Euonymus alatus extract attenuates LPS-induced NF-κB activation via IKKβ inhibition in RAW 264.7 cells

Aim of the study: The present study was performed to investigate the underlying mechanisms of anti-inflammatory effects with the extract of Euonymus alatus (EEA), and specially focused on nuclear factor kappa B (NF-kappa B) signaling pathway by targeting the I kappa B kinase beta (IKK beta). Materials and methods: The effect of EEA for IKKI beta activity was analyzed using an immobilized metal affinity for phosphochemicals (IMAP)-based time-resolved fluorescence resonance energy transfer (TR-FRET) assay. The effect of EEA on lipopolysaccharide (LPS)-induced NF-kappa B activation in murine macrophage RAW 264.7 cells with western blotting and immunofluorescent staining was evaluated. Results: IKK beta studies based on IMAP-TR-FRET showed that EEA possesses a potent IKK beta inhibitory activity with IC50 value of 11.83 mu g/ml. EEA (10, 30 mu g/ml) also attenuated the LPS-induced I kappa B alpha phosphorylation/degradation, NF-kappa B translocation and subsequent NO synthesis in RAW 264.7 cells. Conclusions: These results suggest that EEA abrogates LPS-induced NF-kappa B signaling pathway by targeting the IKK beta in RAW 264.7 cells and these properties may provide a molecular basis for understanding the inhibitory effects of EEA on LPS-mediated inflammation. (C) 2010 Elsevier Ireland Ltd. All rights reserved.