Ginsenoside Rb1 exerts anti-inflammatory effects in vitro and in vivo by modulating toll-like receptor 4 dimerization and NF-kB/MAPKs signaling pathways

Backgound: Ginsenoside Rb1, the main active constituent of Panax ginseng, displays significant anti-in-flammatory activity, although the mechanism has not been clearly unraveled. In this study, Rb1's mechanism of anti-inflammatory effects were investigated. Methods: The flow cytometry and enzyme-linked immunosorbent assay (ELISA) were empolyed to detect pro-inflammatory cytokines release. The related protein and gene expression was investigated by western blotting and qRT-PCR. The dimerization of TLR4 was measured by co-immunoprecipitation and molecular docking as-says. Cellular thermal shift assay was used for the determination of the binding of Rb1 and TLR4. For animal moldels, LPS- or cantharidin-induced acute kidney injury, LPS-induced septic death, and dimethyl benzene-induced ear edema were employed to investigate Rb1's anti-inflammatory activity in vivo. Results: Rb1 significantly decreased inflammatory cytokines release in LPS-stimulated RAW264.7 cells and BMDMs, as well as COX-2 and iNOS amounts. Rb1 reduced LPS-associated calcium influx, ROS production, and NO generation. The NF-kappa B and MAPK axes participated in Rb1's anti-inflammatory effects. Molecular docking simulation indicated Rb1 bound to TLR4 to prevent TLR4 dimerization, as confirmed by co-immunoprecipitation and cellular thermal shift assay. Furthermore, MyD88 recruitment and TAK1 expression were altered by reduced TLR4 dimerization, indicating the TLR4-MyD88-NF-kappa B/MAPK pathways contributed to Rb1's anti-inflammatory process. In animal models, Rb1 markedly alleviated LPS- or cantharidin-induced acute kidney injury, rescued LPS-induced septic mice from death, and inhibited dimethyl benzene-induced mouse ear edema. Conclusion: Overall, these findings demonstrate Rb1 exhibits marked anti-inflammatory effects, suggesting Rb1 represents an optimal molecule for treating inflammatory diseases.