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

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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-κB (NF-κB)-mediated inducible nitric oxide synthase (iNOS, EC1.14.13.39) gene expression were studied in Raw264.7 cells. Methods: Activation of NF-κB was determined by gel mobility shift assay, immunocytochemistry and immunoblot analysis of I-κBα. 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 μM) inhibited lipopolysaccharide (LPS)-inducible nuclear NF-κB activation and nuclear translocation of p65, which was accompanied by inhibition of I-κBα phosphorylation, whereas demethyltraxillagenin was less active. LPS-inducible increase in the iNOS mRNA was 80–90% inhibited by 0.01–1 μM arctigenin, whereas similar extents of inhibition were noted by 50–100 μM demethyltraxillagenin. Immunoblot analysis revealed that arctigenin potently inhibited the induction of iNOS by LPS (IC50≤0.01 μM). The IC50 value of demethyltraxillagenin was ∼50 μM. 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-κBα phosphorylation and nuclear translocation of p65. Potent inhibition of LPS-inducible NO production in macrophages may constitute anti-inflammatory effects of the dibenzylbutyrolactone lignans.