The naturally occurring biflavonoid, ochnaflavone, inhibits LPS-induced iNOS expression, which is mediated by ERK1/2 via NF-kappaB regulation, in RAW264.7 cells.
Ochnaflavone (OC), a naturally occurring biflavonoid with anti-inflammatory activity [S.J. Lee, J.H. Choi, H.W. Chang, S.S. Kang, H.P. Kim. Life Sci. 57(6), 1995, 551-558], was isolated from Lonicera japonica and its effects on inducible nitric oxide synthase (iNOS) gene expression was examined in RAW264.7 cells. U0126, an inhibitor of the extracellular signal-regulated kinase (ERK), significantly down-regulated lipopolysaccharide (LPS)-induced iNOS expression and promoter activity. Transactivation of LPS-stimulated NF-kappaB was inhibited by U0126. These results suggest that the transcription factor NF-kappaB is involved in ERK-mediated iNOS regulation and that activation of the Ras/ERK pathway contributes to the induction of iNOS expression in RAW264.7 cells in response to LPS. OC treatment inhibited the production of nitric oxide in a concentration-dependent manner and also blocked the LPS-induced expression of iNOS. These inhibitory effects were associated with reduced ERK1/2 activity. OC inhibited the phosphorylation of c-Jun NH2-terminal kinase (JNK) and p38 mitogen-activated protein kinase. The findings herein show that the inhibition of LPS-induced ERK1/2 activation may be a contributing factor to the main mechanisms by which OC inhibits RAW264.7. To clarify the mechanistic basis for its ability to inhibit iNOS induction, we examined the effect of OC on the transactivation of the iNOS gene by luciferase reporter activity using the -1588 flanking region. OC potently suppressed reporter gene activity. We also report here, for the first time, that LPS-induced iNOS expression was abolished by OC in RAW264.7 cells through by blocking the inhibition of transcription factor NF-kappaB binding activities. These activities are associated with the down-regulation of inhibitor kappaB (IkappaB) kinase (IKK) activity by OC (6 microM), thus inhibiting LPS-induced phosphorylation as well as the degradation of IkappaBalpha. These findings suggest that the inhibition of LPS-induced NO formation by OC is due to its inhibition of NF-kappaB, which may be the mechanistic basis for the anti-inflammatory effects of OC.
Suh
Seok-Jong
SJ
Department of Biological Science, Sungkyunkwan University and National Research Laboratory for Glycobiology, Chunchun-Dong, Jangan-Gu, Suwon City, Kyunggi 440-746, Republic of Korea.
Chung
Tae-Wook
TW
Son
Min-Jung
MJ
Kim
Sung-Hoon
SH
Moon
Tae Chul
TC
Son
Kun Ho
KH
Kim
Hyun Pyo
HP
Chang
Hyeun Wook
HW
Kim
Cheorl-Ho
CH
D000818:Animals; D044946:Biflavonoids / Q000008:administration & dosage; D002460:Cell Line; D004305:Dose-Response Relationship, Drug; D004338:Drug Combinations; D005419:Flavonoids / Q000008:administration & dosage*; D008070:Lipopolysaccharides / Q000008:administration & dosage*; D008264:Macrophages / Q000187:drug effects / Q000378:metabolism*; D051379:Mice; D019950:Mitogen-Activated Protein Kinase 1 / Q000378:metabolism*; D048052:Mitogen-Activated Protein Kinase 3 / Q000378:metabolism*; D016328:NF-kappa B / Q000378:metabolism*; D052247:Nitric Oxide Synthase Type II / Q000378:metabolism*
D044946:Biflavonoids; D004338:Drug Combinations; D005419:Flavonoids; D008070:Lipopolysaccharides; D016328:NF-kappa B; C090940:ochnaflavone; D052247:Nitric Oxide Synthase Type II; C496317:Nos2 protein, mouse; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3