Regulation of inflammatory mediators in lipopolysaccharide-stimulated RAW 264.7 cells by 2″-hydroxy-3″-en-anhydroicaritin involves down-regulation of NF-κB and MAPK expression

Abstract

2″-hydroxy-3″-en-anhydroicaritin, a flavone, was isolated from the Chinese medicinal herb Epimedium brevicornum for the first time. In our previous study, we have carried out a screening program to identify the anti-inflammatory potentials of 2″-hydroxy-3″-en-anhydroicaritin. In the present study, we further found that this compound regulated lipopolysaccharide (LPS)-induced levels of nitric oxide (NO), and prostaglandin E 2 (PGE 2) (** p < 0.01 or * p < 0.05), and reduced levels of iNOS and COX-2 in RAW 264.7 macrophages in a concentration-dependent manner. We further investigated signal transduction mechanisms to determine how 2″-hydroxy-3″-en-anhydroicaritin affects RAW264.7 macrophages pretreated with 0.5, 2.5, or 12.5 mg/L of 2″-hydroxy-3″-en-anhydroicaritin 1 h prior to treatment with 1 mg/L of LPS. Thirty minutes later, cells were harvested and mitogen-activated protein kinases (MAPK) activation and IκBα were measured by western blotting. Alternatively, the macrophages were fixed and nuclear factor-κB (NF-κB) activation was measured by immunocytochemical analysis. Signal transduction studies showed that the flavone significantly inhibited extracellular signal-regulated kinase (ERK), p38, and c-jun NH2-terminal kinase (JNK) phosphorylation protein expression. The flavone also inhibited p65-NF-κB translocation into the nucleus by IκBα degradation. Therefore, 2″-hydroxy-3″-en-anhydroicaritin may inhibit LPS-induced production of inflammatory cytokines by blocking NF-κB and MAPK signaling in RAW264.7 cells.