Effect Of Amlodipine On Macrophages Lipid-loaded By Oxidized Low Density Lipoprotein And The Caveolin-2Expression

Part Ⅰ. Change of the protein expression of Caveolin-2inRAW264.7macrophages lipid-loaded by oxidized Low DensityLipoprotein.AIM To observe the change of the protein expression of Caveolin-2in lipid-loadedmacrophages induced by oxidized Low Density Lipoprotein(ox-LDL).METHODS After RAW264.7macrophages incubated with50mg/L ox-LDL for48h, Western-blot analysis was used to detect the protein expression of Caveolin-1and Caveolin-2. At the same time, Oil Red O Dye experiment and High PerformanceLiquid Chromatography(HPLC) analysis were used to show the macrophageslipid-loaded.RESULTS After treated with ox-LDL for48h, the protein expression ofCaveolin-1and Caveolin-2was decreased in a time-dependent mannergradually(P<0.05). Oil Red O Dye showed that there were a lot of lipid dropletsaccumulated in RAW264.7macrophages, and HPLC analysis showed that the ratio ofcholesterol ester(CE) to total cholesterol(TC) was increased with45.4%±1.9%, so thecells possessed the characteristic of1ipid-loaded cells.CONCLUSION The protein expression of Caveolin-2was down-regulated byox-LDL in RAW264.7macrophages lipid-loaded. Part Ⅱ. Function of Amlodipine on the protein expression ofCaveolin-1and Caveolin-2and the cellular cholesterol efflux inRAW264.7macrophages lipid-loaded by ox-LDL.AIM To study the role of Amlodipine on anti-atherosclerosis further throughobserving the function of Amlodipine on the protein expression of Caveolin-1andCaveolin-2and the cellular cholesterol efflux in RAW264.7macrophages lipid-loadedby ox-LDL.METHODS RAW264.7macrophages were treated with50mg/L ox-LDL for48h and divided into three groups: the control group, ox-LDL treatment group andAmlodipine+ox-LDL treatment group. Oil Red O Dye experiment and HPLCanalysis showed the lipid droplets in lipid-loaded macrophages. Western-blot analysisand Immunofluorescence(IF) were used to detect the protein expression of Caveolin-2.The protein expression of Caveolin-1was also detected by Western-blot. Liquidscintillation counter checked out the rate of cellular cholesterol efflux. And then therole of Amolodipine on the protein expression of Caveolin-1and Cavolin-2and thecellular cholesterol efflux in RAW264.7macrophages lipid-loaded by ox-LDL couldbe analyzed.RESULTS Oil Red O dye showed that there were a lot of lipid dropletsaccumulated in RAW264.7macrophages, and HPLC analysis showed that the ratio ofCE to TC was increased with45.4%±1.9%. The lipid droplets, TC, freecholesterol(FC), CE and the ratio of CE to TC in the Amlodipine+ox-LDL treatmentgroup were all greatly decreased. Western-blot analysis showed the protein expressionof Caveolin-1and Caveolin-2was decreased in RAW264.7macrophages lipid-loadedby ox-LDL; IF also showed he protein expression of Caveolin-2was decreased. butthis function could be suppressed markedly by Amlodipine treatment inadvance(P<0.05). The rate of intracellular cholesterol efflux decreased obviously inox-LDL treatment group but up-swung by Amlodipine treatment in advance. CONCLUSION Amlodipine treatment in advance could up-regulate the proteinexpression of Caveolin-1and Caveolin-2, and increase the rate of cholesterol efflux inRAW264.7macrophages lipid-loaded by ox-LDL.CONCLUSION The protein expression of Caveolin-2was down-regulated byox-LDL in RAW264.7macrophages lipid-loaded. Amlodipine treatment in advancecould up-regulate the protein expression of Caveolin-1and Caveolin-2, and increasethe rate of cholesterol efflux in RAW264.7macrophages lipid-loaded by ox-LDL.