OxLDL Increase LOX-1, MCP-1and VCAN-1Expression In Huvecs Mediated Through TLR4/NF-κB Signaling Pathway

Background:Leukocyte recruitment to vascular endothelium is a very importantfeature of atherosclerosis,while oxLDL is an important incentive for the process.OxLDL can stimulate endothelial cells to secrete variety inflammatory factors, suchas adhesion molecules, select factors and chemotactic proteins and so on,therebypromoting leukocyte endothelial cell adhesion, migration to the endothelium. As thevery important receptor of oxLDL on endothelial cells，TLR4may be involved in theprocess, but its mechanism is not fully understood, pending further study.PartⅠ Effect of oxLDL on the express of MCP-1、VCAM-1、LOX-1and TLR4in HUVECsObjectives:To indentify the effects of different concentration of oxLDL on theexpression of TLR4,LOX-1,MCP-1and VCAM-1in HUVECs.Methods: After reaching80%confluence, HUVECs were maintained in fresh1640medium without serum to get synchronization growth in G0period,12h later, themedium was replaced with fresh medium without serum containing differentconcentrations of oxLDL(0,20,40,60,80μg/mL) and incubated with cells;24hlater,TLR4, LOX-1, MCP-1and VCAM-1mRNA levels were detected by RT-PCR,and48h later Western blotting were applied to determine the protein levels.Results: OxLDL dose dependently upregulated TLR4, LOX-1, MCP-1and VCAM-1mRNA and protein expression in HUVECs.20μg/mL oxLDL shows obviouslyeffects(P<0.05),and as oxLDL concentration increases,TLR4,LOX-1,MCP-1and VCAM-1mRNA and protein expression level present the concentration dependence uptrend;the80μg/mL group appears the most obviously upregulation(P<0.001) VS0μg/mL group. PartⅡ The effects and mechanisms research of TLR4/NF-κBsignaling pathway in the process of oxLDL upregulated theexpression of LOX-1、MCP-1and VCAM-1in HUVECsObjectives:To indentify the effects and mechanisms of TLR4and NF-κB in theprocess of oxLDL upregulated the expression of LOX-1,MCP-1and VCAM-1inHUVECs.Methods:HUVECs were transfected with siTLR4,then LOX-1,MCP-1andVCAM-1protein levels and NF-κB nuclear translocation were analyzed by Westernblot;HUVECs were treated with PDTC for48h,then TLR4,LOX-1,MCP-1andVCAM-1protein levels were detected by Western blot.Results:Incubation of HUVECs with siRNA obviously reduced LOX-1,MCP-1andVCAM-1protein expression and reduced the nuclear translocation ofNF-κB;incubation of HUVECs with PDTC reduces TLR4,LOX-1,MCP-1andVCAM-1protein expression.Conclusions:OxLDL dose dependently upregulated TLR4,LOX-1,MCP-1andVCAM-1mRNA and protein expression in HUVECs;TLR4/NF-κB signalingpathway plays an important role in the process of oxLDL upregulated the expressionof LOX-1,MCP-1and VCAM-1in HUVECs.