Suppression of inducible nitric oxide synthase expression in RAW 264.7 macrophages by two β-carboline alkaloids extracted from Melia azedarach

We investigated the mechanism of suppression of inducible nitric oxide synthase (iNOS) by two β-carboline alkaloids isolated from Melia azedarach, 4,8-dimethoxy-1-vinyl-β-carboline (compound 1, C-1) and 4-methoxy-1-vinyl-β-carboline (compound 2, C-2). iNOS activity in a cell-free extract of lipopolysaccharide/interferon-γ-stimulated RAW 264.7 cells was found to be markedly increased, and this increase was prevented by C-1 and C-2, accompanied by the parallel reduction in nitrite accumulation in culture medium. However, C-1 and C-2 had no further effect on the iNOS activity prepared from fully lipopolysaccharide/interferon-γ-stimulated RAW 264.7 cells. Treatment with C-1 or C-2 decreased the levels of iNOS protein and mRNA in a concentration-dependent manner. In addition, prostaglandin E₂ production, cyclooxygenase-2 protein and DNA binding of nuclear factor-κB (NF-κB) in lipopolysaccharide-stimulated RAW 264.7 cells were reduced by these compounds. These results indicate that C-1 and C-2 primarily inhibit iNOS and cyclooxygenase-2 activities via the suppression of de novo synthesis of these two enzymes, and that the inhibition of iNOS expression may be associated with the inhibition of NF-κB activation. Taken together, the results suggest that suppression of iNOS and cyclooxygenase-2 induction by lipopolysaccharide is responsible for the anti-inflammatory activity of these alkaloids through selective inhibition of the expression of genes, which play important roles in inflammatory signaling pathways. (C) 2000 Elsevier Science B.V.