The Influence Of Overexpression Of SR-BI On The Cholesterol Trafficking And The Expression Of TNF-a And MCP-1 In J774 Cells

BACKGROUND Atherosclerosis (As) and its related complications is the leading cause of morbidity and mortality for people. The initiation and progression of atherosclerotic lesion, such as fatty streaks, fibrous plaque, atheromatous plaque, complicated lesion, are highly correlated with plasma lipid levels and vascular inflammation. The scavenger receptor class B type I (SR-BI) was the first molecularly well-defined cell-surface high density lipoprotein (HDL) receptor which is principal for atherosclerotic role in vivo. It is reported that it can effectively repress the formation of atheromatous plaque and retard the progression of atherosclerosis. SR-BI is the mainly receptor which is involved in reverse cholesterol transport (RCT) for mediating lipid metabolism. It can be amply found in the initial points (such as monocytes/macrophages in atheromatous plaque) and the end points (such as liver and tissues for producing steroid hormone) of RCT. In the initial points of RCT, it can facilitate the efflux of free cholesterol (FC) from cells to HDL by altering cholesterol distribution for its atherosclerotic effect; And in the end point of RCT, it can mediate hepatic selective uptake of HDL-CE for reducing cholesterol in plasma and stimulating RCT for its atherosclerotic effect. However, Expression of macrophage SR-BI protects mice against atherosclerotic lesion development in apoE deficient mice in vivo without influencing plasma lipids and the efflux of cholesterol. So, the antiatherosclerotic role of macrophage SR-BI may have other important mechanisms besides facilitating the efflux of free cholesterol (FC) from cells to HDL.OBJECTIVE: To investigate the effect of overexpression of SR-BI in different cells on the expression of tumor necrosis factorα(TNF-α) and monocyte chemoattractant protein 1 (MCP-1) by constructing human SR-BI (hSR-BI) eukaryotic expression vector and transfecting hSR-BI eukaryotic expression vector into J774 cells to express hSR-BI transiently, for originally exploring the atherosclerotic mechanism of macrophage SR-BI.METHODS: To gain hSR-BI cDNA and clone complete hSR-BI cDNA by PCR (there are restriction enzyme sites, XbaI and KpnI, in sense primer and antisense primer); the hSR-BI cDNA was restricted with XbaI and KpnI and was inserted into plasmid vector, pcDNA3.1(-), which was opened using XbaI and KpnI; cloned hSR-BI was verified by DNA sequencing; J774 cells and 3T3-L1 cells were transiently transfected to express more hSR-BI detected by RT-PCR and estern blot; the influx and efflux of [3H]Cholesterol from cells were determined by liquid scintillation counting for analyzing the effect of overexpression of SR-BI in J774 cells on FC trafficking; total cholesterol (TC) and FC in cells were determined by high performance liquid chromatography (HPLC) for analyzing the effect of overexpression of SR-BI in J774 cells on cholesterol accumulating; TNF-αand MCP-1 mRNA were determined by RT-PCR for analyzing the impact of overexpression of SR-BI on the expression of cytokines TNF-αand MCP-1.RESULTS:①The human scavenger receptor class B type I (hSR-BI) cDNA was successfully cloned into eukaryotic expression vector, plasmid pcDNA3.1(-), for construction of plasmid pcDNA3.1(-)-hSR-BI. hSR-BI cDNA sequence was verified by DNA sequencing.②The expression of SR-BI was elevated (n=3, P<0.05) in J774 cells transfected with pcDNA3.1(-)-hSR-BI more than those of control and vector. The functions of cholesterol trafficking and cholesterol accumulating in J774 cells transfected with pcDNA3.1(-)-hSR-BI were reinforced relative to control and vector.③The mRNA level of TNF-αwas significantly reduced (n=3, P<0.05) in J774 and 3T3-L1 cells transfected with pcDNA3.1(-)-hSR-BI compared with control and vector, and the recduction can be inhibited by block lipid transport 4 (BLT-4). But, the mRNA level of MCP-1 has no difference among cells respectively transfected with vector and pcDNA3.1(-)-hSR-BI, and control.CONCLUSION: The constructed pcDNA3.1(-)-hSR-BI expression vector was successfully transfected into J774 cells to express hSR-BI transiently. Overexpression of SR-BI in J774 cells can function well as in promotion of cholesterol trafficking and downregulation of TNF-α, significantly. Downregulation of TNF-αmay be related to activation of phosphatidylinositol-3-kinase (PI-3-k) and nitric oxide synthase (NOS) probably mediated by binding of SR-BI to HDL.