Ezetimibe Inhibits OxLDL Uptaken By Reducing SR-BI Expression In RAW264.7

Aim: To investigate the oxLDL uptaken inhibition and the possible mechanism of ezetimibe.Methods: Intracellular lipid droplets and DiI-oxLDL uptake were assayed by oil red O staining, 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate(DiI) lipophilic tracer respectively. The protein expression of Niemann-Pick type C1 Like-1(NPC1L1),Scavenger receptor class B type I(SR-BI) and Sterol regulary element binding protein 1(SREBP-1)was semi-quantified by Western blotting; mRNA level of SREBP-1 and SR-BI was dected through Reverse transcription PCR.Results: Niemann-Pick type C1 Like-1(NPC1L1)existed in RAW264.7. Exposure RAW264.7 to ezetimibe at different concentrations (3, 10, and 30μM) for 24 h or 30μM ezetimibe pretreated for different durations (0, 6, 12 and 24 h) and then be treated with 50 mg/L oxidized low density lipoprotein(ox-LDL) or DiI-oxLDL for further 24 h resulted in diminished number and area of cellular lipid droplets, CE percent of 30μM ezetimibe group lesser than oxLDL group about 47%+0.1%; DiI-oxLDL uptake in 30μM ezetimibe group less 59%+4.5% than DiI-oxLDL group; 30μM ezetimibe pretreated group decrease the protein and mRNA expression of Scavenger receptor class B type I(SR-BI) in dose-dependent and time-dependent, reduced about 12%+1.5% and 80%+6.3% respectively compared to the untreated group but increased about 10%+4.8% and 42%+5.3% compared to the oxLDL group. Pretreated RAW264.7 with ezetimibe decreased SREBP-1 mRNA expression about 18%+0.8% and 22%+2.7% compared to the untreated group and oxLDL group respectively. Treated RAW264.7 with 30μM ezetmibe decreased NPC1L1 and SR-BI about 23%+6.2% and 15%+2.8%. Although the fenofibrate(inhibitor of SR-BI) pretreated RAW264.7 plused ezetimibe also decreased SR-BI expression, there was no additive effects compared with fenofibrate group or ezetimibe group, the DiI-oxLDL uptake in fenofibrate plusing ezetimibe group less than ezetimibe and fenofibrate group(6.6%+0.3% and 8.6%+3.8% respectively). Pretreated RAW264.7 with oleic acid, the inhibitor of SREBP-1, then incubated with ezetimibe for 24h reduced SREBP-1 mRNA expression and there was no significance compared to the oleic acid group.Conclusion:1. NPC1L1 exists in the RAW264.7;2. Ezetimibe decreases cellular cholesterol accumulation in RAW264.7;3. Ezetimibe inhibites oxLDL uptaken by decreasing the SR-BI expression via the inhibition of transcription of SREBP-1.