Effect Of Apolipoprotein M On Cholesterol Uptake And Efflux In Macrophage

Objective:To investigate the role of apolipoprotein M in cholesterol uptake and efflux and its possible mechanism.Methods:Mouse macrophage Ana-1 cells were cultured in vitro with different concentrations of NBD-cholesterol?2.5?M,5?M,10?M?.The supernatant and cell lysates were collected after 0 h,0.5 h,1 h,2 h,4 h,6 h,8 h and 12 h,respectively.QuantiFluor counter measured fluorescence intensities of supernatant and cell lysates to calculate the uptake rate of NBD-cholesterol in Ana-1 cells.The peritoneal macrophages were collected from wild-type mice,ApoM^-/-mice,SR-BI^-/-mice and ApoM^-/-SR-BI^-/-mice.Then peritoneal macrophages were loaded with 10?M NBD-cholesterol.The supernatant and cell lysates were collected after 1,2,4,6 and 12h,respectively.And the fluorescence intensities were measured to calculate the cholesterol uptake rate of peritoneal macrophages.Taking Ana-1 cells as the research object,the lipid loaded cells model in vitro were constructed by incubating Ana-1 cells with 10?M NBD-cholesterol for 4 h.The efflux experiments were divided into four parts:?1?Washed cells were incubated for 0,0.5,1,2,4,6 and 8 h with serum-free medium in the present of apoM⁺HDL?20?g/ml?,apoM^-HDL?20?g/ml?and rh-apoM?10?g/ml?,respectively;?2?Washed cells were incubated for 4 h with 10-100?g/ml rh-apoM,respectively;?3?Washed cells were incubated for 4 h with 20?g/ml apoM⁺HDL?or apoM^-HDL?,and the combination of both rh-apoM and apoM⁺HDL?or apoM^-HDL?,respectively.The supernatant and cells were collected at the appropriate time to calculate the cholesterol efflux rate.Result:In vitro,Ana-1 cells acquired NBD-cholesterol in a concentration-dependent manner at a certain range.The cholesterol uptake by macrophages showed a peak at 6 h,and it was independent with the concentration of cholesterol and macrophage from different genotype mice.In platform stage,the rate of cholesterol uptake in ApoM^-/-SR-BI^-/-mice was significantly lower than that of SR-BI^-/-mice and ApoM^-/-mice?t=6.8,13.66;P<0.05?.In vitro,apoM⁺HDL and apoM^-HDL could enhance cholesterol efflux in Ana-1 cells,and apoM⁺HDL seemed has a stronger role in promoting cholesterol efflux than apoM^-HDL?24.65±0.93%vs 10.85±0.64%;P=0.0003?.rh-apoM could not effectively promote cholesterol efflux,but it could inhibit the ability of apoM^-HDL induced cholesterol efflux.Conclusion:apoM may be involved in the uptake and efflux of cholesterol by macrophages in vitro.rh-apoM could not promote the efflux of cholesterol,but it might be involved in regulating HDL-mediated cholesterol efflux.