Inhibition of lipopolysaccharide-induced inducible nitric oxide synthase expression by a novel compound, mercaptopyrazine, through suppression of nuclear factor-kappaB binding to DNA

doi:10.1016/j.bcp.2004.05.005

Biochemical Pharmacology

Volume 68, Issue 4, 15 August 2004, Pages 719-728

https://doi.org/10.1016/j.bcp.2004.05.005 Get rights and content

Abstract

Macrophage cells in response to cytokines and endotoxins produced a large amount of nitric oxide (NO) by expression of inducible nitric oxide synthase (iNOS), resulting in acute or chronic inflammatory disorders including septic hypotension and atherosclerosis. In the present study, we investigated the effect and the mechanism of mercaptopyrazine (MP) in the induction of iNOS and NO production as a culminating factor for several inflammatory disorders. Pretreatment of MP alleviated the mortality of endotoxemic mice receiving a lethal bolus of lipopolysaccharide (LPS), which was associated with the reduced levels of serum nitrite/nitrate and IL-1β. In RAW264.7 mouse macrophage cells, MP (300 μM) inhibited both protein and mRNA levels of iNOS stimulated by LPS/interferon-γ (IFNγ) up to 50%. The nuclear factor-kappa B (NF-κB)-driven transactivation was also suppressed by MP to the same degree. Treatment of MP reduced the binding of NF-κB to the oligonucleotides containing NF-κB consensus sequence, while it did not affect the translocation of NF-κB to nuclear. Suppression of NF-κB activity by MP was completely reversed by a reducing agent, dithiothreitol, implying that MP might oxidize the sulfhydryl group(s) of DNA binding domain of NF-κB. In conclusion, MP would be one of interesting candidates or chemical moieties of iNOS expression inhibitor via specific suppression of NF-κB binding to DNA, and be useful as a chemopreventive agent or a therapeutic against iNOS-associated inflammatory diseases.

Section snippets

Synthesis of 2-mercaptopyrazine and 2,2′-dithiobispyrazine (DTBP)

To a solution of sodium ethoxide, prepared from sodium (6.9 g, 0.3 mol) and ethanol (150 ml), N,N-dimethylformamide (150 ml) was added. After removing the ethanol by distillation, the residual solution was saturated with hydrogen sulfide. The deep green solution was heated with 2-chloropyrazine (17.25 g, 150 mmol) at 100 °C for 3 h, and solvent was then removed under reduced pressure. The residue was dissolved in water, then acidified with acetic acid to give yellow precipitates, which were extracted

Pretreatment of MP alleviates the LPS-induced mortality

We studied whether MP contains the in vivo protective effects against systemic inflammatory toxicity by LPS. ICR mice that were pretreated with MP for 3 days (100 and 200 mg/kg/day, p.o.) were exposed to lethal doses of LPS (55 mg/kg). Cumulative proportions of mice surviving after lethal dose of LPS are shown in Fig. 2. MP pretreatment of mice prior to LPS injection elevated the survival rate by 70–80% from 40% in LPS alone when examined at 24 h after LPS injection. At 72 h after LPS injection,

Discussion

In this study we demonstrated that MP, a chemically synthesized sulfhydryl group-containing small molecule, inhibited the NO production by suppression of iNOS protein expression in vivo and in vitro. MP blocked the iNOS transcription by interfering with NF-κB binding to the iNOS promoter DNA in a dose-dependent manner. MP did not affect the signaling pathway to the nuclear translocation of NF-κB, which was distinct from other known inhibitors.

The promoter of iNOS contains two consensus

Acknowledgements

This study was supported by grants of FPR02A6-24-110 of 21C Frontier Functional Proteomics Project From Korean Ministry of Science & Technology, and in part by 01-PJ1-PG3-20500-0147 and 02-PJ1-PG1-CH04-0001, Good Health 21 from MOWH, Korea.

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Inhibition of lipopolysaccharide-induced inducible nitric oxide synthase expression by a novel compound, mercaptopyrazine, through suppression of nuclear factor-kappaB binding to DNA

Abstract

Section snippets

Synthesis of 2-mercaptopyrazine and 2,2′-dithiobispyrazine (DTBP)

Pretreatment of MP alleviates the LPS-induced mortality

Discussion

Acknowledgements

Trends Biochem. Sci.

Biochem. Biophys. Res. Commun.

J. Biol. Chem.

J. Biol. Chem.

Biochem. Biophys. Res. Commun.

Comp Biochem. Physiol A Mol. Integr. Physiol

Atherosclerosis

J. Biol. Chem.

J. Biol. Chem.

FEBS Lett.

FEBS Lett.

Nitric oxide synthases: which, where, how, and why?

J. Clin. Invest

Cell biology. NO says yes to mitochondria

Science

Promoter of the mouse gene encoding calcium-independent nitric oxide synthase confers inducibility by interferon gamma and bacterial lipopolysaccharide

J. Exp. Med.

Oxidized low density lipoprotein suppresses activation of NF kappa B in macrophages via a pertussis toxin-sensitive signaling mechanism

J. Biol. Chem.

Activated transcription factor nuclear factor-kappa B is present in the atherosclerotic lesion

J. Clin. Invest

Diabetes-associated sustained activation of the transcription factor nuclear factor-kappaB

Diabetes

NF-kappa B regulates IL-1 beta transcription through a consensus NF-kappa B binding site and a nonconsensus CRE-like site

J. Immunol.

Studies of the mechanism of inhibition of insulin signaling by tumor necrosis factor-alpha

J. Endocrinol.

l-NMMA (a nitric oxide synthase inhibitor) is effective in the treatment of cardiogenic shock

Circulation