Ergolide, sesquiterpene lactone from Inula britannica, inhibits inducible nitric oxide synthase and cyclo-oxygenase-2 expression in RAW 264.7 macrophages through the inactivation of NF-kappa B

1 We investigated the mechanism of suppression of inducible nitric oxide sy nthase (iNOS) and cyclo-oxygenase-2 (COX-2) by ergolide, sesquiterpene lact one from Inula britannica. 2 iNOS activity in cell-free extract of LPS/IFN-gamma -stimulated RAW 264.7 macrophages was markedly attenuated by the treatment with ergolide. Its in hibitory effect on iNOS was paralleled by decrease in nitrite accumulation in culture medium of LPS/IFN-gamma -stimulated RAW 264.7 macrophages in a c oncentration-dependent manner. However, its inhibitory effect does not resu lt from direct inhibition of the catalytic activity of NOS. 3 Ergolide markedly decreased the production of prostaglandin E-2 (PGE(2)) in cell-free extract of LPS/IFN-gamma -stimulated RAW 264.7 macrophages in a concentration-dependent manner, without alteration of the catalytic activ ity of COX-2 itself. 4 Ergolide decreased the level of iNOS and COX-2 protein, and iNOS mRNA cau sed by stimulation of LPS/IFN-gamma in a concentration-dependent manner, as measured by Western blot and Northern blot analysis, respectively. 5 Ergolide inhibited nuclear factor-kappaB (NF-kappaB) activation, a transc ription factor necessary for iNOS and COX-2 expression in response to LPS/I FN-gamma. This effect was accompanied by the parallel reduction of nuclear translocation of subunit p65 of NF-kappaB as well as I kappaB-alpha degrada tion. In addition, these effects were completely blocked by treatment of cy steine, indicating that this inhibitory effect of ergolide could be mediate d by alkylation of NF-kappaB itself or an upstream molecule of NF-kappaB. 6 Ergolide also directly inhibited the DNA-binding activity of active NF-ka ppaB in LPS/IFN-gamma- pretreated RAW 264.7 macrophages. 7 These results demonstrate that the suppression of NF-kappaB activation by ergolide might be attributed to the inhibition of nuclear translocation of NF-kappaB resulted from blockade of the degradation of I kappaB and the di rect modification of active NF-kappaB, leading to the suppression of the ex pression of iNOS and COX-2, which play important roles in inflammatory sign alling pathway.