Oxidized low‐density lipoprotein‐ (Ox‐LDL‐) induced autophagy dysfunction in human vascular endothelial cells contributes to the development of atherosclerosis (AS). Resveratrol (RSV) protects against Ox‐LDL‐induced endothelium injury. The objective of this study was to determine the mechanisms underlying Ox‐LDL‐induced autophagy dysfunction and RSV‐mediated protection in human umbilical vein endothelial cells (HUVECs). The results showed that Ox‐LDL suppressed the expression of sirtuin 1 (SIRT1) and increased LC3‐II and sequestosome 1 (p62) protein levels without altering p62 mRNA levels in HUVECs. Pretreatment with bafilomycin A1 (BafA1) to inhibit lysosomal degradation abrogated the Ox‐LDL‐induced increase in LC3‐II protein level. Ox‐LDL increased colocalization of GFP and RFP puncta in mRFP‐GFP‐tandem fluorescent LC3‐ (tf‐LC3‐) transfected cells. Moreover, Ox‐LDL decreased the expression of mature cathepsin D and attenuated cathepsin D activity. Pretreatment with RSV increased the expression of SIRT1 and LC3‐II and increased p62 protein degradation. RSV induced RFP‐LC3 aggregation more than GFP‐LC3 aggregation. RSV restored lysosomal function and promoted Ox‐LDL degradation in HUVECs. All the protective effects of RSV were blocked after SIRT1 was knocked down. These findings demonstrated that RSV upregulated the expression of SIRT1, restored lysosomal function, enhanced Ox‐LDL‐induced impaired autophagic flux, and promoted Ox‐LDL degradation through the autophagy‐lysosome degradation pathway in HUVECs.

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. 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. 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.