Soyasaponin A1 inhibits the lipid raft recruitment and dimerization of TLR4, MyD88, and TRIF by maintaining cholesterol homeostasis in palmitic acid-stimulated inflammatory Raw264.7 macrophage cell line

[Display omitted] •Soyasaponin A1 (SSA1) inhibits the lipid rafts-mediated TLR4 signal pathway.•SSA1 modify the formation, clustering and size of lipid rafts.•Cholesterol replenishment abrogates the anti-inflammatory effects of SSA1.•SSA1 maintains cholesterol homeostasis by binding to it and affecting its metabolism. The mechanisms of soyasaponin A1 (SS-A1) on antagonizing inflammation via regulating the lipid raft-mediated toll-like receptor 4 (TLR4) signaling are not completely understood. In this study, SS-A1 inhibited palmitic acid (PA)-induced recruitments of TLR4 and its adaptor molecules into lipid rafts in Raw264.7 macrophage cell line by using ultracentrifugation and confocal microscopy. SS-A1 also suppressed the PA-caused dimerization of TLR4 with and its adaptor molecules by applying immunoprecipitation and fluorescence resonance energy transfer. Meanwhile, SS-A1 modified the formation, clustering, and size of lipid rafts and decreased cholesterol in lipid rafts. Cholesterol replenishment abrogated SS-A1’s inhibition on the lipid rafts recruitment and dimerization of TLR4 and its adaptor molecules and its anti-inflammatory activity. Additionally, SS-A1 inhibited the mature sterol regulatory element-binding protein 2 and increases ATP-binding cassette transporter A1. Collectively, SS-A1 inhibits the lipid raft recruitment and dimerization of TLR4 and its adaptor molecules by maintaining cholesterol homeostasis in PA-stimulated inflammatory Raw264.7 macrophage cell line.