Effects Of Midkine On ABCA1 Expression And Cholesterol Efflux In RAW264.7 Macrophages

Objective: Midkine(MK)is a heparin binding growth factor.Recent studies have shown that the level of serum MK is closely associated with the occurrence and development of As-related ischemic cardiovascular disease.However,the atherogenic effect of MK is not clear.Therefore,the aim of this study is to explore the effect and mechanism of MK on RAW264.7 macrophage cholesterol efflux.Methods: RAW264.7 cells were incubation with 50 mg/L ox-LDL for 48 h to differentiate into macrophage before the experiment.1.The cells were loaded with MK(0,50,100,200,400 ng/ml)for 16 h or 200 ng/ml MK for different times(0,8,16,24,48 h).Cellular lipid droplet was measured by Oil Red O staining,intracellular cholesterol levels were observed by NBD-Cholesterol fluorescence labeling,and intracellular lipid content was detected by enzymatic methods.The cholesterol efflux was assessed by liquid scintillation counting(LSC).The expression of ABCA1 was examined by cell immunofluorescence,western blotting and q RT-PCR,respectively.2.Overexpressed the ABCA1 to detect the effect of MK on the intracellular lipid content and the efflux of cholesterol on macrophage.4.Intervention of AMPK-m TOR signaling molecule to detected the effects of MK on the expression of p-AMPK,p-m TOR,ABCA1,cholesterol efflux and intracellular lipid content on macrophage.Results: 1.Compared with Control group,we found that MK reduced the cholesterol efflux rate,promoted the accumulation of intracellular lipids and decreaseed the expression of ABCA1 on RAW264.7 macrophages in a concentration time dependent manner.2.The inhibitory effect of MK on reduction cholesterol efflux was alleviated by the combination of MK and overexpression ABCA1 as compared with MK alone treated group,resulting reducing intracellular lipid accumulation.3.MK inhibited the activation of AMPK-m TOR signaling molecule.The expression of ABCA1 and cholesterol efflux was increased by AICAR treatment as compared with the Control group and MK treatment.In addition,the inhibitory effect of MK on decrease ABCA1 expression,reduction cholesterol efflux was attenuated by the combination of AICAR and MK treatment,resulting obvious reducing intracellular lipid content.Conclusion: 1.MK decreases the expression of ABCA1,inhibits the efflux of cholesterol and promotes the accumulation of lipid on RAW264.7 macrophages.2.AMPK-m TOR signaling molecule is involved in the regulation of MK on the cholesterol metabolism of RAW264.7 macrophages.