Potent inhibition of lipopolysaccharide-inducible nitric oxide synthase expression by dibenzylbutyrolactone lignans through inhibition of I-kappaBalpha phosphorylation and of p65 nuclear translocation in macrophages.
OBJECTIVE
Arctigenin and demethyltraxillagenin, dibenzylbutyrolactone lignans, are phenylpropanoid metabolites with antioxidant and anti-inflammatory activities. The effects of arctigenin and demethyltraxillagenin on the nuclear factor-kappaB (NF-kappaB)-mediated inducible nitric oxide synthase (iNOS, EC1.14.13.39) gene expression were studied in Raw264.7 cells.


METHODS
Activation of NF-kappaB was determined by gel mobility shift assay, immunocytochemistry and immunoblot analysis of I-kappaBalpha. Expression of the iNOS gene was assessed by Northern and Western blot analyses. NO production was monitored by chemiluminescent detection using a nitric oxide analyzer.


RESULTS
Arctigenin (1 microM) inhibited lipopolysaccharide (LPS)-inducible nuclear NF-kappaB activation and nuclear translocation of p65, which was accompanied by inhibition of I-kappaBalpha phosphorylation, whereas demethyltraxillagenin was less active. LPS-inducible increase in the iNOS mRNA was 80-90% inhibited by 0.01-1 microM arctigenin, whereas similar extents of inhibition were noted by 50-100 microM demethyltraxillagenin. Immunoblot analysis revealed that arctigenin potently inhibited the induction of iNOS by LPS (IC50 < 0.01 microM). The IC50 value of demethyltraxillagenin was approximately 50 microM. Production of nitrite and nitrate by LPS in culture medium was also comparably suppressed by the lignans.


CONCLUSIONS
These results demonstrated that arctigenin potently inhibited LPS-inducible iNOS expression in murine macrophages through suppression of I-kappaBalpha phosphorylation and nuclear translocation of p65. Potent inhibition of LPS-inducible NO production in macrophages may constitute anti-inflammatory effects of the dibenzylbutyrolactone lignans.
Cho
Min Kyung
MK
College of Pharmacy, Seoul National University Sillim-dong, Kwanak-gu, Seoul, South Korea.
Park
Jeong Weon
JW
Jang
Young Pyo
YP
Kim
Young Choong
YC
Kim
Sang Geon
SG
D000818:Animals; D015152:Blotting, Northern; D002460:Cell Line; D002467:Cell Nucleus / Q000187:drug effects / Q000378:metabolism; D002470:Cell Survival / Q000187:drug effects; D015342:DNA Probes; D003720:Densitometry; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D005786:Gene Expression Regulation / Q000187:drug effects; D015151:Immunoblotting; D007150:Immunohistochemistry; D017705:Lignans / Q000494:pharmacology* / Q000633:toxicity*; D008070:Lipopolysaccharides / Q000494:pharmacology*; D008264:Macrophages / Q000187:drug effects / Q000378:metabolism*; D051379:Mice; D016328:NF-kappa B / Q000378:metabolism*; D019001:Nitric Oxide Synthase / Q000037:antagonists & inhibitors* / Q000096:biosynthesis; D052247:Nitric Oxide Synthase Type II; D010766:Phosphorylation; D012333:RNA, Messenger / Q000096:biosynthesis / Q000235:genetics; D019892:eIF-2 Kinase / Q000378:metabolism*
D015342:DNA Probes; D004791:Enzyme Inhibitors; D017705:Lignans; D008070:Lipopolysaccharides; D016328:NF-kappa B; D012333:RNA, Messenger; D019001:Nitric Oxide Synthase; D052247:Nitric Oxide Synthase Type II; C496317:Nos2 protein, mouse; D019892:eIF-2 Kinase