Abstract
Purpose
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.
Conclusion
Resveratrol protected HUVECs from oxidative damage caused by ox-LDL. This effect was mediated by Sirt1-dependent autophagy via the AMPK/ Sirt1 pathway.
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References
Jiang X, Yang Z, Chandrakala AN, Pressley D, Parthasarathy S. Oxidized low density lipoproteins–do we know enough about them? Cardiovasc Drugs Ther. 2011;25:367–77.
Mitra S, Goyal T, Mehta JL, Oxidized LDL. LOX-1 and atherosclerosis. Cardiovasc Drugs Ther. 2011;25:419–29.
Martinet W, De Meyer GR. Autophagy in atherosclerosis: a cell survival and death phenomenon with therapeutic potential. Circ Res. 2009;104:304–17.
Liao X, Sluimer JC, Wang Y, Subramanian M, Brown K, Pattison JS, et al. Macrophage autophagy plays a protective role in advanced atherosclerosis. Cell Metab. 2012;15:545–53.
Zhang YL, Cao YJ, Zhang X, Liu HH, Tong T, Xiao GD, et al. The autophagy-lysosome pathway: a novel mechanism involved in the processing of oxidized LDL in human vascular endothelial cells. Biochem Biophys Res Commun. 2010;394:377–82.
Schrijvers DM, De Meyer GR, Martinet W. Autophagy in atherosclerosis: a potential drug target for plaque stabilization. Arterioscler Thromb Vasc Biol. 2011;31:2787–91.
Wang H, Yang YJ, Qian HY, Zhang Q, Xu H, Li JJ. Resveratrol in cardiovascular disease: what is known from current research? Heart Fail Rev. 2012;17:437–48.
Li H, Forstermann U. Resveratrol: a multifunctional compound improving endothelial function. Editorial to: “Resveratrol supplementation gender independently improves endothelial reactivity and suppresses superoxide production in healthy rats” by S. Soylemez et al. Cardiovasc Drugs Ther. 2009;23:425–9.
Schmitt CA, Heiss EH, Dirsch VM. Effect of resveratrol on endothelial cell function: Molecular mechanisms. Biofactors. 2010;36:342–9.
Wood JG, Rogina B, Lavu S, Howitz K, Helfand SL, Tatar M, et al. Sirtuin activators mimic caloric restriction and delay ageing in metazoans. Nature. 2004;430:686–9.
Lee IH, Cao L, Mostoslavsky R, Lombard DB, Liu J, Bruns NE, et al. A role for the NAD-dependent deacetylase Sirt1 in the regulation of autophagy. Proc Natl Acad Sci U S A. 2008;105:3374–9.
Morselli E, Maiuri MC, Markaki M, Megalou E, Pasparaki A, Palikaras K, et al. Caloric restriction and resveratrol promote longevity through the Sirtuin-1-dependent induction of autophagy. Cell Death Dis. 2010;1:e10.
Schrijvers DM, De Meyer GR, Herman AG, Martinet W. Phagocytosis in atherosclerosis: Molecular mechanisms and implications for plaque progression and stability. Cardiovasc Res. 2007;73:470–80.
Gurusamy N, Lekli I, Mukherjee S, Ray D, Ahsan MK, Gherghiceanu M, et al. Cardioprotection by resveratrol: a novel mechanism via autophagy involving the mTORC2 pathway. Cardiovasc Res. 2010;86:103–12.
Wang L, Dong Z, Huang B, Zhao B, Wang H, Zhao J, et al. Distinct patterns of autophagy evoked by two benzoxazine derivatives in vascular endothelial cells. Autophagy. 2010;6:1115–24.
Mizushima N, Yoshimori T, Levine B. Methods in mammalian autophagy research. Cell. 2010;140:313–26.
Morselli E, Maiuri MC, Markaki M, Megalou E, Pasparaki A, Palikaras K, et al. The life span-prolonging effect of sirtuin-1 is mediated by autophagy. Autophagy. 2010;6:186–8.
Kao CL, Chen LK, Chang YL, Yung MC, Hsu CC, Chen YC, et al. Resveratrol protects human endothelium from H(2)O(2)-induced oxidative stress and senescence via SirT1 activation. J Atheroscler Thromb. 2010;17:970–9.
Baur JA. Resveratrol, sirtuins, and the promise of a DR mimetic. Mech Ageing Dev. 2010;131:261–9.
Pacholec M, Bleasdale JE, Chrunyk B, Cunningham D, Flynn D, Garofalo RS, et al. SRT1720, SRT2183, SRT1460, and resveratrol are not direct activators of SIRT1. J Biol Chem. 2010;285(11):8340–51.
Puissant A, Auberger P. AMPK- and p62/SQSTM1-dependent autophagy mediate Resveratrol-induced cell death in chronic myelogenous leukemia. Autophagy. 2010;6:655–7.
Park SJ, Ahmad F, Philp A, Baar K, Williams T, Luo H, et al. Resveratrol ameliorates aging-related metabolic phenotypes by inhibiting cAMP phosphodiesterases. Cell. 2012;148(3):421–33.
Canto C, Gerhart-Hines Z, Feige JN, Lagouge M, Noriega L, Milne JC, et al. AMPK regulates energy expenditure by modulating NAD+ metabolism and SIRT1 activity. Nature. 2009;458:1056–60.
Iwabu M, Yamauchi T, Okada-Iwabu M, Sato K, Nakagawa T, Funata M, et al. Adiponectin and AdipoR1 regulate PGC-1alpha and mitochondria by Ca(2+) and AMPK/SIRT1. Nature. 2010;464:1313–9.
Fullerton MD, Steinberg GR. SIRT1 takes a backseat to AMPK in the regulation of insulin sensitivity by resveratrol. Diabetes. 2010;59:551–3.
Acknowledgments
This work was supported by National Natural Science Foundation of China (30973843) and National Natural Science Foundation of China (30870987).
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Guo, H., Chen, Y., Liao, L. et al. Resveratrol Protects HUVECs from Oxidized-LDL Induced Oxidative Damage by Autophagy Upregulation via the AMPK/SIRT1 Pathway. Cardiovasc Drugs Ther 27, 189–198 (2013). https://doi.org/10.1007/s10557-013-6442-4
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DOI: https://doi.org/10.1007/s10557-013-6442-4