ABSTRACT
BACKGROUND: Lectin-like oxidized low-density lipoprotein-1 (LOX-1) is the major receptor for oxidized low density lipoprotein (ox-LDL) uptake in human umbilical vein endothelial cells (HUVECs). Previously, we found that rapamycin inhibited ox-LDL accumulation in HUVECs, and this effect was related to its role in increasing the activity of autophagy-lysosome pathway. In this study, we determined whether rapamycin could also reduce ox-LDL uptake in HUVECs and investigated the underlying signaling mechanisms. RESULTS: Flow cytometry and live cell imaging showed that rapamycin reduced Dil-ox-LDL accumulation in HUVECs. Furthermore, rapamycin reduced the ox-LDL-induced increase in LOX-1 mRNA and protein levels. Western blotting showed that rapamycin inhibited mechanistic target of rapamycin (mTOR), p70s6k and IκBα phosphorylation triggered by ox-LDL. Flow cytometry implied that mTOR, NF-κB knockdown and NF-κB inhibitors significantly reduced Dil-ox-LDL uptake. Moreover, immunofluorescent staining showed that rapamycin reduced the accumulation of p65 in the nucleus after ox-LDL treatment for 30 h. mTOR knockdown decreased LOX-1 protein production and IκBα phosphorylation induced by ox-LDL. NF-κB knockdown and NF-κB inhibitors reduced LOX-1 protein production, but did not inhibit mTOR phosphorylation stimulated by ox-LDL. CONCLUSIONS: These findings demonstrate that rapamycin reduce mTOR phosphorylation and subsequently inhibit NF-κB activation and suppresses LOX-1, resulting in a reduction in ox-LDL uptake in HUVECs.
Subject(s)
Immunosuppressive Agents/pharmacology , Lipoproteins, LDL/pharmacokinetics , NF-kappa B p50 Subunit/metabolism , Scavenger Receptors, Class E/metabolism , Sirolimus/pharmacology , TOR Serine-Threonine Kinases/metabolism , Atherosclerosis/metabolism , Autophagy/drug effects , Cell Nucleus/metabolism , Cell Proliferation , Disease Progression , Gene Expression Regulation, Enzymologic , Human Umbilical Vein Endothelial Cells/metabolism , Humans , Lysosomes/metabolism , Phosphorylation , Signal Transduction , TransfectionABSTRACT
AIMS: Humanin (HN) is known for its anti-apoptotic functions in neuronal cells. In this study, we sought to investigate the protective effect of [Gly14]-Humanin (HNG) in high glucose (HG)-induced apoptosis of human umbilical vein endothelial cells (HUVECs). METHODS: 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was used to examine cell viability, DNA chromatin morphology was assessed using Hoechst 33342 staining, and the generation of intracellular reactive oxygen species (ROS) was assessed using the fluorescent probe dichlorofluorescein diacetate (DCFH-DA). The expression of poly ADP-ribose polymerase (PARP), the pro-apoptotic protein bax and the anti-apoptotic protein bcl-2 were examined using western blot analysis. The mRNA level of bax and bcl-2 were detected by quantitative Real-Time PCR. RESULTS: Compared with treatment with HG 72h, pretreatment with HNG for 3h significantly increased cell viability (P<0.001), reduced nuclear fluorescence of HUVECs (P<0.05), the levels of cleaved PARP (P<0.05), ROS formation (P<0.05) and the ratio of bax/bcl-2 (P<0.05) compared with treatment with HG for 72h. Quantitative Real-Time PCR showed that mRNA level of bax reduced (P<0.05) and mRNA level of bcl-2 increased (P<0.05) after pretreatment with HNG. CONCLUSIONS: Our results imply that HNG can protect HUVECs from apoptosis induced by HG through the bax/bcl-2 pathway.
