Resveratrol protects HUVECs from oxidized-LDL induced oxidative damage by autophagy upregulation via the AMPK/SIRT1 pathway.
OBJECTIVE
Resveratrol could induce basal autophagy through the activation of sirtuin. In this study, we investigated the effect of resveratrol on oxidative injury of human umbilical endothelial vein cells (HUVECs) induced by oxidized low-density lipoprotein (ox-LDL) and the role of autophagy in this effect.


METHODS
HUVECs were exposed to 100 mg/L ox-LDL for 24 h to cause oxidative injury. The effect of different concentrations of resveratrol on oxidative damage in HUVECs treated with ox-LDL was evaluated by MTT assay and superoxide dismutase (SOD) activity test. The autophagic level in different groups was measured by the protein expression of microtubule-associated protein 1 light chain 3 (LC3) and sequestosome 1 (SQSTM1/P62). Autophagosomes were observed under electron microscope and fluorescence microscope (by MDC staining). The expression of silencing information regulator1 (Sirt1) and AMP activated protein kinaseα1 (AMPK) was investigated by Western blot. Autophagy inhibitor 3-methyladenine (3-MA) and Sirt1 inhibitor 6-Chloro-2,3,4,9-tetrahydro-1H-Carbazole-1-carboxamide (EX527) were used to confirm the role of autophagy in this effect of resveratrol and the pathway involved.


RESULTS
Resveratrol reversed the decreases in cell viability (72.9 ± 1.7 % of the control group) and SOD activity (14.37 ± 0.21 U/ml) caused by ox-LDL at 83.4 ± 1.4 % of the control group and 16.41 ± 0.27 U/ml respectively. This effect accompanied by upregulation of autophagy and increased protein expression of Sirt1 and AMPK phosphorylation on threonine 172 (p-AMPK). Both 3-MA and EX527 abolished the protective effect of resveratrol in cell viability, at 80.4 ± 2.7 % and 73.9 ± 1.1 % of the control group respectively. 3-MA inhibited autophagy activation without any change of Sirt1 expression at both the mRNA and protein level. EX527 suppressed the expression of Sirt1 and diminished the upregulation of autophagy. Addition of 3-MA or EX527 could not affect the protein level of p-AMPK.


CONCLUSIONS
Resveratrol protected HUVECs from oxidative damage caused by ox-LDL. This effect was mediated by Sirt1-dependent autophagy via the AMPK/ Sirt1 pathway.
Department of Pathophysiology, Zhongshan Medical School, Sun Yat-sen University, 74 Zhongshan Er Road, Guangzhou, Guangdong 510080, China.
Hualei
H
Guo
Yanling
Y
Chen
Lizhen
L
Liao
Weikang
W
Wu
D055372:AMP-Activated Protein Kinases / Q000096:biosynthesis*; D000975:Antioxidants / Q000494:pharmacology*; D001343:Autophagy / Q000187:drug effects*; D015153:Blotting, Western; D018929:Cell Culture Techniques; D002462:Cell Membrane / Q000187:drug effects / Q000648:ultrastructure; D002470:Cell Survival / Q000187:drug effects; D003593:Cytoplasm / Q000187:drug effects / Q000648:ultrastructure; D061307:Human Umbilical Vein Endothelial Cells; D006801:Humans; D008077:Lipoproteins, LDL / Q000009:adverse effects*; D046529:Microscopy, Electron, Transmission; D018384:Oxidative Stress / Q000187:drug effects*; D060888:Real-Time Polymerase Chain Reaction; D000077185:Resveratrol; D015398:Signal Transduction / Q000187:drug effects; D056564:Sirtuin 1 / Q000096:biosynthesis*; D013267:Stilbenes / Q000494:pharmacology*; D013482:Superoxide Dismutase / Q000378:metabolism; D015854:Up-Regulation
D000975:Antioxidants; D008077:Lipoproteins, LDL; D013267:Stilbenes; C099252:oxidized low density lipoprotein; D013482:Superoxide Dismutase; D055372:AMP-Activated Protein Kinases; C526180:PRKAA1 protein, human; C447939:SIRT1 protein, human; D056564:Sirtuin 1; D000077185:Resveratrol