Siegesbeckia pubescens Makino inhibits Pam3CSK4-induced inflammation in RAW 264.7 macrophages through suppressing TLR1/TLR2-mediated NF-κB activation

Background: Siegesbeckia pubescens Makino (SP) is one of the important plant origins for the anti-inflammatory Chinese herbal medicine of Siegesbeckiae Herba. The current investigations indicated that the anti-inflammatory effects of SP were associated with the toll-like receptors (TLRs)-mediated nuclear factor-kB (NF-kB) and the mitogen-activated protein kinase (MAPK) signaling pathways. Methods: Raw 264.7 macrophages were pretreated with the 50% ethanol extract of SP (SPE, 50-200 mu g/mL) and then co-treated with Pam(3)CSK(4) (200 ng/mL) for another 12 h. The inhibitory effect of SPE on Pam(3)CSK(4)-stimulated NO release and post-inflammatory cytokines secretions were determined using Griess reagent and Elisa kits, respectively. The influence of SPE on NF-kB and MAPKs signaling relevant proteins was measured by Western blotting analysis, while the intracellular nitric oxide (NO) generation and NF-kB/p65 nuclear translocation were determined using Leica TCS SP8 laser scanning confocal microscope. Moreover, the effect of SPE on luciferase reporter gene in NF-kB-Iuc DNA transfected raw 264.7 cells was determined using the Dual-Glo luciferase assay system kit. Results: SPE dose-dependently (50-200 mu g/mL) attenuated Pam(3)CSK(4)-induced NO release, post-inflammatory cytokines (IL-6,TNF-alpha and MCP-1) secretions and intracellular NO generation in raw 264.7 cells. Biologically, SPE suppressed Pam(3)CSK(4)-induced expressions of cyclooxygenase-2 (COX-2), inducible nitric oxide synthase (iNOS), phosphorylation ofNF-kB/p65 and IkB alpha, but did not significantly show effect on the proteins involved in MAPKs signaling (p38, ERK and JNK). The results were further confirmed by NF-kB-luc reporter gene assay and p65 nuclear translocation assay. Conclusions: In conclusion, SPE ameliorated Pam(3)CSK(4)-induced inflammation in raw 264.7 cells through suppressing TLR 1/2-mediated NF-kB activation.