Suppressive effect of novel aromatic diamine compound on nuclear factor-kappaB-dependent expression of inducible nitric oxide synthase in macrophages.

N1-benzyl-4-methylbenzene-1,2-diamine (BMD) is a novel synthetic compound. In the present study, BMD compound was discovered to inhibit nitric oxide (NO) production in macrophages RAW 264.7. BMD compound attenuated lipopolysaccharide (LPS)-induced synthesis of both mRNA and protein of inducible nitric oxide synthase (iNOS), and inhibited LPS-induced iNOS promoter activity, indicating that the aromatic diamine compound could down-regulate iNOS expression at the transcription level. As a mechanism of the anti-inflammatory action, suppression of BMD compound on nuclear factor (NF)-kappaB activation has been documented. BMD compound exhibited dose-dependent inhibitory effect on LPS-mediated NF-kappaB transcriptional activity in the macrophages. Further, the compound inhibited LPS-mediated nuclear translocation of NF-kappaB p65 and DNA binding activity of NF-kappaB complex, in parallel, but did not affect LPS-mediated degradation of inhibitory kappaBalpha protein (IkappaBalpha). These results indicate that BMD compound could inhibit nuclear localization step of NF-kappaB p65 without affecting IkappaBalpha degradation. Finally, BMD compound could provide an invaluable tool to investigate NF-kappaB-dependent iNOS expression, in addition to its therapeutic potential in NO-associated inflammatory diseases.

Shin HM ，Byung Hak Kim ，Eun Yong Chung ，Jung SH ，Yeong Shik Kim ，Kyung Rak Min ，Kim Y ... -  《EUROPEAN JOURNAL OF PHARMACOLOGY》

Inhibition of inducible nitric-oxide synthase expression by (5R)-5-hydroxytriptolide in interferon-gamma- and bacterial lipopolysaccharide-stimulated macrophages.

(5R)-5-Hydroxytriptolide (LLDT-8) is a novel analog of triptolide that has antiarthritic, hepatoprotective, and antiallogenic transplantation-rejective effects. In the present study, we report that LLDT-8 inhibited nitric oxide (NO) production and inducible nitric-oxide synthase (iNOS) expression in macrophages. LLDT-8 significantly attenuated NO production, in a dose-dependent manner, in primary peritoneal macrophages and a macrophage cell line of Raw 264.7 cells following stimulation with interferon (IFN)-gamma, lipopolysaccharide (LPS), and IFN-gamma plus LPS. It also reduced the production of tumor necrosis factor-alpha from LPS-stimulated Raw 264.7 cells. To further elucidate the mechanism responsible for the inhibition of NO, we examined the effect of LLDT-8 on IFN-gamma and LPS-induced iNOS expression. Indeed, LLDT-8 prevented NO generation by inhibiting iNOS expression at mRNA level and protein level, rather than by interfering its enzymatic activity. In IFN-gamma-stimulated Raw 264.7 cells, LLDT-8 suppressed the gene transcription of signal transducer and activator of transcription 1alpha and interferon regulatory factor (IRF)-1, but it displayed no apparent effect on IFN-gamma receptor level on cell surface. After LPS challenge, LLDT-8 further abrogated the expression of LPS receptor complex, including CD14, Toll-like receptor 4, and myeloid differentiation protein-2; decreased the LPS-induced phosphorylation of stress-activated protein kinase/c-Jun NH(2)-terminal kinase, extracellular signal-regulated kinase 1/2, and p38 mitogen-activated protein kinase (MAPK); retarded the degradation of IkappaBalpha; and ameliorated the DNA binding activity of nuclear factor-kappaB (NF-kappaB) to nuclear proteins that accounts for transcriptional regulation of iNOS. Taken together, these results suggest that LLDT-8 reduces NO production and iNOS expression by inhibiting IFN-gamma-triggered IRF-1 expression and LPS-triggered MAPK phosphorylation and NF-kappaB activation.

Zhou R ，Zheng SX ，Tang W ，He PL ，Li XY ，Yang YF ，Li YC ，Geng JG ，Zuo JP ... -  《JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS》

Down-regulatory effect of quercitrin gallate on nuclear factor-kappa B-dependent inducible nitric oxide synthase expression in lipopolysaccharide-stimulated macrophages RAW 264.7.

Quercetin 3-O-beta-(2''-galloyl)-rhamnopyranoside (QGR) is a naturally occurring quercitrin gallate, a polyphenolic compound isolated from Persicaria lapathifolia (Polygonaceae). In the present study, QGR compound was discovered to have inhibitory effect on nitric oxide (NO) production in lipopolysaccharide (LPS)-stimulated macrophages RAW 264.7. QGR compound attenuated LPS-induced synthesis of both mRNA and protein of inducible nitric oxide synthase (iNOS), in parallel, and inhibited LPS-induced luciferase expression as a reporter of iNOS promoter activity in the macrophages. As a mechanism of the anti-inflammatory action shown by QGR compound, suppression of nuclear factor (NF)-kappaB activation has been documented. QGR compound exhibited inhibitory effect on LPS-mediated NF-kappaB transcriptional activity in macrophages RAW 264.7. Furthermore, the compound inhibited LPS-mediated nuclear translocation of NF-kappaB p65 and DNA binding activity of NF-kappaB complex, in parallel, but did not influence LPS-mediated IkappaBalpha degradation. Taken together, QGR compound suppressed LPS-mediated NF-kappaB activation, specifically to nuclear localization step of NF-kappaB p65, which was attributable to its down-regulatory action on LPS-induced NO production and iNOS expression.

Kim BH ，Cho SM ，Reddy AM ，Kim YS ，Min KR ，Kim Y ... -  《BIOCHEMICAL PHARMACOLOGY》

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.

Oh JH ，Lee TJ ，Park JW ，Kwon TK ... -  《EUROPEAN JOURNAL OF PHARMACOLOGY》

Inhibition of lipopolysaccharide-stimulated NO production by a novel synthetic compound CYL-4d in RAW 264.7 macrophages involving the blockade of MEK4/JNK/AP-1 pathway.

In the present study, a novel synthetic compound 4-(2-(cyclohex-2-enylidene)hydrazinyl)quinolin-2(1H)-one (CYL-4d) was found to inhibit lipopolysaccharide (LPS)-induced nitric oxide (NO) production without affecting cell viability or enzyme activity of expressed inducible NO synthase (iNOS) in RAW 264.7 macrophages. CYL-4d exhibited parallel inhibition of LPS-induced expression of iNOS protein, iNOS mRNA and iNOS promoter activity in the same concentration range. LPS-induced activator protein-1 (AP-1) DNA binding, AP-1-dependent reporter gene activity and c-Jun nuclear translocation were all markedly inhibited by CYL-4d with similar efficacy, whereas CYL-4d produced a weak inhibition of nuclear factor-kappaB (NF-kappaB) DNA binding, NF-kappaB-dependent reporter gene activity and p65 nuclear translocation without affecting inhibitory factor-kappa B alpha (I kappa B alpha) degradation. CYL-4d had no effect on the LPS-induced phosphorylation of extracellular signal-regulated kinase (ERK), p38 mitogen-activated protein kinase (MAPK) and its upstream activator MAPK kinase (MEK) 3, whereas it significantly attenuated the phosphorylation of c-Jun, c-Jun NH(2)-terminal kinase (JNK) and its upstream activator MEK4 in a parallel concentration-dependent manner. Other Toll-like receptors (TLRs) ligands (peptidoglycans, double-stranded RNA, and oligonucleotide containing unmethylated CpG motifs)-induced iNOS protein expression were also inhibited by CYL-4d. Furthermore, the NO production from BV-2 microglial cells as well as rat alveolar macrophages in response to LPS was diminished by CYL-4d. These results indicate that the blockade of NO production by CYL-4d in LPS-stimulated RAW 264.7 cells is attributed mainly to interference in the MEK4-JNK-AP-1 signaling pathway. CYL-4d inhibition of NO production is not restricted to TLR4 activation and immortalized macrophage-like cells.

Lin MW ，Tsao LT ，Chang LC ，Chen YL ，Huang LJ ，Kuo SC ，Tzeng CC ，Lee MR ，Wang JP ... -  《BIOCHEMICAL PHARMACOLOGY》