An ethanolic extract of the aerial part of Siegesbeckia orientalis L. inhibits the production of inflammatory mediators regulated by AP-1, NF-κB and IRF3 in LPS-stimulated RAW 264.7 cells

Herba Siegesbeckiae (HS, the dried aerial part of Siegesbeckia orientalis L.) is a commonly used traditional Chinese medicinal herb for treating inflammatory diseases. HS has been reported to exert anti-inflammatory effects by inhibiting the MAPKs and NF-κB pathways, the downstream effectors of TLR4 signalling. This study aims to further investigate the involvement of TLR4 signalling cascades in the effects of an ethanolic extract of HS (HS for short) on inflammatory mediators in murine macrophages. HS was extracted using 50% ethanol. Lipopolysaccharide (LPS)-stimulated RAW264.7 macrophages were used as the cell model. ELISA was used to detect cytokine/chemokine secretion. Real time-PCR and immunoblotting were used to examine mRNA and protein levels, respectively. We observed that HS dose-dependently inhibited the secretion of PGE₂, MCP-1, MIP-1α and RANTES, and down-regulated mRNA levels of iNOS, COX-2, IL-1β, IL-6, TNF-α, mPGES-1, MCP- 1, MIP-1α and RANTES in LPS-stimulated RAW264.7 cells. HS did not affect the protein levels of TAK1, TBK1, PI3K, Akt, IKK, c-Jun, c-Fos and IRF3, while, dose-dependently decreased levels of their phosphorylated forms. The protein levels of IRAK1 and IRAK4 were upregulated, while those of TRAF6 and TRAF3 were downregulated by HS. Moreover, the nuclear protein levels of AP-1, NF-κB and IRF3 were dose-dependently decreased by HS. These results indicate that suppression of the IRAK4/MAPKs/AP-1, IRAK4/MAPKs/NF- κB, IRAK4/PI3K/NF-κB and TRAF3/TBK1/IRF3 pathways is associated with the inhibitory effects of HS on inflammatory mediators in LPS-stimulated RAW264.7 cells. This study provides a pharmacological basis for the clinical application of this herb in the treatment of inflammatory disorders.