Experiment Of THP-1 Macrophage Foam-cell Formation Through MTOR Signal Pathway Activation Induced By Inflammatory Stress

Objective:To investigate if inflammatory agent-LPS increases the sterol regulatory element binding proteins(SREBPs)cleavage-activating protein(SCAP)-SREBP2 expression by activation of mTOR signal pathway in THP-1 macrophages,upgrading LDLr level,causing foam-cell formation.Methods : 1.THP-1 macrophages were incubated in serum free medium in the absence of 5 ug/ml LDL alone(Control group,CG),or 5ug/ml LDL plus 200ng/ml LPS(LPS group,LG),or 5ug/ml LDL plus LPS 200ng/ml LPS plus 10ng/ml Rapamycin(LPS plus Rapamycin group,LPRG).2.Morphological examination of THP-1 macrophages with Oil Red O staining.3.Expression changes of LDLr,SREBP2,SCAP,S6K1 and mTOR mRNA detected by real time quantitative polymerase chain reaction(PCR).4.Western blotting analysis protein expression changes of LDLr,S6K1 and mTOR.5.Translocation of SCAP-SREBP2 complex from the endoplasmic reticulum(ER)to the Golgi by confocal microscopy.6.The results analyzed by T-test selected to analyze the differences between two groups.Results:1.LPS enhanced transformation of THP-1 macrophages into foam cells by increased uptake of lipid as evidenced by Oil Red O assay.2.The fold changes in LDLr mRNA was 1,1.82±0.35,0.33±0.14 respectively in the CG,LG,LPRG.There was an increase of LDLr mRNA in the LG(P<0.05,compared with the CG),and a decline in the LPRG(P<0.05,compared with the LG).3.The fold changes in SREBP2 mRNA was 1,2.21±0.35,0.74±0.21 respectively in the CG,LG,LPRG.There was an increase of SREBP2 mRNA in the LG(P<0.05,compared with the CG),and a decline in the LPRG(P<0.05,compared with the LG).4.The fold changes in SCAP mRNA was 1,2.15±0.48,0.61±0.33.There was an increase of SCAP mRNA in the LG(P<0.05,compared with the CG),and a decline in the LPRG(P<0.05,compared with the LG).5.The fold changes in S6K1 mRNA was 1,1.26±0.22,0.8±0.5 respectively in the CG,LG,LPRG.There was an increase of S6K1 mRNA in the LG(P<0.05,compared with the CG),and a decline in the LPRG(P<0.05,compared with the LG).6.The fold changes in mTOR mRNA was 1,1.38±0.7,0.3±0.7 respectively in the CG,LG,LPRG.There was an increase of mTOR mRNA in the LG(P<0.05,compared with the CG),and a decline in the LPRG(P<0.05,compared with the LG).7.The expression changes of LDLr protein: there was a upregulation of LDLr protein in the LG(P<0.05,compared with the CG),and a downregulation in the LPRG(P<0.05,compared with the LG).8.The expression changes of S6K1 protein: there was a upregulation of S6K1 protein in the LG(P<0.05,compared with the CG),and a downregulation in the LPRG(P<0.05,compared with the LG).9.The expression changes of mTOR protein: there was a upregulation of mTOR protein in the LG(P<0.05,compared with the CG),and a downregulation in the LPRG(P<0.05,compared with the LG).10.Confocal microscopy demonstrated LPS caused an escape of SCAP-SREBP2 complex from the ER to the Golgi,Rapamycin inhibited the translocation of SCAP-SREBP2 complex from the ER to the Golgi.Conclusions: 1.Inflammatory stress increases SCAP/SREBP2 expression by activation of mTOR signal pathway,resulting in an escape of SCAP-SREBP2 complex from the ER to the Golgi,furthermore elevated LDLr expression and caused foam-cell formation.2.Rapamycin reverse the activation of mTOR signal pathway and decreases lipid deposition in THP-1 macrophages induced by LPS.