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-κB (NF-κB) 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 µg/mL) and then co-treated with Pam₃CSK₄ (200 ng/mL) for another 12 h. The inhibitory effect of SPE on Pam₃CSK₄-stimulated NO release and post-inflammatory cytokines secretions were determined using Griess reagent and Elisa kits, respectively. The influence of SPE on NF-κB and MAPKs signaling relevant proteins was measured by Western blotting analysis, while the intracellular nitric oxide (NO) generation and NF-κB/p65 nuclear translocation were determined using Leica TCS SP8 laser scanning confocal microscope. Moreover, the effect of SPE on luciferase reporter gene in NF-κB-luc DNA transfected raw 264.7 cells was determined using the Dual-Glo luciferase assay system kit.

Results: SPE dose-dependently (50-200 µg/mL) attenuated Pam₃CSK₄-induced NO release, post-inflammatory cytokines (IL-6, TNF-α and MCP-1) secretions and intracellular NO generation in raw 264.7 cells. Biologically, SPE suppressed Pam₃CSK₄-induced expressions of cyclooxygenase-2 (COX-2), inducible nitric oxide synthase (iNOS), phosphorylation of NF-κB/p65 and IκBα, but did not significantly show effect on the proteins involved in MAPKs signaling (p38, ERK and JNK). The results were further confirmed by NF-κB-luc reporter gene assay and p65 nuclear translocation assay.

Conclusions: In conclusion, SPE ameliorated Pam₃CSK₄-induced inflammation in raw 264.7 cells through suppressing TLR 1/2-mediated NF-κB activation.