KB-34, a newly synthesized chalcone derivative, inhibits lipopolysaccharide-stimulated nitric oxide production in RAW 264.7 macrophages via heme oxygenase-1 induction and blockade of activator protein-1

Abstract

Chalcones, a subclass of the flavonoid family, are widely known for their anti-inflammatory and anti-oxidative properties. In the present study, we synthesized the chalcone derivative, KB-34 (3-Phenyl-1-(2,4,6-tris (methoxymethoxy)phenyl)prop-2-yn-1-one), and examined its effect on nitric oxide (NO) production. KB-34 potently inhibited nitrite production in RAW 264.7 macrophages stimulated by lipopolysaccharide (LPS). KB-34 treatment also markedly inhibited inducible nitric oxide synthase (iNOS) expression, as assessed by Western blot and quantitative RT-PCR analyses. Treatment of cells with KB-34 significantly inhibited LPS-induced transcriptional activation by activator protein-1 (AP-1) as determined by luciferase reporter gene assay, whereas nuclear factor-κB (NF-κB) activity was not affected by KB-34, indicating that down-regulation of iNOS gene expression by KB-34 is mainly attributed by blockade of AP-1 activation. We also demonstrated that KB-34 treatment led to an increase in heme oxygenase-1 (HO-1) mRNA and protein expression, mediated by stimulating the expression of nuclear factor-erythroid 2-related factor 2 (Nrf2). Treatment with SnPP, a selective inhibitor of HO-1, reversed the KB-34-mediated inhibition of nitrite production, suggesting that HO-1 plays an important role in the suppression of NO production by KB-34. In contrast, SnPP treatment did not counteract the KB-34-mediated suppression of AP-1 activity, suggesting that inhibition of AP-1 activation by KB-34 is independent of HO-1 induction. Taken together, these results indicate that KB-34 suppresses NO production in LPS-stimulated RAW 264.7 macrophages via simultaneous induction of HO-1 expression and blockade of AP-1 activation. This study reveals that KB-34 would be a promising agent for the treatment of inflammation-associated disease.