ABSTRACT
<p><b>OBJECTIVE</b>To investigate the effects of Ghrelin on the expression of acyl coenzyme A:cholesterol acyltransferases-1 (ACAT-1) in THP-1 derived foam cells.</p><p><b>METHODS</b>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-myristate 13-acetate. Macrophages were then incubated with oxidized LDL (ox-LDL) to generate foam cells. Ghrelin and [D-Lys3]-GHRP-6, the special antagonist of growth hormone secretagogue receptor (GHS-R), were treated during foam cells formation. The ACAT-1 protein and mRNA levels were detected by Western blot and RT-PCR. The effect of variance of cholesterol content was measured by zymochemistry via-fluorospectrophotometer.</p><p><b>RESULTS</b>Ghrelin reduced the content of cholesterol ester in foam cells obviously. ACAT-1 protein and mRNA levels were also decreased. The antagonist of GHS-R inhibited the effects of Ghrelin on ACAT-1 expression in dose-dependent manner. The ACAT-1 mRNA levels of the GHS-R specific antagonist groups (10(-5), 5 x 10(-5), 10(-4) mol/L) were 1.14 +/- 0.04, 1.58 +/- 0.03, 2.40 +/- 0.16, significantly higher than that of the Ghrelin group (0.89 +/- 0.05). And the protein expressions were 1.25 +/- 0.09, 1.77 +/- 0.11, 2.30 +/- 0.09, also higher than that of the Ghrelin group (0.86 +/- 0.08).</p><p><b>CONCLUSIONS</b>Ghrelin might interfere atherosclerosis by down-regulating the expression of ACAT-1 via GHS-R pathway.</p>
Subject(s)
Humans , Acetyl-CoA C-Acetyltransferase , Metabolism , Acyl Coenzyme A , Metabolism , Blotting, Western , Cell Line, Tumor , Cholesterol , Metabolism , Down-Regulation , Foam Cells , Metabolism , Ghrelin , Physiology , RNA, Messenger , Metabolism , Receptors, Ghrelin , Physiology , Reverse Transcriptase Polymerase Chain Reaction , Signal Transduction , SpectrophotometryABSTRACT
<p><b>OBJECTIVE</b>To investigate the expression changes of acyl-coenzyme A: cholesterol acyltransferase 1 (ACAT1) on Chlamydia pneumoniae (C.pn) induced foam cell formation.</p><p><b>METHODS</b>Human monocytic cell line (THP-1) was induced into macrophages by 160 nmol/L phorbol myristate acetate (PMA) for 48 h, and were randomly allocated into four groups: negative control group (50 microg/ml LDL for 48 h); positive control group (50 microg/ml ox-LDL for 48 h); C.pn infection group (50 microg/ml LDL plus 1 x 10(5), 4 x 10(5), 5 x 10(5) and 1 x 10(6) IFU C.pn for 48 h or 1 x 10(6) IFU C.pn for 0, 24, 48 and 72 h); ACAT inhibitor 58-035 plus C.pn infection group (1, 5, 10 microg/ml ACAT inhibitor 58-035 pretreatment for 1 h, 50 microg/ml LDL and 1 x 10(6) IFU C.pn for 48 h). The mRNA and protein expressions of ACAT1 were determined by RT-PCR and Western blot, respectively. Lipid droplets in cytoplasm were observed by oil red O staining. The contents of intracellular cholesteryl esters were detected by enzyme-fluorescence.</p><p><b>RESULTS</b>The mRNA and protein expressions of ACAT1 were significantly up-regulated in positive control cells compared those in negative control cells and further upregulated by C.pn infection in a time-dependent and concentration-dependent manner (all P < 0.05). There were significantly increases in the accumulation of lipid droplets and the ratio of cholesteryl ester to total cholesterol in positive control cells as compared with negative control cells and these were further aggravated by C.pn (at the concentrations of 5 x 10(5) and 1 x 10(6) IFU for 48 h) and C.pn infection induced increases in the accumulation of lipid droplets and the ratio of cholesteryl ester to total cholesterol could be significantly attenuated by ACAT inhibitor 58-035 (all P < 0.05).</p><p><b>CONCLUSION</b>Chlamydia pneumoniae induces THP-1-derived foam cell formation by up-regulating the expression of ACAT1.</p>
Subject(s)
Humans , Cell Line, Tumor , Chlamydophila pneumoniae , Foam Cells , Cell Biology , Metabolism , Monocytes , Cell Biology , Sterol O-Acyltransferase , Metabolism , Up-RegulationABSTRACT
Objective To investigate the influence of tumor necrosis factor-?(TNF-?) on ATP binding cassette transporter A1(ABCA1)in THP-1 macrophage foam cells, and the intervention effect of nuclear factor-?B(NF-?B) inhibitor TPCK on the TNF-?, so as to determine the role of TNF-?/ NF-?B in cellular cholesterol efflux. Methods Foam cells were transformed from THP1 cells. The correlation of cellular cholesterol efflux from foam cells with different concentrations and time stimulated by TNF-? were estimated. Subsequently foam cells were treated with TNF-? at satulated concentration(10.0 ng/ml ), TPCK(10?mol/L),or TPCK(10?mol/L) pretreated for 60 min before TNF-? stimulation. ABCA1 gene expression was analyzed by RT-PCR. ABCA1 protein level was detected by Western blot. Results TNF-? decreased cellular cholesterol efflux of foam cells in concentration-and time-dependent manner. 10 ng/ml of TNF-? down-regulated the levels of both ABCA1 mRNA and protein expressions in time-dependent manner. TPCK was observed to efficiently block the suppressive effect of TNF-? on ABCA1. Conclusions TNF-? decreases cellular cholesterol efflux mainly through the down-regulation of ABCA1. TPCK, an inhibitor of NF-?B activation, is observed to partly block the suppressive effect of TNF-? on ABCA1, suggesting a mechanism involving NF-?B signal transduction. TNF-?/NF-?B might play a critical role in the progression of atherosclerosis by decreasing cellular cholesterol efflux from foam cells.