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‐κB

We investigated the mechanism of suppression of inducible nitric oxide synthase (iNOS) and cyclo‐oxygenase‐2 (COX‐2) by ergolide, sesquiterpene lactone from Inula britannica. iNOS activity in cell‐free extract of LPS/IFN‐γ‐stimulated RAW 264.7 macrophages was markedly attenuated by the treatment with ergolide. Its inhibitory effect on iNOS was paralleled by decrease in nitrite accumulation in culture medium of LPS/IFN‐γ‐stimulated RAW 264.7 macrophages in a concentration‐dependent manner. However, its inhibitory effect does not result from direct inhibition of the catalytic activity of NOS. Ergolide markedly decreased the production of prostaglandin E2 (PGE2) in cell‐free extract of LPS/IFN‐γ‐stimulated RAW 264.7 macrophages in a concentration‐dependent manner, without alteration of the catalytic activity of COX‐2 itself. Ergolide decreased the level of iNOS and COX‐2 protein, and iNOS mRNA caused by stimulation of LPS/IFN‐γ in a concentration‐dependent manner, as measured by Western blot and Northern blot analysis, respectively. Ergolide inhibited nuclear factor‐κB (NF‐κB) activation, a transcription factor necessary for iNOS and COX‐2 expression in response to LPS/IFN‐γ. This effect was accompanied by the parallel reduction of nuclear translocation of subunit p65 of NF‐κB as well as IκB‐α degradation. In addition, these effects were completely blocked by treatment of cysteine, indicating that this inhibitory effect of ergolide could be mediated by alkylation of NF‐κB itself or an upstream molecule of NF‐κB. Ergolide also directly inhibited the DNA‐binding activity of active NF‐κB in LPS/IFN‐γ‐pretreated RAW 264.7 macrophages. These results demonstrate that the suppression of NF‐κB activation by ergolide might be attributed to the inhibition of nuclear translocation of NF‐κB resulted from blockade of the degradation of IκB and the direct modification of active NF‐κB, leading to the suppression of the expression of iNOS and COX‐2, which play important roles in inflammatory signalling pathway. British Journal of Pharmacology (2001) 133, 503–512; doi:10.1038/sj.bjp.0704099