The Experimental Study Of The Effect Of Nifedipine On Cholesterol Efflux In RAW264.7Macrophages

Background and objectionA hallmark of atherosclerosis is the accumulation of cholesterol-laden macrophages(foam cells) in the arterial wall. The formation of foam cells is often caused by either theuncontrolled uptake of modified low-density lipoprotein (LDL) or impaired cholesterolefflux, which represents a key step in the development of atherosclerosis. Reversecholesterol transport (RCT) is a main mechanism in promoting vascular health. DuringRCT, excess cholesterol is transported from the peripheral cells such as macrophages tocirculating high-density lipoprotein (HDL), and then to the liver for subsequent biliary andfecal disposal asbile acids and neutral sterols. At the molecular level, the first step of RCTis that ABCA1effluxes phospholipids and cholesterol from macrophage membranes tolipid-poor apolipoprotein-A-I (ApoA-I) to generate pre-β-HDL. These HDL particlesfurther acquire cholesterol via ABCG1-and SR-BI-mediated efflux pathways.Nifedipine is a calcium channel blocker. Many studies show that nifedipine hasanti-atherogenic effects, but the underlying mechanisms have not been elucidated. In thisstudy, we examined if nifedipine increases macrophage cholesterol efflux, a pathwayknown to inhibit atherogenesis.Methods1. RAW264.7cells were cultured as indicated above, and then labeled with [~3H]cholesterol (0.3μCi/mL) in serum-free DMEM containing50μg/ml ox-LDL and0.2%bovine serum albumin (BSA) for24h. The cells were washed two times with phosphatebuffered saline (PBS) and incubated in2mL of DMEM containing0.2%BSA either withor without nifedipine at1,10, or100nmol/L for48h. The medium was replaced withDMEM containing0.2%BSA in the presence of either lipid-free apoA-I (10μg/mL) orHDL (50μg/mL) for24h. Cholesterol efflux was counted using a liquid scintillationcounter. 2. RAW264.7cells were treated with orwithout nifedipine at1,10, or100nmol/L for24h, then the mRNA expression of ABCA1, ABCG1, SR-BI and LXRα were assayed byquantitative real-time PCR; RAW264.7cells were treated with orwithout nifedipine at1,10,or100nmol/L for48h. Then the mRNA expression of ABCA1, ABCG1, SR-BI and LXRαwere assayed by quantitative real-time PCR Western blotting.3. RAW264.7cells were grown in culture flasks at a density of1×10~7/ml for12h,washed with PBS, and nifedipine at either0,1,10, or100nmol/L was added to the DMEMculture media containing10%fetal bovine serum. The control siRNA or LXRα-siRNAwasadded to each flask, and cultured for96h. Cells were harvested and protein extractsprepared.The proteins were then subjected to Western blot analysis.4. RAW264.7cells were labeled with [~3H] cholesterol (0.3-μCi/mL) in serum-freeDMEM containing50μg/ml ox-LDL and0.2%bovine serum albumin (BSA) for24h. Thecells were washed two times with PBS and incubated in DMEM containing0.2%BSAwith nifedipine at0,1,10, or100-nmol/L. The control siRNA or LXRa siRNA was addedto each culture well and cultured for48h. The medium was replaced with DMEMcontaining0.2%BSA in the presence of either lipid-free apoA-I (10μg/mL) or HDL(50μg/mL) for24h. Cholesterol efflux was counted using a liquid scintillation counter.Results1. Nifedipne at1,10, and100nmol/L increased apoA-I-mediated cholesterol effluxfrom2.55%to5.65%,6.20%, and6.10%; as well as HDL-mediated cholesterol efflux from31.0%to42.5%,46.0%, and43.5%, respectively.2. Nifedipne at1,10, and100nmol/L increased mRNA expression levels of ABCA1,ABCG1, SR-BI, and LXRα (405%,381%,336%;890%,960%,1002%;285%,325%,336%;482%,445%,405%, respectively, p<0.05) in RAW264.7macrophages (p<0.05);Nifedipne at1,10, and100nmol/L increased mRNA expression levels of ABCA1, ABCG1,SR-BI, and LXRα(428%,492%,361%;288%,331%,365%;283%,320%,505%;581%,678%,608%, respectively, p<0.05).3. ABCA1, ABCG1, SR-BI, and LXRα protein expression levels were significantlydown-regulated in RAW264.7macrophages treated with LXRα-siRNA compared with thecontrol siRNA. The expression of ABCA1, ABCG1and SR-BI in RAW264.7macrophagestreated with LXRα-siRNA and nifedipine were lower than those in RAW264.7cells without treatment with nifedipine and LXRa-siRNA.4. LXRa silencing significantly down-regulated ApoA-I-and HDL-mediatedcholesterol efflux (0.95%and16.60%in LXRα-siRNA group versus3.15%and33.50%in the control siRNA group, respectively); nifedipine at1,10, or100nmol/L largelyprevented LXRa silencing-associated reduction in apoA-I-mediated cholesterol efflux(2.96%,2.71%, or2.69%, respectively) and HDL-mediated cholesterol efflux (23.1%,23.5%, or23.9%, respectively), the apoA-I-and HDL-mediated cholesterol efflux inRAW264.7macrophages treated with LXRα-siRNA and nifedipine were lower than that inRAW264.7cells without treatments of LXRα-siRNA and nifedipine.Conclusion1. Nifedipine enhances cholesterol efflux in RAW264.7Macrophages，inhibits uptakeof cholesterol and the formation of foam cells.2. Nifedipine Increases mRNA and Protein Levels of ABCA1, ABCG1, SR-BI, andLXRa in RAW264.7Cells(P<0.05)。3.The effect of nifedipine on the expression of ABCA1, ABCG1, SR-BI, and LXRa inRAW264.7Cells was inhibited by LXRa-siRNA in RAW264.7macrophages4.The effect of nifedipine on cholesterol efflux was inhibited by LXRa-siRNA inRAW264.7macrophages.