Soyasaponin A1 Inhibits the Lipid Raft Recruitment and Dimerization of TLR4, MyD88 and TRIF in Palmitate-Stimulated Inflammatory Macrophages

Abstract Objectives Soyasaponin A1 (SSA1) was previously shown to inhibit the palmitate (PA)-induced inflammation via regulating the toll-like receptor 4 (TLR4) signaling in macrophages. Since the lipid raft recruitment and dimerization of TLR4 and its downstream adaptor molecules are vital for PA-initiated TLR4 signaling, we explored whether this process would be modulated by SSA1. Methods Murine macrophage RAW264.7 were stimulated with PA (200 μmol/L) in the presence or absence of SSA1 (40 μmol/L). The lipid raft fractions were separated by sucrose density gradient ultracentrifugation and immunoblotted with anti-flotillin-1, anti-TLR4, anti-myeloid differentiation primary response protein 88 (MyD88), or anti-Toll/IL-1 receptor domain-containing adaptor inducing interferon-β (TRIF) antibody. Lipid rafts, TLR4, MyD88 and TRIF were fluorescently labeled and analyzed by confocal microscopy to visualize the recruitment of molecules into lipid rafts and investigate the clustering and size of lipid rafts. The complexes of TLR4/MyD88 and TLR4/TRIF were immunoprecipitated by anti-TLR4 antibody first and then immunoblotted by anti-MyD88 or anti-TRIF antibody. Results PA-induced recruitment of TLR4, MyD88 and TRIF into fractions enriched with lipid rafts marker flotillin-1 was inhibited (P < 0.05) by SSA1. Meanwhile, the PA-induced co-localization of TLR4, MyD88, and TRIF with lipid rafts was also decreased (P < 0.05) by SSA1 as visualized by confocal immunofluorescence microscopy. Furthermore, the PA-induced clustering of lipid rafts was diminished (P < 0.05) by SSA1. However, the PA-decreased size of lipid rafts was increased (P < 0.05) by SSA1. The formation of TLR4/MyD88 and TLR4/TRIF complexes was suppressed (P < 0.05) by SSA1, whereas the protein expressions of TLR4, MyD88 and TRIF were not changed (P > 0.05) by SSA1 in PA-stimulated macrophages. Conclusions SSA1 inhibits the recruitment of TLR4 and its adaptor molecules (MyD88 and TRIF) into lipid raft as well as their dimerization (TLR4/MyD88 and TLR4/TRIF) in PA-stimulated inflammatory macrophages. Funding Sources This work was supported by grants from National Natural Science Foundation of China (NSFC).