| IntroductionCardiovascular disease is the main killer of human health and the huge burden of public health. Chinese cardiovascular disease report of 2015 showed that cardiovascular disease, the first cause of death for residents in our country, accounted for 40 percent of total deaths. Atherosclerosis (AS) is the common pathologic basis of cardiovascular disease. Hypercholesterolemia is one of important causes and a risk factor leading to atherosclerosis. Statins, the cholesterol lowering drugs, are the main treatment for atherosclerosis. Statins inhibit the synthesis of hydroxymethylglutaryl coenzyme A (HMG-CoA) reductase to block the synthesis of cholesterol ester and prevent the development of atherosclerosis. However, some studies have shown that the effective of statins is only for 20% to 40% of patients with atherosclerosis. In addition, statins therapy can damage liver and cause rhabdomyolysis. Thus, looking for a new and effective drug for atherosclerosis treatment is very important and urgent.It has been confirmed that the formation of foam cells, the result of cholesterol accumulation in macrophages, is one of the important events in the occurrence of atherosclerosis. Promoting reverse cholesterol transport (RCT) can increase the catabolism of cholesterol to lower cholesterol levels and keep balance of cholesterol in the body. ATP binding cassette subfamily A member 1 (ABCA1), an integral membrane protein, plays an important role in RCT. ABCA1 facilitates cholesterol efflux from macrophages to the extracellular cholesterol acceptor-apolipoprotein Al (ApoA1), by using energy from ATP hydrolysis. Mutation in ABCA1 causes Tangier disease (TD) and family HDL deficiency (FHD), resulting in near absent HDL levels and cholesterol accumulation in tissues. In the general population, several common single nucleotides of ABCA1 gene are closely related to blood lipid level and the severity of atherosclerosis. These results suggest that ABCA1 functions as a key role in lowing cholesterol level and preventing the development of atherosclerosis.Liver X Receptor a (LXRa) is a member of the nuclear receptor superfamily of ligand-activated transcription factor, which is mainly expressed in tissue with active lipid metabolism. LXRa can make low density lipoprotein receptor (LDLR) ubiquitin to cause the LDLR degradation, which inhibit the intake of much low density lipoprotein cholesterol. Also, LXRa deficient mice fed a high-cholesterol diet developed massive hepatic accumulation of cholesterol, whereas wild-type mice were highly resistant to cholesterol feeding. These experiments in vivo and in vitro showed that LXRa palys a key role in keeping cholesterol balance and has anti-atherogenic effects. In addition, studies have showed that ABCA1 is one of target genes of LXRa. LXRa activation resulted in enhancing ABCA 1 expression and leading to an increased cholesterol availability to efflux to apoA1 and HDL. Thus, The LXRa-ABCA1 pathway represents a powerful means to stimulate cholesterol efflux from macrophages and therefore strongly influence the progression of atherosclerotic plaque development.Lipoxin A4 (LXA4) is a kind of metabolites generated from arachidonic acid via LOX enzymes. LXA4 inhibites expression of inflammatory genes and promotes inflammation retrogression, which make it function as "braking signals" in inflammation. Recent studies have indicated that LXA4 has many biological effects in cardiovascular diseases. LXA4 could prevent the endothelial cell hyperpermeability induced by lipopolysaccharides (LPS) in human umbilical vein endothelial cells (HUVECs) through inhibiting nuclear factor kappa B (NF-κB) expression and its related cytokines through receptor-dependent. Also, LXA4 may have the potential to prevent atherosclerosis by confering a protective phenotypic switch and inhibiting proliferation and migration induced by plateletderived growth factor (PDGF) in vascular smooth muscle cells (VSMCs). In addition, the latest research demonstrated that aspirin can induce LXA4 concentration in liver and arachidonic acid favorably affect plasma cholesterol level. These research results support the notion that LXA4 may associate with the occurrence and development of cardiovascular diseases, such as atherosclerosis and coronary heart disease in particular.Although there are some correlation between LXA4 and cholesterol level, the regulatory role of LXA4 on cholesterol remains unclear. The regulating mechanism of LXA4 on cholesterol has not yet been explored. In this study, THP-1 macrophage derived foam cells are used to idetify the effect of LXA4 on cholesterol metabolism. We found that LXA4 could markedly up-regulate ABCA1 expression and promote cholesterol efflux through enhancing LXRα expression in THP-1 macrophage-derived foam cells.Materials and Methods1. Cell culture(1) The cuture of THP-1 cells:Human monocytic THP-1 cells were purchased from the American Type Culture Collection (Manassas, VA, USA) and cultured in Roswell Park Memorial Institute (RPIM) 1640 medium containing 10% fetal calf serum, 100μg/ml streptomycin and 100U/ml penicillin under standard culture conditions (5% CO2,37℃).(2) The differentiation of faom cells:The cells were transformed into macrophages by the incubation with 100ng/ml of phorbol 12-myristate 13-acetate(PMA) Macrophages were differentiated into foam cells by addition of 50μg/ml of Ox-LDL in serum-free RPMI 1640 medium for 48h.(3) Transfection of siRNA:Cells were seeded in 6-or 12-well plates and grown to 80%-90% confluence before use. SiRNA was specific for LXRa and control siRNA were transfected to THP-1 macrophages-derived cells according to lipofectamine RNAiMAX transfection manufacturer’s instruction.2. Preparation of Ox-LDLNative LDL (200 mg protein/mL; Sigma-Aldrich, St. Louis, MO, USA) was oxidized by exposure to CuSO4 (5mM free Cu2+) in phosphate-buffered saline (PBS) at 37℃ for 24h. The cells were treated with 200mM ethylenediaminetetraacetic acid (EDTA) to terminate oxidation reaction. Freshly prepared Ox-LDL was dialyzed with PBS to remove Cu2+at 37℃ for 48h and then was sterilized through a 0.22μm filter. Protein content was determined by using a bicinchoninic acid (BCA) protein assay kit with the use of bovine serum albumin as the standard. The Ox-LDL was maintained in 50 mM Tris-HCl,0.15 M NaCl, and 2.0 mM EDTA at pH 7.4 and was used within 10 days of preparation.3. RNA isolation and real-time quantitative PCR Analysis (qPCR)Total RNA from THP-1 macrophage-derived foam cells were extracted using TRIzol reagent according to the manufacturer’s instructions. Real-time quantitative PCR was performed on an ABI 7500 Fast Real Time PCR system (Applied Biosystems, Foster City, CA,USA) using SYBR Green detection chemistry. Melt curve analyses of all real-time PCR products were performed and shown to produce a single DNA duplex. All samples were measured in triplicate and the mean value was considered for comparative analysis. Quantitative measurements were determined using the △Ct method and GAPDH expression was used as the internal control.4. Western blot analysesProteins were extracted from THP-1 macrophage-derived foam cells using protein extraction kit and quantified using the BCA protein assay kit. Protein samples were subjected to western blot analyses (8% or 10% polyacrylamide gel electrophoresis; 50μg protein per lane) using rabbit polyclonal anti-ABC A1 antibodies, rabbit polyclonal anti-LXRa antibodies and β-actin-specific antibodies. The proteins were visualized using a chemiluminescence method (ECL Plus Western blot Detection System; Amerisham Biosciences, Foster City, CA, USA).5. Transfection with small interfering RNA (siRNA)The small interfering RNAs (siRNAs) against LXRa and an irrelevant 21-nucleotide siRNA, as a negative control, were purchased from Ribo Biotech Co.Ltd(Guangzhou, Guangdong, China). THP-1 macrophage-derived foam cells (2×106cells/well) were transfected using lipofectamine RNAiMAX transfection reagent (Invitrogen) for 48h according to the manufacturer’s instructions. After 48h of transfection, real-time RT-PCR and western blotting were performed.6. High-performance liquid chromatography analysis of cellular cholesterol levelsCells were washed with PBS three times. The appropriate volume (usually 1mL) of 0.5% NaCl was added about 50-200μg cellular proteins per mL. Cells were sonicated using an ultrasonic processor for 2min. The protein concentration in the cell solution was measured using a BCA kit. A 0.1 mL aliquot of cell solution (containing 5-20μg of protein) was used to measure the free cholesterol, and another aliquot was used for total cholesterol detection. Free cholesterol was dissolved in isopropanol (1mg cholesterol/mL) and stored at-20℃ as a stock solution. A cholesterol standard calibration solution ranging from 0 to 40μg of cholesterol per mL was obtained by diluting the cholesterol stock solution in the same cell lysed buffer.A 0.1 mL aliquot of each sample (cholesterol standard calibration solutions, or cell solutions) was supplemented with 10μL of the reaction mixture, which included 500 mmol/L MgCl2,500 mmol/L Tris-HCl (pH 7.4),10 mmol/L dithiothreitol, and 5% NaCl. A total of 0.4 U of cholesterol oxidase in 10μL 0.5% NaCl was added to each tube for free cholesterol determination or 0.4 U cholesterol oxidase plus 0.4 U of cholesterol esterase for a total cholesterol measurement. The total reaction solution in each tube was incubated at 37℃ for 30min, and 100μL methanol:ethanol (1:1) was added to stop the reaction. Each solution was kept cold for 30 min to allow protein precipitation and then centrifuged at 1500 r/min for 10min at 15℃. Ten microliters of supernatant was applied onto a System Chromatographer (PerkinElmer Inc) that included a PerkinElmer series 200 vacuum degasser, a pump, a PerkinElmer series 600LINK, and a PerkinElmer series 200 UV/vis detector, and a Disovery C-18 HLPC column(Supelco Inc). The column was eluted using isopropanol:n-heptane: acetonitrile (35:13:52) at a flow rate of 1mL/min for 8min. The absorbance at 216nm was monitored, and the data were analyzed with TotalChrom software from PerkinElmer.7. Cellular cholesterol efflux experimentsCells were incubated with RPMI 1640 medium containing 0.2 μCi/ml [3H] cholesterol. Then cells were washed with PBS and incubated with RPMI 1640 medium containing 0.1%(wt/vol) BSA overnight to allow equilibration of [3H] cholesterol in all cellular pools. Cells were washed with PBS and incubated in 2ml efflux medium containing RPMI 1640 and 0.1% BSA with or without 25μg/ml of human plasma ApoAl for 12h. Medium and cell-associated [3H] cholesterol were then measured by liquid scintillation counting. Percent efflux was calculated using the following equation:[total media counts/(total cellular counts+total media counts)]× 100%.8. Statistical analyseData are reported as means±S.D. The date were compared by one-way analysis of variance and the Student’s t-test, using the Statistical Package for the Social Sciences (version13.0) software (SPSS, Inc., Chicago, IL, USA). Statistical significance was obtained when p values were less than 0.05.Results1. LXA4 up-regulates ABCA1 expression in THP-1 macrophage-derived foam cells.The development of atherosclerosis is a result of the interaction between the macrophages, endothelial cells and smooth muscle cells. Recent reseaches have revealed that LXA4 can influence inflammatory response of endothelial cell and migration and proliferation of smooth muscle cell, which indicate that LXA4 is associated with the development of atherosclerosis. ABCA1 is a crucial cholesterol transporter, which is widely expressed and is abundant in macrophages. Increasing ABCA1 activity can elevate plasma HDL levels. The effect of LXA4 on ABCA1 remains unclear. Thus, We first examined the effect of LXA4 on ABCA1 expression in THP-1 macrophage-derived foam cells by real-time quantitative PCR and Western blot assays. LXA4 obviously increased ABCA1 mRNA and protein expression in a dose-dependent manner in THP-1 macrophage-derived foam cells.2. LXRa is enhanced by LXA4 in THP-1 macrophage-derived foam cells.LXRa is a member of the nuclear receptor superfamily of ligand-activated transcription factors, which is an important regulator of cholesterol, free fatty acid, and glucose metabolism. In order to explore the effect of LXA4 on cholesterol metabolism, we next detected whether LXRa expression can be affected by LXA4 in THP-1 macrophage-derived foam cells by real-time quantitative PCR and western blot analysis. LXA4 markedly increased LXRa expression at both transcriptional levels and translational levels in a dose-dependent manner.3. LXRa is involved in LXA4-induced ABCA1 increase in THP-1 macrophage-derived foam cells.The previous study has proved that LXRa agonists stimulate ABCA1 gene expression in human macrophages, which suggested that ABCA1 gene is one of the target gene of LXRa. Furthermore, here we showed that LXA4 could increase ABCA1 and LXRa expression in THP-1 macrophage-derived foam cells. Thus we speculated that LXA4 may upregulate ABCA1 expression through the LXRa pathway in THP-1 macrophage-derived foam cells. We then examined the effect of LXRa siRNA on regulation of ABCA1 with and without LXA4 treatment. In comparison to the control siRNA, treatment with siRNA targeting LXRa decreased LXRa protein expression by 88% in THP-1 macrophage-derived foam cells. After LXRa siRNA treatment, the basal expression of ABCA1 is decreased, which indicated LXRa can mediate ABCA1 expression. In addition, the up-regulation of ABCA1 expression by LXA4 treatment was markedly reversed by treating with LXRa siRNA.4. LXA4 contributes to cellular cholesterol content and cholesterol efflux.ABCA1 promotes free cholesterol efflux from macrophages into ApoAl to form HDL, which is central to prevent atherosclerosis progression. And here we indicated that expression levels of ABCA1 could be increased by LXA4 and completely abolished by siRNA-targeted silencing of LXRa. Therefore, we next examined the effect of LXA4 on celluar cholesterol content and apoAl-specific cholesterol efflux and whether the LXRa pathway is involved in this process in THP-1 macrophage-derived foam cells. Cellular cholesterol content was decreased while cholesterol efflux was increased when cells were treated with LXA4. After LXRa siRNA treatment, inhibition of cellular cholesterol content and promotion of cholesterol efflux by LXA4 treatment were markedly abolished.Conclusion1.LXA4 markedly enhances ABCA1 mRNA and protein expression in a dose-dependent manner in THP-1 macrophage-derived foam cells.2. LXA4 obviously increases LXRa expression at both transcriptional levels and translational levels in a dose-dependent manner.3. LXA4 increases ABCA1 expression through enhancing LXRa expression in THP-1 macrophage-derived foam cells. The up-regulation of ABCA1 expression by LXA4 treatment is markedly accentated by LXRa siRNA treatment.4. LXA4 decreases cellular cholesterol content and increases cholesterol efflux via LXRa pathway in THP-1 macrophage-derived foam cells. After LXRa siRNA treatment, inhibition of cellular cholesterol content and promotion of cholesterol efflux by LXA4 treatment are markedly abolished. |
PDF Full Text Request