not detectable in contralateral area and in sham-operated brains. The time course of iNOS protein, enzymatic activity and NO concentration in the penumbra but not in the core matched the process of infarct maturation. Treatment with iNOS inhibitor aminoguanidine (100 mg.kg(-1), i.p.) at 6 and 12 h after reperfusion inhibited iNOS activity by 88.0 +/- 10.4% and reduced NO concentration by 48.5 +/- 8.3% in the penumbra, and lessened infarct size by 48.8 +/- 7.2%. The iNOS activity and NO level in the core were not affected by the administration of aminoguanidine. These results suggest that iNOS expression in the ischemic penumbra is involved in the recruitment of penumbra into infarction and thereby contributing to the enlargement of infarct.     

Endogenous bacteria-triggered inducible nitric oxide synthase activation protects the ovariectomized rat stomach

Under experimental circumstances, ovariectomy attenuates gastric mucosal injury where nitric oxide (NO)-mediated pathways are involved. In this study, we have examined the changes in constitutive (cNOS) and inducible NO synthase (iNOS) enzyme activities (assessed by the citrulline assay), and the role of endogenous bacteria in ovariectomy-provoked mucosal defence. Gastric lesions were induced by indomethacin (50 mg/kg, s.c.) over a 4 h period in sham-operated and ovariectomized female Wistar rats. Groups of animals received the wide-spectrum antibiotic ampicillin (800 mg/kg/day, p.o., for 3 days), and others were injected with bacterial endotoxin (E. coli, 3 mg/kg, i.v., 5 h before autopsy). We found that ovariectomy increased iNOS and decreased cNOS activity (resulting an elevated total gastric NOS level), and protected the stomach, effects reversed by ampicillin treatment. In ovary-intact rats, administration of bacterial endotoxin enhanced gastric iNOS activity and reduced lesion-formation. These results suggest that ovariectomy improves gastric mucosal defence perhaps by endogenous bacteria-triggered induction of iNOS.     

The constituents of licorice (Glycyrrhiza uralensis) differentially suppress nitric oxide production in interleukin-1β-treated hepatocytes

Licorice (Glycyrrhizae radix) is the roots and stolons of Glycyrrhiza uralensis Fischer or Glycyrrhiza glabra Linnaeus in the Japanese Pharmacopoeia. Glycyrrhizae radix has been widely used as a sweetener and a traditional medicine. A Glycyrrhizae radix extract contains many constituents and has antispasmodic, antitussive, anti-ulcer, and anti-inflammatory effects. However, reports comparing the anti-inflammatory effects of these constituents are very few. Here, we purified several constituents from the roots and stolons of G. uralensis and examined and compared their anti-inflammatory effects by monitoring the levels of the inflammatory mediator, nitric oxide (NO), in interleukin (IL)-1β-treated rat hepatocytes. From the G. uralensis extract, we purified the main constituent glycyrrhizin and the constituents that are characteristic of G. uralensis (chalcones and flavanones). These constituents suppressed NO production in IL-1β-treated rat hepatocytes, and isoliquiritigenin showed the greatest suppression activity. Isoliquiritigenin, isoliquiritin, and liquiritigenin significantly decreased both protein and mRNA for the inducible nitric oxide synthase. These constituents reduced the levels of mRNAs encoding tumor necrosis factor α and IL-6. In contrast, although glycyrrhizin is abundant, it showed a 100-fold lower potency in NO suppression. Therefore, both glycyrrhizin and the minor constituents (isoliquiritigenin, isoliquiritin, and liquiritigenin) may be responsible for the anti-inflammatory effects of G. uralensis. It is also implied that these constituents may have a therapeutic potential for inflammatory hepatic disorders.