Effects Of PCSK9 On The Cholesterol Efflux And SREBP-2 Expression In THP-1-derived Lipid-loaded Cells

Background Proprotein convertase subtilisin/kexin 9 (PCSK9) is one kind of serum protein that regulate lipid metabolism discovered in 2003. A fairly large number of studies have confirmed it was closely associated with atherosclerosis (AS) form. Serum PCSK9 levels could up-regulate the level of low-density lipoprotein cholesterol (LDL-C) in plasma on account of the degration of low-density lipoprotein receptors (LDLR) PCSK9 is mainly by regulating the plasma LDL-C levels to participate in the AS. Inhibition of PCSK9 produces an increase in surface LDLR and increases removal of LDL from the circulation. PCSK9 inhibitors have shown significant efficacy at lowering LDL and are well-tolerated, with no apparent muscle-related side effects. PCSK9 inhibitors are expected to become the most development prospects lipid-lowering drugs after statin drugs. In 2015, there have been two PCSK9 inhibitors approved. PCSK9 promote the AS formation, however, the specific mechanism is not clear, and the study of effects of PCSK9 on the cholesterol efflux in THP-1-derived lipid-loaded cells have rare reported at home and abroad.Objective To study the effects of PCSK9 on the cholesterol efflux and sterol regulatory element-binding protein 2 (SREBP-2) expression in THP-1-derived lipid-loaded cells.Methods1. Establishing the THP-1-derived lipid-loaded cell model. THP-1 cells were cultivated in vitro and induced to macrophages by PMA. Macrophages were treated with 50mg/l Ox-LDL for 24 hours. Cells were stained by oil red O to observe intracellular lipid droplets. Setting up blank control group (macrophages), the experimental group (macroph ages were treated with 50mg/l Ox-LDL for 24 hours). Extraction of protein, with BCA kit protein quantitatively. To extract protein sample on the high performance liquid chromatography, then detection cell total cholesterol (TC), free cholesterol (FC), cholesterol ester (CE), the percentage of CE/TC. Establishing THP-1-derived lipid-loaded cell model.2. The effect of 22-NBD-cholesterol on THP-1-derived lipid-loaded cells. Different concentrations of 22-NBD-cholesterol (0.1 mg/1,1 mg/1,5 mg/1) incubation cells. To observe cells on cholesterol absorption by inverted fluorescence microscope, and in 0,2,4,6,8,10,12 hours with multifunctional enzyme mark to detect 22-NBD-cholesterol levels in cells.3. Effects of PCSK9 on the cholesterol efflux in THP-1-derived lipid-loaded cells. Experiment design is divided into blank control group (macrophages), negative control group (THP-1-derived lipid-loaded cell), 5 experimental group (THP-1-derived lipid-loaded cell+0.01 ug/ml,0.05 ug/ml,0.1 ug/ml,0.5 ug/ml,1 ug/ml PCSK9). Groups of cells were treated with 22-NBD-cholesterol, high-density lipoprotein (HDL), with multifunctional enzyme mark to detect 22-NBD-cholesterol levels in culture medium and cells. According to the formula to calculate the rate of cholesterol efflux.4. Effects of PCSK9 with Atorvastatin on the cholesterol efflux in THP-1-derived lipid-loaded cells. Experiment is divided into blank control group (macrophages), negative control group (THP-1-derived lipid-loaded cells), three experimental group (THP-1-derived lipid-loaded cells+0.1 ug/ml PCSK9,0.1 ug/ml PCSK9+0.1 umol/1 Atorvastatin,0.1 ug/ml PCSK9+10 umol/1 Atorvastatin). Cells cultured with 22-NBD-cholesterol (5mg/L) serum free medium. Add preconfigured PCSK9 and Atorvastatin to each group, then treated with 50 mg/L HDL serum free medium. After 6 hours, with multifunctional enzyme mark to detect 22-NBD-cholesterol levels in culture medium and cells. According to the formula to calculate the rate of cholesterol efflux.5. SREBP-2 protein expression:experimental is divided into blank control group (macrophages), negative control group (THP-1-derived lipid-loaded cells), experimental group (THP-1-derived lipid-loaded cells+0.1 ug/ml PCSK9). Using western blot method to detect each group SREBP-2 protein expression levels.Results1. THP-1 cells induced to macrophages, then macrophages were treated with Ox-LDL for 24 hours. Using high performance liquid chromatograp hy to measure the percentage of CE/TC was 36.18 ±5.11%, more than 20% and less than 50%, THP-1-derived lipid-loaded cells model succe ssfully established.2. THP-1-derived lipid-loaded cells were treated with different concentra tions of 22-NBD-cholesterol. Compared with 0.1mg/l 22-NBD-cholesterol group and 1 mg/l 22-NBD-cholesterol group,the cells of 5 mg/l 22-NBD-cholesterol group intake most cholesterol, and at 6 hours into the plateau (P< 0.05).3. After dealing with different concentrations of PCSK9, the cholesterol efflux rate of THP-1-derived lipid-loaded cells decreased with increasing concentration. Compared with negative control group, the cholesterol efflux rate of experimental group was significantly decreased (P< 0.05). The cholesterol efflux rate of 0.lug/ml PCSK9 group was 20.60± 2.07%, compared with other experimental group, the difference was statistically significance (P< 0.05).4. After dealing with PCSK9 and Atorvastatin, the cholesterol efflux rate of experimental group was significantly decreased compared with negative control group (P<0.05). In experimental group, the cholesterol efflux rate of 0. lug/ml PCSK9+10 umol/L Atorvastatin group was 10.24 ±1.44%, and compared with 0.1 ug/ml PCSK9 group, the difference was statistically significance (P< 0.05).5. Compared with the blank control group, SREBP-2 protein expression was up-regulated in THP-1-derived lipid-loaded cells after dealing with PCSK9, the difference was statistically significant (P< 0.05).Conclusion1. PCSK9 can effectively inhibit the cholesterol efflux of THP-1-derived lipid-loaded cells. PCSK9 with Atorvastatin can inhibit the cholesterol efflux of THP-1-derived lipid-loaded cells more efficiently.2. PCSK9 can effectively inhibit the cholesterol efflux of THP-1-derived lipid-loaded cells, which was contributed to the up-regulation of SREBP-2 repression at some degree.