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.     

Inhibition of inducible nitric oxide synthase and cyclooxygenase II by Platycodon grandiflorum saponins via suppression of nuclear factor-kappaB activation in RAW 264.7 cells

Saponins are glycosidic compounds present in many edible and inedible plants. They exhibit potent biological activities in mammalian systems, including several beneficial effects such as anti-inflammation and immunomodulation. In this study, we investigated the effects of seven platycodin saponins on the activities of inducible nitric oxide synthase (iNOS) and cyclooxygenase II (COX-2) in lipopolysaccharide (LPS)-induced RAW 264.7 macrophages. We found that 2"-O-acetyl polygalacin D (S1), platycodin A (S2), platycodin D (S3), and polygalacin D (S6) inhibited LPS-induced NO production in a concentration-dependent manner. Furthermore, these compounds inhibited the expression of LPS-induced iNOS and COX-2 protein and mRNA without an appreciable cytotoxic effect on RAW 264.7 macrophages, and could suppress induction by LPS of pro-inflammatory cytokines such as prostaglandin E2 (PGE2). Treatment with these compounds of RAW 264.7 cells transfected with a reporter construct indicated a reduced level of LPS-induced nuclear factor-kappaB (NF-kappaB) activity and effectively lowered NF-kappaB binding as measured by electrophoretic mobility shift assay (EMSA). The suppression of NF-kappaB activation appears to occur through the prevention of inhibitor kappaB (IkappaB) degradation. In vivo, platycodin saponin mixture (PS) and S3 protected mice from the lethal effects of LPS. The 89% lethality induced by LPS/galactosamine was reduced to 60% and 50% when PS and S3, respectively, were administered simultaneously with LPS. These results suggest that the main inhibitory mechanism of the platycodin saponins may be the reduction of iNOS and COX-2 gene expression through blocking of NF-kappaB activation.     

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.     

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.     

CME‐1, a novel polysaccharide,suppresses iNOS expression in lipopolysaccharide‐stimulated macrophages through ceramide‐initiated protein phosphatase 2A activation

CME‐1, a novel water‐soluble polysaccharide purified from Ophiocordyceps sinensis mycelia, has anti‐oxidative, antithrombotic and antitumour properties. In this study, other major attributes of CME‐1, namely anti‐inflammatory and immunomodulatory properties, were investigated. Treating lipopolysaccharide (LPS)‐stimulated RAW 264.7 cells with CME‐1 concentration‐dependently suppressed nitric oxide formation and inducible nitric oxide synthase (iNOS) expression. In the CME‐1‐treated RAW 264.7 cells, LPS‐induced IκBα degradation and the phosphorylation of p65, Akt and mitogen‐activated protein kinases (MAPKs), including extracellular signal‐regulated kinase, c‐Jun N‐terminal kinase and p38, were reduced. Treatment with a protein phosphatase 2A (PP2A)‐specific inhibitor, significantly reversed the CME‐1‐suppressed iNOS expression; IκBα degradation; and p65, Akt and MAPK phosphorylation. PP2A activity up‐regulation and PP2A demethylation reduction were also observed in the cells. Moreover, CME‐1‐induced PP2A activation and its subsequent suppression of LPS‐activated RAW 264.7 cells were diminished by the inhibition of ceramide signals. LPS‐induced reactive oxygen species (ROS) and hydroxyl radical formation were eliminated by treating RAW 264.7 cells with CME‐1. Furthermore, the role of ceramide signalling pathway and anti‐oxidative property were also demonstrated in CME‐1‐mediated inhibition of LPS‐activated primary peritoneal macrophages. In conclusion, CME‐1 suppressed iNOS expression by up‐regulating ceramide‐induced PP2A activation and reducing ROS production in LPS‐stimulated macrophages. CME‐1 is a potential therapeutic agent for treating inflammatory diseases.     

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.     

Differential regulation of NO availability from macrophages and endothelial cells by the garlic component S-allyl cysteine

Garlic has been used as a traditional medicine for prevention and treatment of cardiovascular diseases. However, the molecular mechanism of garlic's pharmacological action has not been clearly elucidated. We examined here the effect of garlic extract and its major component, S-allyl cysteine (SAC), on nitric oxide (NO) production by macrophages and endothelial cells. The present study demonstrates that these reagents inhibited NO production through the suppression of iNOS mRNA and protein expression in the murine macrophage cell line RAW264.7, which had been stimulated with LPS and IFNgamma. The garlic extract also inhibited NO production in peritoneal macrophages, rat hepatocytes, and rat aortic smooth muscle cells stimulated with LPS plus cytokines, but it did not inhibit NO production in iNOS-transfected AKN-1 cells or iNOS enzyme activity. These reagents suppressed NF-kappaB activation and murine iNOS promoter activity in LPS and IFNgamma-stimulated RAW264.7 cells. In contrast, these reagents significantly increased cGMP production by eNOS in HUVEC without changes in activity, protein levels, and cellular distribution of eNOS. Finally, garlic extract and SAC both suppressed the production of hydroxyl radical, confirming their antioxidant activity. These data demonstrate that garlic extract and SAC, due to their antioxidant activity, differentially regulate NO production by inhibiting iNOS expression in macrophages while increasing NO in endothelial cells. Thus, this selective regulation may contribute to the anti-inflammatory effect and prevention of atherosclerosis by these reagents.     

Lactoferrin activates macrophages via TLR4-dependent and -independent signaling pathways

Lactoferrin (LF) is a component of innate immunity and is known to interact with accessory molecules involved in the TLR4 pathway, including CD14 and LPS binding protein, suggesting that LF may activate components of the TLR4 pathway. In the present study, we have asked whether bovine LF (bLF)-induced macrophage activation is TLR4-dependent. Both bLF and LPS stimulated IL-6 production and CD40 expression in RAW 264.7 macrophages and in BALB/cJ peritoneal exudate macrophages. However, in macrophages from congenic TLR4(-/-) C.C3-Tlr4(lps-d) mice, CD40 was not expressed while IL-6 secretion was increased relative to wild-type cells. The signaling components NF-kappaB, p38, ERK and JNK were activated in RAW 264.7 cells and BALB/cJ macrophages after bLF or LPS stimulation, demonstrating that the TLR4-dependent bLF activation pathway utilizes signaling components common to LPS activation. In TLR4 deficient macrophages, bLF-induced activation of NF-kappaB, p38, ERK and JNK whereas LPS-induced cell signaling was absent. We conclude from these studies that bLF induces limited and defined macrophage activation and cell signaling events via TLR4-dependent and -independent mechanisms. bLF-induced CD40 expression was TLR4-dependent whereas bLF-induced IL-6 secretion was TLR4-independent, indicating potentially separate pathways for bLF mediated macrophage activation events in innate immunity.     

Inhibitory effects of Irigenin from the rhizomes of Belamcanda chinensis on nitric oxide and prostaglandin E(2) production in murine macrophage RAW 264.7 cells

In the present study, we investigated antiinflammatory effects of six flavonoids isolated from the rhizomes of Belamcanda chinensis (Iridaceae) in RAW 264.7 macrophages. The results indicated that irigenin concentration dependently inhibited lipopolysaccharide (LPS)-induced nitric oxide (NO) and prostaglandin (PG) E(2) production. Furthermore, this compound inhibited the expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase (COX)-2 proteins and mRNAs without an appreciable cytotoxic effect. Treatment of the transfectant RAW 264.7 cells with irigenin reduced the level of nuclear factor-kappaB (NF-kappaB) activity, also effectively lowered NF-kappaB binding measured by electrophoretic mobility shift assay (EMSA), which was associated with decreased p65 protein levels in the nucleus. On the basis of the above data, we suggest that the effect of irigenin in decreasing LPS-induced NO and PGE(2) synthesis is due to diminish the mRNA and protein expression of iNOS and COX-2, respectively, also may be due to under the suppression of NF-kappaB activation. Therefore, irigenin isolated from the rhizomes of Belamcanda chinensis could be offered as a leading compound for anti-inflammation.     

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.     

Suppression of cytokine production in lipopolysaccharide-stimulated mouse macrophages by novel cationic glucosamine derivative involves down-regulation of NF-kappaB and MAPK expressions

Exposure of macrophages to bacterial lipopolysaccharide (LPS) induces release of proinflammatory cytokines that play crucial roles in chronic inflammation. Glucosamine has reported to possess anti-inflammatory properties and currently is the oral supplement of choice for the management of inflammation related complications including osteoarthritis. In this study, quaternized amino glucosamine (QAGlc), a newly synthesized cationic glucosamine (Glc) derivative was found to inhibit LPS-stimulated production of IL-1beta, IL-6, TNF-alpha, and PGE(2) in RAW264.7, mouse macrophages more potently than its starting material Glc. Since production of cytokines is regulated mainly via activation of NF-kappaB and regulation of mitogen-activated protein kinases (MAPKs), we examined if QAGlc could be responsible for the suppression of NF-kappaB pathway and MAPKs. We used reporter gene assay and Western blotting to examine the effects of QAGlc on activation and translocation of NF-kappaB. Further, QAGlc-mediated inhibition of NF-kappaB was accompanied with a suppression of its translocation. Apparently, QAGlc was shown to attenuate LPS-induced activation of p38 MAPK and JNK in RAW264.7 cells suggesting that inhibition of MAPK-mediated LPS signaling also contribute to suppression of cytokine production following stimulation of macrophages with LPS.     

Hydrogen sulfide inhibits nitric oxide production and nuclear factor-kappaB via heme oxygenase-1 expression in RAW264.7 macrophages stimulated with lipopolysaccharide

Hydrogen sulfide (H(2)S), a regulatory gaseous molecule that is endogenously synthesized by cystathionine gamma-lyase (CSE) and/or cystathionine beta-synthase (CBS) from L-cysteine (L-Cys) metabolism, is a putative vasodilator, and its role in nitric oxide (NO) production is unexplored. Here, we show that at noncytotoxic concentrations, H(2)S was able to inhibit NO production and inducible NO synthase (iNOS) expression via heme oxygenase (HO-1) expression in RAW264.7 macrophages stimulated with lipopolysaccharide (LPS). Both H(2)S solution prepared by bubbling pure H(2)S gas and NaSH, a H(2)S donor, dose dependently induced HO-1 expression through the activation of the extracellular signal-regulated kinase (ERK). Pretreatment with H(2)S or NaHS significantly inhibited LPS-induced iNOS expression and NO production. Moreover, NO production in LPS-stimulated macrophages that are expressing CSE mRNA was significantly reduced by the addition of L-Cys, a substrate for H(2)S, but enhanced by the selective CSE inhibitor beta-cyano-L-alanine but not by the CBS inhibitor aminooxyacetic acid. While either blockage of HO activity by the HO inhibitor, tin protoporphyrin IX, or down-regulation of HO-1 expression by HO-1 small interfering RNA (siRNA) reversed the inhibitory effects of H(2)S on iNOS expression and NO production, HO-1 overexpression produced the same inhibitory effects of H(2)S. In addition, LPS-induced nuclear factor (NF)-kappaB activation was diminished in RAW264.7 macrophages preincubated with H(2)S. Interestingly, the inhibitory effect of H(2)S on NF-kappaB activation was reversed by the transient transfection with HO-1 siRNA, but was mimicked by either HO-1 gene transfection or treatment with carbon monoxide (CO), an end product of HO-1. CO treatment also inhibited LPS-induced NO production and iNOS expression via its inactivation of NF-kappaB. Collectively, our results suggest that H(2)S can inhibit NO production and NF-kappaB activation in LPS-stimulated macrophages through a mechanism that involves the action of HO-1/CO.