S1PR1is Involved In HDL Mediating Cholesterol Efflux Of THP-1-derived Macrophage And Mechanism

background and objective: S1P(sphingosine-1-phosphate), anintermediate of sphingomyelin and lysophosphatide metabolism,is a far concerning lipidsignal molecule currently,which plays a significant role in the occurrence anddevelopment of many cardiovascular diseases,such as atherosclerosis (AS).S1P exists inmany places in body,including plasma,lymph,erythrocyte,leukocyte,platelet and soon,having a variety of biological functions,which are performed by itself directly induingcellular effects,or by its specific receptors affecting cellular signal transductionpathway.S1PR(S1P receptors),belonging to GPCR(G protein coupled receptors),havebeen found5kinds of subtypes so far,namely S1PR1~5,and S1PR1is mainly associatedwith Gi.Different GPCR mediate different biological effects.Therefore,S1P can have animpact on many biological activities through combining S1PR.However,until now it hasnot been reported that S1PR1can influence the cholesterol metabolism inmacrophage.This article will explore that S1PR1participate in the HDL mediatingcholesterol efflux of THP-1-derived macrophage and its corresponding mechanism,putting forward new theoretical basis for overall comprehending the antiatherogenicmechanism of S1PR1.Methods:1. THP-1mononuclear cells were induced by Oxidized low densitylipoprotein (oxLDL，60μg/ml) for24h, THP-1derived macrophages were incubated with50μg/ml HDL and VPC23019(an inhibitor of S1PR1), the content of cholesterol weredetermined by oil red O and High performance liquid chromatograph.2. THP-1derivedmacrophages were incubated with50μg/ml HDL and VPC23019(an inhibitor of S1PR1，10μM) for12h, and then add BODIPY-Cholesterol overnight incubation, the content ofcholesterol was determined by High performance liquid chromatograph，and then add50μg/ml HDL overnight incubation, the accumulation change of cholesterol in cellmembrane were observed under5Μ BODIPY fluorescent mark.3. THP-1derived macrophages were treated with VPC23019(10μM) and siRNA for24h, the proteinexpression level of ABCA1was determined by Western Blot.4. The protein expressionlevel of LXRα through S1PR1-PI3K-Akt Signaling pathways were determined by S1P、VPC23019、LY294002and MK2206.5. The protein expression level of ABCA1throughS1PR1-PI3K-Akt-LXRα Signaling pathways were determined by T0901317、LY294002、MK2206and22S-HC.6.THP-1mononuclear cells were induced by S1P、siRNA and VPC23019(an inhibitor of S1PR1) to determine the influence of Aktphosphorylation by S1P/S1PR1.7. THP-1mononuclear cells were induced by Oxidizedlow density lipoprotein (oxLDL), and then add S1P、VPC23019（an inhibitor ofS1PR1）、T0901317（an agonists of LXRα）and22S-HC（a Selective inhibitors of LXRα）to determine the accumulation change of cholesterol in cell membrane by S1PR1andLXRα.Conclusions:1. S1PR1is involved in cholesterol efflux from THP-1macrophage induced byHDL.2. S1P1through PI3K-Akt-LXR signal transduction pathways promotes cholesterolefflux from THP-1macrophage.