Potent inhibition of lipopolysaccharide-inducible nitric oxide synthase expression by dibenzylbutyrolactone lignans through inhibition of I-kappa B alpha phosphorylation and of p65 nuclear translocation in macrophages

Aims: 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-kappaB alpha. 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 muM) inhibited lipopolysaccharide (LPS)-inducible nuclear NF-kappaB activation and nuclear translocation of p65, which was accompanied by inhibition of I-kappaB alpha phosphorylation, whereas demethyltraxillagenin was less active. LPS-inducible increase in the iNOS mRNA was 80-90% inhibited by 0.01-1 muM arctigenin, whereas similar extents of inhibition were noted by 50-100 muM demethyltraxillagenin. Immunoblot analysis revealed that arctigenin potently inhibited the induction of iNOS by LPS (IC50 less than or equal to 0.01 muM). The IC50 value of demethyltraxillagenin was similar to 50 muM. Production of nitrite and nitrate by LPS in culture medium was also comparably suppressed by the lignans. Conclusion: These results demonstrated that arctigenin potently inhibited LPS-inducible iNOS expression in murine macrophages through suppression of I-kappaB alpha phosphorylation and nuclear translocation of p65. Potent inhibition of LPS-inducible NO production in macrophages may constitute anti-inflammatory effects of the dibenzylbutyrolactone lignans. (C) 2002 Elsevier Science B.V. All rights reserved.