Involvement of heme oxygenase-1 in the anti-inflammatory activity of Chrysanthemum boreale Makino extracts on the expression of inducible nitric oxide synthase in RAW264.7 macrophages

Abstract

Aim of the study: This study is to elucidate the involvement of anti-inflammatory heme oxygenase-1 (HO-1) in the inhibitory activity of a Chrysanthemum boreale Makino (CB) extract on nitric oxide (NO) production and inducible nitric oxide synthase (iNOS) expression in lipopolysaccharide (LPS)-stimulated RAW264.7 macrophages.

Materials and methods: Cell viability and NO assay were performed. In addition, iNOS expression was detected by Western blotting and real-time PCR. HO-1 expression was also evaluated by Western blotting, and blocking HO-1 activity on NO production was performed.

Results: The CB extract at the highest concentration (100 μg/ml) significantly inhibited NO production by approximately 90% and suppressed iNOS protein expression by approximately 84.8% compared to LPS-stimulated cells. Furthermore, the CB extract (100 μg/ml) inhibited iNOS mRNA expression in a concentration-dependant manner and suppressed iNOS mRNA expression by 94.8%. The CB extract induced the expression of HO-1 in a dose-dependent manner, and blocking HO-1 activity abolished the inhibitory effects of the CB extract. Moreover, the addition of carbon monoxide such as tricarbonyl dichlororuthenium (II) dimmer (RuCO), a byproduct derived from heme degradation, mimicked the inhibitory action of low concentrations of CB extract.

Conclusion: These results suggest that a CB extract has potent anti-inflammatory activity in RAW264.7 macrophages involving the induction of HO-1.