巨噬细胞诱导型一氧化氮合酶的表达调节机制

一氧化氮是一种重要的巨噬细胞免疫效应分子,它参与免疫调节和宿主防御反应.一氧化氮的生成主要由诱导型一氧化氮合酶调节,然而诱导型一氧化氮合酶表达的调节机制及信号通路尚不完全清楚.     

姜黄素对急性肺损伤大鼠肺组织诱导型一氧化氮合酶和内皮型一氧化氮合酶表达的影响

目的:探讨姜黄素对内毒素(LPS)致急性肺损伤(ALI)模型大鼠肺组织中诱导型一氧化氮合酶(iNOS)和内皮型一氧化氮合酶(eNOS)表达的影响。方法:雄性SD大鼠采用随机数字表法分为对照组、急性肺损伤模型组及姜黄素治疗组。采用一次性气管内滴注内毒素4 mg/kg制备急性肺损伤模型大鼠,治疗组于造模前15 min腹腔注射姜黄素200 mg/kg,于造模后3、6、12 h及24 h取肺组织,观察并比较各组肺组织形态学改变,并检测肺湿/干重比、肺含水量和肺组织中NO的含量,real-time PCR及免疫印迹法检测iNOS和eNOS的mRNA和蛋白表达。结果:模型组大鼠肺湿/干重比、肺组织含水量及NO含量均较对照组明显升高,iNOS的mRNA和蛋白表达量较对照组显著上调,而eNOS的mRNA和蛋白表达量较对照组显著下调。与模型组相比,姜黄素治疗组肺湿/干重比、肺含水量、NO含量均明显降低,iNOS的mRNA和蛋白表达量明显下调,eNOS的mRNA和蛋白表达量明显上调。结论:姜黄素可下调内毒素致急性肺损伤模型大鼠肺组织iNOS表达,并匕调eNOS表达。     

氨甲蝶呤抑制小鼠巨噬细胞一氧化氮合酶表达

研究氨甲蝶呤（MTX）在小鼠巨噬细胞（RAW264.7)对一氧化氮合酶（NOS）表达的抑制作用。本试验应用内毒素（LPS）、干扰素（IFNr)、MTX、二氨基－羟基嘧啶（DAHP），四氢生物喋呤（BH4）刺激8h后，用Northern杂交检测NOS mRNA水平和GTP－CH-1 mRNA水平表达，并用Griess reaction方法检测NO2^-/NO3^-浓度，用Duch方法测定GTP－CH－1活性值。我们发现，MTX在小鼠巨噬细胞（RAW264.7)对NOS mRNA水平和GTP－HC－1 mRNA水平表达有轻度抑制作用，对NO2^-/NO3^-浓度和GTP－CH－1活性抑制作用较弱。MTX和DAHP联合应用不仅对NOS mRNA水平和GTP－CH－1 mRNA水平表达抑制作用较强，而且对NO2^-/NO3^-浓度和GTP－CH－1活性抑制较大。BH4的加入可以减少MTX和DAHP对一氧化氮（NO)生成的抑制作用。     

P38丝裂原活化蛋白激酶对帕金森病模型小鼠黑质诱导型一氧化氮合酶和前列腺素E2表达的影响

目的:研究P38丝裂原活化蛋白激酶(P38MAPK)在1-甲基-4-苯基-1, 2, 3, 6四氢吡啶(MPTP)所致帕金森病(PD)模型小鼠中对黑质诱导型一氧化氮合酶(iNOS)和前列腺素E2(PGE2)的调控作用.方法:将小鼠随机分为 MPTP模型组,腹腔注射MPTP;抑制剂组,每次注射MPTP前1h腹腔注射P38MAPK特异性抑制剂SB 203580;对照组,注射与模型组和抑制剂组等量生理盐水和DMSO.行为学观察,采用免疫组织化学和免疫蛋白印迹法观察黑质酪氨酸羟化酶(TH)、 iNOS、 PGE2和磷酸化P38MAPK (p-P38MAPK)的表达.结果:模型组小鼠出现PD典型的行为学症状,TH阳性细胞和蛋白水平下降61%～65%, p-P38MAPK、 iNOS和PGE2阳性细胞及蛋白水平显著增加;经SB 203580处理后,上述变化均明显减轻.结论: P38MAPK对PD模型小鼠黑质iNOS和PGE2表达可能有重要调控作用,SB 203580对PD小鼠具有一定神经保护作用.     

大鼠坐骨神经损伤后诱导型一氧化氮合酶表达的变化

为了探讨大鼠坐骨神经损伤后再生过程中诱导型一氧化氮合酶(iNOS)表达的变化及意义,本研究采用大鼠坐骨神经切断缝合模型,分别于术后1、3、7、14、21及28d取吻合口远端的神经,采用免疫组织化学和实时荧光定量聚合酶链反应(RT-PCR)方法检测损伤神经远端iNOSmRNA及其蛋白的表达水平。结果显示:假手术对照组坐骨神经中未见明显的iNOS阳性产物,iNOSmRNA表达极低。实验组神经损伤后iNOSmRNA及其蛋白的表达水平均明显增高(P<0.01),iNOS阳性产物的吸光度(A)值在术后7d达高峰。iNOSmRNA表达在术后1、3、7d维持较高水平,此后则明显下降。上述结果说明大鼠坐骨神经损伤后神经纤维中iNOS的表达增加,iNOS可能在周围神经损伤后的再生过程中起着一定的作用。     

动脉硬化性脑梗塞患者载脂蛋白E基因多态性的研究

动脉硬化性脑梗塞患者载脂蛋白Ｅ基因多态性的研究鄢盛恺周新李秀玲余敦礼哈黛文载脂蛋白Ｅ（ａｐｏｌｉｐｏｐｒｏｔｅｉｎＥ，ａｐｏＥ）是一种含有２９９个氨基酸结合有磷脂的糖蛋白，由位于人第１９号染色体上的单一结构基因所编码。ＡｐｏＥ结构基因具有多态性，同一...     

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。     

内皮型、诱导型一氧化氮合酶在乳腺癌中的表达

目的 :研究内皮型一氧化氮合酶 (eNOS)、诱导型一氧化氮合酶 (iNOS)在乳癌中表达及与淋巴结转移的关系。方法 :采用免疫组化S P法检测 60例乳癌中eNOS和iNOS的表达。结果 :eNOS和iNOS阳性在乳癌中表达率分别为 75 0 %和71 7%。在淋巴结转移组和无淋巴结转移组中eNOS阳性表达率分别为 66 7%和 83 3 % ,两组间差异无统计学意义 (χ2 =2 2 2 ,P >0 0 5) ,而iNOS在淋巴结转移和无转移组中阳性表达率分别为 53 3 %和 90 0 % ,两组间差异有统计学意义 (χ2 =9 93 ,P <0 0 1 )。结论 :内皮型、诱导型一氧化氮合酶在乳腺癌中高表达 ;iNOS的表达与乳腺癌的淋巴转移相关     

SA-30诱导的小鼠子宫内诱导型一氧化氮合酶的变化

目的　检测精子膜抗原 SA- 30对小鼠子宫内诱导型一氧化氮合酶 (i NOS)含量及分布的影响 ,从而了解 SA - 30的抗生育机制。　方法　 SA- 30免疫雌性昆明小鼠后 ,应用免疫组织化学方法 ,对子宫内的 i NOS进行了检测。结果　 SA - 30免疫后对间情期小鼠子宫内的 i NOS没有明显影响 ,无论是免疫组还是对照组 ,在子宫内膜基质中均有大量阳性细胞分布 ,两者无明显差异 ;在妊娠第 3d时 ,对照组的小鼠子宫中有较多 i NOS表达 ,阳性标记主要集中在内膜上皮及固有膜中的子宫腺上 ,经 SA- 30免疫的小鼠子宫中 ,i NOS表达则显著减少 ,整个内膜标记极弱 ,只在子宫肌层内有少量 i NOS阳性细胞分布。　结论　 SA - 30可能通过使妊娠早期小鼠子宫内 i NOS表达显著减少 ,而影响小鼠胚胎植入及早期胚胎的发育     

寒冷应激对小鼠脑内一氧化氮及一氧化氮合酶的影响研究

目的探讨寒冷应激(-10℃、10h)对小鼠脑内一氧化氮(NO)及一氧化氮合酶(NOS)的影响及意义.方法将40只雄性昆明品系小鼠随机分为对照组和实验组,每组20只.寒冷应激实验结束后取脑组织检测一氧化氮含量及一氧化氮合酶活性.结果对照组NO及NOS分别为21.12±8.68μmol/gprot、2.21±0.57U/mgprot.实验组为16.03±6.98μmol/gprot、2.84±0.79U/mgprot.寒冷应激后NO水平降低并有统计学意义(t=2.58,P<0.05),NOS活性增高并有统计学意义(t=-2.72,P<0.05).应激后NO与NOS呈显著性负相关(r=-0.461,P<0.05).结论神经递质NO及NOS参与了中枢神经寒冷应激反应,且NO水平降低、NOS活性增高,可能对中枢神经细胞主要起保护作用.在寒冷应激后期吸入适量NO可能增强脑神经元对寒冷应激的耐受性.     

Failure of L-NAME to cause inhibition of nitric oxide synthesis: Role of inducible nitric oxide synthase

We addressed the hypothesis that administration of nitric oxide synthase inhibitor, N^G-nitro-L-arginine methyl ester (L-NAME) does not result in a sustained suppression of nitric oxide (NO) synthesis, because of a compensatory expression of inducible nitric oxide synthase (iNOS). L-NAME was administered in the drinking water (0.1–1.0 mg/ml) for 7 days to guinea pigs and rats. Nitric oxide synthesis was assessed by [1] ex vivo formation of nitrite in blood vessels and intestine [2] tissue levels of cGMP [3] iNOS gene expression by RT-PCR [4] NADPH diaphorase staining [5] direct assessment of NO release in tissue explants using a microelectrode/electrochemical detection system. Chronic L-NAME administration elevated intestinal cGMP and nitrite levels in guinea pigs (p<0.05). In rats, intestinal nitrite levels were comparable in control and L-NAME treatment groups, whereas direct assessment of NO release defined a marked increase in the L-NAME group. Chronic L-NAME resulted in an induction of iNOS gene expression in rats and guinea pigs and novel sites of NADPH diaphorase staining in the intestine. We conclude that iNOS expression is responsible for a compensatory increase or normalization of NO synthesis during sustained administration of L-NAME.accepted by G. Letts     

低氧对人脐静脉内皮细胞一氧化氮和内皮素-1的生成及诱导型一氧化氮合酶mRNA表达的影响

目的：通过观察低氧对人脐静脉内皮细胞(HUVECs)分泌一氧化氮(NO）、内皮素-1(ET-1)以及诱导型一氧化氮合酶(iNOS)mRNA 表达的影响，探讨低氧性肺动脉高压(HPH)形成的机制。 方法： 在离体培养人脐静脉内皮细胞基础上，采用硝酸还原酶和放射免疫分析方法检测了低氧（3％O2）培养6 h、12 h和24 h人脐静脉内皮细胞分泌NO和ET-1的变化，同时运用半定量RT-PCR对iNOS mRNA表达情况进行分析。 结果： 低氧组各时点培养液中NO2-/NO3-和ET-1水平显著高于常氧组(P<0.01)，同时观察到iNOS基因mRNA的表达也相应增高(P<0.01)。 结论： 低氧能刺激人脐静脉内皮细胞生成释放NO和ET-1, NO生成增多与低氧正调iNOS基因mRNA的表达有关。     

Vasopressin release during endotoxaemic shock in mice lacking inducible nitric oxide synthase

We tested the hypothesis that nitric oxide (NO) arising from the action of inducible nitric oxide synthase (iNOS) is responsible for the deficiency in vasopressin (AVP) release and consequent hypotension during endotoxaemic shock. Wild-type (WT) and iNOS knockout mice (iNOS^–/–) were given either saline or Escherichia coli lipopolysaccharide (LPS, 1.0 mg/kg i.v., final volume 0.03 ml). Mean arterial blood pressure (MAP) was measured and plasma AVP levels determined before and after LPS or saline injection. In WT mice, MAP was significantly lower 2 h after LPS administration and remained low for the remainder of the 6-h observation period. AVP plasma levels were increased at the 2nd and 4th h of the experiment, returning thereafter to basal levels. Conversely, LPS injection in iNOS iNOS^–/– mice elicited a sustained increase in plasma AVP concentration and attenuated the fall in blood pressure. These data indicate that NO arising from the iNOS plays an important inhibitory role in AVP release during endotoxaemia and may be responsible for the hypotension occurring during this vasodilatory shock.     

An inducible nitric oxide synthase polymorphism is associated with the risk of recurrent depressive disorder

Evidence indicates that depressive disorder is a heterogenic disease, and oxidative stress, inflammation and impairment of neurogenesis play a role in its aetiology. Moreover, there are data suggesting that genetic factors affect the development of depression. Nitric oxide (NO) is a biological molecule with both a beneficial and a detrimental role in brain. One of the three enzymes generating NO is inducible nitric oxide synthase (iNOS). Recent studies have shown that depressed patients are characterised by excessive NO production. In addition, iNOS inhibitors are effective in depression treatment. This study investigated the importance of a functional single nucleotide polymorphism (SNP), −1026C/A, located in the promoter region of the human NOS2A gene, for the risk of recurrent depressive disorder (RDD) vulnerability. The study was carried out in a group of 181 patients with RDD and 149 ethnically matched controls. Genotyping was performed by direct sequencing of the polymerase chain reaction (PCR) products. The genotype distribution of the −1026C/A polymorphism between depressed patients and healthy controls was significantly different. Individuals who were homozygous for the CC genotype exhibited an increased risk of developing RDD. In conclusion we cautiously conclude that polymorphism in the NOS2A gene promoter may play a role in the background of RDD.     

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.     

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。     

Spontaneous recovery of injured Achilles tendon in inducible nitric oxide synthase gene knockout mice

Objective and Design: To determine if inducible nitric oxide synthase (iNOS) gene could affect Achilles tendon healing using iNOS gene knockout mice. Methods: 21 iNOS knockout (iNOS^−/−) mice and 8 of the wild type (iNOS^+/+) mice were utilized in this study. Group 1: iNOS^+/+ mice (n = 8), group 2: iNOS^−/− mice (n = 11) and group 3: iNOS^−/− with a NOS inhibitor, (aminoguanidine, 500 mg/kg/day, via an intraperitoneal mini-osmotic pump for 7 days, n = 10). The right Achilles tendon was transected in all mice and harvested on day 7 for cross-sectional area and biomechanical properties. Serum nitrate concentration of the mice was measured by gas chromatography mass spectrometry (GC/MS). Results: A significant reduction in cross-sectional area of the healing Achilles tendon was observed in group 3 mice compared to group 2 mice (p < 0.01). The serum nitrate concentration in both group 2 and group 3 mice was lower than that in group 1 mice (p < 0.01) iNOS gene deletion and inhibition of NOS did not affect the biomechanical properties of the healing tendons. Conclusions: iNOS gene is not solely responsible for the beneficial effects of nitric oxide (NO) on tendon healing. Received: 30 March 2005; returned for revision 4 August 2005; returned for final revision 27 September 2005; accepted by M. Parnham 28 September 2005     

Effect of skin sensitizers on inducible nitric oxide synthase expression and nitric oxide production in skin dendritic cells: role of different immunosuppressive drugs

Nitric oxide (NO) is involved in the pathogenesis of acute and chronic inflammatory conditions, namely in allergic contact dermatitis (ACD). However, the mechanism by which NO acts in ACD remains elusive. The present study focuses on the effects of different contact sensitizers (2,4-dinitrofluorbenzene, 1,4-phenylenediamine, nickel sulfate), the inactive analogue of DNFB, 2,4-dichloronitrobenzene, and two irritants (sodium dodecyl sulphate and benzalkonium chloride) on the expression of the inducible isoform of nitric oxide synthase (iNOS) and NO production in skin dendritic cells. It was also studied the role of different immunosuppressive drugs on iNOS expression and NO production. Only nickel sulfate increased the expression of iNOS and NO production being these effects inhibited by dexamathasone. In contrast, cyclosporin A and sirolimus, two other immunosuppressive drugs tested, did not affect iNOS expression triggered by nickel.     

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。