Magnolol suppresses NF-kappaB activation and NF-kappaB regulated gene expression through inhibition of IkappaB kinase activation

The mis-regulation of nuclear factor-kappa B (NF-kappaB) signal pathway is involved in a variety of inflammatory diseases that leds to the production of inflammatory mediators. Our studies using human U937 promonocytes cells suggested that magnolol, a low molecular weight lignan isolated from the medicinal plant Magnolia officinalis, differentially down-regulated the pharmacologically induced expression of NF-kappaB-regulated inflammatory gene products MMP-9, IL-8, MCP-1, MIP-1alpha, TNF-alpha. Pre-treatment of magnolol blocked TNF-alpha-induced NF-kappaB activation in different cell types as evidenced by EMSA. Magnolol did not directly affect the binding of p65/p50 heterodimer to DNA. Immunoblot analysis demonstrated that magnolol inhibited the TNF-alpha-stimulated phosphorylation and degradation of the cytosolic NF-kappaB inhibitor IkappaBalpha and the effects were dose-dependent. Mechanistically, a non-radioactive IkappaB kinases (IKK) assay using immunoprecipitated IKKs protein demonstrated that magnolol inhibited both intrinsic and TNF-alpha-stimulated IKK activity, thus suggesting a critical role of magnolol in abrogating the phosphorylation and degradation of IkappaBalpha. The involvement of IKK was further verified in a HeLa cell NF-kappaB-dependent luciferase reporter system. In this system magnolol suppressed luciferase expression stimulated by TNF-alpha and by the transient transfection and expression of NIK (NF-kappaB-inducing kinase), wild type IKKbeta, constitutively active IKKalpha and IKKbeta, or the p65 subunit. Magnolol was also found to inhibit the nuclear translocation and phosphorylation of p65 subunit of NF-kappaB. In line with the observation that NF-kappaB activation may up-regulate anti-apoptotic genes, it was shown in U937 cells that magnolol enhanced TNF-alpha-induced apoptotic cell death. Our results suggest that magnolol or its derivatives may have potential anti-inflammatory actions through IKK inactivation.     

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.     

Fas-FasL interaction modulates nitric oxide production in Trypanosoma cruzi-infected mice

During acute Trypanosoma cruzi infection in mice, many leucocytes undergo apoptosis. Although apoptosis has been ascribed to increased levels of nitric oxide (NO) and Fas-FasL interaction, the importance of this phenomenon in modulating the host response against T. cruzi is unknown. Herein, the role of NO- and Fas-FasL-induced apoptosis in modulating the immune response to T. cruzi was evaluated using mice deficient in Fas expression (MRL/MpJ-Fas lpr) and inducible nitric oxide synthase (iNOS) knockout mice (iNOS-/-). The results showed that besides decreasing apoptosis induction after infection, impairment of the Fas-FasL interaction resulted in decreased NO production, as a consequence of enhanced T helper 2 (Th2) cytokine production. Differently, blockage of NO-induced apoptosis resulted in uncontrolled cytokine production, rather than a biased Th2 cytokine pattern. Together, these results suggested that Fas and FasL-induced apoptosis could be implied in modulation of the immune response against T. cruzi by interfering with cytokine and NO production during the acute phase of the infection.     

Early 2-mercaptoethanol-resistant antibody production in endotoxin-treated rats

Experiments are reported concerning both natural and specific antibody production in endotoxin-treated rats.     

Glucocorticoids enhance concanavalin A-induced mitogenic response through the inhibition of nitric oxide production.

Glucocorticoids (GC) are known to inhibit mitogen-induced proliferation of T cells. In this study we show two experimental situations where the addition of GC increases lymphocyte proliferation. It has been reported by different authors that rat spleen (SPL) cells proliferate poorly after concanavalin A (Con A) activation. These poor responses have been related to the suppressor activity of macrophages. Similarly, it is known that T-cell proliferation is depressed in the presence of an excess of macrophages in the culture. Here we show that in both experimental situations, the inclusion of dexamethasone (DEX), a synthetic glucocorticoid, in the culture medium enhances the Con A-stimulated proliferation. We provide evidence that this effect is a consequence of the inhibition of nitric oxide (NO) synthesis by the hormone. Furthermore, we also demonstrate that rat SPL cells are inefficient antigen-presenting cells (APC) because of their spontaneous high production of NO. Taken together our results suggest that the effects of GC on T-cell activation may be to promote or inhibit proliferation depending on the level of endogenous NO synthesis. The possible significance of these results is briefly discussed.     

L-Arginine availability modulates local nitric oxide production and parasite killing in experimental trypanosomiasis

Nitric oxide (NO) is an important effector molecule of the immune system in eliminating numerous pathogens. Peritoneal macrophages from Trypanosoma brucei brucei-infected mice express type II NO synthase (NOS-II), produce NO, and kill parasites in the presence of L-arginine in vitro. Nevertheless, parasites proliferate in the vicinity of these macrophages in vivo. The present study shows that L-arginine availability modulates NO production. Trypanosomes use L-arginine for polyamine synthesis, required for DNA and trypanothione synthesis. Moreover, arginase activity is up-regulated in macrophages from infected mice from the first days of infection. Arginase competes with NOS-II for their common substrate, L-arginine. In vitro, arginase inhibitors decreased urea production, increased macrophage nitrite production, and restored trypanosome killing. In vivo, a dramatic decrease in L-arginine concentration was observed in plasma from infected mice. In situ restoration of NO production and trypanosome killing were observed when excess L-arginine, but not D-arginine or L-arginine plus N(omega)-nitro-L-arginine (a NOS inhibitor), was injected into the peritoneum of infected mice. These data indicate the role of L-arginine depletion, induced by arginase and parasites, in modulating the L-arginine-NO pathway under pathophysiological conditions.     

LY294002 inhibits LPS-induced NO production through a inhibition of NF-kappaB activation: independent mechanism of phosphatidylinositol 3-kinase

Phosphatidylinositol 3-kinase (PI3K) is critical player in cell proliferation and survival. The effects of LY294002 and wortmannin, inhibitors of PI3K, on nitric oxide (NO) production and inducible nitric oxide synthase (iNOS) expression in lipoploysaccharide (LPS)-induced Raw 264.7 cells were investigated. Significant inhibition of LPS-induced protein kinase B (PKB, Akt) phosphorylation occurred at 25 microM LY294002 or 0.5 microM wortmannin. At the same concentrations, LY294002, but not wortmannin, significantly inhibited NO production and iNOS expression. LY303511, an inactive analogue of LY294002, also inhibited NO production and iNOS expression. In addition, LY294002 and LY303511 significantly inhibited the DNA binding activity of NF-kappaB and NF-kappaB dependent reporter gene expression. These results suggest that LY294002 inhibits iNOS expression at least in part via inhibition of NF-kappaB activation, independent of PI3K.     

Myocardial microcirculation stereological changes in rats subjected to nitric oxide synthesis inhibition

This work aims to study stereological changes in intramyocardial blood vessels in rats submitted to nitric oxide (NO) synthesis inhibition within different periods. NO synthesis inhibition was achieved by administration of L-NAME (50 mg/kg/day); control and L-NAME rats were sacrificed 25 and 40 days after experimentation. Light microscopy and stereology [according to references 7, 13 and 14] were used for analyzing the myocardium. Arterial blood pressure and cardiac weight increased by 74.5% and 57.8% after 25 days and by 90.2% and 34.6% after 40 days, respectively. Comparing the L-NAME rats with corresponding controls revealed that the volume density of the vessels decreased by 31.3% after 40 days, and the length density by 53.5% after 25 days and by 25.7% after 40 days. The mean cross-sectional area of the vessels increased by 154.6% after 25 days. In this study on intramyocardial vessels, we observed an important decrease of the length density in L-NAME animals. Likewise, the volume density also decreased significantly in L-NAME animals. The mean cross sectional area of the vessels, which normally increases during cardiac growth between 25 and 40 days, was precociously increased in L-NAME animals at 25 days, suggesting that these animals suffer from a precocious increase of the heart (including blood vessels) due to pressure overload. Stereology of cardiac microvessels revealed remodeling of these vessels in rats under NO synthesis inhibition. Although these changes may be caused by NO inhibition and not by arterial hypertension, further comparative studies on different models of arterial hypertension are needed to confirm this hypothesis.     

Central nitric oxide inhibition modifies metabolic adjustments induced by exercise in rats

The influence of the central nervous system on metabolic function is of interest in situations deviating from basal states, such as during exercise. Our previous study in rats demonstrated that central nitric oxide (NO) blockade increases metabolic rate, reducing mechanical efficiency during exercise. To assess the role of brain nitric oxide in the plasma glucose, lactate and free fatty acids (FFAs) concentrations of rats submitted to an incremental exercise protocol on a treadmill until fatigue, 1.43 micromol (2 microl) of N(omega)-nitro-l-arginine methyl ester (L-NAME, n=6), a NO synthase inhibitor, or 2 microl of 0.15M NaCl (SAL, n=6) was injected into the lateral cerebral ventricle (icv) of male Wistar rats immediately before exercise (starting at 10 m/min, with increments of 1m/min every 3 min until fatigue, 10% inclination). Blood samples were collected through a chronic jugular catheter at rest and during exercise until fatigue. During exercise, the L-NAME-treated animals had the following metabolic response compared to controls: (1) an increased hyperglycemic response during the first 60% of time to fatigue; (2) higher plasma lactate levels; and (3) a significant transitory increase in plasma free fatty acids during the dynamic phase of exercise that returned to basal levels earlier than controls during the steady state phase of exercise. In addition L-NAME-treated rats fatigued earlier than controls. The data indicate that the inhibition of the brain nitrergic system induced by icv L-NAME treatment disrupted the accuracy of the neural mechanism that regulates plasma glucose and free fatty acids mobilization during exercise in rats.     

一氧化氮对香烟烟雾提取物诱导大鼠肺泡巨噬细胞核因子кB活化的影响

目的:探讨一氧化氮(NO)对香烟烟雾提取物(CSE)诱导的大鼠肺泡巨噬细胞(AM)中核因子κB(NF—κB)活化的影响及机 制。方法:将大鼠AM与不同浓度的NO前体左旋精氨酸(L-Arg)或iNOS特异性抑制剂N6-(1-亚氨乙基)赖氨酸(L-NIL)及CSE共同培养,用免疫细胞化学染色法检测NF-κB,用Western blot检测I-κB蛋白含量,用Griess法测定培养上清液中NO的水平。结果:CSE可使NF-κB活化细胞的百分率增加,I-κB的水平下降。加入CSE和低浓度L-Arg培养的AM,NF-κB活化细胞的百分率显著高于只加入CSE的AM;而I-κB的水平显著低于只加入CSE的AM。加入CSE和高浓度L-Arg培养的AM,NF-κB活化细胞的百分率显著低于只加入CSE的AM,而I-κB的水平无显著变化。加入CSE和不同浓度的L-NIL培养的AM,NF-κB活化细胞的百分率显著低于只加入CSE的AM;而I-κB的水平则显著高于只加入CSE的AM,并呈浓度依赖(P<0.01)。结论:内源性NO对香烟烟雾所致NF-κB的活化具有双向调控作用。     

NO在脂联素抑制高脂血症大鼠血小板聚集中的作用

目的: 探讨一氧化氮(NO)信号转导通路在脂联素抑制高脂血症血小板聚集机制中的作用。方法: 采用成年大鼠饲以高脂饲料14周,分离其血小板并以重组脂联素(rAPN)孵育。采用免疫荧光、Western blotting等方法观察检测血小板聚集、NO含量、超氧化物含量、内皮型一氧化氮合酶(eNOS)/诱导型一氧化氮合酶(iNOS)的表达和抗氧化物活性。结果: 采用rAPN处理能抑制高脂血症诱导的血小板聚集(P<0.05),并导致血小板NO的生成显著减少。同时,在高脂血症血小板中,采用rAPN处理还能显著减少超氧化物的生成(降低62%, P<0.05) 并增强其抗氧化能力(增加38%, P<0.05)。此外,高脂血症诱导的eNOS磷酸化的降低和iNOS表达的增加在rAPN处理后被显著逆转(P<0.05, P<0.01)。结论: 脂联素是一种抑制高脂血症血小板聚集的脂肪细胞因子,其机制与减少超氧化物水平、增加抗氧化物活性和阻断iNOS的表达有关。     

Verapamil modulates LPS-induced cytokine production via inhibition of NF-kappa B activation in the liver

Objective: To investigate the effect of verapamil on Lipopolysaccharide (LPS)-induced cytokines [tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6) and interleukin-10 (IL-10)] and nuclear factor kappa B (NF-κ B) in the liver. Methods and Materials: Adult male Sprague-Dawley rats were randomly divided into seven groups of eight rats each: control rats treated with saline (0.9 % NaCl); rats treated with saline and then challenged intraperitoneally with LPS (10 mg/kg); rats treated intraperitoneally with different levels of verapamil (1, 2.5, 5, 10 mg/kg) and then challenged with LPS (10 mg/kg); and rats treated only with verapamil (10 mg/kg). TNF-α, IL-6, IL-10 and NF-κ B in the liver tissues were investigated as well as the serum levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) one hour after LPS injection. Results: LPS alone stimulated production of TNF-α, IL-6 and IL-10, and activated NF-κ B in the liver. Pretreatment with verapamil before LPS challenge reduced acute liver injury, down-regulated production of LPS-induced pro-inflammatory cytokines (TNF-α and IL-6), up-regulated production of anti-inflammatory cytokines (IL-10) and inhibited NF-κ B activation in the liver in a dose-dependent manner. Conclusion: Verapamil can attenuate acute liver injury by down-regulating the production of TNF-α and IL-6 and up-regulating IL-10 in the liver, possibly via inhibition of NF-κ B. Received 8 August 2005; returned for revision 25 September 2005; returned for final revision 18 November 2005; accepted by M. Katori 6 December 2005     

The inhibition of nitric oxide synthase suppresses LPS- and psychological-stress-induced fever in rats

The purpose of this study was to assess the effects of a non-selective nitric oxide synthase (NOS) inhibitor on changes in fever response due to injection of lipopolysaccharide (LPS) or on stress fever caused by exposure to an open field in freely moving biotelemetered rats. N(omega)-nitro-L-arginine methyl ester (L-NAME), an inhibitor of all NOS-isoforms, was injected intraperitoneally (ip) at a dose of 50 mg/kg just before intraperitoneal injection of LPS at a dose of 50 microg/kg or exposure to open field. L-NAME at a dose of 50 mg/kg had no effect on normal day-time body temperature (T(b)) and normal night-time T(b). The same dose of L-NAME administered intraperitoneally caused a significant attenuation of LPS-induced fever. The thermal index calculated for rats pretreated with L-NAME and injected with LPS was reduced by approximately 75% compared to that calculated for saline-pretreated and LPS-injected rats. To examine the effect of NOS inhibition on psychological-stress-induced elevation in T(b), rats were injected intraperitoneally with L-NAME and then immediately exposed to open field for 60 min. After exposure to the open field, rats not treated with NOS inhibitor responded with a rapid rise in T(b), and it was accompanied with an increase of motor activity. L-NAME significantly suppressed the stress fever without any effect on changes in motor activity. Presented data provide clear evidence that NO formation is involved in LPS- and psychological-stress-induced fevers in rats.     

Endothelial function and nitric oxide production in rats adapted to intermittent hypoxia

Adaptation to intermittent hypoxic hypoxia did not affect the endothelium-dependent relaxation of the aorta in rats, but significantly increased the relaxation of their tail artery. Following the adaptation, the NO level fell in the liver, intestine, and kidneys and remained unchanged in the spleen. Adaptation to hypoxia presumably limits NO synthesis and/or release in the vascular endothelium or enhances the capacity of this oxide to bind in a physiologically active depot. Translated fromByulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 120, N ^o 11, pp. 495–498, November, 1995 Presented by Yu. A. Vladimirov, Member of the Russian Academy of Medical Sciences     

Entamoeba histolytica modulates the nitric oxide synthase gene and nitric oxide production by macrophages for cytotoxicity against amoebae and tumour cells.

Nitric oxide (NO) is the major cytotoxic molecule produced by activated macrophages for cytotoxicity against Entamoeba histolytica trophozoites. In the present study, we determined whether E. histolytica infection and soluble amoebic proteins affected macrophage cytotoxicity against amoebae and tumour cells by modulating the inducible NO synthase gene (iNOS) and NO (measured as nitrite, NO2-) and tumour necrosis factor-alpha (TNF-alpha) production. Amoebic liver abscess-derived macrophages [days 10, 20, 30 post-infection (p.i.)] stimulated with interferon-gamma (IFN-gamma) and lipopolysaccharide (LPS) showed increased cytotoxicity against L929 cells (TNF-alpha-sensitive), but were refractory for killing amoebae and P815 cells (both NO-sensitive), concomitant with low NO2- production (< 4 microM/10(6) cells). In contrast, peritoneal and spleen macrophages at 10 and 20 days p.i. activated with IFN-gamma and LPS demonstrated increased killing of amoebae, and L929 and P815 cells concomitant with high NO2- production (> 12 microM/10(6) cells). Pretreatment of mouse bone marrow-derived macrophages with amoebic proteins suppressed IFN-gamma and LPS-induced amoebicidal (33%) and tumoricidal (44-49%) activities, with a corresponding decrease in TNF-alpha (56%) and NO (41%) production as well as TNF-alpha (41%) and iNOS (27%) mRNA by Northern blot analyses as compared to untreated activated controls. Inhibition of prostaglandin E2 (PGE2) biosynthesis in abscess and naive macrophages pretreated with amoebic proteins augmented IFN-gamma- and LPS-induced killing of L929 cells and TNF-alpha production, but failed to increase killing of P815 cells and amoebae as well as iNOS mRNA levels or NO production. These results suggest that E. histolytica selectively induces dysfunction of macrophage cytotoxicity by modulating iNOS mRNA expression and NO production independent from TNF-alpha and PGE2 allowing the parasites to survive within the host by impairing host immune responses.     

Combined inhibition of indoleamine 2,3-dioxygenase and nitric oxide synthase modulates neurotoxin release by interferon-gamma-activated macrophages

We evaluated the synthesis of nitric oxide (NO) and of the neurotoxic kynurenine metabolites 3OH-kynurenine and quinolinic acid (QUIN) in interferon-gamma (IFN-gamma)-activated macrophages of the murine BACl.2F5 cell line with the aim of investigating the roles of mononuclear phagocytes in inflammatory neurological disorders. IFN-gamma induced indoleamine 2,3-dioxygenase (IDO) and NO synthase (NOS) and increased the synthesis of 3OH-kynurenine, QUIN, and NO that accumulated in the incubation medium where they reached neurotoxic levels. Macrophage exposure to norharmane, an IDO inhibitor, resulted in a decreased formation of not only the kynurenine metabolites but also NO. The inhibition of NO synthesis could not be ascribed to reduced NADPH availability or decreased NOS induction. Norharmane inhibited NOS activity also in coronary vascular endothelial cells and in isolated aortic rings. Our findings suggest that activated macrophages release large amounts of neurotoxic molecules and that norharmane may represent a prototype compound to study macrophage involvement in inflammatory brain damage.     

Decreased renal haemodynamic response to inhibition of nitric oxide synthase in subtotally nephrectomized rats

To assess the renal haemodynamic response to manipulations of the nitric oxide (NO) system, we examined subtotally nephrectomized (SNX) rats and control rats (CON) 28 days after their operation. Bolus infusions of the NO synthase inhibitor N ^G-nitro-l-arginine (l-NA) were given intravenously at doses of 2 mg/kg and 10 mg/kg. Blood pressure was measured intra-arterially, glomerular filtration rate was measured by inulin clearance and fractional changes in renal blood flow (RBF) were determined by a Doppler flow probe. Both doses of l-NA caused a similar and dose dependent increase in mean blood pressure in both SNX and CON rats. In contrast, the decrease in RBF and the increase in the renovascular resistance index (RVRI) was less in SNX rats as compared to CON rats (RBF = –70.1±2.2% of baseline vs –52.7±5.2%, P<0.01; RVRI = +177±9% of baseline vs +243±24%, P<0.05). These changes were not affected by autonomic blockade (hexamethonium), or by blockade of the angiotensin II receptor (Losartan). The exogenous NO donor sodium nitroprusside (0.5 and 1.5 g · kg^–1 · min^–1) lowered mean blood pressure to a similar degree in SNX and CON rats; in contrast, RVRI decreased less in SNX rats (86.9±9.2% of baseline) than in CON rats (68.2±4.6%, P<0.05). We conclude that the reaction of the renal vasculature to manipulations of the NO system is altered in the SNX rats. The data suggest that in the remnant kidney, renovascular resistance is less dependent on endogenous NO and the vascular bed is less sensitive to exogenous NO.