香菇多糖对巨噬细胞一氧化氮和一氧化氮合酶活性的影响

目的研究香菇多糖(LTN)诱导巨噬细胞的一氧化氮(NO)生成和一氧化氮合酶(iNOS)的活性,探讨LTN的免疫调节作用机理.方法采用Griess反应和荧光法测定不同剂量的LTN作用小鼠腹腔巨噬细胞后NO的生成量和iNOS活性.观察mRNA转录抑制剂、蛋白质合成抑制剂和iNOS抑制剂对巨噬细胞NO的生成和iNOS活性的影响.结果LTN能使小鼠腹腔巨噬细胞NO生成增加,iNOS活性增高,并呈作用剂量依赖关系.3种抑制剂均能抑制LTN诱导的小鼠腹腔巨噬细胞N0的生成和iNOS活性.结论LTN能刺激小鼠腹腔巨噬细胞提高iNOS活性和NO的生成.提示LTN的免疫调节作用机制可能与LTN刺激巨噬细胞NO生成有关.     

香菇多糖对巨噬细胞一氧化氦和一氧化氦合酶活性的影响

目的 研究香菇多糖(LTN)诱导巨噬细胞的一氧化氮(NO)生成和一氧化氮合酶(iNOS)的活性，探讨LTN的免疫调节作用机理。方法 采用Griess反应和荧光法测定不同剂量的LTN作用小鼠腹腔巨噬细胞后NO的生成量和iNOS活性。观察mRNA转录抑制剂、蛋白质合成抑制剂和iNOS抑制剂对巨噬细胞NO的生成和iNOS活性的影响。结果 LTN能使小鼠腹腔巨噬细胞NO生成增加，iNOS活性增高，并呈作用剂量依赖关系。3种抑制剂均能抑制LTN诱导的小鼠腹腔巨噬细胞NO的生成和iNOS活性。结论 LTN能刺激小鼠腹腔巨噬细胞提高iNOS活性和NO的生成。提示LTN的免疫调节作用机制可能与LTN刺激巨噬细胞NO生成有关。     

花色素苷诱导小鼠巨噬细胞一氧化氮生成及其机制

目的研究花色素苷对小鼠腹腔巨噬细胞一氧化氮(NO)合成的诱导作用及其机制。方法用CCK-8试剂检测花色素苷对小鼠脾细胞增殖的影响;硝酸盐还原酶法检测小鼠腹腔巨噬细胞NO含量;荧光法检测一氧化氮合酶(NOS)活性;RT-PCR检测iNOS mRNA的表达。结果花色素苷可促进小鼠脾细胞增殖,诱导小鼠腹腔巨噬细胞合成NO,提高NOS活性,其中矢车菊素-3-葡萄糖苷能够诱导iNOS mRNA的表达。结论花色素苷能够促进脾细胞的增殖,诱导小鼠腹腔巨噬细胞合成NO,使巨噬细胞激活,具有一定的免疫调节活性。     

细脚拟青霉多糖诱导小鼠巨噬细胞一氧化氮生成和iNOS mRNA的表达

目的研究单一组分细脚拟青霉多糖(Paecilomyces tenuipespolysaccharide,PTPS)诱导小鼠腹腔巨噬细胞的免疫调节活性及其作用机制。方法应用水提、乙醇沉淀、DEAE-纤维素色谱,对PTPS进行提取并分级;用CCK-8试剂检测PTPS对小鼠脾细胞的增殖作用,硝酸盐还原酶法检测小鼠腹腔巨噬细胞一氧化氮(NO)的生成量和RT-PCR半定量法检测iNOS mRNA的表达。结果从细脚拟青霉菌丝体中分离出含糖量为92%的中性多糖PTPS,可促进脾细胞的增殖,并在一定浓度下显示剂量依赖效应;能明显诱导小鼠腹腔巨噬细胞NO的生成和iNOS mRNA的表达。结论PTPS通过诱导小鼠巨噬细胞NO合成和iNOS的转录,表明其具有免疫调节和潜在的抗肿瘤活性。     

灵芝多糖肽对小鼠腹腔巨噬细胞一氧化氮产生的影响

目的　研究灵芝多糖肽 (GLPP)对小鼠腹腔巨噬细胞一氧化氮产生的影响并探讨其作用机制。方法　以Griess法 ,观察GLPP对LPS诱导小鼠腹腔巨噬细胞一氧化氮(NO)产生的影响 ;以免疫组化法检测诱导型一氧化氮合成酶 (iNOS)的表达 ,观察GLPP对iNOS的影响。结果　GLPP(2 5～ 2 0 0mg·kg-1)灌胃给药 5d或体外给药 (3 12 5～ 2 0 0mg·L-1)均可促进巨噬细胞NO释放 ,但对LPS刺激NO的释放影响不大 ;GLPP(10 0mg·kg-1)灌胃给药 5d或体外给药 (10mg·L-1)均可使巨噬细胞iNOS含量增加。结论　GLPP可增加小鼠腹腔巨噬细胞NO产生 ,其机制可能与其促进巨噬细胞iNOS合成有关。     

库拉索芦荟多糖对小鼠腹腔巨噬细胞一氧化氮生成的影响

目的研究库拉索芦荟多糖对小鼠腹腔巨噬细胞一氧化氮(NO)生成的影响。方法用G riess法测定巨噬细胞一氧化氮的生成量。结果库拉索芦荟多糖在25～400μg/mL浓度范围可显著促进正常巨噬细胞的NO生成,在50～400μg/mL浓度范围可抑制LPS激活的巨噬细胞的NO生成。结论库拉索芦荟多糖对小鼠腹腔巨噬细胞一氧化氮的生成具有双向调节作用。     

库拉索芦荟多糖对小鼠腹腔巨噬细胞一氧化氮生成的影响

目的研究库拉索芦荟多糖对小鼠腹腔巨噬细胞一氧化氮(NO)生成的影响。方法用G riess法测定巨噬细胞一氧化氮的生成量。结果库拉索芦荟多糖在25~400μg/mL浓度范围可显著促进正常巨噬细胞的NO生成,在50~400μg/mL浓度范围可抑制LPS激活的巨噬细胞的NO生成。结论库拉索芦荟多糖对小鼠腹腔巨噬细胞一氧化氮的生成具有双向调节作用。     

酵母多糖对小鼠腹腔巨噬细胞产生一氧化氮和白细胞介素-1的影响

目的探讨酵母多糖对小鼠腹腔巨噬细胞产生一氧化氮 (NO)和白细胞介素 1(IL 1)的影响。方法将不同剂量的酵母多糖加入体外培养的小鼠腹腔巨噬细胞中 ,取细胞培养上清液根据Griess反应检测NO-2 的量 ,间接反映巨噬细胞产生NO的生成量 ,并用溴化四唑蓝 (MTT)比色法检测上清液中IL 1的生成量。结果酵母多糖可明显促进小鼠腹腔巨细胞产生NO和IL 1,NO的生成量呈现剂量依赖关系。结论酵母多糖可诱导小鼠腹腔巨噬细胞产生NO和IL 1,可能是酵母多糖调节机体免疫功能、杀伤病原微生物和抗肿瘤的重要途径     

12味止血中药对脂多糖诱导小鼠巨噬细胞产生一氧化氮的抑制作用

目的:研究12味止血中药对脂多糖(LPS)诱导小鼠巨噬细胞产生一氧化氮(NO)的抑制作用,并探讨其止血机制。方法:以1μg.mL-1LPS刺激小鼠巨噬细胞RAW264.7,22h后以Griess法测定NO的终产物亚硝酸盐的含量,观察LPS对NO生成的影响。结果:12味止血药均可抑制LPS诱导小鼠巨噬细胞RAW264.7中NO的生成。三七、地榆、仙鹤草、槐花、艾叶、蒲黄、侧柏叶及白茅根的抑制作用显著(P<0.05)。结论:本实验为12味止血中药对脂多糖诱导小鼠巨噬细胞产生NO的抑制作用提供了一定的理论依据。     

糖萜素对小鼠腹腔巨噬细胞产生一氧化氮的影响

目的:探讨糖萜素对小鼠腹腔巨噬细胞产生NO的影响。方法:在饲料中添加不同剂量糖萜素喂养NIH小鼠,以基础饲料为对照,检测环磷酰胺免疫抑制组与非抑制组不同时间段小鼠腹腔巨噬细胞产生NO的含量。结果:含糖萜素饲料组于不同时间测得NO含量均较基础饲料组显著增高(P<0.05)。免疫抑制状态下,含糖萜素饲料组NO含量也较基础饲料组高(P<0.05)。体外糖萜素不能直接影响NO的生成。结论:糖萜素能显著提高小鼠体内巨噬细胞产生NO。体外则不能直接影响NO的生成     

Modulation of prostaglandin biosynthesis by nitric oxide and nitric oxide donors

The biosynthesis and release of nitric oxide (NO) and prostaglandins (PGs) share a number of similarities. Two major forms of nitric-oxide synthase (NOS) and cyclooxygenase (COX) enzymes have been identified to date. Under normal circumstances, the constitutive isoforms of these enzymes (constitutive NOS and COX-1) are found in virtually all organs. Their presence accounts for the regulation of several important physiological effects (e.g. antiplatelet activity, vasodilation, and cytoprotection). On the other hand, in inflammatory setting, the inducible isoforms of these enzymes (inducible NOS and COX-2) are detected in a variety of cells, resulting in the production of large amounts of proinflammatory and cytotoxic NO and PGs. The release of NO and PGs by the inducible isoforms of NOS and COX has been associated with the pathological roles of these mediators in disease states as evidenced by the use of selective inhibitors. An important link between the NOS and COX pathways was made in 1993 by Salvemini and coworkers when they demonstrated that the enhanced release of PGs, which follows inflammatory mechanisms, was nearly entirely driven by NO. Such studies raised the possibility that COX enzymes represent important endogenous "receptor" targets for modulating the multifaceted roles of NO. Since then, numerous papers have been published extending the observation across various cellular systems and animal models of disease. Furthermore, other studies have highlighted the importance of such interaction in physiology as well as in the mechanism of action of drugs such as organic nitrates. More importantly, mechanistic studies of how NO switches on/off the PG/COX pathway have been undertaken and additional pathways through which NO modulates prostaglandin production unraveled. On the other hand, NO donors conjugated with COX inhibitors have recently found new interest in the understanding of NO/COX reciprocal interaction and potential clinical use. The purpose of this article is to cover the advances which have occurred over the years, and in particular, to summarize experimental data that outline how the discovery that NO modulates prostaglandin production has impacted and extended our understanding of these two systems in physiopathological events.     

Feedback inhibition of nitric oxide synthase activity by nitric oxide.

1. A murine macrophage cell line, J774, expressed nitric oxide (NO) synthase activity in response to interferon-gamma (IFN-gamma, 10 u ml-1) plus lipopolysaccharide (LPS, 10 ng ml-1). The enzyme activity was first detectable 6 h after incubation, peaked at 12 h and became undetectable after 48 h. 2. The decline in the NO synthase activity was not due to inhibition by stable substances secreted by the cells into the culture supernatant. 3. The decline in the NO synthase activity was significantly slowed down in cells cultured in a low L-arginine medium or with added haemoglobin, suggesting that NO may be involved in a feedback inhibitory mechanism. 4. The addition of NO generators, S-nitroso-acetyl-penicillamine (SNAP) or S-nitroso-glutathione (GSNO) markedly inhibited the NO synthase activity in a dose-dependent manner. The effect of NO on the enzyme was not due to the inhibition of de novo protein synthesis. 5. SNAP directly inhibited the inducible NO synthase extracted from activated J774 cells, as well as the constitutive NO synthase extracted from the rat brain. 6. The enzyme activity of J774 cells was not restored after the removal of SNAP by gel filtration, suggesting that NO inhibits NO synthase irreversibly.     

Mitochondrial nitric oxide synthase

Mitochondria produce nitric oxide (NO) through a Ca(2+)-sensitive mitochondrial NO synthase (mtNOS). The NO produced by mtNOS regulates mitochondrial oxygen consumption and transmembrane potential via a reversible reaction with cytochrome c oxidase. The reaction of this NO with superoxide anion yields peroxynitrite, which irreversibly modifies susceptible targets within mitochondria and induces oxidative and/or nitrative stress. In this article, we review the current understanding of the roles of mtNOS as a crucial biochemical regulator of mitochondrial functions and attempt to reconcile apparent discrepancies in the literature on mtNOS.     

Role of endogenous nitric oxide in the nitric oxide donor-induced plasma extravasation of mouse skin.

The role of endogenous nitric oxide (NO) and prostanoids in the increase in microvascular permeability induced by NO donors was investigated in the mouse skin by a dye leakage method. Subcutaneous (s.c.) injection of 1-hydroxy-2-oxo-3-(3-aminopropyl)-3-isopropyl-1-triazene (NOC 5), 1-hydroxy-2-oxo-3,3-bis(2-aminoethyl)-1-triazene (NOC 18) and sodium nitroprusside dose-dependently increased local dye leakage. While indomethacin inhibited the dye leakage elicited by these NO donors, N(G)-nitro-L-arginine methyl ester (L-NAME) inhibited the effect of NOC 5 and NOC 18 but not of sodium nitroprusside. These results suggest that endogenous NO, in addition to the prostanoid biosynthesis, is involved in the dermal microvascular permeability increase induced by the NOC series NO donors.     

Influence of nitric oxide derived from neuronal nitric oxide synthase on glomerular filtration

The neuronal isoform of nitric oxide synthase (nNOS) has been localized to specific regions of the kidney, including the thick ascending limb of the loop of Henle and the macula densa. Because of this discrete localization in the renal cortex, nitric oxide (NO) produced by nNOS has been suggested to play an important role in the regulation of macula densa-mediated arteriole tone and therefore could play an important role in the regulation of whole-kidney glomerular filtration rate (GFR). We hypothesized that selective blockade of nNOS would decrease GFR. Renal hemodynamics were measured before and after acute selective blockade of nNOS by 50 mg/kg 7-nitroindazole (7-NI) in anesthetized rats. Administration of 7-NI had no significant effect on basal blood pressure (from 105 +/- 3 to 101 +/- 2 mm Hg), renal blood flow [from 6.08 +/- 0.39 to 6.31 +/- 0.33 ml/min/gram of kidney weight (gkw)], or total renal vascular resistance (from 18.1 +/- 1.6 to 16.4 +/- 1.0 mm Hg/ml/min/gkw) but decreased GFR by 26% (from 1.36 +/- 0.15 to 1.00 +/- 0.13 ml/min/gkw; p < 0.02), urinary flow rate by 28% (from 24.7 +/- 1.8 to 17.8 +/- 2.2 microl/min; p < 0.05), and sodium excretion by 22% (from 5.55 +/- 0.53 to 4.30 +/- 0.52 microEq/min; p < 0.05). However, fractional sodium excretion was not changed by nNOS inhibition. There were no such changes in vehicle-treated time controls. We conclude that, in the renal cortex, NO produced by nNOS plays an important role in the regulation of whole-kidney GFR and excretion in normal, sodium-replete rats.     

Cardiovascular diseases and the nitric oxide pathway

Fifteen years after its discovery, NO has fully reached an established position in physiology, medicine and therapeutics. It is difficult to find a biological function or a pathological condition where NO does not play a relevant role. Discoveries in the NO field have historically evolved from cardiovascular research, although its influences have already covered nearly all the medical specialties. This review analyzes, step by step, the pathway through which NO is synthesized in the cells of the cardiovascular system and the main physiological and pathological routes it undergoes once it is released. We focus on various diseases affecting the cardiovascular system (atherosclerosis, hypertension, diabetes mellitus and septic shock). We describe in detail those steps of the NO pathway in which anomalies have been detected and may account for the pathophysiology of these diseases. In atherosclerosis, hypertension and diabetes mellitus, the endothelial form of NOS is upregulated, but is very sensitive to environmental conditions, such as substrate or cofactor deficiencies or increases in LDL or glucose. In this situation NOS synthesizes superoxide anion instead of NO leading to oxidative and nitrosative stress. In diabetes mellitus and, very importantly, in septic shock, the inducible form of NOS is highly upregulated. Overproduction of NO appears to underlie the hypotension and tissue damage of septicemia and the destruction of beta-cells in diabetes mellitus. New knowledge of the role of NO in these diseases has started to influence therapeutic design. We also review the current status of research on NO-based therapies.     

Inhaled nitric oxide in the neonatal period

Pulmonary hypertension of the newborn is an important cause of hypoxaemia, particularly in the at term or near term neonate. It can occur as a primary condition or secondary to a variety of other diseases. Endogenous nitric oxide is an important modulator of vascular tone in pulmonary circulation. Initial uncontrolled studies indicated that inhalation of nitric oxide resulted in a reduction in pulmonary hypertension, with improvement in oxygenation, but no change in the systemic vascular resistance. There have now been a number of randomised trials performed exploring the efficacy of inhaled nitric oxide. These trials have demonstrated that in at term or near term infants, inhaled nitric oxide reduces the combined end point of death or the need for extracorporeal membrane oxygenation. The significant effect seems due to the reduced extracorporeal membrane oxygenation requirement. No such beneficial effect has been consistently reported in infants with congenital diaphragmatic hernia. Randomised trials have failed to highlight long-term positive results in preterm infants. Inhaled nitric oxide has side effects, although those due to nitrogen dioxide and methaemoglobin formation can be minimised by appropriate nitric oxide delivery. It is important to use the smallest effective nitric oxide dose, continuous nitric oxide and nitrogen dioxide monitoring and frequent methaemoglobin analyses. Careful patient selection should be undertaken, avoiding those at high risk of haemorrhagic complications. Longer term follow-up studies are required to determine the real risk:benefit ratio of inhaled nitric oxide treatment.     

Inhaled nitric oxide in the neonatal period

Pulmonary hypertension of the newborn is an important cause of hypoxaemia, particularly in the at term or near term neonate. It can occur as a primary condition or secondary to a variety of other diseases. Endogenous nitric oxide is an important modulator of vascular tone in pulmonary circulation. Initial uncontrolled studies indicated that inhalation of nitric oxide resulted in a reduction in pulmonary hypertension, with improvement in oxygenation, but no change in the systemic vascular resistance. There have now been a number of randomised trials performed exploring the efficacy of inhaled nitric oxide. These trials have demonstrated that in at term or near term infants, inhaled nitric oxide reduces the combined end point of death or the need for extracorporeal membrane oxygenation. The significant effect seems due to the reduced extracorporeal membrane oxygenation requirement. No such beneficial effect has been consistently reported in infants with congenital diaphragmatic hernia. Randomised trials have failed to highlight long-term positive results in preterm infants. Inhaled nitric oxide has side effects, although those due to nitrogen dioxide and methaemoglobin formation can be minimised by appropriate nitric oxide delivery. It is important to use the smallest effective nitric oxide dose, continuous nitric oxide and nitrogen dioxide monitoring and frequent methaemoglobin analyses. Careful patient selection should be undertaken, avoiding those at high risk of haemorrhagic complications. Longer term follow-up studies are required to determine the real risk:benefit ratio of inhaled nitric oxide treatment.     

Cyclo(dehydrohistidyl-l-tryptophyl) inhibits nitric oxide production by preventing the dimerization of inducible nitric oxide synthase

Dimerization of inducible NOS has been known to be a potential therapeutic target for iNOS-mediated pathologies. Cyclic dipeptides are among the simplest peptides commonly found as by-products of food processing or metabolites of microorganisms. In this study, we found that cyclo(dehydrohistidyl-l-tryptophyl) (CDHT), a cyclic dipeptide from an unidentified fungal strain Fb956, prevents iNOS dimerization in activated microglial BV-2 cells. CDHT inhibited NO production with an IC50 of 6.5 microM in LPS-treated BV-2 cells. Western blot analysis and iNOS activity measurement of fractions from size-exclusion chromatography of cell lysates indicated that CDHT inhibits dimerization of iNOS, while it has no effect on iNOS expression or enzyme activity. The CDHT inhibition of iNOS dimerization was confirmed by partially denaturing SDS-PAGE analysis. In contrast, CDHT did not affect cGMP production in endothelial HUVEC cells, which indicates no inhibition of endothelial NOS activity. These results reveal that CDHT, one of the simplest and cyclic dipeptides, selectively inhibits NO production by inhibiting iNOS dimerization, and could be a useful therapeutic agent for inflammation-mediated diseases.