Anti-inflammatory activity of c-phycocyanin in lipopolysaccharide-stimulated RAW 264.7 macrophages

C-phycocyanin (C-PC), found in blue green algae, is often used as a dietary nutritional supplement. C-PC has been found to have an anti-inflammatory activity and exert beneficial effect in various diseases. However, little is known about its mechanism of action. Overproduction of nitric oxide (NO) derived from inducible nitric oxide synthase (iNOS) plays an important role in the pathogenesis of inflammation. The aim of this study was to determine whether C-PC inhibits production of nitrite, an index of NO, and iNOS expression in lipopolysaccharide (LPS)-treated RAW 264.7 macrophages. Our results indicated that C-PC significantly inhibited the LPS-induced nitrite production and iNOS protein expression accompanied by an attenuation of tumor necrosis factor-alpha (TNF-alpha) formation but had no effect on interleukin-10 production in macrophages. Furthermore, C-PC also suppressed the activation of nuclear factor-kappaB (NF-kappaB) through preventing degradation of cytosolic IkappaB-alpha in LPS-stimulated RAW 264.7 macrophages. Thus, the inhibitory activity of C-PC on LPS-induced NO release and iNOS expression is probably associated with suppressing TNF-alpha formation and nuclear NF-kappaB activation, which may provide an additional explanation for its anti-inflammatory activity and therapeutic effect.     

Acetylbritannilatone suppresses NO and PGE2 synthesis in RAW 264.7 macrophages through the inhibition of iNOS and COX-2 gene expression

In order to elucidate the mechanism of anti-inflammatory effect of 1-o-acetylbritannilatone (ABL) isolated from Inula Britannica-F, we investigated ABL for its ability to inhibit the inflammatory factor production in RAW 264.7 macrophages. The studies showed that ABL not only inhibited LPS/IFN-gamma-mediated nitric oxide (NO) production and inducible nitric synthase (iNOS) expression, but also decreased LPS/IFN-gamma-induced prostaglandin E2 (PGE2) production and cyclo-oxygenase-2 (COX-2) expression in a concentration-dependent manner. EMSA demonstrated that ABL inhibited effectively the association of NF-kappaB, which is necessary for the expression of iNOS and COX-2, with its binding motif in the promoter of target genes. These data suggest that ABL suppress NO and PGE2 synthesis in RAW 264.7 macrophages through the inhibition of iNOS and COX-2 gene expression, respectively. The anti-inflammatory effect of ABL involves blocking the binding of NF-kappaB to the promoter in the target genes and inhibiting the expression of iNOS and COX-2.     

Fucoidan induces nitric oxide production via p38 mitogen-activated protein kinase and NF-kappaB-dependent signaling pathways through macrophage scavenger receptors

It has been reported that ligands of the macrophage scavenger receptor (MSR) induce a range of cellular responses including urokinase-type plasminogen activator and the production of inflammatory cytokines. Although nitric oxide (NO) is an important regulatory molecule in physiological functions such as vascular homeostasis, neurotransmission, and host defense, the effect of MSR ligands on NO production from macrophages was unknown. Here, we demonstrate that the MSR ligand, fucoidan, but neither oxidized low-density lipoprotein, acetylated LDL, maleylated bovine serum albumin nor dextran sulfate induces activation of inducible nitric oxide synthase (iNOS) promoter or NO production in RAW264.7 cells. Furthermore, we investigated the molecular mechanism by which fucoidan induces iNOS promoter activation. Using different inhibitors, we showed that the stimulation of fucoidan was mediated by both the p38 mitogen-activated protein kinase and the NF-kappaB-dependent pathways. Although these two pathways were independent, heat shock protein 90 (HSP90) played a significant role in both pathways. Our previous study showed that HSP90 directly interacts with the cytoplasmic domain of MSR. These results provide the evidence that HSP90 bound to the cytoplasmic domain of MSR is implicated in MSR-mediated signal transduction. Moreover, fucoidan-induced NO production by peritoneal macrophages from MSR-knockout (MSR-/-) mice significantly decreases compared with those from wild-type mice. This is the first indication that MSR transduces the signal of fucoidan to iNOS gene expression.     

Antimicrobial peptide P18 inhibits inflammatory responses by LPS- but not by IFN-γ-stimulated macrophages

Antimicrobial peptide P18 markedly inhibited the expression of inducible nitric oxide synthase (iNOS), tumor necrosis factor-alpha (TNF-alpha) and interleukin-1 beta (IL-1beta) in lipopolysaccharide (LPS)-stimulated RAW264.7 macrophage cells, whereas magainin 2 did not inhibit these activities. P18 dose-dependently reduced nitric oxide (NO) production by LPS-stimulated RAW 264.7 macrophage cells, with complete inhibition at 20 microg P18 ml(-1). In contrast, P18 had no effect on NO production and the expression of iNOS mRNA and iNOS protein by interferon-gamma (IFN-gamma)-stimulated RAW264.7 cells, suggesting P18 selectively inhibits LPS-stimulated inflammatory responses in macrophages. An LAL assay showed that P18 has strong LPS-neutralizing activity, indicating that P18 inhibits the inflammatory responses in LPS-stimulated macrophages by direct binding to LPS. Collectively, our results indicate that P18 has promising therapeutic potential as a novel anti-inflammatory as well as antimicrobial agent.     

Structure-activity relationship studies on chalcone derivatives. the potent inhibition of chemical mediators release

Some chalcones exert potent anti-inflammatory activities. 2',5'-Dialkoxychalcones and 2',5'-dihydroxy-4-chloro-dihydrochalcone inhibited nitric oxide (NO) production in lipopolysaccharide (LPS)/interferon-gamma (IFN-gamma)-activated N9 microglial cells and in LPS-activated RAW 264.7 macrophage-like cells have been demonstrated in our previous reports. These compounds also suppressed the inducible NO synthase (iNOS) expression and cyclooxygenase-2 (COX-2) activity in RAW 264.7 cells. In an effort to continually develop potent anti-inflammatory agent, a series of chalcones were prepared by Claisen-Schmidt condensation of appropriate acetophenones with appropriate aromatic aldehyde and then evaluated their inhibitory effects on the activation of mast cells, neutrophils, macrophages, and microglial cells. Most of the 2',5'-dihydroxychaclone derivatives exhibited potent inhibitory effects on the release of beta-glucuronidase and lysozyme from rat neutrophils stimulated with formyl-Met-Leu-Phe (fMLP)/cytochalasin B (CB). Some chalcones showed potent inhibitory effects on superoxide anion generation in rat neutrophils in response to fMLP/CB. Compounds 1 and 5 exhibited potent inhibitory effects on NO production in macrophages and microglial cells. Compound 11 showed inhibitory effect on NO production and iNOS protein expression in RAW 264.7 cells. The present results demonstrated that most of the 2',5'-dihydroxychaclones have anti-inflammatory effects. The potent inhibitory effect of 2',5'-dihydroxy-dihydrochaclones on NO production in LPS-activated macrophage, probably through the suppression of iNOS protein expression, is proposed to be useful for the relief of septic shock.     

Astaxanthin inhibits nitric oxide production and inflammatory gene expression by suppressing I(kappa)B kinase-dependent NF-kappaB activation

Astaxanthin, a carotenoid without vitamin A activity, has shown anti-oxidant and anti-inflammatory activities; however, its molecular action and mechanism have not been elucidated. We examined in vitro and in vivo regulatory function of astaxanthin on production of nitric oxide (NO) and prostaglandin E2 (PGE2) as well as expression of inducible NO synthase (iNOS), cyclooxygenase-2, tumor necrosis factor-alpha (TNF-alpha), and interleukin-1beta (IL-1beta). Astaxanthin inhibited the expression or formation production of these proinflammatory mediators and cytokines in both lipopolysaccharide (LPS)-stimulated RAW264.7 cells and primary macrophages. Astaxanthin also suppressed the serum levels of NO, PGE2, TNF-alpha, and IL-1beta in LPS-administrated mice, and inhibited NF-kappaB activation as well as iNOS promoter activity in RAW264.7 cells stimulated with LPS. This compound directly inhibited the intracellular accumulation of reactive oxygen species in LPS-stimulated RAW264.7 cells as well as H2O2-induced NF-kappaB activation and iNOS expression. Moreover, astaxanthin blocked nuclear translocation of NF-kappaB p65 subunit and I(kappa)B(alpha) degradation, which correlated with its inhibitory effect on I(kappa)B kinase (IKK) activity. These results suggest that astaxanthin, probably due to its antioxidant activity, inhibits the production of inflammatory mediators by blocking NF-kappaB activation and as a consequent suppression of IKK activity and I(kappa)B-alpha degradation.     

Induction of heme oxygenase-1 mediates the anti-inflammatory effects of the ethanol extract of Rubus coreanus in murine macrophages

Foods of plant origin, especially fruits and vegetables, draw increased attention because of their potential benefits to human health. The aim of the present study was to determine in vitro anti-inflammatory activity of four different extracts obtained from the fruits of Rubus coreanus (aqueous and ethanol extracts of unripe and ripe fruits). Among the four extracts, the ethanol extract of unripe fruits of R. coreanus (URCE) suppressed nitric oxide (NO) and prostaglandin E(2) (PGE(2)) production in lipopolysaccharide (LPS)-stimulated RAW264.7 murine macrophages. We also demonstrated that URCE by itself is a potent inducer of heme oxygenase-1 (HO-1). Inhibition of HO-1 activity by tin protoporphyrin, a specific HO-1 inhibitor, suppressed the URCE-induced reductions in the production of NO and PGE(2) as well as the expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase 2 (COX-2). Our data suggest that URCE exerts anti-inflammatory effects in macrophages via activation of the HO-1 pathway and helps to elucidate the mechanism underlying the potential therapeutic value of R. coreanus extracts.     

Anti-inflammatory effect of soyasaponins through suppressing nitric oxide production in LPS-stimulated RAW 264.7 cells by attenuation of NF-κB-mediated nitric oxide synthase expression

The anti-inflammatory properties of soyasaponins (especially soyasaponins with different chemical structures) have scarcely been investigated. We investigated the inhibitory effects of five structural types of soyasaponins (soyasaponin A¹, A², I and soyasapogenol A, B) on the induction of nitric oxide (NO) and inducible NO synthase (iNOS) in murine RAW 264.7 cells activated with lipopolysaccharide (LPS). Soyasaponin A¹, A² and I (25-200 μg/mL) dose-dependently inhibited the production of NO and tumor necrosis factor α (TNF-α) in LPS-activated macrophages, whereas soyasapogenol A and B did not. Furthermore, soyasaponin A¹, A² and I suppressed the iNOS enzyme activity and down-regulated the iNOS mRNA expression both in a dose-dependent manner. The reporter gene assay revealed that soyasaponin A¹, A² and I decreased LPS-induced nuclear factor kappa B (NF-κB) activity. Soyasaponin A¹, A² and I exhibit anti-inflammatory properties by suppressing NO production in LPS-stimulated RAW 264.7 cells through attenuation of NF-κB-mediated iNOS expression. It is proposed that the sugar chains present in the structures of soyasaponins are important for their anti-inflammatory activities. These results have important implication for using selected soyasaponins towards the development of effective chemopreventive and anti-inflammatory agents.     

Lipoteichoic acid-induced nitric oxide synthase expression in RAW 264.7 macrophages is mediated by cyclooxygenase-2, prostaglandin E2, protein kinase A, p38 MAPK, and nuclear factor-kappaB pathways

We recently reported that lipoteichoic acid (LTA), a cell wall component of the gram-positive bacterium Staphylococcus aureus, stimulated inducible nitric oxide synthase (iNOS) expression, nitric oxide (NO) release, and cyclooxygenase-2 (COX-2) expression in RAW 264.7 macrophages. This study was carried out to further investigate the roles of COX-2 and prostaglandin E2 (PGE2) in LTA-induced iNOS expression and NO release in RAW 264.7 macrophages. Treatment of RAW 264.7 macrophages with LTA caused a time-dependent increase in PGE2 release. LTA-induced iNOS expression and NO release were inhibited by a non-selective COX inhibitor (indomethacin), a selective COX-2 inhibitor (NS-398), an adenylyl cyclase (AC) inhibitor (dideoxyadenosine, DDA), and a protein kinase A (PKA) inhibitor (KT-5720). Furthermore, both PGE2 and the direct PKA activator, dibutyryl-cAMP, also induced iNOS expression in a concentration-dependent manner. Stimulation of RAW 264.7 macrophages with LTA, PGE2, and dibutyryl-cAMP all caused p38 MAPK activation in a time-dependent manner. LTA-mediated p38 MAPK activation was inhibited by indomethacin, NS-398, and SB 203580, but not by PD 98059. The PGE2-mediated p38 MAPK activation was inhibited by DDA, KT-5720, and SB 203580, but not by PD 98059. LTA caused time-dependent activation of the nuclear factor-kappaB (NF-kappaB)-specific DNA-protein complex formation. The LTA-induced increase in kappaB-luciferase activity was inhibited by indomethacin, NS-398, KT-5720, and a dominant negative mutant of p38 alphaMAPK (p38 alphaMAPK DN). These results suggest that LTA-induced iNOS expression and NO release involve COX-2-generated PGE2 production, and AC, PKA, p38 MAPK, and NF-kappaB activation in RAW 264.7 macrophages.     

Osteopontin is induced by nitric oxide in RAW 264.7 cells

Nitric oxide (NO) produced by macrophages is thought to contribute to various pathological conditions. Osteopontin (OPN) is a phosphorylated glycoprotein produced principally by macrophages. OPN inhibits inducible nitric oxide synthase (iNOS), which generates large amounts of NO production. However, the relationship between NO and endogenous OPN in activated macrophages has not yet been elucidated. We therefore examined expression of endogenous iNOS and OPN in a murine macrophage cell line, RAW 264.7 cells, by treating the cells with lipopolysaccharide (LPS) and interferon-gamma (IFN-gamma). Treatment of cells with LPS and IFN-gamma resulted in an increase of iNOS mRNA to maximum at 12 h after stimulation. In contrast, OPN mRNA was induced more slowly than iNOS mRNA. Induction of both iNOS and OPN mRNA in RAW 264.7 cells was markedly suppressed by addition of the specific iNOS inhibitor S-2-aminoethyl isothiourea dihydrobromide. The NOS inhibitor NG-methyl-L-arginine also suppressed induction of OPN mRNA but hardly affected iNOS mRNA expression. The NO-releasing agent spermine-NONOate but not peroxynitrite enhanced induction of OPN mRNA. These results suggest that NO directly up-regulates the endogenous OPN in macrophages stimulated with LPS and IFN-gamma. This up-regulation of endogenous OPN may represent a negative feedback system acting to reduce iNOS expression.     

Rosmarinic acid inhibits the formation of reactive oxygen and nitrogen species in RAW264.7 macrophages

Antioxidant action of Rosmarinic acid (Ros A), a natural phenolic ingredient in many Lamiaceae herbs such as Perilla frutescens, sage, basil and mint, was analyzed in relation to the Ikappa-B activation in RAW264.7 macrophages. Ros A inhibited nitric oxide (NO) production and inducible nitric oxide synthase (iNOS) protein synthesis induced by lipopolysaccharide (LPS), and also effectively suppressed phorbol 12-myristate 13-acetate (PMA)-induced superoxide production in RAW264.7 macrophages in a dose-dependent manner. Peroxynitrite-induced formation of 3-nitrotyrosine in bovine serum albumin and RAW264.7 macrophages were also inhibited by Ros A. Moreover, Western blot analysis demonstrated that LPS-induced phosphorylation of Ikappa-Balpha was abolished by Ros A. Ros A can act as an effective protector against peroxynitrite-mediated damage, and as a potent inhibitor of superoxide and NO synthesis; the inhibition of the formation of reactive oxygen and nitrogen species are partly based on its ability to inhibit the serine phosphorylation of Ikappa-Balpha.     

Antioxidant enzymes suppress nitric oxide production through the inhibition of NF-kappa B activation: role of H(2)O(2) and nitric oxide in inducible nitric oxide synthase expression in macrophages.

Reactive molecules O(-)(2), H(2)O(2), and nitrogen monoxide (NO) are produced from macrophages following exposure to lipopolysaccharide (LPS) and involved in cellular signaling for gene expression. Experiments were carried out to determine whether these molecules regulate inducible nitric oxide synthase (iNOS) gene expression in RAW264.7 macrophages exposed to LPS. NO production was inhibited by the antioxidative enzymes catalase, horseradish peroxidase, and myeloperoxidase but not by superoxide dismutase (SOD). In contrast, the NO-producing activity of LPS-stimulated RAW264.7 cells was enhanced by the NO scavengers hemoglobin (Hb) and myoglobin. The antioxidant enzymes decreased levels of iNOS mRNA and protein in LPS-stimulated RAW264.7 cells, whereas the NOS inhibitor N(G)-monomethyl-L-arginine as well as Hb increased the level of iNOS protein but not mRNA, indicating that NO inhibits iNOS protein expression. NF-kappa B was activated in LPS-stimulated RAW264.7 cells and the activation was significantly inhibited by antioxidant enzymes, but not by Hb. Similar results were obtained using LPS-stimulated rodent peritoneal macrophages. Extracellular O(-)(2) generation by LPS-stimulated macrophages was suppressed by SOD, but not by antioxidative enzymes, while accumulation of intracellular reactive oxygen species was inhibited by antioxidative enzymes, but not by SOD. Exogenous H(2)O(2) induced NF-kappa B activation in macrophages, which was inhibited by catalase and pyrroline dithiocarbamate (PDTC). H(2)O(2) enhanced iNOS expression and NO production in peritoneal macrophages when added with interferon-gamma, and the effect of H(2)O(2) was inhibited by catalase and PDTC. These findings suggest that H(2)O(2) production from LPS-stimulated macrophages participates in the upregulation of iNOS expression via NF-kappa B activation and that NO is a negative feedback inhibitor of iNOS protein expression.     

Effects of 6-formylpterin, a xanthine oxidase inhibitor and a superoxide scavenger, on production of nitric oxide in RAW 264.7 macrophages

As well as superoxide generated from neutrophils, nitric oxide (NO) produced by inducible nitric oxide synthase (iNOS) in macrophages plays an important role in inflammation. We previously showed that 6-formylpterin, a xanthine oxidase inhibitor, has a superoxide scavenging activity. In the present study, to elucidate other pharmacological activities of 6-formylpterin, we investigated the effects of 6-formylpterin on production of nitric oxide (NO) in the murine macrophage cell line RAW 264.7 stimulated by lipopolysaccharide (LPS) and interferon-gamma (INF-gamma). 6-Formylpterin suppressed the expression of iNOS, and it also inhibited the catalytic activity of iNOS, which collectively resulted in the inhibition of NO production in the stimulated macrophages. However, 6-formylpterin did not scavenge the released NO from an NO donor, S-nitroso-N-acetylpenicillamine (SNAP). These results indicate that 6-formylpterin inhibits pathological NO generation from macrophages during inflammation, but that it does not disturb the physiological action of NO released from other sources.     

Anethum graveloens flower extracts inhibited a lipopolysaccharide-induced inflammatory response by blocking iNOS expression and NF-κB activity in macrophages

Inflammation is a system used by a host to defend against the presence of bacteria, viruses, or yeasts. Toll-like receptors (TLRs) in the plasma membranes of macrophages are activated when they recognize the molecular structure of a virus or bacterium. Lipopolysaccharide (LPS), an outer cell-wall component of Gram-negative bacteria, initiates an inflammatory process via TLR4. We investigated the effect of the extract of Anethum graveloens flowers (AGFs) on LPS-mediated inflammation in RAW 264.7 cells. The extract markedly suppressed nitric oxide generation in a concentration-dependent manner in LPS-stimulated RAW 264.7 cells. It inhibited inducible nitric oxide synthase (iNOS) and the mRNA expression of cytokines such as interleukin-1 beta and interleukin-6 in LPS-stimulated RAW 264.7 cells. It also inhibited iNOS protein levels in LPS-stimulated RAW 264.7 cells. In addition, AGF decreased the LPS-induced phosphorylation of mitogen-activated protein kinases in LPS-stimulated RAW 264.7 cells. AGF inhibited the phosphorylation of Akt, an upstream molecule of the nuclear factor kappa B (NF-κB) pathway, and thus inhibited NF-κB activity in LPS-stimulated RAW 264.7 cells. These results suggest that AGF exerts an anti-inflammatory effect in LPS-stimulated RAW 264.7 cells by inhibiting iNOS expression and blocking the NF-κB pathway.