Inhibitors from the rhizomes of Alpinia officinarum on production of nitric oxide in lipopolysaccharide-activated macrophages and the structural requirements of diarylheptanoids for the activity

The 80% aqueous acetone extract from the rhizomes of Alpinia officinarum, a Chinese medicinal herb, were found to inhibit nitric oxide (NO) production in lipopolysaccharide (LPS)-activated mouse peritoneal macrophages. Through bioassay-guided separation, two diarylheptanoids [7-(4'-hydroxy-3'-methoxyphenyl-1-phenylhept-4-en-3-one and 3,5-dihydroxy-1,7-diphenylheptane] and a flavonol constituent (galangin) substantially inhibited LPS-induced NO production with IC50 values of 33-62 microM. To clarify structure-activity relationships of diarylheptanoids, related diarylheptanoids from Curcuma zedoaria were examined. Results indicate that the double bond or enone moiety at the 1-7 positions is important for the activity.     

Effects of stilbene constituents from rhubarb on nitric oxide production in lipopolysaccharide-activated macrophages

Two new anthraquinone glucosides [chrysophanol 8-O-beta-D-(6'-galloyl)-glucopyranoside, aloe-emodin 1-O-beta-D-glucopyranoside] together with various known stilbenes and their glucosides, anthraquinone glucosides, and a naphthalene glucoside were isolated from the rhizome of Rheum undulatum L. Three stilbenes (rhapontigenin, piceatannol, resveratrol), a naphthalene glucoside (torachrysone 8-O-beta-D-glucopyranoside), and two stilbene glucoside gallates (rhaponticin 2'-O-gallate, rhaponticin 6'-O-gallate) showed inhibitory activity of NO production in lipopolysaccharide-activated macrophages, (IC50 = 11-69 microM). The oxygen functions (-OH,-OCH3) at the benzene ring were found to be essential to show the activity. Whereas, the glucoside moiety reduced the activity, while the alpha,beta-double bond did not affect the activity. Furthermore, the active stilbenes (rhapontigenin, piceatannol, resveratrol) inhibited iNOS induction.     

Antioxidant constituents from rhubarb: structural requirements of stilbenes for the activity and structures of two new anthraquinone glucosides

The methanolic extracts from five kinds of rhubarb were found to show scavenging activity for DPPH radical and .O2-. Two new anthraquinone glucosides were isolated from the rhizome of Rheum undulatum L. together with two anthraquinone glucosides, a naphthalene glucoside, and 10 stilbenes. In the screening test for radical scavenging activity of rhubarb constituents, stilbenes and a naphthalene glucoside showed activity, but anthraquinones and sennosides did not. In addition, most stilbenes inhibited lipid peroxidation of erythrocyte membrane by tert-butyl hydroperoxide. Detailed examination of the scavenging effect on various related compounds suggested the following structural requirements; 1) phenolic hydroxyl groups are essential to show the activity; 2) galloyl moiety enhances the activity; 3) glucoside moiety reduces the activity; 4) dihydrostilbene derivatives maintain the scavenging activity for the DPPH radical, but they show weak activity for .O2-. In addition, several stilbenes with both the 3-hydroxyl and 4'-methoxyl groups inhibited xanthine oxidase.     

Synthesis and inhibitory effect of cis-guggulsterone on lipopolysaccharide-induced production of nitric oxide in macrophages

Guggulsterone is a bioactive plant sterol naturally found in migratory plants. It exists in various forms, and its active compounds include the isomers cis-guggulsterone (E-GS) and trans-guggulsterone (Z-GS). In this study, the anti-inflammatory effects of these two isomeric pregnadienedione steroids were investigated against lipopolysaccharide-induced inflammatory reaction in RAW264.7 mouse macrophages. Our results showed that both guggulsterones inhibited the production of NO in macrophages treated with lipopolysaccharide, with IC50 values ranging from 3.0 to 6.7 μM. E-GS exerted higher efficacy than Z-GS, and its anti-inflammatory effects was mediated through inhibition of iNOS and COX-2 expression.     

Effect of nitric oxide on phagocytic activity of lipopolysaccharide-induced macrophages: possible role of exogenous L-arginine

Among the antimicrobial mechanisms associated with macrophages, NO produced by iNOS plays a major role in intracellular killing, but the relationship between NO and phagocytic activity after injection of inflammatory agents into the peritoneal cavity is not clear. The aim of the present study was to investigate the effect of nitric oxide (NO) on macrophage function after treatment with intraperitoneal lipopolysaccharide (LPS) and the role of exogenous L-arginine administration in this event. Six experimental groups and one control group, each consisting of seven Wistar rats were used: Group I: Control; Group II: LPS; Group III: LPS+L-arginine; Group IV: LPS+L-arginine+Aminoguanidine; Group V: LPS+Aminoguanidine; Group VI: L-arginine; Group VII: Aminoguanidine. Macrophage phagocytic activity and total plasma nitrite levels were increased in the LPS group. In the LPS+L-arginine group, both the phagocytic activity and total plasma nitrite levels showed large increases. Administration of aminoguanidine (AG), a specific iNOS inhibitor, abolished macrophage phagocytic activity and total plasma nitrite levels in the LPS and LPS+L-arginine groups. As a result, we showed that NO produced by macrophages has a role not only in intracellular killing, but also in phagocytic activity.     

Syntheses of 1,2,3-triazolyl salicylamides with inhibitory activity on lipopolysaccharide-induced nitric oxide production

A 28-membered 1,2,3-triazolyl salicylamide library was synthesized via a Cu(I)-catalyzed azide-alkyne 1,3-dipolar cycloaddition and evaluated for their abilities to inhibit NO production in LPS-activated RAW264.7 macrophage cells. Among 28 analogues, 29g showed a significant inhibitory activity (IC₅₀ = 12.8 μM). The inhibitory effects of 29g on LPS-mediated NO production in macrophage cells appeared to be associated with the suppression of iNOS expression.     

Organ sites of lipopolysaccharide-induced nitric oxide production in the anesthetized rat.

The objective of this research was to determine the amount and timing of nitric oxide (NO, nitrogen monoxide) gas produced by the lungs, intestinal mucosa, and organ surfaces facing the peritoneal cavity after iv injection of a bacterial toxin, lipopolysaccharide (LPS). Some of the deleterious effects of LPS on organ function have been attributed to NO or strong oxidants formed locally from NO. Medical-grade air was used as an inspiratory air source (50 strokes/min x 3 ml/stroke) or was pumped through the ileal lumen or peritoneal cavity (20 strokes/min x 3 ml/stroke). The air was collected at intervals of 15-30 min for 3 h after LPS and analyzed for authentic NO gas by chemiluminescence. LPS (5 mg/kg) or saline was injected iv. Sodium nitroprusside (SNP) was injected to determine the appearance of its NO released into the perfused compartments. Blood pressure, plasma nitrate plus nitrite (NO(x)), and total plasma leukocytes were measured as other manifestations of LPS effects. NO began to increase in the pulmonary expired air 90 min after LPS and continued to increase for the remainder of the experiment. The final pulmonary post-LPS [NO] was about 20-fold greater than the [NO] before LPS. LPS had no effect on intraluminal or intraperitoneal [NO]. The saline injection had no effect on [NO] in any compartment. SNP injection increased NO entry into all three air-perfused compartments. Thus, NO from an exogenous tissue source was not prevented from being detected. Blood pressure was decreased by LPS only during the pulmonary perfusion. There were no significant effects of LPS on leukocytes or plasma NO(x). LPS decreased blood pressure and leukocytes and increased plasma NO(x) when air perfusion was not done. It was concluded that different organs can produce LPS-induced NO at markedly different rates and times. However, some aspect of the experimental technique of air perfusion could alter the effects of LPS.     

Diarylheptanoids from Curcuma kwangsiensis and their inhibitory activity on nitric oxide production in lipopolysaccharide-activated macrophages

Eleven diarylheptanoids (1-11) were isolated from rhizomes of Curcuma kwangsiensis, together with seven known compounds. Their structures were elucidated by 1D and 2D NMR, circular dichroism (CD), and accurate mass measurements. Inhibitory effects of the isolated compounds on nitric oxide production in lipopolysaccaride-activated macrophages were evaluated. Compounds 1, 2, and 3 showed strong inhibitory activity on NO production with IC(50) values of 3.13, 2.81 and 2.41 μM, respectively.     

Nimesulide inhibits lipopolysaccharide-induced production of superoxide anions and nitric oxide and iNOS expression in alveolar macrophages

The study was designed to investigate the effect of nimesulide on lipopolysaccharide (LPS)-induced proinflammatory oxidants production by rat alveolar macrophages (AMs). Effects of LPS and nimesulide on antioxidant defense and the expression of inducible nitric oxide synthase (iNOS) were also studied. It was found that nimesulide could scavenge superoxide anions (O2*-), nitric oxide (NO*) and total oxidant burden induced by LPS in AMs in vitro. Approximately 850 nmoles of nimesulide had activity equivalent to one IU of superoxide dismutase (SOD). Further, to confirm the in vitro observation, Male Wistar rats were orally administered with nimesulide (9 mg/kg b. wt. twice daily) for one week followed by intratracheal instillation of 2 microg LPS to stimulate lung inflammation. AMs from bronchoalveolar lavage fluid were collected 18 h after instillation of LPS. Nimesulide pretreatment could inhibit O2*-, NO() and lipid peroxidation in AMs. Nimesulide also suppressed LPS-induced iNOS expression in AMs in vivo and in vitro. Nimesulide could also normalize LPS-induced changes in the levels of superoxide dismutase (SOD), glutathione reductase (GR) and reduced glutathione (GSH) in AMs. Inhibition in production of oxidants in LPS-challenged AMs by nimesulide could be one of the pathways for its anti-inflammatory action.     

Regulation of nitric oxide production from macrophages by lipopolysaccharide and catecholamines.

Catecholamines are elaborated in stress responses to mediate vasoconstriction, and elevate systemic vascular resistance and blood pressure. They are elaborated in disorders such as sepsis, cocaine abuse, and cardiovascular disease. The aim of the study was to determine whether catecholamines affect nitric oxide (NO) production, as NO is a vasodilator and counteracts the harmful effects of catecholamines. RAW264.7 macrophage cells were cultured with lipopolysaccharide (LPS)+/-epinephrine, norepinephrine, and dopamine at 5x10(-6)M concentrations for 24h. Supernatants were harvested for measuring NO by spectrophotometry using the Greiss reagent and cells were harvested for detecting inducible NO synthase (iNOS) by Western blot. NO production in RAW 264.7 macrophages was increased significantly by addition of LPS (0.5-10ng/ml) in a dose-dependent fashion. The NO production induced by LPS was further enhanced by epinephrine and norepinephrine, and to a lesser extent by dopamine. These increases in NO correlated with expression of iNOS protein in these cells. The enhancing effect of iNOS synthesis by epinephrine and norepinephrine on LPS-induced macrophages was down regulated by beta-adrenoceptor antagonist, propranolol, and dexamethasone. The results suggest that catecholamines have a synergic effect on LPS in induction of iNOS synthesis and NO production, and this may mediate some of the vascular effects of infection. These data support a novel role for catecholamines in disorders such as septic shock and cocaine use, and indicate that beta-adrenoceptor antagonists and glucocorticoids may be used therapeutically for modulation of the catecholamine-NO axis in disease states.     

Effects of resveratrol-related hydroxystilbenes on the nitric oxide production in macrophage cells: structural requirements and mechanism of action

NF-kappaB that plays an important role in iNOS expression is one of the targets of various potential anti-inflammatory agents including resveratrol. Resveratrol contains a structural similarity with estrogen, and there has been speculation about resveratrol as estrogen agonist. In this study, the mechanism and structural requirements of resveratrol and related hydroxystilbenes for the inhibition of LPS-induced nitric oxide production were studied in macrophage cells (RAW 264.7 and J774) by comparing its effect on LPS-induced NF-kappaB translocation and nitric oxide production, and by considering the possibility of involvement of an estrogen receptor. LPS-induced nitric oxide production was inhibited only when cells were treated with resveratrol prior to stimulation with LPS, suggesting that resveratrol does not affect the enzyme itself. A higher concentration of resveratrol than needed for the inhibition of nitric oxide production was required for the inhibition of NF-kappaB mobilization or iNOS expression. Estrogen and diethylstilbesterol, an estrogen agonist, caused only weak inhibition of nitric oxide production, and the effects of resveratrol were not noticeably blocked by ICI-182780, an estrogen antagonist. Structure-activity analysis of resveratrol and nine hydroxystilbenes suggests that the structural balance between oxygen functional groups on the benzene rings is important for their activity. Our results suggest that resveratrol might act on other cellular targets as well as NF-kappaB at the initial stage of gene expression. Unique structural features of hydroxystilbenes are needed for suppression of nitric oxide production and it is unlikely that estrogen receptor is involved in it.     

Potentiation by high potassium of lipopolysaccharide-induced nitric oxide production from cultured astrocytes

Uptake of K+ is an important role of astrocytes to maintain physiological lower extracellular K+ concentration in the CNS. In this study, the effect of high K+ concentration was examined on the cellular function of astrocytes from embryonic rat brain in primary culture. Nitric oxide (NO) production induced by lipopolysaccharide (LPS) was measured as an index of cellular function of astrocytes. Increasing KCl concentration to about 40 mM did not directly evoke NO production, but doubled the level of LPS (1 ng/ml)-induced NO production. K-gluconate showed a similar enhancing effect although the degree of enhancement was about half of that of KCl. Neither NaCl nor Na-gluconate showed any effect. The K(+)-channel blocker, 4-aminopyridine, but not tetraethylammonium or apamin, inhibited the enhancing effect of KCl. The LPS-induced iNOS protein expression determined by immunoblotting analysis was enhanced by high K+ treatment. The level of iNOS mRNA determined by real-time RT-PCR technique was also augmented by the presence of 40 mM KCl. These results indicate that the elevation of extracellular K+ concentration regulates astrocytic cell functions through a mechanism involving K(A)-type K(+)-channels and that potentiation of NO production by high K+ is due to the augmentation of iNOS mRNA and iNOS protein levels.     

Inhibitory effect of the alkyl side chain of caffeic acid analogues on lipopolysaccharide-induced nitric oxide production in RAW264.7 macrophages

Caffeic acid esters, one of the components of propolis, are known to show a variety of biological effects such as anti-tumor, anti-oxidant, and anti-inflammatory activities. Although, the anti-inflammatory activities of caffeic acid esters have been studied by analyzing their structure, the detailed mechanisms of their activities remain unclear. Thus, in this study, we examined the function of the ester functional group and the alkyl side chain (alcoholic part) and transformed caffeic acid to several derivatives. The inhibitory effect of these derivatives on NO production in murine macrophage RAW264.7 cells was dependent on the length and size of the alkyl moiety, and undecyl caffeate was the most potent inhibitor of NO production. In addition, individual experiments using undecanol, caffeic acid, undecanol plus caffeic acid, and undecyl caffeate showed that the connection between caffeic acid and the alkyl chain is critical for activity. Amide and ketone derivatives showed that not only the ester functional group but also the amide and ketone functional groups exhibit an inhibitory effect on NO production.     

Structural requirements of flavonoids for nitric oxide production inhibitory activity and mechanism of action

To clarify the structure-activity relationships of flavonoids for nitric oxide (NO) production inhibitory activity, we examined the inhibitory effects of 73 flavonoids on NO production in lipopolysaccharide-activated mouse peritoneal macrophages. Among those flavonoids, apigenin (IC(50)=7.7 microM), diosmetin (8.9 microM), and tetra-O-methylluteolin (2.4 microM), and hexa-O-methylmyricetin (7.4 microM) were found to show potent inhibitory activity, and the results suggested the following structural requirements of flavonoids: (1) the activities of flavones were stronger than those of corresponding flavonols; (2) the glycoside moiety reduced the activity; (3) the activities of flavones were stronger than those of corresponding flavanones; (4) the flavones and flavonols having the 4'-hydroxyl group showed stronger activities than those lacking the hydroxyl group at the B ring and having the 3',4'-dihydroxyl group; (5) the flavonols having the 3',4'-dihydroxyl group (catechol type) showed stronger activities than those having the 3',4',5'-trihydroxyl group (pyrogallol type); (6) the 5-hydroxyl group tended to enhance the activity; (7) methylation of the 3-, 5-, or 4'-hydroxyl group enhanced the activity; (8) the activities of isoflavones were weaker than those of corresponding flavones; (9) methylation of the 3-hydroxyl group reduced the cytotoxicity. In addition, potent NO production inhibitors were found to inhibit induction of inducible nitric oxide synthase (iNOS) without iNOS enzymatic inhibitory activity.     

Analysis of the effects of nitric oxide and oxygen on nitric oxide production by macrophages

The interactions between NO and O(2) in activated macrophages were analysed by incorporating previous cell culture and enzyme kinetic results into a novel reaction-diffusion model for plate cultures. The kinetic factors considered were: (i) the effect of O(2) on NO production by inducible NO synthase (iNOS); (ii) the effect of NO on NO synthesis by iNOS; (iii) the effect of NO on respiratory and other O(2) consumption; and (iv) the effects of NO and O(2) on NO consumption by a possible NO dioxygenase (NOD). Published data obtained by varying the liquid depth in macrophage cultures provided a revealing test of the model, because varying the depth should perturb both the O(2) and the NO concentrations at the level of the cells. The model predicted that the rate of NO(2)(-) production should be nearly constant, and that the net rate of NO production should decline sharply with increases in liquid depth, in excellent agreement with the experimental findings. In further agreement with available results for macrophage cultures, the model predicted that net NO synthesis should be more sensitive to liquid depth than to the O(2) concentration in the headspace. The main reason for the decrease in NO production with increasing liquid depth was the modulation of NO synthesis by NO, with O(2) availability playing only a minor role. The model suggests that it is the ability of iNOS to consume NO, as well as to synthesize it, that creates very sensitive feedback control, setting an upper bound on the NO concentration of approximately 1 microM. The effect of NO consumption by other possible pathways (e.g., NOD) would be similar to that of iNOS, in that it would help limit net NO production. The O(2) utilized during enzymatic NO consumption is predicted to make the O(2) demands of activated macrophages much larger than those of unactivated ones (where iNOS is absent); this remains to be tested experimentally.     

Involvement of protein kinase C in the inhibition of lipopolysaccharide-induced nitric oxide production by thapsigargin in RAW 264.7 macrophages

This study explored the effects of inhibition of endoplasmic reticulum (ER) Ca²⁺-ATPase on lipopolysaccharide (LPS)-induced protein kinase C (PKC) activation, nuclear factor-κB (NF-κB) translocation, inducible nitric oxide synthase (iNOS) expression and nitric oxide (NO) production in RAW 264.7 macrophages. Thapsigargin (TG) irreversibly inhibits ER Ca²⁺-ATPase and LPS-induced NO production is reduced even after washout. TG also attenuated LPS-stimulated iNOS expression by using immunoblot analysis. However, another distinct fully reversible ER Ca²⁺-ATPase inhibitor, 2,5-di-tert-butylhydroquinone (DBHQ), ionophore A23187 and ionomycin could exert a similar effect to TG in increasing intracellular calcium concentration; however, these agents could not mimic TG in reducing iNOS expression and NO production. LPS increased PKC- and -β activation, and TG pretreatment attenuated LPS-stimulated PKC activation. Not did pretreatment with DBHQ, A23187 and ionomycin reduce LPS-stimulated PKC activation. Furthermore, NF-κB-specific DNA–protein-binding activity in the nuclear extracts was enhanced by treatment with LPS, and TG pretreatment attenuated LPS-stimulated NF-κB activation. None of DBHQ, A23187 and ionomycin pretreatment reduced LPS-stimulated NF-κB activation. These data suggest that persistent inhibition of ER Ca²⁺-ATPase by TG would influence calcium release from ER Ca²⁺ pools that was stimulated by the LPS activated signal processes, and might be the main mechanism for attenuating PKC and NF-κB activation that induces iNOS expression and NO production.     

Saccharated colloidal iron enhances lipopolysaccharide-induced nitric oxide production in vivo

We investigated the effects of iron on the production of nitric oxide (NO), inducible NO synthase (iNOS), and plasma cytokines induced by lipopolysaccharide (LPS) in vivo. Male Wistar rats were preloaded with a single intravenous injection of saccharated colloidal iron (Fesin, 70 mg iron/kg body weight) or normal saline as a control, and then given an intraperitoneal injection of LPS (5.0 mg/kg body weight). Rats, preloaded with iron, had evidence of both iron deposition and strong iNOS induction in liver Kupffer cells upon injection of LPS; phagocytic cells in the spleen and lung had similar findings. LPS-induced NO production in iron-preloaded rats was significantly higher than control rats as accessed by NO-hemoglobin levels measured by ESR (electron spin resonance) and NOx (nitrate plus nitrite) levels. Western blot analysis showed that iron preloading significantly enhanced LPS-induced iNOS induction in the liver, but not in the spleen or lung. LPS-induced plasma levels of IL-6, IL-1beta, and TNF-alpha were also significantly higher in iron-preloaded rats as shown by ELISA, but IFN-gamma levels were unchanged. We conclude that colloidal-iron phagocytosed by liver Kupffer cells enhanced LPS-induced NO production in vivo, iNOS induction in the liver, and release of IL-6, IL-1beta, and TNF-alpha.     

Dehydroepiandrosterone inhibits lipopolysaccharide-induced nitric oxide production in BV-2 microglia

Levels of dehydroepiandrosterone (DHEA) and its sulfated derivative (DHEAS) decline during aging and reach even lower levels in Alzheimer's disease (AD). DHEA is known to exhibit a variety of functional activities in the CNS, including an increase of memory and learning, neurotrophic and neuroprotective effects, and the reduction of risk of age-related neurodegenerative disorders. However, the influence of DHEA on the immune functions of glial cells is poorly understood. In this study, we investigated the effect of DHEA on activated glia. The production of inducible nitric oxide synthase (iNOS) was studied in lipopolysaccharide (LPS)-stimulated BV-2 microglia, as a model of glial activation. The results showed that DHEA but not DHEAS significantly inhibited the production of nitrite in the LPS-stimulated BV-2 cell cultures. Pretreatment of BV-2 cells with DHEA reduced the LPS-induced iNOS mRNA and protein levels in a dose-dependent manner. The LPS-induced iNOS activity in BV-2 cells was decreased by the exposure of 100 microM DHEA. Moreover, DHEA suppressed iNOS gene expression in LPS-stimulated BV-2 cells did not require de novo synthesis of new proteins or destabilize of iNOS mRNA. Since DHEA is biosynthesized by astrocytes and neurons, our findings suggest that it might have an important regulatory function on microglia.     

Modulation of nitric oxide synthase activity in macrophages

L-Arginine is converted to the highly reactive and unstable nitric oxide (NO) and L-citrulline by an enzyme named nitric oxide synthase (NOS). NO decomposes into other nitrogen oxides such as nitrite (NO(2) (-)) and nitrate (NO(2) (-)), and in the presence of superoxide anion to the potent oxidizing agent peroxynitrite (ONOO(-)). Activated rodent macrophages are capable of expressing an inducible form of this enzyme (iNOS) in response to appropriate stimuli, i.e., lipopolysaccharide (LPS) and interferon-gamma (IFNgamma). Other cytokines can modulate the induction of NO biosynthesis in macrophages. NO is a major effector molecule of the anti-microbial and cytotoxic activity of rodent macrophages against certain micro-organisms and tumour cells, respectively. The NO synthesizing pathway has been demonstrated in human monocytes and other cells, but its role in host defence seems to be accessory. A delicate functional balance between microbial stimuli, host-derived cytokines and hormones in the microenvironment regulates iNOS expression. This review will focus mainly on the known and proposed mechanisms of the regulation of iNOS induction, and on agents that can modulate NO release once the active enzyme has been expressed in the macrophage.