Investigation On The Molecular Mechanism That Soyasaponins Exert The Anti-inflammatory Activity By Regulating The Lipid Raft-Mediated Toll-like Receptor(TLR) 4 Signaling Pathway

Background and objectivesNon-communicable diseases(NCDs)are a group of chronic diseases that seriously threats public health.The etiologies of NCDs are various and complicated.Inflammation is one of the common pathological factors that promote the occurrence and development of NCDs.Therefore,anti-inflammation has been recognized as an important strategy to prevent and treat NCDs.Many food-derived phytochemicals could exert anti-inflammatory effects against NCDs.Soyasaponins(SS)are a family of phytochemicals derived from soybeans and their products.SS have been found to have anti-inflammatory,anti-oxidative,and many other biological activities.We and others have found that SS exhibit anti-inflammatory properties by regulating lipid raftmediated toll-like receptor 4(TLR4)signaling pathway,but its molecular mechanism has not been fully elucidated.In this study,two inflammatory cell models were used to explore the regulatory effects of SS on the lipid raft-mediated TLR4 signaling pathway.This study aimed to clarify the anti-inflammatory molecular mechanism of SS and provide new ideas and theories for rational use of SS to prevent and treat inflammationmediated NCDs.Method1.Inflammatory cell models and interventions:Raw264.7 macrophages were incubated with 1μg/mL lipopolysaccharide(LPS)for 30 min or 200 μmol/L palmitic acid(PA)for 5 min to establish the inflammatory models.The cells were pretreated with 40 μmo1/L SS-A1 or SS-A2 for 2 h.Meanwhile,methyl β cyclodextrin(MβCD)acted as the positive control which was used to pretreat cells in a concentration of 20 μmol/L for 30 min.2.The mRNA expressions of inflammatory enzymes(inos and Cox2)and proinflammatory cytokines(Il1b,Il6,and Tnfa)were determined by Q-PCR.3.The protein expressions levels of TLR4,MyD88,TRIF,p65,SREBP2,and ABCA1 were determined by Western blot.4.The recruitment and dimerization of TLR4 and its adaptor molecules(MyD88 and TRIF)in lipid rafts were detected by sucrose gradient ultracentrifugation,confocal microscopy,immunoprecipitation and fluorescence energy resonance transfer.5.The formation,clustering and size of lipid rafts were visualized by confocal microscopy.The levels of cholesterol in lipid rafts and cells were measured by Amplex red cholesterol assay kit.6.Cholesterol replenishment was performed to verify the effect of cholesterol on the regulatory roles of SS in the regulation of lipid raft-mediated TLR4 signaling pathway.Result1.SS(A1 and A2)decreased the elevated mRNA expression of inflammatory enzymes(inos and Cox2)and pro-inflammatory cytokines(Il1b,Il6,and Tnfa)in LPSor PA-stimulated inflammatory Raw264.7 macrophages(p<0.05).2.SS(A1 and A2)inhibited the LPS-or PA-stimulated phosphorylation of p65(p<0.05),but did not affect the protein expression of TLR4,MyD88 and TRIF(p<0.05).3.SS(A1 and A2)inhibited the LPS-or PA-induced recruitment and dimerization of TLR4,MyD88 and TRIF in lipid raft in inflammatory Raw264.7 macrophages(p<0.05).4.SS(A1 and A2)inhibited the LPS-or PA-induced formation,clustering,size change and cholesterol elevation of lipid raft in Raw264.7 macrophages(p<0.05).The LPS-increased cholesterol levels in lipid raft and cells were reduced by 47.9%and 52.4%,respectively by SS-A1,and by 52.2%and 58.8%,respectively by SS-A2.The PA-elevated cholesterol levels in lipid raft and cells were reduced by 56.4%and 51.0%,respectively by SS-A1,and by 57.2%and 47.5%,respectively by SS-A2.5.Cholesterol replenishment abrogated the anti-inflammatory effects of SS(A1 and A2)in LPS-or PA-stimulated inflammatory macrophages(p<0.05).6.SS(A1 and A2)inhibited LPS-or PA-induced maturation of SREBP2 and alleviated the inhibitory effects of LPS or PA on ABCA1 expression in Raw264.7 macrophages(p<0.05).7.No significantly statistical difference was found between the anti-inflammatory activities of SS(A1 and A2)in both LPS-and PA-induced inflammatory cell models(p>0.05).Conclusion1.SS(A1 and A2)alleviate inflammation by regulating the lipid raft-mediated TLR4 signaling pathway.2.SS(A1 and A2)maintain the cholesterol homeostasis,improve the status of lipid raft,and thus regulate the TLR4 signaling pathway.3.Both SS-A1 and SS-A2 have anti-inflammatory properties in LPS-and PAstimulated inflammatory macrophages models but exhibit no difference.