Expression Of Cyclophilin A In Lipid-Loaded Macrophages Induced By Oxidized Low Density Lipoprotein And Probucol And Its Effect On Cellular Cholesterol Accumulation

Part I . Effect of Cyclophilin A on Cellular Cholesterol Accumulation in Lipid-Loaded Macrophages Induced by Oxidized Low Density LipoproteinAIM To observe the protein expression of Cyclophilin A in lipid-loaded macrophages induced by oxidized low density lipoprotein and its effect on cellular cholesterol accumulation.METHODS RAW264.7 mouse macrophages were treated with 75mg/L ox-LDL for different time (control, 24 hours, 48 hours and 72hours) .Western-blot analysis and Immunofluorescence were used to detect protein expression of Cyclophilin A and Caveolin-1.Oil Red O Dye experiment was used to show the lipid droplets in lipid-loaded macrophages. High Performance Liquid Chromatography(HPLC) analysis was peformed to determine the contant of cellular total cholesterol and cholesterol ester. Cholesterol efflux was determined by FJ-2107P type liquid scintillator.RESULTS Western-blot analysis showed that the protein expression of Cyclophilin A was significantly decreased in a time-dependent manner , and so was Caveolin-1.Immunofluorescence showed that Cyclophilin A and Caveolin-1 were obviously decreased from the protein expression in macrophages. Oil Red O Dye showed that there were a lot of lipid droplets accumulated in RAW264.7 mouse macrophages with ox-LDL treatment after 48 hours. High Performance Liquid Chromatography(HPLC) analysis was peformed the content of cellular total cholesterol and cholesterol ester were increased greatly .The rate of intracellular cholesterol efflux was also decreased obviouslyin a time-dependent manner.CONCLUSION The protein expression of Cyclophilin A and Caveolin-1 were down-regulated in lipid-loaded macrophages while the content of cellular total cholesterol and cholesterol ester were increased greatly.Part II . Function of Probucol on Lipid-Loaded Cellular Cholesterol Accumulation Derived from Macrophages Induced by Ox-LDL and on theExpression of Cyclophilin AAIM To study the function of Probucol on lipid-loaded cellular cholesterol accumulation derived from RAW264.7 macrophages induced by ox-LDL and on the protein expression of Cyclophilin A and Caveolin-1.METHODS Oil Red O Dye experiment showed the lipid droplets in lipid-loaded macrophages.High Performance Liquid Chromatography(HPLC) analysis was peformed the content of cellular total cholesterol and cholesterol ester. The rate of cholesterol efflux was determined by FJ-2107P type liquid scintillator.Western-blot analysis and Immuno -fluorescence were used to detect the protein expression of Cyclophilin A and Caveolin-1. Immunoprecipitation was used to show the interaction of Cyclophilin A and Caveolin-1 in RAW264.7 Cells induced by ox-LDL after immunoprecipitated with Cyclophilin A.RESULTS Oil Red O dye showed that there were a lot of lipid droplets accumulated in RAW264.7 mouse macrophages with 75mg/L ox-LDL treatment after 48 hours while with 75mg/L Probucol treatment at the same time the lipid droplets were greatly decreased. High Performance Liquid Chromatography(HPLC) analysis was peformed the content of cellular total cholesterol and cholesterol ester were significantly increased with ox-LDL treatment but decreased after Probucol treatment. The rate of intracellular cholesterol...