Rapamycin Inhibits The Uptake Of Oxidized Low Density Lipoprotein In Human Umbilical Vein Endothelial Cells Via The MTOR/NF-κB/LOX-1 Pathway.

Objective:Previously, we found that rapamycin could inhibit oxidized low density lipoprotein(ox-LDL) accumulation in human umbilical vein endothelial cells(HUVECs), and this effect was related to its role in increasing the activity of autophagy-lysosome pathway. Lectin-like oxidized low-density lipoprotein-1(LOX-1) is the major receptor for ox-LDL uptake in HUVECs. In this study, we sought to ascertain if rapamycin could also reduce ox-LDL uptake in HUVECs and investigate the underlying signaling mechanisms.Methods:MTT ASsay was applied to examine cell viability infected by rapamycin concentration course, flow cytometry was used to examine the uptake of Dil-ox-LDL infected by rapamycin concentration course, live cell imaging was applied to observe real-time change of Dil-ox-LDL uptake. Quantitive real-time PCR and western blot were applied to examine the m RNA and protein expression of LOX-1. Transfection was used to knockdown m TOR and NF-κB, NF-κB inhibitors PDTC and Bay were used to inhibit NF-κB activity respectively. The expression of m TOR, IκBα and phosphorylation of m TOR, IκBα were determined by western blot. Flow cytometry was applied to examine the uptake of Dil-ox-LDL infected by m TOR, NF-κB si RNA and inhibitors. The p65 translocation was studied by immunofluorescence. The upstream and downstream relationship was determined by western blot.Results:1. MTT assay showed that 5~80n M rapamycin did no harm to cell viability, flow cytometry and live cell imaging showed that rapamycin reduced Dil-ox-LDL uptake in HUVECs.2. Rapamycin concentration-dependently reduced the ox-LDL-induced increases in the m RNA and protein level of LOX-1, rapamycin could also concentration-dependently reduce the expression of LOX-1 protein.3. Western blotting showed that ox-LDL could induce m TOR phosphorylation(p<0.001), rapamycin inhibited m TOR and p70s6 k phosphorylation triggered by ox-LDL(p<0.01). Flow cytometry implied that m TOR knockdown significantly reduced Dil-ox-LDL uptake in HUVECs(p<0.05).4. Western blot and immunofluorescent staining showed that ox-LDL could induce IκBα phosphorylation and p65 nuclear translocation, rapamycin inhibited IκBα phosphorylation triggered by ox-LDL(p<0.01) and reduced p65 nuclear translocation, rapamycin could also concentration-dependently inhibit IκBα phosphorylation. Flow cytometry showed that NF- κ B knockdown and inhibitors significantly reduced Dil-ox-LDL uptake in HUVECs(p<0.01).5. Western blot showed that m TOR, NF-κB knockdown and NF-κB inhibitors decreased LOX-1 protein production(p<0.001) and IκBα phosphorylation(p<0.01) induced by ox-LDL, NF-κB knockdown and NF-κB inhibitors reduced LOX-1 protein production(p<0.001), but couldn’t reduce m TOR phosphorylation stimulated by ox-LDL.Conclusion:Rapamycin inhibits the uptake of oxidized low density lipoprotein in human umbilical vein endothelial cells via the m TOR/NF-κB/LOX-1 pathway. This study may reveal new therapeutic strategies for atherosclerosis, based on modulating m TOR/NF-κB/LOX-1 pathway.