Momordicin Acclerated Cholesterol Efflux From THP-1 Macrophage-derived Foam Cells Through Activating LXRα/ABCA1 Pathway

Objective:To explore the effects of momordicin on cholesterol outflow from THP-1 macrophage derived foam cells and expression change of ATP-binding cassette A1(ABCA1)and liver X receptorα(LXRα)in these cells.Mehtods:Firstly,momordicin was extracted from fresh Momordica by using homogenation,centrifugation,ultrafiltration and chromatographic technique.Meanwhile,THP-1 cells were cultured in RPMI-1640 medium(containing 10%FBS)and then differentiated into macrophages induced by phorbol 12-myristate 13-acetate(PMA)for 24h in a 37°C incubator.Afterwards,the viability of THP-1-derived macrophages after treated with different concentration(0,0.5,1.5,4.5,5mg/ml)of momordicin for24h were detected by MTT assay.And the THP-1-derived foam cells for follow-up experiment were obtained by inducing THP-1-derived macrophages differentiation by incubation with 0.2μCi/ml[~3H]cholesterol and 50 mg/ml ox-LDL for 24h.In the further experiments,the effects of momordicin on THP-1-derived foam cells cholesterol regulations were measured by high performance liquid chromatography(HPLC)after cells treated by different concentration(0,0.5,1.5,4.5mg/ml)momordicin for 24h or 4.5mg/ml momordicin for different time(0,3,6,12,24h)represented by levels of intracellular total cholesterol,cholesterol ester,and free cholesterol in the cells.And the effect of momordicin on changes of mRNA and protein expression of ABCA1 and LXR-αin THP-1-derived foam cells were also detected by Real-time PCR and Western blot.Finally,the THP-1-derived foam cells transfected with specific siRNA target for LXRαwere used for identifying the role of LXRαin the effect of momordicin on up-regulation of ABCA1 and chlesterol efflux from cells.Result:1.The THP-1 foam cells were disposed to various concentrations of momordicin(0、0.5、1.5、4.5mg/ml)for 24h,0.5mg/ml of momordicin couldn’t conspicuous accelerate choleseterol outflow to extra cell,1.5mg of momordicin could accelerate choleseterol efflux to extra cell was 1.45flods respectively compared with control group(P<0.05).4.5mg/ml of momordicin dramatically accelerate choleseterol efflux to extra cell was1.69 flods respectively compared with control group(P<0.05)Subsequently,4.5mg/ml of momordicin was used to stimulate the chlesterol-loaded THP-1 macrophages for varying times(0.3.6.12.24h).4.5mg of momordicin could accelerate choleseterol efflux to extra cell was 1.11 and 1.14 flods respectively compared with control group for 3h and 6h.(P<0.05),while cholesterol efflux was 1.51 flods after 12h incubation(P<0.05),and was 1.81 flods after 24h(P<0.01)2.The expression of ABCA1 in THP-1 macrophages at both mRNA levelsandprotein.Thecellsweretreatedwithdifferent concengtrations(0,0.5,1.5,4.5mg/ml)of momordicin for 24 hours.ABCA1was measured by real-time PCR and wstern blot each in m RNA and protein levels.compared with the control group,the expression of ABCA1in protein and mRNA levels was obvious augment under the disposed of1.5(P<0.05)and 4.5mg/ml(P<0.01),while 0.5mg/ml of momordicin failed to enhance the expression of ABCA1.Furtermore,4.5mg/ml of momordicin was used to stimulate the cells for different time(0,3,6,12 and 24h).the reltive expression of ABCA1mRNA and proteinwasn’t obvious augment compared with the control group for 3h.The reltive expression of ABCA1 was obvious increased for6h,while significantly increased for 12h and 24h.3.The expression of LXRαin THP-1 macrophages at both mRNA levelsandprotein.thecellsweretreatedwithdifferent concengtrations(0,0.5,1.5,4.5mg/ml)ofmomordicinfor24hours.compared with the control group,the expression of LXRαin mRNA levels and protein levels was significantly increased under the stmulation of 1.5(P<0.05)and 4.5mg/ml(P<0.01),while 0.5mg/ml of momordicin failed to enhance the expression of LXRα.4.The expression of ABCA1 after the silence of LXRαwere respctively applied.compared with the control group,it observably inhibited the dowm-regulation influence of momordicin on the expreesion of ABCA1 in mRNA and protein levels(P<0.05).5.THP-1 macrophage-derived foam cells were transfected with control or LXRαsiRNA,incubating with or without momordicin for24h.compared with the control group,LXRαsiRNA group inhition of cellular cholesterol efflux.with the momodicin group,momordicin with LXRαsiRNA group dramatically reduce choleseterol efflux to extra cell.ConclusionMomordicinacceleratecholesteroleffluxfromTHP-1macrophage-derived foam cells through activating LXRα/ABCA1 pathway.