Ginsenoside Rh 2 Alleviates LPS-Induced Inflammatory Responses by Binding to TLR 4 /MD-2 and Blocking TLR 4 Dimerization.

Lipopolysaccharide (LPS) triggers a severe systemic inflammatory reaction in mammals, with the dimerization of TLR ₄ /MD-2 upon LPS stimulation serving as the pivotal mechanism in the transmission of inflammatory signals. Ginsenoside Rh ₂ (G-Rh ₂ ), one of the active constituents of red ginseng, exerts potent anti-inflammatory activity. However, whether G-Rh ₂ can block the TLR ₄ dimerization to exert anti-inflammatory effects remains unclear. Here, we first investigated the non-cytotoxic concentration of G-Rh ₂ on RAW 264.7 cells, and detected the releases of pro-inflammatory cytokines in LPS-treated RAW 264.7 cells, and then uncovered the mechanisms involved in the anti-inflammatory activity of G-Rh ₂ through flow cytometry, fluorescent membrane localization, Western blotting, co-immunoprecipitation (Co-IP), molecular docking and surface plasmon resonance (SPR) analysis in LPS-stimulated macrophages. Our results show that G-Rh ₂ stimulation markedly inhibited the secretion of LPS-induced interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α) and nitric oxide (NO). Additionally, G-Rh ₂ blocked the binding of LPS with the membrane of RAW 264.7 cells through direct interaction with TLR ₄ and MD-2 proteins, leading to the disruption of the dimerization of TLR ₄ and MD-2, followed by suppression of the TLR ₄ /NF-κB signaling pathway. Our results suggest that G-Rh ₂ acts as a new inhibitor of TLR ₄ dimerization and may serve as a promising therapeutic agent against inflammation.