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.     

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.     

EP2 and EP4 receptors on muscularis resident macrophages mediate LPS-induced intestinal dysmotility via iNOS upregulation through cAMP/ERK signals

Intestinal resident macrophages play an important role in gastrointestinal dysmotility by producing prostaglandins (PGs) and nitric oxide (NO) in inflammatory conditions. The causal correlation between PGs and NO in gastrointestinal inflammation has not been elucidated. In this study, we examined the possible role of PGE(2) in the LPS-inducible inducible NO synthase (iNOS) gene expression in murine distal ileal tissue and macrophages. Treatment of ileal tissue with LPS increased the iNOS and cyclooxygenase (COX)-2 gene expression, which lead to intestinal dysmotility. However, LPS did not induce the expression of iNOS and COX-2 in tissue from macrophage colony-stimulating factor-deficient op/op mice, indicating that these genes are expressed in intestinal resident macrophages. iNOS and COX-2 protein were also expressed in dextran-phagocytized macrophages in the muscle layer. CAY10404, a COX-2 inhibitor, diminished LPS-dependent iNOS gene upregulation in wild-type mouse ileal tissue and also in RAW264.7 macrophages, indicating that PGs upregulate iNOS gene expression. EP(2) and EP(4) agonists upregulated iNOS gene expression in ileal tissue and isolated resident macrophages. iNOS mRNA induction mediated by LPS was decreased in the ileum isolated from EP(2) or EP(4) knockout mice. In addition, LPS failed to decrease the motility of EP(2) and EP(4) knockout mice ileum. EP(2)- or EP(4)-mediated iNOS expression was attenuated by KT-5720, a PKA inhibitor and PD-98059, an ERK inhibitor. Forskolin or dibutyryl-cAMP mimics upregulation of iNOS gene expression in macrophages. In conclusion, COX-2-derived PGE(2) induces iNOS expression through cAMP/ERK pathways by activating EP(2) and EP(4) receptors in muscularis macrophages. NO produced in muscularis macrophages induces dysmotility during gastrointestinal inflammation.     

连翘脂素对LPS诱导RAW264.7细胞炎症反应的影响

为研究连翘脂素的抗炎效应及其抗炎机制,以地塞米松作为阳性对照,建立脂多糖(LPS)诱导小鼠巨噬细胞RAW264.7炎症模型,检测炎症因子的释放及相关蛋白和mRNA的表达,以期提高对连翘脂素抗炎作用的全面认识并为连翘脂素临床开发提供有力的科学依据。实验采用Griess法检测细胞上清液中NO含量,ELISA法检测TNF-α和IL-6的含量,Westernblot法检测iNOS、COX-2蛋白的表达,RT-qPCR法检测iNOS、COX-2mRNA的表达。与LPS组比较,连翘脂素组和地塞米松组可以明显降低LPS诱导的RAW264.7细胞释放NO、TNF-α和IL-6的量,并呈现浓度依赖关系。Westrenblot和RT-qPCR结果显示连翘脂素能抑制LPS诱导的iNOS、COX-2的蛋白表达以及mRNA的表达,并呈浓度依赖关系。实验研究表明连翘脂素能够明显抑制LPS诱导的RAW264.7细胞炎症因子的释放,iNOS、COX-2蛋白及mRNA的表达从而抑制炎症反应。     

Role of the cyclic AMP-protein kinase A pathway in lipopolysaccharide-induced nitric oxide synthase expression in RAW 264.7 macrophages. Involvement of cyclooxygenase-2.

The signaling pathway for lipopolysaccharide (LPS)-induced nitric oxide (NO) release in RAW 264.7 macrophages involves the protein kinase C and p38 activation pathways (Chen, C. C., Wang, J. K., and Lin, S. B. (1998) J. Immunol. 161, 6206-6214; Chen, C. C., and Wang, J. K. (1999) Mol. Pharmacol. 55, 481-488). In this study, the role of the cAMP-dependent protein kinase A (PKA) pathway was investigated. The PKA inhibitors, KT-5720 and H8, reduced LPS-induced NO release and inducible nitric oxide synthase (iNOS) expression. The direct PKA activator, Bt(2)cAMP, caused concentration-dependent NO release and iNOS expression, as confirmed by immunofluorescence studies. The intracellular cAMP concentration did not increase until after 6 h of LPS treatment. Two cAMP-elevating agents, forskolin and cholera toxin, potentiated the LPS-induced NO release and iNOS expression. Stimulation of cells with LPS or Bt(2)cAMP for periods of 10 min to 24 h caused nuclear factor-kappaB (NF-kappaB) activation in the nuclei, as shown by detection of NF-kappaB-specific DNA-protein binding. The PKA inhibitor, H8, inhibited the NF-kappaB activation induced by 6- or 12-h treatment with LPS but not that induced after 1, 3, or 24 h. The cyclooxygenase-2 (COX-2) inhibitors, NS-398 and indomethacin, attenuated LPS-induced NO release, iNOS expression, and NF-kappaB DNA-protein complex formation. LPS induced COX-2 expression in a time-dependent manner, and prostaglandin E(2) production was induced in parallel. These results suggest that 6 h of treatment with LPS increases intracellular cAMP levels via COX-2 induction and prostaglandin E(2) production, resulting in PKA activation, NF-kappaB activation, iNOS expression, and NO production.     

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.     

β-Phenylethyl and 8-methylsulphinyloctyl isothiocyanates, constituents of watercress, suppress LPS induced production of nitric oxide and prostaglandin E2 in RAW 264.7 macrophages

Beta-phenylethyl (PEITC) and 8-methylsulphinyloctyl isothiocyanates (MSO) represent two phytochemical constituents present in watercress Rorripa nasturtium aquaticum, with known chemopreventative properties. In the present investigation, we examined whether PEITC and MSO could modulate the inflammatory response of Raw 264.7 macrophages to bacterial lipopolysaccharide (LPS) by assessment of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) expression. Overproduction of both nitric oxide (NO) and prostaglandins (PGE) has been associated with numerous pathological conditions including chronic inflammation and cancer. Our results demonstrate that LPS (1 microg/ml approximately 24 h) induced nitrite and prostaglandin E2 (PGE-2) synthesis in Raw 264.7 cells was attenuated by both isothiocyanates (ITCs) in a concentration-dependent manner. Both PEITC and MSO decreased (iNOS) and (COX-2) protein expression levels leading to reduced secretion of both pro-inflammatory mediators. Interestingly, the reduction in both iNOS and COX-2 expression were associated with the inactivation of nuclear factor-kappaB and stabilization of IkappaBalpha. Taken together our data gives further insight into the possible chemopreventative properties of two dietary derived isothiocyanates from watercress.     

草木犀石油醚提取物对小鼠巨噬细胞促炎介质的影响

目的研究草木犀石油醚提取物在体外的抗炎作用。方法采用小鼠巨噬细胞系RAW264．7建立炎症细胞模型,加入10μg/L的LPS培养液和不同浓度的草木犀石油醚提取物进行干预。ELISA法检测上清液中TNF-α,IL-1β,IL-6和NO的分泌量;实时荧光定量RT-PCR检测TNF-α,iNOS和COX-2的mRNA表达;Western印迹法检测COX-2蛋白的表达。结果草木犀提取物干预后细胞所分泌的炎性介质（TNF—α,IL-1β,IL-6和NO）与模型组相比均显著降低（P〈0．01）,并存在剂量依赖关系;RT-PCR结果显示干预后细胞TNF-α,iNOS和COX-2的mRNA表达水平显著降低（P〈0．01）,也存在剂量依赖关系;Western印迹结果显示草木犀石油醚提取物及地塞米松干预后COX-2蛋白水平明显降低（P〈0．01）。结论草木犀的石油醚提取物通过下调LPS诱导的巨噬细胞表达炎性介质而发挥其体外抗炎作用,且其下调作用呈剂量依赖性。     

Upregulation of iNOS by COX-2 in muscularis resident macrophage of rat intestine stimulated with LPS

We investigated the effect of lipopolysaccharide (LPS) on the induction of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) in muscularis resident macrophages of rat intestine in situ. When the tissue was incubated with LPS for 4 h, mRNA levels of iNOS and COX-2 were increased. The majority of iNOS and COX-2 proteins appeared to be localized to the dense network of muscularis resident macrophages immunoreactive to ED2. LPS treatment also increased the production of nitric oxide (NO), PGE(2), and PGI(2). The increased expression of iNOS mRNA by LPS was suppressed by indomethacin but not by N(G)-monomethyl-L-arginine (L-NMMA). The increased expression of COX-2 mRNA by LPS was affected neither by indomethacin nor by L-NMMA. Muscle contractility stimulated by 3 microM carbachol was significantly inhibited in the LPS-treated muscle, which was restored by treatment of the tissue with L-NMMA, aminoguanidine, indomethacin, or NS-398. Together, these findings show that LPS increases iNOS expression and stimulates NO production in muscularis resident macrophages to inhibit smooth muscle contraction. LPS-induced iNOS gene expression may be mediated by autocrine regulation of PGs through the induction of COX-2 gene expression.     

A New EGFR Inhibitor from Ficus benghalensis Exerted Potential Anti-Inflammatory Activity via Akt/PI3K Pathway Inhibition

Inflammation is a critical defensive mechanism mainly arising due to the production of prostaglandins via cyclooxygenase enzymes. This study aimed to examine the anti-inflammatory activity of fatty acid glucoside (FAG), which is isolated from Ficus benghalensis against lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophages. The cytotoxic activity of the FAG on RAW 264.7 macrophages was evaluated with an MTT assay. The levels of PGE2 and NO and the activity of iNOS, COX-1, and COX-2 enzymes in LPS-stimulated RAW 264.7 cells were evaluated. The gene expression of IL-6, TNF-α, and PGE2 was investigated by qRT-PCR. The expression of epidermal growth factor receptor (EGFR), Akt, and PI3K proteins was examined using Western blotting analysis. Furthermore, molecular docking of the new FAG against EGFR was investigated. A non-cytotoxic concentration of FAG increased NO release and iNOS activity, inhibited COX-1 and COX-2 activities, and reduced PGE2 levels in LPS-stimulated RAW 264.7 cells. It diminished the expression of TNF-α, IL-6, PGE2, EGFR, Akt, and PI3K. Furthermore, the molecular docking study proposed the potential direct binding of FAG with EGFR with a high affinity. This study showed that FAG is a natural EGFR inhibitor, NO-releasing, and COX-inhibiting anti-inflammatory agent via EGFR/Akt/PI3K pathway inhibition.     

Ca2+/calmodulin-dependent protein phosphatase calcineurin mediates the expression of iNOS through IKK and NF-kappaB activity in LPS-stimulated mouse peritoneal macrophages and RAW 264.7 cells

Ca(2+) and Ca(2+)/calmodulin-dependent protein phosphatase calcineurin (CN) have been known to play crucial roles in immune response and inflammation. Using mouse peritoneal macrophages and RAW 264.7 macrophage cells, we demonstrated that LPS mobilized intracellular free Ca(2+) and induced CN phosphatase activity. iNOS expression and NO secretion in response to LPS were suppressed by Ca(2+) antagonists (TMB-8, BAPTA/AM, and nifedipine) and CN inhibitor (cyclosporin A). Transient expression of constitutively active CN in mouse peritoneal macrophages and RAW 264.7 macrophages strongly activated NF-kappaB, a key mediator of iNOS expression. We also found that CN mediates NF-kappaB activation via IkappaB-alpha hyperphosphorylation and degradation. Overexpression of dominant negative mutant of IKKalpha and -beta demonstrates that only IKKbeta is the target for CN. These results indicate that CN is required for full iNOS expression and the effective activation of NF-kappaB in RAW 264.7 and peritoneal macrophages.     

Involvement of mitogen-activated protein kinase homologues in the regulation of lipopolysaccharide-mediated induction of cyclo-oxygenase-2 but not nitric oxide synthase in RAW 264.7 macrophages.

In RAW 264.7 macrophages lipopolysaccharide (LPS) stimulated the activation of p42 and p44 MAP kinases and their upstream activator mitogen-activated protein (MAP) kinase kinase (MAPKK), and induced the 69-kDa isoform of cyclo-oxygenase-2 (COX-2) and the 130-kDa isoform of nitric oxide synthase (iNOS). PD 098059, a specific inhibitor of the activation of MAPKK, prevented LPS-mediated activation of MAPKK (IC50 = 3.0 +/- 0.1 microM, n = 3) and p42/44 MAP kinases and substantially reduced the induction of COX-2 by approximately 40%-70%, but was without effect upon the induction of iNOS. In parallel, LPS also stimulated the activation of p38 MAP kinase and the MAPKAP kinase-2, a downstream target of p38 MAP kinase. SB 203580, a specific inhibitor of p38 MAP kinase prevented the activation of p38 MAP kinase (IC50 = 3.3 +/- 1.4 microM, n = 3) and MAPKAP kinase-2 by LPS and reduced the induction of COX-2 by approximately 50-90%, with no significant effect upon iNOS expression. These studies indicate the involvement of both the classical p42/44 MAP kinases and p38 MAP kinase in the regulation of COX-2 but not iNOS induction following exposure to LPS.     

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.     

Cross-regulation of iNOS and COX-2 by its products in murine macrophages under stress conditions.

Exposure of macrophages to heat shock induces rapid synthesis of heat shock proteins (HSPs) which are important for cell homeostasis. Prostaglandins (PGs) and nitric oxide (NO) are important cell regulatory molecules. We have therefore investigated the interactions between these molecules in the LPS-induced expression of iNOS and COX-2 and in the mitochondrial activity of macrophages. Cultures of the murine macrophage cell line, J774, were exposed to heat shock (43 degrees C, 30 min) and stimulated with LPS (1 microg/ml), concomitantly or after 8h of cell recovery. NO production was measured by Griess reaction; PGE(2) by ELISA; HSP70, iNOS and COX-2 by immunobloting; mitochondrial activity by MTT assay. Heat shock induced HSP70, but not iNOS or COX-2 whereas LPS induced iNOS and COX-2 but not HSP70. When heat shock and LPS were given concomitantly, iNOS but not COX-2 expression was reduced. When a period of 8h was given between heat shock and LPS stimulation, iNOS, COX-2, PGE(2) and NO levels were significantly increased. Under these conditions, the expression of COX-2 was reduced by L-NAME (NO-synthesis inhibitor) and of iNOS by nimesulide (PGs-synthesis inhibitor). Such cross-regulation was not observed in cells at 37 degrees C. These treatments significantly reduced MTT levels in cells at 37 degrees C but not in cells submitted to heat shock. These results suggest that HSPs and cross-regulation of iNOS and COX-2 by their products might be of relevance in the control of cell homeostasis during stress conditions.     

Anti-inflammatory activity of myricetin from Diospyros lotus through suppression of NF-κB and STAT1 activation and Nrf2-mediated HO-1 induction in lipopolysaccharide-stimulated RAW264.7 macrophages

Diospyros lotus is traditionally used for the treatment of diabetes, diarrhea, tumor, and hypertension. The purpose of this study was to investigate the anti-inflammatory effect and underlying molecular mechanisms of myricetin in lipopolysaccharide (LPS)-stimulated RAW264.7 macrophages. Myricetin dose-dependently suppressed the production of pro-inflammatory mediators (NO, iNOS, PGE₂, and COX-2) in LPS-stimulated RAW264.7 macrophages. Myricetin administration decreased the production of NO, iNOS, TNF-α, IL-6, and IL-12 in mice. Myricetin decreased NF-κB activation by suppressing the degradation of IκBα, nuclear translocation of p65 subunit of NF-κB, and NF-κB DNA binding activity in LPS-stimulated RAW264.7 macrophages. Moreover, myricetin attenuated the phosphorylation of STAT1 and the production of IFN-β in LPS-stimulated RAW264.7 macrophages. Furthermore, myricetin induced the expression of HO-1 through Nrf2 translocation. In conclusion, these results suggest that myricetin inhibits the production of pro-inflammatory mediators through the suppression of NF-κB and STAT1 activation and induction of Nrf2-mediated HO-1 expression in LPS-stimulated RAW264.7 macrophages.     

Inhibition of nitric oxide synthase inhibitors and lipopolysaccharide induced inducible NOS and cyclooxygenase-2 gene expressions by rutin, quercetin, and quercetin pentaacetate in RAW 264.7 macrophages

Several natural flavonoids have been demonstrated to perform some beneficial biological activities, however, higher-effective concentrations and poor-absorptive efficacy in body of flavonoids blocked their practical applications. In the present study, we provided evidences to demonstrate that flavonoids rutin, quercetin, and its acetylated product quercetin pentaacetate were able to be used with nitric oxide synthase (NOS) inhibitors (N-nitro-L-arginine (NLA) or N-nitro-L-arginine methyl ester (L-NAME)) in treatment of lipopolysaccharide (LPS) induced nitric oxide (NO) and prostaglandin E2 (PGE2) productions, inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) gene expressions in a mouse macrophage cell line (RAW 264.7). The results showed that rutin, quercetin, and quercetin pentaacetate-inhibited LPS-induced NO production in a concentration-dependent manner without obvious cytotoxic effect on cells by MTT assay using 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide as an indicator. Decrease of NO production by flavonoids was consistent with the inhibition on LPS-induced iNOS gene expression by western blotting. However, these compounds were unable to block iNOS enzyme activity by direct and indirect measurement on iNOS enzyme activity. Quercetin pentaacetate showed the obvious inhibition on LPS-induced PGE2 production and COX-2 gene expression and the inhibition was not result of suppression on COX-2 enzyme activity. Previous study demonstrated that decrease of NO production by L-arginine analogs effectively stimulated LPS-induced iNOS gene expression, and proposed that stimulatory effects on iNOS protein by NOS inhibitors might be harmful in treating sepsis. In this study, NLA or L-NAME treatment stimulated significantly on LPS-induced iNOS (but not COX-2) protein in RAW 264.7 cells which was inhibited by these three compounds. Quercetin pentaacetate, but not quercetin and rutin, showed the strong inhibitory activity on PGE2 production and COX-2 protein expression in NLA/LPS or L-NAME/LPS co-treated RAW 264.7 cells. These results indicated that combinatorial treatment of L-arginine analogs and flavonoid derivates, such as quercetin pentaacetate, effectively inhibited LPS-induced NO and PGE2 productions, at the same time, inhibited enhanced expressions of iNOS and COX-2 genes.     

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.