4-Hydroxykobusin inhibits the induction of nitric oxide synthase by inhibiting NF-kappaB and AP-1 activation

We recently isolated a novel lignan, 4-hydroxykobusin from Geranium thunbergii (Liu et al., Arch. Pharm. Res., 29, 1109-1113, 2006). Here, we studied its effect on the expression of inducible nitric oxide synthase (iNOS) gene in RAW264.7 cells. 4-Hydroxykobusin inhibited nitric oxide (NO) production in a concentration-dependent manner and blocked the lipopolysaccharide (LPS)-induced expression of inducible nitric oxide synthase (iNOS). To identify the mechanistic basis for its inhibition of iNOS induction, we examined the effect of 4-hydroxykobusin on the transactivation of iNOS gene by luciferase reporter activity using -1.59 kb flanking region. The lignan suppressed the reporter gene activity and the LPS-induced reporter activations of nuclear factor-kappaB (NF-kappaB) and activator protein-1 (AP-1) were also significantly blocked by 4-hydroxykobusin. These findings suggest that the inhibition of LPS-induced NO formation by 4-hydroxykobusin is due to its inhibition of NF-kappaB and AP-1 activation.     

N-[3,4-dimethoxycinnamoyl]-anthranilic acid (tranilast) suppresses microglial inducible nitric oxide synthase (iNOS) expression and activity induced by interferon-gamma (IFN-gamma).

1. Microglial cells up-regulate inducible nitric oxide synthase (iNOS) expression in response to various pro-inflammatory stimuli including interferon-gamma (IFN-gamma), allowing for the release of nitric oxide (NO). Tranilast (N-[3,4-dimethoxycinnamoyl]-anthranilic acid) is an antiallergic compound with suppressive effects on the activation of monocytes. 2. Here, we show that N9 murine microglial cells express iNOS mRNA and protein and release nitric oxide into the culture medium in response to IFN-gamma (200 u x ml(-1)) as measured by Northern and Western blot analyses and Griess assay. 3. Exposure to non-toxic doses of tranilast (30-300 microM) leads to a concentration-dependent inhibition of IFN-gamma-induced (200 u x ml(-1)) iNOS mRNA and protein expression. This is paralleled by a suppression of NO-release into the cell culture medium. 4. Inhibition of IFN-gamma-induced iNOS mRNA expression by tranilast is paralleled by an inhibition of nuclear factor-kappaB (NF-kappaB) activation and phosphorylation of inhibitory kappaB (IkappaB) as determined by Western blot analyses and NF-kappaB reporter gene assay. 5. These results suggest that tranilast-mediated suppression of microglial iNOS activity induced by IFN-gamma involves the inhibition of NF-kappaB-dependent iNOS mRNA expression.     

Withaferin A inhibits iNOS expression and nitric oxide production by Akt inactivation and down-regulating LPS-induced activity of NF-kappaB in RAW 264.7 cells

Induction of inducible nitric oxide synthase (iNOS) expression and nitric oxide (NO) production is thought to have beneficial immunomodulatory effects in acute and chronic inflammatory disorders. In Raw 264.7 cells stimulated with lipopolysaccharide (LPS) to mimic inflammation, withaferin A inhibited LPS-induced expression of both iNOS protein and mRNA in a dose-dependent manner. To investigate the mechanism by which withaferin A inhibits iNOS gene expression, we examined activation of mitogen-activated protein kinases (MAPKs) and Akt in Raw 264.7 cells. We did not observe any significant changes in the phosphorylation of p38 MAPK in cells treated with LPS alone or LPS plus withaferin A. However, LPS-induced Akt phosphorylation was markedly inhibited by withaferin A, while the phosphorylation of p42/p44 extracellular signal-regulated kinases (ERKs) was slightly inhibited by withaferin A treatment. Withaferin A prevented IkappaB phosphorylation, blocking the subsequent nuclear translocation of nuclear factor-kappaB (NF-kappaB) and inhibiting its DNA binding activity. LPS-induced p65 phosphorylation, which is mediated by extracellular signal-regulated kinase (ERK) and Akt pathways, was attenuated by withaferin A treatment. Moreover, LPS-induced NO production and NF-kappaB activation were inhibited by SH-6, a specific inhibitor of Akt. Taken together, these results suggest that withaferin A inhibits inflammation through inhibition of NO production and iNOS expression, at least in part, by blocking Akt and subsequently down-regulating NF-kappaB activity.     

Amentoflavone inhibits the induction of nitric oxide synthase by inhibiting NF-κB activation in macrophages

Amentoflavone is a bi-flavonoid compound with anti-fungal and anti-inflammatory activities. We isolated amentoflavone from Selaginella tamariscina (Selaginellaceae) and studied its effects on nuclear factor-kappaB (NF-kappaB)-mediated inducible nitric oxide synthase (iNOS) gene expression in RAW 264.7 cells. Amentoflavone inhibited the production of nitric oxide in a concentration-dependent manner and also blocked the lipopolysaccharide (LPS)-induced expression of inducible nitric oxide synthase (iNOS). To clarify the mechanistic basis for its inhibition of iNOS induction, we examined the effect of amentoflavone on the transactivation of iNOS gene by luciferase reporter activity using -1.59 kb flanking region. Amentoflavone potently suppressed the reporter gene activity. The LPS-induced activation of NF-kappaB was also found to be significantly blocked by amentoflavone, but AP-1 activation was unaffected. Furthermore, the nuclear translocation of p65 by LPS was inhibited by amentoflavone. NF-kappaB activation is controlled by the phosphorylation and subsequent degradation of I-kappaBalpha, and the cytosolic degradation of I-kappaBalpha was found to be inhibited by amentoflavone. These findings suggest that the inhibition of LPS-induced NO formation by amentoflavone is due to its inhibition of NF-kappaB by blocking I-kappaBalpha degradation, which may be the mechanistic basis of the anti-inflammatory effects of amentoflavone.     

Suppression by a sesquiterpene lactone from Carpesium divaricatum of inducible nitric oxide synthase by inhibiting nuclear factor-kappaB activation

Excessive nitric oxide (NO) produced by inducible NO synthase (iNOS) acts as a causative regulator in various inflammatory disease states. Carpesium divaricatum has been used in Korean traditional herbal medicine for its antipyretic, analgesic, vermifugic, and anti-inflammatory properties. We investigated the molecular mechanism for the suppression of lipopolysaccharide/interferon-gamma (LPS/IFN-gamma)-induced NO production in RAW 264.7 macrophages by the sesquiterpene lactone 2beta,5-epoxy-5,10-dihydroxy-6alpha-angeloyloxy-9beta-isobutyloxy-germacran-8alpha,12-olide (C-1), which has been identified recently as a new compound from C. divaricatum. C-1 decreased NO production in LPS/IFN-gamma-stimulated RAW 264.7 cells in a concentration-dependent manner, with an IC50 of approximately 2.16 microM; however, it had no direct effect on the iNOS activity of fully LPS/IFN-gamma-stimulated RAW 264.7 cells. Furthermore, treatment with C-1 led to a decrease in iNOS protein and mRNA. These effects appear to be due to inhibition of nuclear factor-kappaB (NF-kappaB) activation through a mechanism involving stabilization of the NF-kappaB/inhibitor of the kappaB (I-kappaB) complex, since inhibition of NF-kappaB DNA binding activity by C-1 was accompanied by a parallel reduction of nuclear translocation of subunit p65 of NF-kappaB and I-kappaBalpha degradation. Taken together, the results suggest that the ability of C-1 to inhibit iNOS gene expression may be responsible, in part, for its anti-inflammatory effects.     

Gigantol isolated from the whole plants of Cymbidium goeringii inhibits the LPS-induced iNOS and COX-2 expression via NF-kappaB inactivation in RAW 264.7 macrophages cells

During our efforts to find bioactive natural products with anti-inflammatory activity, we isolated gigantol from the whole plants of Cymbidium goeringii (Orchidaceae) by activity-guided chromatographic fractionation. Gigantol was found to have potent inhibitory effects on LPS-induced nitric oxide (NO) and prostaglandin E (2) (PGE (2)) production in RAW 264.7 cells. Consistent with these findings, gigantol suppressed the expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) at the protein and mRNA levels in RAW 264.7 cells in a concentration-dependent manner. Our data also indicate that gigantol is a potent inhibitor of tumor necrosis factor-alpha (TNF-alpha), interleukin-1beta (IL-1beta) and interleukin-6 (IL-6) release and influenced the mRNA expression levels of these cytokines in a dose-dependent manner. Furthermore, a reporter gene assay for nuclear factor kappa B (NF-kappaB) and an electromobility shift assay (EMSA) demonstrated that gigantol effectively inhibited the activation of NF-kappaB, which is necessary for the expression of iNOS, COX-2, TNF-alpha, IL-1beta and IL-6. Thus, our studies suggest that gigantol inhibits LPS-induced iNOS and COX-2 expression by blocking NF- kappaB activation.     

Styraxoside A isolated from the stem bark of Styrax japonica inhibits lipopolysaccharide-induced expression of inducible nitric oxide synthase and cyclooxygenase-2 in RAW 264.7 cells by suppressing nuclear factor-kappa B activation

In the present study, the effects of terpenes (styraxosides A and B) and lignans (egonol, masutakeside I, and styraxlignolide A) isolated from the stem bark of Styrax japonica Sieb. et Zucc. (styracaceae) were evaluated on lipopolysaccharide (LPS)-induced nitric oxide (NO) and prostaglandin E2 (PGE2) production by the RAW 264.7 macrophage cell line. Of the tested compounds, styraxoside A was found to most potently inhibit the productions of NO and PGE2, and also significantly reduced the release of tumor necrosis factor-alpha (TNF-alpha) and interleukin-1beta (IL-1beta). Consistent with these observations, the protein expression levels of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) and the mRNA expression levels of iNOS, COX-2, TNF-alpha and IL-1beta were found to be inhibited by styraxoside A in a concentration-dependent manner. Furthermore, styraxoside A inhibited the LPS-induced DNA binding activity of nuclear factor-kappaB (NF-kappaB). Taken together, our data indicate that styraxoside A inhibits LPS-induced iNOS, COX-2, TNF-alpha, and IL-1beta expressions through the down-regulation of NF-kappaB-DNA binding activity.     

Methanol extract of Xanthium strumarium L. possesses anti-inflammatory and anti-nociceptive activities

As an attempt to identify bioactive natural products with anti-inflammatory activity, we evaluated the effects of the methanol extract of the semen of Xanthium strumarium L. (MEXS) on lipopolysaccharide (LPS)-induced nitric oxide (NO), prostaglandin E2 (PGE2) and tumor necrosis factor-alpha (TNF-alpha) production in RAW 264.7 cells. Our data indicate that MEXS is a potent inhibitor of NO, PGE2 and TNF-alpha production. Consistent with these findings, the expression levels of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) protein and iNOS, COX-2 and TNF-alpha mRNA were down-regulated in a concentration-dependent manner. Furthermore, MEXS inhibited nuclear factor kappa B (NF-kappaB) DNA binding activity and the translocation of NF-kappaB to the nucleus by blocking the degradation of inhibitor of kappa B-alpha (IkappaB-alpha). We further evaluated the anti-inflammatory and anti-nociceptive activities of MEXS in vivo. MEXS (100, 200 mg/kg/d, p.o.) reduced acute paw edema induced by carrageenin in rats, and showed analgesic activities in an acetic acid-induced abdominal constriction test and a hot plate test in mice. Thus, our study suggests that the inhibitions of iNOS, COX-2 expression, and TNF-alpha release by the methanol extract of the semen of Xanthium strumarium L. are achieved by blocking NF-kappaB activation, and that this is also responsible for its anti-inflammatory effects.     

Equol inhibits nitric oxide production and inducible nitric oxide synthase gene expression through down-regulating the activation of Akt

In the present study, we report the inhibitory effect of equol on nitric oxide (NO) production and inducible nitric oxide synthase (iNOS) gene expression in murine macrophages. In vivo administration of equol (i.p.) attenuated NO production by peritoneal adherent cells isolated from lipopolysaccharide (LPS)-treated mice. Equol dose-dependently inhibited the LPS-induced production of NO in isolated peritoneal adherent cells and RAW 264.7 cells. The mRNA expression of iNOS was also blocked by equol in LPS-stimulated RAW 264.7 cells. Further study demonstrated that the LPS-induced activation of Akt was suppressed by equol in RAW 264.7 cells while the activation of ERK, SAPK/JNK and p38 MAP kinase was not affected. Equol also blocked LPS-induced NF-kappaB activation. Moreover, the LPS-induced NO production and NF-kappaB activation was inhibited by LY294002, a specific inhibitor of phosphatidylinositol 3-kinase/Akt pathway, in RAW 264.7 cells. These results suggest that equol might inhibit NO production and iNOS gene expression, at least in part, by blocking Akt activation and subsequent down-regulation of NF-kappaB activity.     

Inhibitory effects of cynaropicrin and related sesquiterpene lactones from leaves of artichoke (Cynara scolymus L.) on induction of iNOS in RAW264.7 cells and its high-affinity proteins

The methanolic extract of the leaves of artichoke (Cynara scolymus L.) was found to inhibit nitric oxide (NO) production in lipopolysaccharide (LPS)-stimulated RAW264.7 cells. Among the constituents of the extract, six sesquiterpene lactones (cynaropicrin, grosheimin, 11β,13-dihydrocynaropicrin, 3β-hydroxy-8α-[(S)-3-hydroxy-2-methylpropionyloxy]guaia-4(15),10(14),11(13)-trien-1α,5α,6βH-12,6-olide, 3β-hydroxy-8α-[2-methoxymethyl-2-propenoyloxy]guaia-4(15),10(14),11(13)-trien-1α,5α,6βH-12,6-olide, and deacylcynaropicrin) inhibited NO production and/or inducible nitric oxide synthase (iNOS) induction. The acyl group having an α,β-unsaturated carbonyl group at the 8-position and the α-methylene-γ-butyrolactone moiety were important for the strong inhibitory activity. Our results suggested that these sesquiterpene lactones inhibited the LPS-induced iNOS expression via the suppression of the JAK-STAT signaling pathway in addition to the κNF-κB signaling pathway. With regard to the target molecules of the sesquiterpene lactones, high-affinity proteins of cynaropicrin were purified from the cell extract. ATP/ADP translocase 2 and tubulin were identified and suggested to be involved in the cytotoxic effects of cynaropicrin, although the target molecules for the inhibition of iNOS expression were not clarified.

     

Wogonin, baicalin, and baicalein inhibition of inducible nitric oxide synthase and cyclooxygenase-2 gene expressions induced by nitric oxide synthase inhibitors and lipopolysaccharide

We previously reported that oroxylin A, a polyphenolic compound, was a potent inhibitor of lipopolysaccharide (LPS)-induced expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2). In the present study, three oroxylin A structurally related polyphenols isolated from the Chinese herb Huang Qui, namely baicalin, baicalein, and wogonin, were examined for their effects on LPS-induced nitric oxide (NO) production and iNOS and COX-2 gene expressions in RAW 264.7 macrophages. The results indicated that these three polyphenolic compounds inhibited LPS-induced NO production in a concentration-dependent manner without a notable cytotoxic effect on these cells. The decrease in NO production was in parallel with the inhibition by these polyphenolic compounds of LPS-induced iNOS gene expression. However, these three compounds did not directly affect iNOS enzyme activity. In addition, wogonin, but not baicalin or baicalein, inhibited LPS-induced prostaglandin E2 (PGE2) production and COX-2 gene expression without affecting COX-2 enzyme activity. Furthermore, N-nitro-L-arginine (NLA) and N-nitro-L-arginine methyl ester (L-NAME) pretreatment enhanced LPS-induced iNOS (but not COX-2) protein expression, which was inhibited by these three polyphenolic compounds. Wogonin, but not baicalin or baicalein, similarly inhibited PGE2 production and COX-2 protein expression in NLA/LPS or L-NAME/LPS-co-treated RAW 264.7 cells. These results indicated that co-treatment with NOS inhibitors and polyphenolic compounds such as wogonin effectively blocks acute production of NO and, at the same time, inhibits expression of iNOS and COX-2 genes.     

Prenylflavones from Psoralea corylifolia inhibit nitric oxide synthase expression through the inhibition of I-kappaB-alpha degradation in activated microglial cells

The overproduction of nitric oxide (NO) by inducible nitric oxide synthase (iNOS) switches the function of NO from a physiological neuromodulator to a neurotoxic effector in central nervous system (CNS) after brain injury. From the methanol extracts of Psoralea corylifolia, we purified two inhibitors of NO production in lipopolysaccharide (LPS)-activated microglia by activity guided purification along with two inactive compounds. The active compounds were identified as a chromenoflavanone [7,8-dihydro-8-(4-hydroxyphenyl)-2,2-dimethyl-2H,6H-benzo-(1,2-b:5,4-b')dipyran-6-one] (1) and 4-hydroxylonchocarpin (2). And the inactive two compounds were identified as bavachinin (3) and bavachalcone (4) by spectral analysis. The compound 2 was isolated first time from this plant. Compounds 1 and 2 inhibited the production of NO in LPS-activated microglia in a dose dependent manner (IC(50)'s were 11.4, 10.2 microM, respectively). They also suppressed the expression of protein and mRNA of iNOS in LPS-activated microglial cells at 10 muM as observed in Western blot analysis and RT-PCR experiment. Furthermore they inhibited the degradation of I-kappaB-alpha in activated microglia. These results imply that compounds 1 and 2 can be lead compounds for the development of neuroprotective drug with the inhibitory activity of NO overproduction by activated microglial cells.     

Rengyolone inhibits inducible nitric oxide synthase expression and nitric oxide production by down-regulation of NF-kappaB and p38 MAP kinase activity in LPS-stimulated RAW 264.7 cells

Nitric oxide (NO) is recognized as a mediator and regulator of inflammatory responses. Rengyolone, a cyclohexylethanoid isolated from the fruits of Forsythia koreana, exhibits anti-inflammatory activity with unknown mechanism. In this study, we found that rengyolone has a strong inhibitory effect on the production of nitric oxide (NO) and tumor necrosis factor-alpha (TNF-alpha). Rengyolone also inhibited inducible nitric oxide synthase (iNOS) gene expression and cyclooxygenase 2 (COX-2) by lipopolysaccharide (LPS). In order to explore the mechanism responsible for the inhibition of iNOS gene expression by rengyolone, we investigated its effect on LPS-induced nuclear factor-kappaB (NF-kappaB) activation. The LPS-induced DNA binding activity of NF-kappaB was significantly inhibited by rengyolone, and this effect was mediated through inhibition of the degradation of inhibitory factor-kappaBalpha and phosphorylation of p38 MAP kinase. Furthermore, rengyolone suppressed the expression of ICE protein in IL-1beta-treated D10S cells. Taken together, these results suggest that rengyolone attenuates the inflammation through inhibition of NO production and iNOS expression by blockade of NF-kappaB and p38 MAPK activation in LPS-stimulated RAW 264.7 cells.