| Objective:Sphingosine-1-phosphate(S1P),a sphingosine blood-derived chemical signaling molecule released by blood cells and endothelial cells,regulates immune and protects blood vessels.Recent studies have shown that S1 P is closely related to the cholesterol efflux from macrophage,but its specific role and mechanism is not yet clear.Therefore,the purpose of this study is to investigate the effect of S1 P on macrophage cholesterol accumulation and its possible mechanisms.Methods : 1.THP-1 cells were treated with 60μg/ml oxidized low-density lipoprotein(ox-LDL)for different time(0,12,24,48h)to determine the lipid content.Cellular lipid droplet was measured by Oil Red O staining.Cellular cholesterol content was measured by high performance liquid chromatography(HPLC).2.THP-1 derived foam cells were treated with different concentration of S1P(0,0.5,1.0,2.0 μM)for 24 h or 1μM S1 P under different time(0,6,12,24h).The expression of SR-BI(scavenger receptor,class B,type I,SR-BI)and ABCA1(ATP binding cassette transporter A1,ABCA1)in cells were detected by Western blot.3.Intervention the expression of S1P1/3 receptors by VPC23019,Western blot and quantitative real-time PCR(qRT-PCR)were used to detect the expression of ABCA1 and SR-BI protein and mRNA,respectively.4.According to the appropriate S1 P concentration and the corresponding processing time on the last steps of the experiment results are processed,THP-1 monocytes were preincubated with phorbol-12-myristate-13-acetate(PMA)and then oxidized low-density lipoprotein(ox-LDL)to induce THP-1 derived foam cell formation,and the distribution and location of cholesterol in cells were observed under microscope,then the oil red O staining was used to investigate the accumulation of intracellular lipid.3.Western blotting was used to detect the levels of phosphorylated SIRT1 and LXRα.4.The inhibitors and agonists were used to interfere with the expression of LXRα/SIRT1,respectively.Western blotting was used to detect the expression of p-LXRα,p-SIRT1.Oil red O staining was used to detect the lipid accumulation in THP-1 derived foam cells.Results:1.60μg/ml ox-LDL treatment significantly increased lipid accumulation and cholesterol content in THP-1 derived foam cells.2.S1 P effects the expression of SR-BI/ABCA1 in a time-dependent manner.3.Activating or Blocking S1 P receptors by FTY720 or VPC230193,respectively,can directly affect the expression of ABCA1/SR-BI in THP-1 derived foam cells.4.LXRα/SIRT1 inhibitor can significantly inhibit the expression of ABCA1 in THP-1 cells,while the activation of LXRα/SIRT1 inhibits the expression of SR-BI.The detection of SIRT1 and LXRα phosphorylation results showed that SIRT1/LXRα signaling molecules participate in the regulation of ABCA1 and SR-BI,and through the up-regulation of ABCA1 expression involved in cholesterol reverse transport and thus affect the cholesterol accumulation.2.S1 P essentially activated Silent mating type information regulation 2 homolog-1-liver X receptor α(LXRα)signal pathway in THP-1-derived foam cells.Blocking S1 P receptors by VPC230193 can directly affect the expression of ABCA1/SR-BI.3.Nicotinamide can effectively inhibit SIRT1,and this process also abolishes the S1P-induced down-regulated the expression of ABCA1.4.LXRα inhibitor(K1327)significantly inhibited ABCA1 expression in THP-1 cells and increased intracellular lipid accumulation.SIRT1 agonist(Resveratrol)inhibits the expression of SR-BI,and to a certain extent inhibits the up-regulation of SR-BI by the LXRα agonist(T0901327),resulting in a decrease in intracellular lipid accumulation.4.Ox-LDL treatment down-regulated the expression of p-SIRT1 and p-LXRα while S1 P increasing the expression of p-SIRT1 and p-LXRα.Conclusion:S1P reduces cholesterol accumulation in THP-1 derived macrophages by up-regulating ABCA1 expression and inhibiting SR-BI expression,and LXRα/SIRT1 signaling molecules are involved in the regulation of S1 P on the cholesterol accumulation of THP-1 derived macrophages. |
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