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-kappaB.
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-gamma-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-gamma-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 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 activity of COX-2 itself. Ergolide decreased the level of iNOS and COX-2 protein, and iNOS mRNA caused by stimulation of LPS/IFN-gamma in a concentration-dependent manner, as measured by Western blot and Northern blot analysis, respectively. Ergolide inhibited nuclear factor-kappaB (NF-kappaB) activation, a transcription factor necessary for iNOS and COX-2 expression in response to LPS/IFN-gamma. This effect was accompanied by the parallel reduction of nuclear translocation of subunit p65 of NF-kappaB as well as IkappaB-alpha 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-kappaB itself or an upstream molecule of NF-kappaB. Ergolide also directly inhibited the DNA-binding activity of active NF-kappaB in LPS/IFN-gamma-pretreated RAW 264.7 macrophages. 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 IkappaB and the direct modification of active NF-kappaB, leading to the suppression of the expression of iNOS and COX-2, which play important roles in inflammatory signalling pathway.
Whan Han
J
J
College of Pharmacy, Sungkyunkwan University, Suwon 440-746, Korea.
Gon Lee
B
B
Kee Kim
Y
Y
Woo Yoon
J
J
Kyoung Jin
H
H
Hong
S
S
Young Lee
H
H
Ro Lee
K
K
Woo Lee
H
H
D021581:Active Transport, Cell Nucleus / Q000187:drug effects; D000704:Analysis of Variance; D000818:Animals; D000893:Anti-Inflammatory Agents / Q000494:pharmacology*; D000972:Antineoplastic Agents, Phytogenic / Q000494:pharmacology; D002384:Catalysis; D002478:Cells, Cultured; D051546:Cyclooxygenase 2; D004247:DNA / Q000187:drug effects / Q000378:metabolism; D015232:Dinoprostone / Q000378:metabolism; D015870:Gene Expression / Q000187:drug effects*; D007371:Interferon-gamma / Q000494:pharmacology; D038143:Inula; D007444:Inulin / Q000737:chemistry*; D007527:Isoenzymes / Q000096:biosynthesis / Q000235:genetics; D007783:Lactones / Q000494:pharmacology*; D008070:Lipopolysaccharides / Q000494:pharmacology; D008264:Macrophages / Q000187:drug effects* / Q000201:enzymology; D051379:Mice; D016328:NF-kappa B / Q000037:antagonists & inhibitors* / Q000378:metabolism; D019001:Nitric Oxide Synthase / Q000096:biosynthesis / Q000235:genetics; D052247:Nitric Oxide Synthase Type II; D009573:Nitrites / Q000378:metabolism; D010936:Plant Extracts / Q000737:chemistry*; D011451:Prostaglandin-Endoperoxide Synthases / Q000096:biosynthesis / Q000235:genetics; D012333:RNA, Messenger / Q000187:drug effects / Q000378:metabolism; D012717:Sesquiterpenes / Q000494:pharmacology*
D000893:Anti-Inflammatory Agents; D000972:Antineoplastic Agents, Phytogenic; D007527:Isoenzymes; D007783:Lactones; D008070:Lipopolysaccharides; D016328:NF-kappa B; D009573:Nitrites; D010936:Plant Extracts; D012333:RNA, Messenger; D012717:Sesquiterpenes; C099797:ergolide; D007371:Interferon-gamma; D007444:Inulin; D004247:DNA; D019001:Nitric Oxide Synthase; D052247:Nitric Oxide Synthase Type II; C496317:Nos2 protein, mouse; D051546:Cyclooxygenase 2; D011451:Prostaglandin-Endoperoxide Synthases; D015232:Dinoprostone