慢性低O2高CO2肺动脉高压大鼠血浆NO和肺动脉NOS及其基因表达的变化

目的：探讨一氧化氮体系在慢性低O₂高CO₂肺动脉高压形成中的作用。方法：雄性Sprague-Dawley大鼠分为正常对照组和4周低O₂高CO₂肺动脉高压组。测定血浆NO含量,免疫组化法检测肺细小动脉cNOS和iNOS活性,原位杂交法检测其cNOS mRNA和iNOS mRNA的表达。结果：肺动脉高压组血浆NO含量、肺细小动脉cNOS活性和cNOS mRNA表达显著低于对照组（均P<0.01）,而iNOS活性和iNOS mRNA表达明显高于对照组（均P<0.01）。结论：低O₂高CO₂时肺动脉NOS活性和NOS mRNA表达的改变引起的NO变化参与了肺动脉高压的形成。     

内源性H2S对LPS所致大鼠急性肺损伤时肺动脉高压的影响及其与一氧化氮的相互作用

目的： 探讨硫化氢（H2S）对脂多糖（LPS）所致急性肺损伤时肺动脉高压（PAH）的影响及H2S/胱硫醚-γ-裂解酶（CSE）体系和一氧化氮（NO）/一氧化氮合酶（NOS）体系在其发生机制中的相互作用。 方法： 将72只大鼠随机分为生理盐水（NS）对照组、LPS组、LPS+L-NAME组、LPS+PPG组，检测给药后2、4、6、8 h的平均肺动脉压（mPAP），以及4、8 h血浆H2S、NO含量和iNOS、cNOS活性、肺组织NO含量和iNOS、cNOS、CSE活性，免疫组化法测定肺组织iNOS蛋白表达，并结合肺光镜形态等指标综合评价肺损伤程度。 结果： LPS组各时点的mPAP显著高于对照组，给药后4、8 h，NO含量、iNOS活性和蛋白表达升高，cNOS活性及H2S含量、CSE活性降低，肺组织损伤较重。预先给予L-NAME可减轻LPS所致上述指标的改变。而预先给予PPG可加重LPS所致肺损伤，但对cNOS活性无明显影响。 结论： LPS使内源性H2S减少导致mPAP升高； H2S/CSE体系与NO/NOS体系共同参与LPS所致急性肺损伤时PAH形成的调控机制，在其中呈相互的负性调节作用。     

慢性乙型肝炎患者体内一氧化氮和一氧化氮合酶水平的研究

目的　探讨慢性乙型肝炎(慢乙肝)患者体内一氧化氮(NO)和一氧化氮合酶(NOS)水平及其意义。方法　检测37例慢乙肝患者NO、NOS[诱生型NOS(iNOS)和结构型NOS(cNOS) ]、肝功能、乙型肝炎病毒(HBV)DNA和HBV基因型(GT)并做出统计分析。结果　慢乙肝患者组与正常对照组比较,NO和iNOS的浓度均明显升高(P <0 0 5 ) ;丙氨酸转氨酶(ALT)异常组与正常对照组及ALT正常组比较:NO和iNOS的浓度均明显升高(P <0 0 5 ) ;ALT正常组与正常对照组比较:NO的浓度明显升高(P <0 0 5 ) ;cNOS在各组间比较差异无统计学意义。在慢乙肝患者中:NO和iNOS浓度与ALT水平呈明显正相关(r=0 36 7,r=0 4 74 )。NO和NOS与HBVDNA指标间均无明显相关关系。不同基因型组之间,NO和NOS的浓度差异无统计学意义(P >0 0 5 )。结论　在慢乙肝患者中,存在NO和iNOS水平升高的现象。慢乙肝患者ALT升高时,NO浓度高,对机体损伤重;慢乙肝患者ALT正常者,NO对机体无明显的损伤。NO和NOS与HBVDNA是相对独立的检测指标。NO水平与不同HBVGT患者病情及预后不同无明显关系。     

Augmentation of nitric oxide production by gamma interferon in a mouse vascular endothelial cell line and its modulation by tumor necrosis factor alpha and lipopolysaccharide

The effect of gamma interferon (IFN-gamma), tumor necrosis factor alpha (TNF-alpha), and lipopolysaccharide (LPS) on nitric oxide (NO) production in the mouse vascular aortic endothelial cell line END-D was examined. LPS, TNF-alpha, and a low concentration of IFN-gamma inhibited NO production in END-D cells, while a high concentration of IFN-gamma definitely enhanced it. The NO production induced by a high concentration of IFN-gamma was further augmented by using IFN-gamma in combination with LPS or TNF-alpha. In sequential incubations of LPS and IFN-gamma, the enhancement of NO production required prior treatment with IFN-gamma. Stimulation of END-D cells with a high concentration of IFN-gamma led to the expression of inducible NO synthase (iNOS). The augmentation of NO production by IFN-gamma alone or in combination with LPS or TNF-alpha was completely blocked by several inhibitors of iNOS. It was strongly suggested that a high concentration of IFN-gamma itself enhanced NO production in END-D cells through inducing the expression of iNOS. LPS and TNF-alpha exclusively modulated the activity of iNOS once its expression was triggered by IFN-gamma. On the other hand, a low concentration of IFN-gamma, LPS, and TNF-alpha reduced NO production through down-regulating constitutive NOS (cNOS). The differential regulation of cNOS- and iNOS-mediated NO production by IFN-gamma, TNF-alpha, and LPS is discussed.     

Constitutively synthesized nitric oxide is a physiological negative regulator of mammalian angiogenesis mediated by basic fibroblast growth factor

We recently reported that the systemically administered nitric oxide synthase (NOS) inhibitor Nw-nitro-L-arginine methyl ester, L-NAME, administered before, during and after the angiogenic treatment stimulated angiogenesis induced by basic fibroblast growth factor, bFGF, in the rat. This suggests that suppression of constitutively expressed NOS, cNOS, plus inducible NOS, iNOS, and thus reduced production of nitric oxide, NO, was the stimulating factor. In those studies, the rat mesenteric-window angiogenesis assay was used. Moreover, the systemic administration of a NO releaser inhibited bFGF-mediated angiogenesis. Using the same experimental system, we have now studied whether the inhibition of cNOS alone in adult animals under physiological conditions, i.e. prior to the administration of the angiogenic stimulation with bFGF, affected the subsequent angiogenic response. cNOS constitute endothelial cell NOS (ecNOS) and neuronal NOS (nNOS). L-NAME or its inactive enantiomer Nw-nitro-D-arginine methyl ester, D-NAME, were given continuously in the drinking water (1.0 g/L) during 14 days prior to the start of the treatment with bFGF. The treatment with L-NAME significantly enhanced the subsequent angiogenic response. NO synthesized under physiological conditions by ecNOS in endothelial cells and platelets or nNOS in platelets may thus act as a first constitutional angiostatic factor in bFGF-mediated mammalian angiogenesis.     

LPS induces inflammatory responses in human aortic vascular smooth muscle cells via Toll-like receptor 4 expression and nitric oxide production

Inflammation is an important event in the development of vascular diseases such as hypertension, atherosclerosis, and restenosis. In addition, the stimulation of Toll-like receptor 4 (TLR4) by lipopolysaccharide (LPS) induces the release of critical proinflammatory cytokines that activate potent immune responses. In this study, LPS was found to induce TLR4 expression and increased nitric oxide (NO) production by increasing the expression of inducible nitric oxide synthase (iNOS). Furthermore, LPS was found to induce interleukin (IL)-8 and vascular endothelial growth factor (VEGF) production, as well as intracellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) expression. Taken together, these results indicate that LPS induces inflammatory responses in HASMC. Moreover, NOS inhibitor (L-NAME) and anti-TLR 4mAb reduced the LPS-induced NO, IL-8 and VEGF production and ICAM-1 expression. Additionally, TLR4 expression was reduced by NOS inhibitor. Taken together, these results indicate that LPS-induced inflammatory responses are regulated by TLR4 expression and NO production.     

抗血小板药物对缺血再灌注后一氧化氮合酶的影响

目的:评价急性心肌梗死（acute myocardial infarction, AMI）再灌注后一氧化氮合酶(NOS)的变化及抗血小板药物对其影响。 方法:中华小型猪24只，随机分成对照组、氯吡格雷与阿司匹林合用治疗组、替罗非斑治疗组和假手术组,每组8只。冠状动脉结扎3 h , 松解1 h制备AMI再灌注模型。采用硝酸还原酶法检测AMI前、后和再灌注后血一氧化氮 (NO) 的含量;采用催化L-Arg法和逆转录-聚合酶链反应（RT-PCR）的方法观察正常、再流和无再流区心肌组织内NOS及其mRNA的表达。 结果:（1）替罗非斑组可提高血NO水平(P<0.05-0.01)，增加再流区心肌组织中cNOS活性及其mRNA表达，减少再流区心肌组织中iNOS活性及其mRNA表达(均P<0.05-0.01)。而氯吡格雷与阿司匹林合用不能提高血NO水平，增加再流区心肌组织中cNOS活性及其mRNA表达，仅能减少再流区心肌组织中iNOS活性及其mRNA表达。 结论:替罗非斑可能通过保护内皮细胞起到了减少无再流的作用，而氯吡格雷与阿司匹林合用并不能保护内皮功能，仅能减轻再灌注后的炎性反应。     

体外反搏对心肌梗死犬一氧化氮系统的影响

目的:探讨体外反搏对心肌梗死犬一氧化氮(NO)、一氧化氮合酶(NOS)和其基因表达的影响。方法:19只健康杂种犬随机分为对照组、缺血组和缺血+反搏组(反搏组)3组,采用开胸结扎冠状动脉左前降支的方法建立心肌缺血模型,用改良硝酸还原酶法测定心肌缺血前后血清NO含量、以及心肌组织的NO含量和NOS比活性,采用免疫组化方法检测缺血区心肌组织的NOS亚型即诱导型NOS(iNOS)和内皮型NOS(eNOS)的蛋白合成,用原位杂交方法检测构成型NOS(cNOS)信使核糖核酸(mRNA)的基因表达。结果:在冠状动脉结扎前和结扎后60min,3组犬血清NO含量均无明显差异(P＞0.05);结扎后120min和180min时,反搏组犬血清NO含量明显高于缺血组(P＜0.05)。正常组和反搏组犬心肌组织NO含量和NOS比活性均大于缺血组(P＜0.05)。免疫组化结果表明心肌缺血时iNOS蛋白合成增多,而eNOS蛋白合成减少;体外反搏对iNOS有抑制作用,对eNOS有促进作用。此外心肌缺血时cNOSmRNA的表达明显减少,反搏可促进cNOSmRNA的表达。结论:体外反搏促进NO的产生可能是其抗心肌缺血性损伤的重要机制之一。     

肢体缺血预适应的小肠保护作用及与一氧化氮/内皮素-1的关系

目的：观察一氧化氮/内皮素-1(NO/ET-1)失衡与肢体缺血再灌注(I/R)后小肠损伤的关系，以及缺血预适应(IPC)对NO/ET-1系统的调节作用。 方法： 雄性Wistar大鼠18只，随机分为对照（control）组，缺血再灌注（IR）组和缺血预适应（IPC+IR）组，每组6只，分别测定血浆和小肠组织二氨氧化酶(DAO)、一氧化氮(NO)、内皮素-1(ET-1)、NO/ET-1比值的含量变化及小肠组织的髓过氧化物酶(MPO)、DNA双链百分率(ratio of DNA chain %)、总一氧化氮合酶（tNOS）、诱导型一氧化氮合酶（iNOS）、结构型一氧化氮合酶（cNOS）的水平；免疫组化法检测小肠组织的诱导型一氧化氮合酶（iNOS）、内皮型一氧化氮合酶（eNOS）的表达。 结果： IR组血浆和小肠组织NO、ET-1，血浆DAO及组织MPO均明显高于对照组，而 NO/ET-1的比值，组织DAO及DNA双链百分率明显少于对照组；小肠粘膜iNOS的表达及总NOS活性高于对照组，cNOS（主要为eNOS）的表达少于对照组。IPC+IR组血浆和小肠组织NO、ET-1，血浆DAO及组织MPO均明显少于IR组，而 NO/ET-1的比值，组织DAO及DNA双链百分率却明显高于IR组；小肠粘膜iNOS的表达及总NOS活性少于IR组，cNOS（主要为eNOS）的表达高于IR组。 结论： 肢体I/R后小肠损伤与NO/ET-1失衡有关，IPC对肢体IR继发的小肠粘膜损伤的拮抗作用可能通过对NO/ET-1系统的调节作用而介导，此时内皮源的NO产生增加，非内皮源的NO产生减少。     

高原低氧对大鼠下丘脑谷氨酸、天门冬氨酸和NOS的影响

目的:观察高原低氧大鼠下丘脑谷氨酸(Glu)、天门冬氨酸(Asp)和一氧化氮合酶(NOS)的变化。方法:应用氨基酸测定和NADPH-d组化法,检测高原低氧模型大鼠下丘脑Glu、Asp含量和NADPH-d阳性神经元的数量。结果:高原低氧大鼠下丘脑Glu、Asp含量明显增多,室旁核、视上核可见密集深染的NADPH-d阳性神经元;用NMDA受体拮抗剂氯氨酮(Ketamine)和AP-V对高原低氧大鼠进行预处理后置于低压氧舱,观察到大鼠下丘脑室旁核、视上核NADPH-d阳性神经元数明显少于相应时间的高原低氧组(P＜0.01)。结论:NMDA受体可能参与了高原低氧引起的下丘脑NOS的表达。     

A defect in HIV-1 transgenic murine macrophages results in deficient nitric oxide production

HIV transgenic mice bearing multiple copies of a noninfectious (Deltagag/pol) proviral DNA were tested for the systemic production of nitric oxide (NO). Serum levels of NO metabolites were reduced about 50% in HIV transgenic mice compared with nontransgenic sibling mice. This difference persisted when NO production was induced with peritoneal injections of bacterial endotoxin (LPS). Peritoneal inflammatory macrophages, but not resident peritoneal macrophages, derived from HIV-1 transgenic mice and activated in vitro with LPS and IFN-gamma (or tumor necrosis factor alpha and IFN-gamma) also produced about 50% less NO than did macrophages harvested from nontransgenic littermates. Isogenic, transgenic mice bearing mutated nef or vpr genes had normal serum levels of NO metabolites and their macrophages produced normal levels of NO when stimulated. An explanation for the reduced NO response of HIV[Vpr+Nef+] macrophages was not apparent from measured levels of iNOS expression, viral gene expression, or arginase activity in activated macrophages. Inhibition of nitric oxide synthase (NOS) isoforms with L-NAME or aminoguanidine blocked time-dependent increases in HIV gene expression in activated macrophages cultured ex vivo. Inhibition with L-NAME occurred despite high levels of NO generated by iNOS, and exogenously supplied NO induced HIV gene expression only weakly, suggesting that cNOS had the greater influence on proviral gene induction. This system is presented as a model of HIV-1 proviral gene expression and dysfunction in macrophages.     

炎性细胞因子及脂多糖诱导产生的一氧化氮对人血管内皮细胞的损伤作用

目的:探讨炎症过程诱生的一氧化氮(NO)对内皮细胞的损伤及其作用机制。方法:黄递酶法及Griess法检测可诱导性NO合酶(iNOS)活性、NO₂^-/NO₃^-水平,RT-PCR技术分析iNOSmRNA的表达;同时观察NO产生后对内皮细胞的损伤作用。结果:IL-1β2×10⁵U/L、TNF-α5×10⁵U/L、γ-INF2×10⁵U/L联用LPS(10mg/L)可诱导出高浓度NOS合成及NO产生,比单用这些细胞因子或LPS诱导的量高两倍多,iNOSmRNA的表达水平也显著增加;同时MDA及LDH释放率明显增加,细胞存活率下降,并伴随细胞受损的形态学改变。而单用上述细胞因子或LPS,以及降低剂量或缩短处理时间,其诱生的NOS及NO与正常对照相比P＞0.05;但MDA及LDH释放率仍增加明显。使用2倍剂量的这些炎性细胞因子或延长处理时间到48h,与标准剂量及24h组相比NOS和NO的增加虽不显著,但细胞生长受限更明显。NOS抑制剂能阻止NO的产生及其对细胞的损伤作用。结论:炎性细胞因子及脂多糖可激活iNOS大量表达诱生高浓度的NO,对内皮细胞具有明显的氧化损伤作用。     

Myometrial constitutive nitric oxide synthase expression is increased during human pregnancy

Nitric oxide (NO), derived from L-arginine by the action of nitric oxide synthase (NOS), is a mediator of many diverse biological activities, including vasodilation, neurotransmission and inhibition of platelet adhesion. A role for NO in the maintenance of rat and rabbit pregnancy is supported by a variety of studies. A recent study in women demonstrated that myometrial inducible NOS (iNOS) expression was greater in the early third trimester than either the late third trimester or in the non-pregnant condition, suggesting that increased iNOS expression is involved in the maintenance of human pregnancy. Constitutive NOS (cNOS) expression was not determined. The aim of this study was to compare constitutive NOS (both eNOS and bNOS) expression in the human non-pregnant uterus, preterm pregnant uterus (25-34 weeks gestation) and term pregnant uterus (>37 weeks gestation) using immunohistochemistry and Western blotting. Preterm pregnant samples were taken from women with a variety of pathologies necessitating early delivery. We found that eNOS and bNOS protein concentrations were greater in the preterm pregnant myometrium than non-pregnant myometrium. eNOS, but not bNOS, protein concentration was lower in myometrial samples obtained at term compared with those obtained preterm. We conclude that the constitutive isoforms of NOS are also up-regulated in human pregnancy and may play a role in the maintenance of myometrial quiescence.     

Myenteric denervation in gastric carcinogenesis: differential modulation of nitric oxide and annexin-A1

This study evaluated the properties of endogenous nitric oxide synthases (NOS) and annexin-A1 (ANXA1) and determined how they can be exploited in the N-methyl-N-nitro-N-nitrosoguanidine (MNNG)-induced gastric carcinogenesis and myenteric denervation model. Male Wistar rats were treated with MNNG and/or aminoguanidine (AG) for 20 weeks. In another set of experiments, rats with nondenervated and denervated stomachs were treated with MNNG or water for 28 weeks. Fragments of the pyloric region were processed for histopathology, NOS activity, and immunohistochemistry to explore the activity and expression of constitutive (cNOS) and inducible (iNOS) NO synthase and their relationship with annexin-A1 (ANXA1) expression. NO inhibition by AG increased the percentage of animals with adenocarcinomas (~29%) compared with the untreated MNNG group (~4%). Myenteric denervation did not alter NOS activity. cNOS activity was significantly greater in nondernervated and denervated stomachs with or without lesions (P<0.001) than iNOS activity (P<0.01), as confirmed by immunohistochemistry. Further, cNOS activity in normal stomachs and outside the lesion area was considerably higher than inside it (P<0.01). By densitometric analysis of nondenervated and denervated stomachs, ANXA1 expression was modulated in epithelial and inflammatory cells (mast cells and neutrophils), wherein significant alterations were induced by lesion development and myenteric denervation. In conclusion, NO protects against the development of gastric adenocarcinomas. The pattern of ANXA1 expression was not associated with NOS activity or expression, suggesting that NO and ANXA1 act in gastric tumors in disparate pathways.     

诱生型一氧化氮合酶抑制剂硫酸甲基异硫脲对内毒素血症大鼠肠系膜淋巴管一氧化氮合酶表达的影响

目的 :研究内毒素血症中诱生型一氧化氮合酶 (induciblenitricoxidesyhthases,iNOS)抑制剂硫酸甲基异硫脲(S Methy1isothioureasulfate,SMT)对大鼠肠系膜淋巴管一氧化氮合酶 (nitricoxidesyhthase,NOS)表达的影响。探讨在内毒素血症中一氧化氮 (nitricoxide,NO)对肠系膜淋巴微循环的影响。方法 :利用NADPH 黄递酶组织化学染色方法观察NOS在大鼠肠系膜淋巴管的表达 ,用硝酸还原酶法测定血清中NO的含量。结果 :脂多糖(lipopolysaccharide,LPS)组大鼠随时间延长肠系膜淋巴管染色强度明显加深 ,血清NO含量显著升高。LPS SMT组染色强度比LPS组降低 ,血清NO含量显著下降。结论 :在内毒素血症中 ,SMT可能通过抑制NOS的活性而起保护作用。     

Non-lethal Sepsis and Nitric Oxide Inhibition in the Guinea Pig

Nitric oxide (NO) is synthesized from arginine by three distinct isoforms of nitric oxide synthase (NOS). Two of these isoforms, endothelial NOS (eNOS, type III NOS) and neuronal NOS (nNOS, type I NOS) are expressed in a constitutive manner and are responsible for regulating physiological functions. The induction of the third isoform of nitric oxide synthase (iNOS) by inflammatory processes and the subsequent overproduction of NO is thought to contribute to the tissue damage that occurs in a number of diseases having an inflammatory component, such as sepsis. As a model of sepsis, non-lethal endotoxemia in Hartley guinea pigs was induced using three serotypes of lipopolysaccharide (LPS, 30 mg/kg, i.p.). Each stimulated an increase in total plasma nitrites 6 h following their administration. Treatment orally with two inhibitors of NOS, L-nitroarginine methyl ester (L-NAME), a non-selective inhibitor and N-iminoethyl-L-lysine (L-NIL), a selective iNOS inhibitor, 30 min after the induction of sepsis, inhibited (p < 0.05) the increase in plasma nitrites with ED₅₀ values of 7 ± 1 and 0.4 ± 0.03 mg/kg, respectively, suggesting that NOS inhibitors may be useful in the treatment of human sepsis, or other diseases where excess NO levels have been implicated, such as asthma, arthritis and inflammatory bowel disease.     

罗格列酮对改善糖尿病大鼠心肌微血管内皮细胞功能的影响

目的探讨罗格列酮对糖尿病大鼠心肌微血管内皮细胞功能的影响。方法腹腔注射小剂量链脲佐菌素结合高能量饲料制备2型糖尿病大鼠模型，随机分为未治疗组与治疗组：罗格列酮4mg／（kg·d）。16周后定量测定血管内皮损伤标志物：可溶性血管内皮细胞蛋白C受体（sEPCR）、可溶性血栓调节蛋白（sTM）、血管性血友病因子（VWF）和一氧化氮（NO）的血浆浓度。分光光度法及免疫组化法分别测定心肌组织内NO浓度、NO合酶（NOS）的活性及内皮型NO合酶（cNOS）在微血管内皮细胞的表达。结果罗格列酮治疗组血糖及血浆中sEPCR、sTM、vWF显著低于未治疗组，但高于非糖尿病对照组。与未治疗组比较，罗格列酮治疗组心肌组织中NO、结构型NOS（cNOS）含量增高，eNOS阳性反应产物量增多，而诱导型NOS（iNOS）含量降低。结论罗格列酮可以降低糖尿病大鼠血糖，减轻内皮细胞损伤与改善心肌微血管内皮细胞功能，有助于减少糖尿病时心肌微血管病的发生与发展。     

Nitric oxide in health and disease of the respiratory system

During the past decade a plethora of studies have unravelled the multiple roles of nitric oxide (NO) in airway physiology and pathophysiology. In the respiratory tract, NO is produced by a wide variety of cell types and is generated via oxidation of l-arginine that is catalyzed by the enzyme NO synthase (NOS). NOS exists in three distinct isoforms: neuronal NOS (nNOS), inducible NOS (iNOS), and endothelial NOS (eNOS). NO derived from the constitutive isoforms of NOS (nNOS and eNOS) and other NO-adduct molecules (nitrosothiols) have been shown to be modulators of bronchomotor tone. On the other hand, NO derived from iNOS seems to be a proinflammatory mediator with immunomodulatory effects. The concentration of this molecule in exhaled air is abnormal in activated states of different inflammatory airway diseases, and its monitoring is potentially a major advance in the management of, e.g., asthma. Finally, the production of NO under oxidative stress conditions secondarily generates strong oxidizing agents (reactive nitrogen species) that may modulate the development of chronic inflammatory airway diseases and/or amplify the inflammatory response. The fundamental mechanisms driving the altered NO bioactivity under pathological conditions still need to be fully clarified, because their regulation provides a novel target in the prevention and treatment of chronic inflammatory diseases of the airways.