肝硬化大鼠全胃肠外营养时一氧化氮和一氧化氮合酶活性的变化及意义

目的 观察一氧化氮（NO）、一氧化氮合酶（NOS）活性在肝硬化大鼠全胃肠外营养（TPN）时的变化,并探讨其意义。方法 Wistar大鼠分为3组：正常组8只,肝硬化对照组6只,肝硬化TPN组7只。测定血清NO浓度、肝脏NO含量和肝脏NOS比活性,观察肝脏β－BADPH黄递酶组化染色。结果 除TPN组一只大鼠因输液过快,肺水肿死亡外,其余大鼠均成活。血清NO浓度、肝脏NOS比活性,肝硬化对照组比正常组升高明显（P＜0．05）,肝硬化TPN级比肝硬化对照组升高明显（P＜0．05）。肝脏NO含量,肝硬化对照组比对照组升高明显（P＜0．05）,但肝硬化TPN组比肝硬化对照组降低明显（P＜0．05）。肝脏β－NADPH黄递酶组化染色,正常组为－,肝硬化对照组为＋,肝硬化TPN组为＋＋。结论 NO可能是一种抗肝损伤因子,损伤因子存在时,表现为肝脏NOS活性增强,当肝脏NO含量减少时,表现为肝脏损害的加重。     

肝硬化患者血浆一氧化氮与GMP-140水平的变化

目的：观察肝硬化患者血浆一氧化氮（ＮＯ）与可溶性α－颗粒膜蛋白（ＧＭＰ－１４０）水平的变化。方法：分别采用硝酸还原酶法和酶联免疫分析法,测定２９例肝硬化（无腹水）、２３例肝硬化（有腹水）患者和３０例健康人的血浆ＮＯ２＾－／ＮＯ３＾－水平及ＧＭＰ－１４０含量。结果：肝硬化患者有腹水组和无腹水组较健康对照组血浆ＮＯ２＾－／ＮＯ３＾－水平均有显著性增高（Ｐ〈０．０１）;肝硬化有腹水组较健康对照组ＧＭＰ－１４０含量显著性增高（Ｐ〈０．０１）,无腹水且亦增高但无显著性意义。结论：提示ＮＯ和粘附分子可能参与肝硬化的发生发展过程。     

内毒素对正常大鼠和肝硬化大鼠肝脏一氧化氮合酶活性及局部一氧化氮水平的影响

观察内毒素血症时肝硬化大鼠和正常大鼠肝脏一氧化氨台酶(NOS)活性以及NO水平的变化．探讨两者在肝硬化大鼠对内毒素高敏感性中的作用。方法：肝硬化大鼠模型由四氯化碳台并乙醇诱导．内毒素4mg／kg体重经尾静脉注射。分别于注射后2、6、12小时测定血清咎丙转氨酶(ALT)、谷草转氨酸(AST）肝脏组织中NOS活性以及N0代谢产物NO^-／NO^-水平。结果：肝硬化大鼠对内毒素损伤敏感性增高。正常大鼠在内毒素作用2小时后NOS活性和NO水平均显著增高．并持续至12d,时,但肝硬化大鼠肝脏NOS活性则未见显著变化,NO水平在12小时才开始增高。结论：肝硬化时肝脏NOS对内毒素刺激的敏感性降低,NO产生减少,可能是导致肝硬化肝脏对内毒素损伤高敏感性的原因之一。     

清肠栓对溃疡性结肠炎大鼠一氧化氮和一氧化氮合酶活性的影响

目的：观察不同剂量清肠栓治疗溃疡性结肠（UC）的作用及其对一氧化氮（NO）和一氧化氮合酶（NOS）活性的影响。方法：将动物随机分成高、中、低剂量清肠栓组、SASP组、模型组、空白组,除空白组外其余5组动物分别用三硝基苯磺酸（TNBS）建立大鼠溃疡性结肠炎模型,连续用药2周后取结肠组织,采用改良的G法、分光光度法分别测定其NO含量和NOS活性。结果：模型组大鼠NO、NOS含量较空白组明显降低,其他各组均有不同程度的增高,尤其以清肠栓高剂量组的增高为明显。结论：TNBS急性损伤使结肠组织的NO、NOS活性发生改变,可能是UC发生的重要机制。中药复方清扬栓具有调控NO及NOS活性的作用,是有效治疗UC的可能机制之一。     

卡托普利对缺血再灌注兔心肌一氧化氮及其合酶活性的影响

目的：探讨卡托普利（ｃａｐｔｏｐｒｉｌ）对局部缺血再灌注免心肌保护作用的机制。 方法：将１８只新西兰兔随机分３组（每组ｎ＝６）,对照组左冠状动脉前降支阻断３０分,再灌注９０分;卡托普利组阻断前３０分静脉注射卡托普利每 ２０分 ２ ｍｇ／ｋｇ,再灌注时再持续静脉注射卡托普利每 ９０分１ｍｇ／ｋｇ,假手术组环左冠状动脉前降支置线但不阻断血流。观察心肌一氧化氮合酶（ＮＯＳ）同工酶活性、过氧化物歧化酶活性、丙二醛含量、肌酸激酶含量及右心房血一氧化氮（ＮＯ）的变化,监测心肌功能。 结果：缺血再灌注心肌原生型ＮＯＳ（ＣＮＯＳ）活性（Ｐ＜０．００１）及总ＮＯＳ活性（Ｐ＜０．０１）显著下降,ＮＯ产生减少（Ｐ＜０．０５～０．０１）,卡托普利组缺血再灌注期间ＮＯ水平高于对照组（Ｐ＜０．０１）,再灌注３０分心肌ＣＮＯＳ活性（Ｐ＜０．０１）及总ＮＯＳ活性（Ｐ＜０．０５）显著高于对照组,心肌损害较对照组减轻。 结论：ＮＯ产生不足是心肌再灌注损伤的重要因素,卡托普利通过调节ＮＯＳ活性,维持正常ＮＯ水平起到保护作用。     

肝硬化大鼠内脏血管壁NOS分布的免疫组化研究

目的：观察肝硬化门静脉高压大鼠内脏动、静脉血管壁一氧化氮合酶（ＮＯＳ）的分布及染色强度变化,探讨ＮＯ在门静脉高压形成机制中的作用。方法：采用免疫组化染色法,应用两种ＮＯＳ特异性抗体,分别观察内皮型（ｅＮＯＳ）和诱生型（ｉＮＯＳ）ＮＯＳ的变化特点,并结合计算机图象分析系统对染色强度进行量化处理。结果：肝硬化大鼠肠系膜上动脉（ＳＭＡ）ｉＮＯＳ和ｅＮＯＳ染色强度与对照组相比均显著增加（Ｐ＜０．０１）,其中以ｅＮＯＳ增加更为明显。而两组门静脉（ＰＶ）ＮＯＳ染色强度则无明显差异（Ｐ＞０．０５）。肝硬化组ＳＭＡ的ＮＯＳ染色强度明显高于ＰＶ（Ｐ＜０．０５）。结论：ＮＯＳ在内脏血管表达增多,以及在ＳＭＡ的表达高于ＰＶ,提示ＮＯ可能主要通过扩张内脏动脉、增加内脏血流量而参与门静脉高压的形成。     

缺血期补充硝酸甘油对离体大鼠心脏心肌缺血再灌注损伤的作用

目的：观察离体大鼠心脏缺血期补充一氧化氮（ＮＯ）供体对缺血再灌注损伤的影响。　　方法：将２６只离体大鼠心脏缺血３０分，再灌注６０分。分为两组，用药组（１５只）及对照组（１１ 只）。用药组于缺血期给予４．４×１０－ ３ ｍｍ ｏｌ／Ｌ硝酸甘油（ｎｉｔｒｏｇｌｙｃｅｒｉｎ，ＮＴＧ，一种ＮＯ供体），碳酸氢盐缓冲液灌注。对照组仅给予碳酸氢盐缓冲液灌注。全部心脏均测定ＮＯ释放量、肌酸激酶漏出量及（或）心脏功能。　　结果：用药组大鼠心脏，使用ＮＴＧ表现为两种效应。其中部分大鼠心脏（非心室颤动组，ｎ＝ ７），ＮＴＧ增加肌酸激酶漏出量，减弱再灌注期心脏功能的恢复，伴随缺血期ＮＯ释放量的增加。对另一部分大鼠心脏（心室颤动组，ｎ＝ ８），ＮＴＧ减少肌酸激酶的释放，但心脏于再灌注期持续心室颤动，缺血期ＮＯ释放量无明显增加。　　结论：缺血期给予同一剂量ＮＴＧ对心肌缺血再灌注损伤产生双重效应，既可增加心肌损伤，又可减轻心肌损伤。     

急性心肌梗死大鼠心脏一氧化氮合酶表达的改变

目的 通过建立大鼠心肌梗死模型，观察急性心肌梗死对大鼠心脏内皮型一氧化氮合酶mRNA和诱导型一氧化氮合酶蛋白表达的影响。方法48只健康成年SD大鼠（体重200～250g）随机分为假手术组和缺血组，取1、2、8和24h四个不同时间点观察。采用开胸结扎冠状动脉左前降支建立心肌缺血模型，逆转录聚合酶链反应检测大鼠心肌梗死后1、2及24h三个时段缺血心肌内皮型一氧化氮合酶mRNA的表达；免疫组织化学染色检测冠状动脉结扎后8h缺血心肌诱导型一氧化氮合酶蛋白的表达。结果冠状动脉结扎后2h，缺血组大鼠缺血心肌组织内皮型一氧化氮合酶mRNA表达下降（P〈0．05），并持续至结扎后24h；结扎后24h组内皮型一氧化氮mRNA的表达与结扎后2h组相比无显著性差异（P〉0．05）。冠状动脉结扎后8h，梗死区存活心肌组织细胞诱导型一氧化氮合酶蛋白大量表达，而假手术组未见诱导型一氧化氮合酶蛋白表达。结论正常大鼠心肌组织有内皮型一氧化氮合酶基因表达，无诱导型一氧化氮合酶蛋白表达。在心肌梗死早期缺血心肌内皮型一氧化氮合酶mRNA表达减少。心肌急性缺血刺激早期诱导大鼠缺血心肌组织诱导型一氧化氮合酶蛋白大量表达。     

Non-Anticoagulant Heparin Increases Endothelial Nitric Oxide Synthase Activity: Role of Inhibitory Guanine Nucleotide Proteins

Heparin, which is widely used clinically, has recently been shown to have specific properties affecting the vascular endothelium. We hypothesized that heparin stimulates endothelial nitric oxide synthase (eNOS) activity by a mechanism independent of its anticoagulant properties and dependent on an inhibitory guanine nucleotide regulatory protein (Gi). We determined the effect of both heparin andN-acetyl heparin (Non-Hep), a heparin derivative without anticoagulant properties, on eNOS activity in cultured bovine aortic endothelial cells and on endothelium-dependent relaxation in isolated vascular rings. The eNOS activity was determined by measuring both citrulline and nitric oxide (NO) metabolite formation. Heparin and Non-Hep dose-dependently increased basal eNOS activity (ED₅₀1.0μg/ml or 0·15 U/ml), an effect that was significantly inhibited by pertussis toxin (100 ng/ml), a Gi-protein inhibitor. Agonist-stimulated (acetylcholine, 10μ ) eNOS activity was potentiated following pre-treatment with both heparin and Non-Hep and reversed by pertussis toxin. Heparin and Non-Hep induced a dose-dependent relaxation in preconstricted thoracic aortic rings, an effect that was significantly inhibited by pertussis toxin, endothelial inactivation (following treatment with sodium deoxycholate) andN^G-nitro- -arginine-methyl ester (L-NAME). We conclude that heparin and non-anticoagulant heparin induce endothelium-dependent relaxation following activation of eNOS by a mechanism involving a Gi-protein. Administration of heparin derivatives without anticoagulant properties may have therapeutic implications for the preservation of eNOS in conditions characterized by endothelial dysfunction.     

内源性一氧化氮合成酶抑制物在冠心病中的表达及意义研究

目的:探讨冠心病患者血中内源性一氧化氮合成酶(NOS)抑制物非对称性二甲基精氨酸(ADMA)与冠心病的关系及其临床意义。方法:随机选择37例冠心病患者和18例正常人,分别作为冠心病组和正常对照组,均经选择性冠脉造影确定冠状动脉有无病变及病变程度,测定血清中ADMA、对称性二甲基精氨酸(SDMA)和L-精氨酸水平及血脂水平。结果:冠心病组血清中NOS抑制物ADMA水平较正常对照组明显增高(P<0.01);冠心病组血清中的内源性ADMA水平随着冠状动脉病变的加重略有增高的趋势(P>0.05);冠心病组与正常对照组之间的SDMA和L-精氨酸的水平无明显差异(P>0.05);ADMA水平与冠状动脉病变程度有正相关关系(r=0.28,P<0.05)。结论:内源性NOS抑制物ADMA的异常增高与冠心病发病有关,ADMA水平增高可能是冠心病的一项值得关注的危险因素。     

反流性食管炎时食管一氧化氮合酶表达的实验研究

目的：初步探讨反流性食管炎时食管ＮＯＳ的表达。方法：采用幽门半缝扎＋贲门肌切开术制备反流性食管炎动物模型,采用ＮＡＤＰＨ－ｄ组化染色观察食管ＮＯＳ表达,测定食管组织ＮＯ含量。结果：食管炎组大多呈ＮＯＳ强阳性反应,阳性率明显高于正常对照组（Ｐ＜０．００１）,食管组织ＮＯ含量也明显高于正常对照组（术后２４小时比较Ｐ＜０．０１,术后４８小时,７２小时比较均Ｐ＜０．０５）。结论：胃－食管反流可引起食管上皮粘膜ＮＯＳ的过度表达,过多的ＮＯ可能发挥着重要的作用。     

吡喹酮对血吸虫病肝纤维化小鼠血清NO及肝内iNOS活性的影响

目的:研究吡喹酮治疗血吸虫病肝纤维化小鼠前后血清一氧化氮及肝内诱导型一氧化氮合酶活性的变化。方法:制备血吸虫病肝纤维化小鼠模型,应用免疫组化染色方法和多媒体彩色病理图文分析系统定量观察吡喹酮治疗前后肝脏诱导型一氧化氮合酶含量的变化,并通过化学还原反应检测血清一氧化氮的水平变化。结果:吡喹酮治疗组肝内iNOS和血清NO的含量均低于感染组,P均小于0.01。结论:吡喹酮可显著降低血吸虫病肝纤维化小鼠肝脏诱导型一氧化氮合酶活性及血清NO含量而发挥抗血吸虫病肝纤维化的作用。     

Nitric Oxide Synthase (NOS) Does Not Contribute to Simulated Ischaemic Preconditioning in an Isolated Rat Cardiomyocyte Model

It is widely accepted that nitric oxide (NO) is a trigger and mediator of late ischaemic preconditioning (IP), however its role in classic (protection observed within 2-4 hours after the IP stimulus) IP is less certain. In addition, the contribution of cardiomyocyte nitric oxide synthase (NOS) activation to NO production in ischaemia is unknown. The aim of this study was therefore to investigate the role of NOS, NO, reactive oxygen species (ROS) and cGMP in IP in an isolated cardiomyocyte model. METHODS: Adult rat cardiomyocytes were isolated by collagenase perfusion. Hypoxia was induced by covering pelleted cardiomyocytes with mineral oil. The IP protocol was one 10 min hypoxia/20 min reoxygenation cycle, followed by 2 hr sustained hypoxia. Non-IP cells were subjected to 2 hr sustained hypoxia only. The contribution of NO was investigated by NOS inhibition (L-NAME 50 microM) or by pre-treatment of cells with a NO donor (SNP 100 microM), and that of ROS by inclusion of ROS scavengers (MPG and N-acetyl-cysteine) or pre-treatment with H(2)O(2). End-points were cellular cGMP content and cell viability as assessed by trypan blue exclusion (TBE) and cell morphology. RESULTS: IP significantly improved myocyte viability (54% increase in TBE) at the end of sustained hypoxia. Treatment of cells with L-NAME and ROS scavengers during either the IP protocol or during sustained hypoxia had no effect on cell viability after 2 hr hypoxia, whereas viability of non-IP cells treated with L-NAME during sustained hypoxia improved significantly. cGMP levels were reduced in IP cells. Pre-treatment with SNP and H(2)O(2) did not mimic IP. CONCLUSIONS: IP conferred cardioprotection in isolated cardiomyocytes. Protection in this model was not due to activation of cardiomyocyte NOS or ROS production. However, NOS activation induced by sustained hypoxia, appeared to be harmful to non-IP cells.     

Diminished Expression of Constitutive Nitric Oxide Synthases in the Kidney of Spontaneously Hypertensive Rat

In kidney, nitric oxide (NO) synthesized by nitric oxide synthase (NOS) regulates sodium and water excretion, and renal medullary blood flow. The expression of constitutive NOS, endothelial NOS (eNOS) and neuronal NOS (nNOS), were assessed in kidney of the spontaneously hypertensive rat (SHR) and the normotensive Wistar Kyoto (WKY) rat by Western blot analysis and immunocytochemistry. Neuronal NOS expression was observed in the cortex and eNOS was detected only in the inner medulla of both WKY and SHR. In SHR, expression of eNOS was attenuated to 35.1 ± 10.8%, while expression of nNOS was only 57.5 ± 5.7% of the levels seen in WKY rat. Immunocytochemical studies revealed decreased staining of nNOS in the macula densa, collecting ducts and in the glomerulus of SHR compared to WKY rat. Endothelial NOS immunoreactivity was restricted to vascular structures of the inner intima cells and smooth muscle cells, and was markedly reduced in the vasculature of SHR. The decreased renal blood flow observed in SHR may be linked to a diminished expression of eNOS and nNOS, underscoring the importance of these enzymes in the pathophysiology and maintenance of genetic hypertension.     

Pathogenic Role of Endothelial Nitric Oxide Synthase (eNOS/NOS-III) in Cerulein-Induced Rat Acute Pancreatitis

Nitric oxide (NO) is a potent pancreatic vasodilator, yet the pathogenic role of NO in acute pancreatitis remains controversial. NO is generated from L-arginine by NO synthase (NOS), classified into three isozymes: neuronal (nNOS), inducible (iNOS), and endothelial NOS (eNOS). The purpose of the present study was to investigate the role of NO/NOS isozymes in the pathogenesis of cerulein-induced acute pancreatitis in rats. Acute pancreatitis was induced in male Wistar rats by two subcutaneous injections of cerulein (20 μg/kg). N ^G-Nitro-L-arginine methyl ester (L-NAME: a nonselective NOS inhibitor) or aminoguanidine (a relatively selective iNOS inhibitor) was given orally, while tetrahydrobiopterin (BH₄), a critical cofactor for NOS, was administered intraperitoneally 30 min before the first cerulein injection. Cerulein given repeatedly twice produced acute pancreatitis, with concomitant increases in the serum amylase level, pancreas weight, myeloperoxidase activity, lipid peroxidation and microvascular permeability. Prior administration of L-NAME, but not aminoguanidine, significantly prevented these changes, in a dose-dependent manner, and this effect was antagonized by the coadministration of L-arginine, a precursor of NO. The expression of dimetric eNOS in the pancreas was markedly suppressed by cerulein injections, together with a decrease in NO production, but the response was partially but significantly reversed by the prior administration of BH₄. The increases in the serum amylase level and pancreas weight, as well as the lipid peroxidation induced by cerulein, were significantly attenuated by the administration of BH₄. L-NAME had no effect on pancreatic secretion induced by cerulein. These results suggest that the uncoupled eNOS, probably caused by the decrease in endogenous BH₄ availability, plays a deleterious role in the pathogenesis of cerulein-induced acute pancreatitis.     

Nitric Oxide and Superoxide Anion in Low-Grade Esophagitis Induced by Acid and Pepsin in Rabbits

It has been suggested that free radicals are involved in esophagitis. To study the role and potential interaction of superoxide anion and nitric oxide (NO) in low-grade esophagitis, we perfused acidified pepsin (30 min every 12 hr) for seven days in rabbits treated with different agents to modulate the generation of these radicals. Measurements included macroscopic and microscopic damage, superoxide anion generation, mucosal nitric oxide synthase activity, and peroxynitrite formation. Low-grade esophagitis was associated with increased nitric oxide synthase mucosal activity and mucosal damage was dose-dependently increased by treatment with the NO synthase inhibitor N ^G-nitro-l-arginine. Superoxide anion was scarcely generated in the mucosa, but this was not accompanied by any change in the activity of mucosal superoxide dismutase. Treatment with superoxide dismutase did not improve mucosal damage. Generation of peroxynitrites was not detected. In conclusion, nitric oxide is involved in the mucosal defense of the esophagus against acid- and pepsin-induced damage. Superoxide anion generation seems irrelevant in the induction of low-grade esophagitis and not sufficient to interact with nitric oxide to generate measurable mucosal peroxynitrite radicals.     

Superoxide Anion and Nitric Oxide in High-Grade Esophagitis Induced by Acid and Pepsin in Rabbits

It has been proposed that free radicals are involved in the pathogenesis of esophageal mucosal damage induced by acid and pepsin. Recent data have suggested that nitric oxide (NO) is involved in the mucosal defense of the esophagus and that superoxide anion plays a minor role in low-grade esophagitis. To study the role and potential interaction of NO and superoxide anion in an experimental model of high-grade esophagitis, acidified pepsin was perfused (45 min/12 hr) for five days in rabbits with different agents to modulate the generation of these radicals. Measurements included both macroscopic and microscopic mucosal damage, superoxide anion generation, NO synthase mucosal activity, and peroxynitrite formation. High-grade esophagitis was associated with mucosal superoxide anion generation. Treatment with exogenous superoxide dismutase completely prevented mucosal damage. The perfusion of acidified pepsin in the lumen of the esophagus was initially associated with increased NO synthase mucosal activity but decreased with the progression of damage. Generation of peroxynitrites was present in those cases with severe damage. Treatment with NO-modifying agents did not induce consistent modification of mucosal damage. It is concluded that superoxide anion is involved in the induction of high-grade esophagitis and that it interacts with nitric oxide to generate peroxynitrite radicals in this model. Superoxide dismutase but not NO-donor-modifying agents might have a therapeutic role in preventing severe esophageal mucosal damage induced by acid and pepsin.     

r-干扰素对血管平滑肌细胞一氧化氮合酶mRNA表达的影响

探讨r-干扰素 (INF -r)对培养血管平滑肌细胞 (SMC)诱生型一氧化氮合酶 (iNOS)mRNA表达的影响。 0 5%血清培养 4 8h后的大鼠主动脉SMC ,加入 150 μ/ml的INF -r入培养基中 ,于 0、1、3、6和 12h获取细胞标本 ,提取总RNA ,采用Northern杂交检测iNOSmRNA在SMC中的表达水平。结果经INF -r刺激后 ,SMC中iNOSmRNA的表达水平明显增高 ,3h达高峰为对照水平的 8倍 ,12h后渐降。INF -r可刺激血管SMCiNOSmRNA表达水平上调 ,其作用可能与感染性休克的发生及SMC增殖受抑制等有关     

一氧化氮合酶抑制剂对深低温停循环后脑再灌注损伤的保护作用

目的 :研究一氧化氮合酶抑制剂 (L- NAME)对深低温停循环 (DHCA)后脑再灌注损伤的保护作用。　　方法 :8只幼猪随机分为实验组 (n=4)和对照组 (n=4) ,建立体外循环 (CPB) ,行 DHCA90分后恢复灌流 12 0分 ,实验组动物静脉注射 L- NAME1.5 mg/ kg,随后在 6 0分内再给予相同的剂量。　　结果 :实验组 Na - K 三磷酸腺苷酶、超氧化物歧化酶、谷胱甘肽过氧化物酶活力明显高于对照组 ,丙二醛含量低于对照组。病理检查及电子显微镜超微结构显示实验组与对照组细胞损伤存在显著性差异 (P<0 .0 5 )。　　结论 :L- NAME对 DHCA后脑再灌注损伤有保护作用。