Withaferin A inhibits iNOS expression and nitric oxide production by Akt inactivation and down-regulating LPS-induced activity of NF-kappaB in RAW 264.7 cells.
Induction of inducible nitric oxide synthase (iNOS) expression and nitric oxide (NO) production is thought to have beneficial immunomodulatory effects in acute and chronic inflammatory disorders. In Raw 264.7 cells stimulated with lipopolysaccharide (LPS) to mimic inflammation, withaferin A inhibited LPS-induced expression of both iNOS protein and mRNA in a dose-dependent manner. To investigate the mechanism by which withaferin A inhibits iNOS gene expression, we examined activation of mitogen-activated protein kinases (MAPKs) and Akt in Raw 264.7 cells. We did not observe any significant changes in the phosphorylation of p38 MAPK in cells treated with LPS alone or LPS plus withaferin A. However, LPS-induced Akt phosphorylation was markedly inhibited by withaferin A, while the phosphorylation of p42/p44 extracellular signal-regulated kinases (ERKs) was slightly inhibited by withaferin A treatment. Withaferin A prevented IkappaB phosphorylation, blocking the subsequent nuclear translocation of nuclear factor-kappaB (NF-kappaB) and inhibiting its DNA binding activity. LPS-induced p65 phosphorylation, which is mediated by extracellular signal-regulated kinase (ERK) and Akt pathways, was attenuated by withaferin A treatment. Moreover, LPS-induced NO production and NF-kappaB activation were inhibited by SH-6, a specific inhibitor of Akt. Taken together, these results suggest that withaferin A inhibits inflammation through inhibition of NO production and iNOS expression, at least in part, by blocking Akt and subsequently down-regulating NF-kappaB activity.
Department of Immunology, School of Medicine, Keimyung University, 194 DongSan Dong Jung-Gu, Taegu, 700-712, South Korea.
Jung Hwa
JH
Oh
Tae-Jin
TJ
Lee
Jong-Wook
JW
Park
Taeg Kyu
TK
Kwon
D000818:Animals; D002460:Cell Line; D004305:Dose-Response Relationship, Drug; D015536:Down-Regulation / Q000187:drug effects; D004875:Ergosterol / Q000008:administration & dosage / Q000031:analogs & derivatives* / Q000494:pharmacology; D048049:Extracellular Signal-Regulated MAP Kinases / Q000187:drug effects / Q000378:metabolism; D007249:Inflammation / Q000188:drug therapy / Q000503:physiopathology; D008070:Lipopolysaccharides; D008264:Macrophages / Q000187:drug effects / Q000378:metabolism; D051379:Mice; D016328:NF-kappa B / Q000187:drug effects / Q000378:metabolism; D009569:Nitric Oxide / Q000096:biosynthesis*; D052247:Nitric Oxide Synthase Type II / Q000187:drug effects* / Q000378:metabolism; D010766:Phosphorylation / Q000187:drug effects; D051057:Proto-Oncogene Proteins c-akt / Q000187:drug effects* / Q000378:metabolism; D012333:RNA, Messenger / Q000187:drug effects / Q000378:metabolism; D051996:Transcription Factor RelA / Q000187:drug effects / Q000378:metabolism; D054358:Withanolides; D048051:p38 Mitogen-Activated Protein Kinases / Q000187:drug effects / Q000378:metabolism
D008070:Lipopolysaccharides; D016328:NF-kappa B; D012333:RNA, Messenger; D051996:Transcription Factor RelA; D054358:Withanolides; D009569:Nitric Oxide; D052247:Nitric Oxide Synthase Type II; D051057:Proto-Oncogene Proteins c-akt; D048049:Extracellular Signal-Regulated MAP Kinases; D048051:p38 Mitogen-Activated Protein Kinases; C009684:withaferin A; D004875:Ergosterol