Ginsenoside Rh₂ Alleviates LPS-Induced Inflammatory Responses by Binding to TLR₄/MD-2 and Blocking TLR₄ Dimerization

Lipopolysaccharide (LPS) triggers a severe systemic inflammatory reaction in mammals, with the dimerization of TLR₄/MD-2 upon LPS stimulation serving as the pivotal mechanism in the transmission of inflammatory signals. Ginsenoside Rh₂ (G-Rh₂), one of the active constituents of red ginseng, exerts potent anti-inflammatory activity. However, whether G-Rh₂ can block the TLR₄ dimerization to exert anti-inflammatory effects remains unclear. Here, we first investigated the non-cytotoxic concentration of G-Rh₂ 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-Rh₂ 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-Rh₂ stimulation markedly inhibited the secretion of LPS-induced interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α) and nitric oxide (NO). Additionally, G-Rh₂ blocked the binding of LPS with the membrane of RAW 264.7 cells through direct interaction with TLR₄ and MD-2 proteins, leading to the disruption of the dimerization of TLR₄ and MD-2, followed by suppression of the TLR₄/NF-κB signaling pathway. Our results suggest that G-Rh₂ acts as a new inhibitor of TLR₄ dimerization and may serve as a promising therapeutic agent against inflammation.