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。     

Blastomyces dermatitidis yeast cells inhibit nitric oxide production by alveolar macrophage inducible nitric oxide synthase

The ability of pathogens to evade host antimicrobial mechanisms is crucial to their virulence. The dimorphic fungal pathogen Blastomyces dermatitidis can infect immunocompetent patients, producing a primary pulmonary infection that can later disseminate to other organs. B. dermatitidis possesses a remarkable ability to resist killing by alveolar macrophages. To date, no mechanism to explain this resistance has been described. Here, we focus on macrophage production of the toxic molecule nitric oxide as a potential target of subversion by B. dermatitidis yeast cells. We report that B. dermatitidis yeast cells reduce nitric oxide levels in the supernatants of activated alveolar macrophages. This reduction is not due to detoxification of nitric oxide, but rather to suppression of macrophage nitric oxide production. We show that B. dermatitidis yeast cells do not block upregulation of macrophage inducible nitric oxide synthase (iNOS) expression or limit iNOS access to its arginine substrate. Instead, B. dermatitidis yeast cells appear to inhibit iNOS enzymatic activity. Further investigation into the genetic basis of this potential virulence mechanism could lead to the identification of novel antifungal drug targets.     

Cell-specific effects of pentoxifylline on nitric oxide production and inducible nitric oxide synthase mRNA expression.

Cytokine-stimulated astrocytes and macrophages are potent producers of nitric oxide (NO), a free radical proposed to play an important role in organ-specific autoimmunity, including demyelinating diseases of the central nervous system. The aim of this study was to investigate effects of pentoxifylline (PTX), a phosphodiesterase inhibitor with immunomodulatory properties, on NO production and inducible NO synthase (iNOS) mRNA expression in rat astrocytes and macrophages. We have shown that PTX affects cytokine (interferon-gamma, IFN-gamma; interleukin-1, IL-1; tumour-necrosis factor-alpha, TNF-alpha)-induced NO production in both cell types, but in the opposite manner--enhancing in astrocytes and suppressive in macrophages. While PTX did not have any effect on enzymatic activity of iNOS in activated cells, expression of iNOS mRNA was elevated in astrocytes and decreased in macrophages treated with cytokines and PTX. Treatment with PTX alone affected neither NO production nor iNOS mRNA levels in astrocytes or macrophages. This study indicates involvement of different signalling pathways associated with iNOS induction in astrocytes and macrophages, thus emphasizing complexity of regulation of NO synthesis in different cell types.     

Microenvironmental regulation of inducible nitric oxide synthase expression and nitric oxide production in mouse bone marrow-derived mast cells

In addition to its well-known role in relaxation of vascular smooth muscle, NO modulates immune responses in a concentration- and location-specific manner. For MC, it is well accepted that exogenous NO regulates their function. However, there are inconsistencies in the literature of whether MC express NOS and make NO. MC progenitors mature in peripheral tissues, but the factors that influence MC maturation and their specific phenotype, such as whether they express NOS, are not well understood. To study microenvironmental conditions that could be "permissive" for NOS expression, we cultured BMMC in various conditions--BMMC(IL-3), BMMC(SCF/IL-3), or BMMC(SCF/IL-4)-for >3 weeks and examined NOS expression. We detected Nos2 mRNA in BMMC(SCF/IL-4) but not BMMC(IL-3) or BMMC(SCF/IL-3). After stimulation with IFN-γ and/or LPS, NOS2 expression and NO production were detected in BMMC(SCF/IL-4) but rarely detected in BMMC cultured with other conditions. Confocal microscopic analysis showed that NOS2 expression induced by IFN-γ colocalized in CD117(+) BMMC. NO production, after activation with IFN-γ and LPS in BMMC(SCF/IL-4), was abrogated by pretreatment with the NOS2-specific inhibitor. In addition to NOS2 expression, BMMC(SCF/IL-4) were distinguished from BMMC(IL-3) in heparin and MMCP expression. Thus, MC progenitors that develop in SCF + IL-4 can be induced to express NOS2 after receiving appropriate signals, such as IFN-γ, and subsequently produce NO. Microenvironmental conditions during their development can influence whether MC are capable of NOS expression and of NO production.     

Anti-inflammatory effect of Mentha longifolia in lipopolysaccharide-stimulated macrophages: Reduction of nitric oxide production through inhibition of inducible nitric oxide synthase

Abstract

Mentha longifolia is an aromatic plant used in flavoring and preserving foods and as an anti-inflammatory folk medicine remedy. The present study assessed the effects of M. longifolia extracts, including essential oil and crude methanol extract and its fractions (ethyl acetate, butanol and hexane), on nitric oxide (NO) production and inducible NO synthase (iNOS) mRNA expression in lipopolysaccharide (LPS)-stimulated J774A.1 cells using real-time polymerase chain reaction (PCR). The cytotoxic effects of the extracts on the cells were examined and non-cytotoxic concentrations (<0.2?mg/ml) were used to examine their effects on NO production and iNOS mRNA expression. Only the hexane fraction that contained high levels of phenolic and flavonoid compounds at concentrations from 0.05–0.20?mg/ml significantly reduced NO production in LPS-stimulated cells (p?<?0.001). Real-time PCR analysis indicated the ability of this fraction at the same concentrations to significantly decrease iNOS as well as TNFα mRNA expression in the cells (p?<?0.001). All extracts were able to scavenge NO radicals in a concentration-dependent manner. At concentrations greater than 0.2?mg/ml, total radicals were 100% scavenged. In conclusion, M. longifolia possibly reduces NO secretion in macrophages by scavenging NO and inhibiting iNOS mRNA expression, and also decreases TNFα pro-inflammatory cytokine expression, thus showing its usefulness in the inflammatory disease process.     

Morphine-6beta-glucuronide modulates the expression of inducible nitric oxide synthase

The immunomodulatory effects of morphine are well established; however, suprisingly little is known about the immunomodulatory properties of the major metabolites of morphine. The present study tests the hypothesis that expression of inducible nitric oxide synthase (iNOS) is modulated by the administration of the morphine metabolite, morphine-6-glucuronide. The initial study using rats shows that morphine-6-glucuronide administration (0, 1.0, 3.163, 10 mg/kg s.c.) results in a pronounced reduction in lipopolysaccharide (LPS)-induced expression of iNOS (inducible nitricoxide synthease) in spleen, lung, and liver tissue as measured by western blotting. Morphine-6-glucuronide also produces a reduction in the level of plasma nitrite/nitrate, the more stable end-product of nitric oxide degradation. In a subsequent study, administration of the opioid receptor antagonist, naltrexone (0.1 mg/kg) prior to the injection of morphine-6-glucuronide (10 mg/kg) blocks the morphine-6-glucuronide induced reduction of iNOS expression and plasma nitrite/nitrite levels indicating that the effect is mediated via the opioid-receptor. This study provides the first evidence that morphine-6-glucuronide alters the expression of iNOS.     

Sulphasalazine inhibits macrophage activation: inhibitory effects on inducible nitric oxide synthase expression, interleukin-12 production and major histocompatibility complex II expression

The anti-inflammatory agent sulphasalazine is an important component of several treatment regimens in the therapy of ulcerative colitis, Crohn's disease and rheumatoid arthritis. Sulphasalazine has many immunomodulatory actions, including modulation of the function of a variety of cell types, such as lymphocytes, natural killer cells, epithelial cells and mast cells. However, the effect of this agent on macrophage (M phi) function has not been characterized in detail. In the present study, we investigated the effect of sulphasalazine and two related compounds - sulphapyridine and 5-aminosalicylic acid - on M phi activation induced by bacterial lipopolysaccharide (LPS) and interferon-gamma (IFN-gamma). In J774 M phi stimulated with LPS (10 microg/ml) and IFN-gamma (100 U/ml), sulphasalazine (50-500 microM) suppressed nitric oxide (NO) production in a concentration-dependent manner. The expression of the inducible NO synthase (iNOS) was suppressed by sulphasalazine at 500 microM. Sulphasalazine inhibited the LPS/IFN-gamma-induced production of both interleukin-12 (IL-12) p40 and p70. The suppression of both NO and IL-12 production by sulphasalazine was superior to that by either sulphapyridine or 5-aminosalicylic acid. Although the combination of LPS and IFN-gamma induced a rapid expression of the active forms of p38 and p42/44 mitogen-activated protein kinases and c-Jun terminal kinase, sulphasalazine failed to interfere with the activation of any of these kinases. Finally, sulphasalazine suppressed the IFN-gamma-induced expression of major histocompatibility complex class II. These results demonstrate that the M phi is an important target of the immunosuppressive effect of sulphasalazine.     

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.     

LPS对大鼠雪旺氏细胞iNOS表达的影响

目的：探讨内毒素脂多糖（Lipopolysaccharide,LPS）对大鼠雪旺氏细胞（Schwann cells,Scs）诱导型一氧化氮合酶（Inducible nitric-oxide synthase,iNOS）基因表达及一氧化氮（Nitric oxide,NO）生成的影响。方法：用不同浓度（1、10、100μg/ml）和同一浓度不同时间（1、2、4、6小时）的LPS刺激雪旺氏细胞,分别用RT-PCR和亚硝酸盐含量测定观察细胞iNOS mRNA的表达量和细胞培养液中亚硝酸盐的水平,同时用免疫荧光细胞化学染色检测iNOS的细胞定位。结果：用LPS10μg/ml刺激2小时后,iNOS mRNA的表达增加,4小时表达活性最高。细胞上清中的亚硝酸盐含量高峰在6小时。免疫细胞化学证明LPS诱导雪旺氏细胞iNOS的表达定位在胞浆。结论：LPS可在转录水平上诱导雪旺氏细胞iNOS mRNA表达,促进NO的合成,提示雪旺氏细胞在周围神经系统炎症过程中可能发挥免疫调节作用。     

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     

大鼠额叶皮质损害后诱导型iNOS阳性细胞的变化

目的：一氧化氮在脑内的许多生功能中起重要作用,并参与脑损伤的病理生理过程。本研究旨在探讨实验性脑损伤后的NOS阳性细胞的来源及成份。方法：利用机械油吸法制成大鼠额叶皮质损伤动物模型,应用NADPH-d组织化学及GFAP免疫组化法观察损伤后1、3、7、14、21、30及60d皮质损害区NOS阳性细胞的类型和变化。结果：损害后1d即见皮质损害区底部和两侧nNOS阳性细胞增加,损害底部出现诱导型胶质细胞,尤以胼胝体中更为密集。这种反应3-7d时逐渐增强,2周时最明显,以后随时间推移及损害的修复而降低,研究发现部分iNOS阳性细胞与GFAP者共存,提示系反应性胶质细胞,未见eNOS阳性细胞上调现象。结论：皮质受损时,出现2种不同表型的NOS阳性细胞且上调,即出现诱导型神经元nNOS和诱导型iNOS胶质细胞。究其来源各不相同,但均能合成NO,NO主要集中在损伤部位的周围。     

Inhibition of the induction of the inducible nitric oxide synthase in murine brain microglial cells by sodium salicylate.

The induction of the inducible nitric oxide synthase (iNOS) has been proposed to play a role in a variety of inflammatory diseases. Sodium salicylate (NaSal) is the most commonly used anti-inflammatory agent. We investigated whether NaSal can diminish the induction of iNOS in murine brain microglial cells. In primary cultures, interferon-gamma (IFN-gamma) or lipopolysaccharide (LPS) separately did not stimulate nitric oxide (NO) production, whereas IFN-gamma combined with LPS synergistically induced iNOS. NaSal inhibited both the production of NO and expression of iNOS in microglial cells. Synergy between IFN-gamma and LPS was mainly dependent on tumour necrosis factor-alpha (TNF-alpha) secretion as the increase of the induction of the iNOS by IFN-gamma plus LPS was associated with the increase of TNF-alpha secretion and IFN-gamma plus LPS-induced TNF-alpha secretion by microglial cells was decreased by the treatment with NaSal. These results suggest a possible use of NaSal in managing inflammation of the central nervous system through inhibition of the iNOS induction.     

Pathological consequences of inducible nitric oxide synthase expression in hippocampal slice cultures

The generation of toxic concentrations of nitric oxide by the inducible nitric oxide synthase expressed in microglia and other brain cell types is frequently invoked as a causative factor in neurodegeneration. Experiments were carried out on slice cultures of rat hippocampus to test this hypothesis. Exposure of the slices to bacterial lipopolysaccharide plus interferon-gamma led to a time-dependent expression of functional inducible nitric oxide synthase that was found only in microglia. Microglial activation by other means, such as physical damage, was not associated with inducible nitric oxide synthase expression. Damage and cell death in slices expressing inducible nitric oxide synthase was evaluated over a period of 6 days, but none was found. Consistent with this result, cGMP measurements indicated that the average local nitric oxide concentration remained in the low nanomolar range. When the microglial population was expanded to a density three-fold above normal by applying granulocyte-macrophage colony stimulating factor, however, lipopolysaccharide plus interferon-gamma provoked neurodegeneration that could be blocked by an inducible nitric oxide synthase inhibitor. The associated nitric oxide concentration in the slices was saturating for guanylyl cyclase-coupled nitric oxide receptors, signifying at least 10 nM. It is concluded that inducible nitric oxide synthase is expressed in microglia only in response to specific stimuli involving the innate immune system, and that the resulting level of nitric oxide in intact brain tissue is normally too low to inflict damage directly. Quantities of nitric oxide sufficient to contribute directly or indirectly to pathology could be produced should the density of microglia become high enough, although caution must be exercised in extrapolating this finding to the human brain in vivo.