Effects And Mechanisms Of Ghrelin Inhibit Macrophage-derived Foam Cell Formation

PartⅠRegulation of Ghrelin on the Expression of ATP-bindingCassette Transporters A1/G1 in Macrophage-Derived Foam CellsBackground:ATP-binding cassette transporter A1(ABCA1)and ATP-bindingcassette transporter G1(ABCG1)are involved in cholesterol removal from the macrophagefoam cells,and form the critical part of a process termed as reverse cholesterol transport,and therefore play critical roles in foam cell formation.Ghrelin,an endocrine peptide newlyidentified mainly in stomach epithelium,stimulates food intake in humans.Recently,manyresults imply a beneficial role of this hormone in the development of atherosclerosis.Theaim of this study is to investigate the regulation of ghrelin on the expression ofATP-binding Cassette transporters A1 and G1(ABCA1/ABCG1)during foam cellsformation.Methods:The human monocytic leukemia cell line(THP-1)was chosen in our study.The differentiation of THP-1 cells into macrophages was induced by using phorbolmyristate acetate(PMA).Macrophages were then incubated with oxidized LDL(ox-LDL)to generate foam cells.Ghrelin of different concentrations were treated at different timepoints during foam cells formation.The ABCA1/ABCG1 protein and mRNA expressionwere detected by Western blotting and RT-PCR.The effect of variance of cholesterolcontent was measured by zymochemistry via-fiuorospectrophotometer. Results:Ghrelin reduced the content of lipid droplet in foam cells,and increased theefflux of intracellular cholesterol significantly.Ghrelin increased ABCA1 protein mass andmRNA level in a dose-dependent manner.Compared with untreated cells,treatment ofTHP-1 derived foam cells with 10^-7mol/L ghrelin resulted in a significant 1.6-fold increasein ABCA1 protein expression.And 2- and 2.8-fold increase at 10^-6and 10^-5mol/L ghrelin.The changements of ABCA1 mRNA level were the same as ABCA1.Ghrelin also increasedABCG1 protein mass and mRNA level in a dose-dependent manner.Conclusion:Ghrelin might inhibit foam cells formation by up-regulating theexpression of ABCA1 and ABCG1.PartⅡGhrelin inhibit foam cell formation via a GrowthHormone Secretagogue Receptor-Dependent PathwayBackground:Ghrelin,a endogenous ligand of the growth hormone secretagoguereceptor(GHS-R),revealed cardioprotective effects in both experimental models andhuman.There is far less imformation information on mechanisms that produceantiatherogenic effects.Acyl-coenzyme A:cholesterol acyltransferase 1(ACAT-1)andABCA1 have been implicated in regulating cellular cholesterol homeostasis and thereforeplay critical roles in foam cell formation.We demonstrated that ghrelin coulddown-regulated the expression of ACAT-1 and up-regulated ABCA1/G1 simultaneously,and hypothesize that ghrelin can inhibit foam cell formation by regulating the expression ofACAT-1 and ABCA1 via the signaling pathway of GHS-R.The aim of this part of study isto investigate the expression of ACAT-1 and ABCA1/G1 with ghrelin and variousconcentration of GHS-R antagonist in macrophage derived foam cells.Methods:The human monocytic leukemia cell line(THP-1)was chosen in our study.The differentiation of THP-1 cells into macrophages was induced by phorbol 12-myristate13-acetate(PMA).Macrophages were then incubated with oxidized LDL(ox-LDL)togenerate foam cells.Ghrelin and [D-Lys3]-GHRP-6,the special antagonist of growth hormone secretagogue receptor(GHS-R),were treated during foam cells formation.TheACAT-1 and ABCA1/G1 protein and mRNA levels were detected by Western blotting andRT-PCR.The effect of variance of cholesterol content was measured by zymochemistryvia-fluorospectrophotometer.Results:Ghrelin reduced the content of cholesteryl ester in foam cells obviously.ACAT-1 protein and mRNA levels were also decreased.The antagonist of GHS-R inhibitedthe effects of ghrelin on ACAT-1 expression in a dose-dependent manner.Ghrelin increasedthe cholesterol efflux obviously.ABCA1/G1 protein and mRNA levels were also increased.The antagonist of GHS-R inhibited the effects of ghrelin on ABCA1 and ABCG1expression in a dose-dependent manner.Conclusions:Ghrelin might interfere foam cells formation by simultaneouslydown-regulating the expression of ACAT-1 and up-regulating the expression of ABCA1/G1via a GHS-R pathway.PartⅢStudy of the Peroxisome Proliferator-activated Receptor ysignaling pathway in the Inhibition of Foam Cell Formation by GhrelinBackground:To investigate the effects of ghrelin on the expression of peroxisomeproliferator-activated receptorγin foam cells formation.And to investigate the effects ofghrelin on the expression of ACAT-1 and ABCA1/G1 in THP-1 derived foam cells.Methods:The human monocytic leukemia cell line(THP-1)was chosen in our study.The differentiation of THP-1 cells into macrophages was induced by using PMA.Macrophages were then incubated with oxidized LDL(ox-LDL)to generate foam cells.The PPARγmRNA level and protein expression were detected by RT-PCR and Westernblotting.Macrophage were incubated with ghrelin and antagonist of PPARγ,and then wereincubated with oxidized LDL(ox-LDL)to generate foam cells.The ACAT-1 and ABCA1/G1 mRNA level and protein expression were detected by RT-PCR and Westernblotting.The effect of variance of cholesterol content was measured by zymochemistry viafluorospectrophotometer.Results:Ghrelin increased PPARγprotein expression in a dose-dependent manner.The changements of PPARγmRNA level were the same as protein expression.Ghrelinreduced cholesteryl ester obviously.ACAT-1 protein and mRNA levels were also decreased.The antagonist of PPARγinhibited the effects of ghrelin on ACAT-1 expression in adose-dependent manner.Ghrelin increased the cholesterol efflux obviously.ABCA1/G1protein and mRNA levels were also increased.The antagonist of PPARγinhibited theeffects of ghrelin on ABCA1/G1 expression in a dose-dependent manner.Conclusion:Ghrelin could increase the expression of PPARγin a dose-dependentmanner during foam cell formation.The PPARγinhibitor could interfere the expression ofACAT-1 and ABCA1/G1 regulated by ghrelin.The PPARγsignaling pathway mightparticipate in the inhibition of foam cells formation by ghrelin.