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Frontiers of Medicine

ISSN 2095-0217

ISSN 2095-0225(Online)

CN 11-5983/R

Postal Subscription Code 80-967

2018 Impact Factor: 1.847

Front. Med.    2018, Vol. 12 Issue (6) : 697-706
Resveratrol reduces intracellular reactive oxygen species levels by inducing autophagy through the AMPK-mTOR pathway
Jun Song1,2,3, Yeping Huang1, Wenjian Zheng4, Jing Yan1, Min Cheng5, Ruxing Zhao2, Li Chen2, Cheng Hu1(), Weiping Jia1()
1. Shanghai Diabetes Institute, Shanghai Key Laboratory of Diabetes Mellitus, Shanghai Key Clinical Center for Metabolic Diseases, Shanghai Jiao Tong University Affiliated Sixth People’s Hospital, Shanghai 200233, China
2. Department of Endocrinology, Qilu Hospital of Shandong University, Jinan 250012, China
3. Department of Endocrinology, Shanghai East Hospital, Tongji University School of Medicine, Shanghai 200120, China
4. Department of Geriatrics, Qingdao Haici Medical Treatment Group, Qingdao 266000, China
5. Huangdao Disease Prevention and Control Center, Qingdao 266555, China
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Oxidative stress induced by free fatty acid aggravates endothelial injury, which leads to diabetic cardiovascular complications. Reduction of intracellular oxidative stress may attenuate these pathogenic processes. The dietary polyphenol resveratrol reportedly exerts potential protective effects against endothelial injury. This study determined whether resveratrol can reduce the palmitic acid (PA)-induced generation of reactive oxygen species (ROS) and further explored the underlying molecular mechanisms. We found that resveratrol significantly reduced the PA-induced endothelial ROS levels in human aortic endothelial cells. Resveratrol also induced endothelial cell autophagy, which mediated the effect of resveratrol on ROS reduction. Resveratrol stimulated autophagy via the AMP-activated protein kinase (AMPK)-mTOR pathway. Taken together, these data suggest that resveratrol prevents PA-induced intracellular ROS by autophagy regulation via the AMPK-mTOR pathway. Thus, the induction of autophagy by resveratrol may provide a novel therapeutic candidate for cardioprotection in metabolic syndrome.

Keywords resveratrol      reactive oxygen species      AMPK      mTOR      autophagy     
Corresponding Authors: Cheng Hu,Weiping Jia   
Just Accepted Date: 12 September 2018   Online First Date: 13 November 2018    Issue Date: 03 December 2018
 Cite this article:   
Jun Song,Yeping Huang,Wenjian Zheng, et al. Resveratrol reduces intracellular reactive oxygen species levels by inducing autophagy through the AMPK-mTOR pathway[J]. Front. Med., 2018, 12(6): 697-706.
Fig.1  Resveratrol decreased palmitic acid (PA)-induced ROS overproduction and improved endothelial dysfunction in HAECs. (A) PA dose-dependently increased intracellular ROS levels. HAECs were incubated by increasing amount of PA. Intracellular ROS was stained by CM-H2DCFDA. (B) Resveratrol dramatically inhibited PA-induced ROS production. HAECs were incubated with resveratrol and PA and then stained with CM-H2DCFDA. (C) Flow cytometry was conducted in PA- and resveratrol-treated HAECs. (D) Resveratrol reversed PA-induced reduction in superoxide dismutase (SOD) level. HAECs were incubated with different doses of resveratrol and PA. SOD activity in different groups was measured, and data are shown as the percentage of control. (E) Resveratrol significantly reduced PA-induced superoxide formation. Treated HAECs with resveratrol and/or PA were stained with DHE probe. Representative images and quantitative analysis of the staining are shown. (F) Resveratrol increased eNOS phosphorylation and decreased iNOS expression. p-eNOS, t-eNOS, iNOS, and GAPDH levels were examined by Western blot. Significantly different (*P<0.05, **P<0.01, ***P<0.001) from control; significantly different (##P<0.01, ###P<0.001) from PA incubation.
Fig.2  Resveratrol-activated autophagy in HAECs. (A) PA exhibited dual effects on autophagy activation. After incubation with various concentrations of PA, Western blots were obtained to test the levels of LC3, p62, and GAPDH in treated HAECs. Representative blots and quantification data are shown. (B) Resveratrol greatly increased LC3II/LC3I ratio and decreased p62 expression dose-dependently. Western blots were conducted to evaluate the protein levels of LC3, p62, or GAPDH in HAECs with different doses of resveratrol and PA treatments. Representative blots and quantitative data of three independent experiments were exhibited. (C) Immunostainings of LC3 in PA and resveratrol-treated HAECs are shown. (D) Lysosomal inhibitor Baf promoted the expression of p62 and LC3II/LC3I ratio, but enhanced turnover of LC3-II was found under co-treatment with resveratrol and Baf. HAECs were treated with resveratrol and Baf. Western blots were performed to test the LC3, p62, and GAPDH levels. Representative blots and quantitative data of three independent experiments were expressed. Significantly different (*P<0.05, **P<0.01, ***P<0.001) from control; significantly different (#P<0.05, ##P<0.01, ###P<0.001) from PA incubation.
Fig.3  Autophagy was involved in the decrease in ROS induced by resveratrol. (A) Atg5 siRNAs were transfected in HAECs, followed by resveratrol treatment with or without PA. Intracellular ROS accumulation was detected by CM-H2DCFDA staining. Representative images are shown, and quantitative data were calculated from three independent experiments. (B) HAECs were treated with PA, resveratrol, and 3-MA. Intracellular ROS accumulation was stained by CM-H2DCFDA. *P<0.05; ***P<0.001.
Fig.4  AMPK activation induced autophagy and improved endothelial dysfunction. (A) AMPK pathway activation significantly unregulated the LC3II/LC3I expression ratio and inhibited p62 expression. Western blot was used to detect LC3, p62, and GAPDH expression levels in treated HAECs. Representative blots are shown, and quantitative data were calculated from three independent experiments. (B) AMPK siRNA suppressed LC3II/LC3I ratio while increasing p62 expression. The expression of AMPK was silenced by specific siRNA, and the transfected cells were treated by PA. Western blot was used to detect LC3, p62, and GAPDH expression levels. Representative blots are shown, and quantitative data were calculated from three independent experiments. (C) AMPK siRNA significantly decreased eNOS phosphorylation. AMPK protein was silenced by AMPK siRNA, followed by treatment with resveratrol and/or PA. Representative blots of LC3, p62, and GAPDH are shown, and quantitative data were calculated from three independent experiments.
Fig.5  AMPK-mTOR pathway was involved in autophagy regulation. (A) Resveratrol increased AMPK phosphorylation but suppressed the phosphorylation of mTOR in a dose-dependent manner. Western blots were used to measure p-AMPK, AMPK, p-mTOR, and mTOR protein levels in PA- and resveratrol-treated cells. (B) Compound C inhibited the basal and resveratrol-induced autophagy flux with the reduction in LC3-II/LC3-I proportion but an increase in p62 level. Western blot was conducted to detect p-AMPK, AMPK, LC3, p62, and GAPDH expression levels in resveratrol- and compound C-treated HAECs. Representative blots and quantitative results are shown. Significantly different (*P<0.05; **P<0.01) from control; significantly different (#P<0.05;##P<0.01; ###P<0.001) from PA incubation.
Fig.6  Resveratrol activated autophagy and reduced ROS in vivo. (A and B) The C57BL/6 mice fed with high-fat diet received saline, resveratrol, or CQ treatments, and the expression levels of autophagy markers in the aortas were compared. Representative blots and quantitative data are shown. Significantly different (*P<0.05; **P<0.01) from the CD subgroup; significantly different (#P<0.05) from the HFD subgroup. (C) Schematic of the mechanism involved in resveratrol-mediated decrease of intracellular ROS.
1 Incalza MA, D'Oria R, Natalicchio A, Perrini S, Laviola L, Giorgino F. Oxidative stress and reactive oxygen species in endothelial dysfunction associated with cardiovascular and metabolic diseases. Vascul Pharmacol 2018; 100: 1–19 pmid: 28579545
2 Morales CR, Pedrozo Z, Lavandero S, Hill JA. Oxidative stress and autophagy in cardiovascular homeostasis. Antioxid Redox Signal 2014; 20(3): 507–518 pmid: 23641894
3 Saad MI, Abdelkhalek TM, Saleh MM, Kamel MA, Youssef M, Tawfik SH, Dominguez H. Insights into the molecular mechanisms of diabetes-induced endothelial dysfunction: focus on oxidative stress and endothelial progenitor cells. Endocrine 2015; 50(3): 537–567 pmid: 26271514
4 Carrizzo A, Forte M, Damato A, Trimarco V, Salzano F, Bartolo M, Maciag A, Puca AA, Vecchione C. Antioxidant effects of resveratrol in cardiovascular, cerebral and metabolic diseases. Food Chem Toxicol 2013; 61: 215–226 pmid: 23872128
5 Bonnefont-Rousselot D. Resveratrol and cardiovascular diseases. Nutrients 2016; 8(5): E250 pmid: 27144581
6 Baxter RA. Anti-aging properties of resveratrol: review and report of a potent new antioxidant skin care formulation. J Cosmet Dermatol 2008; 7(1): 2–7 pmid: 18254804
7 Xu M, Xue W, Ma Z, Bai J, Wu S. Resveratrol reduces the incidence of portal vein system thrombosis after splenectomy in a rat fibrosis model. Oxid Med Cell Longev 2016; 2016:7453849 pmid: 27433290.
8 Han SY, Choi YJ, Kang MK, Park JH, Kang YH. Resveratrol suppresses cytokine production linked to FcεRI-MAPK activation in IgE-antigen complex-exposed basophilic mast cells and mice. Am J Chin Med 2015; 43(8): 1605–1623 pmid: 26621445
9 Diaz-Gerevini GT, Repossi G, Dain A, Tarres MC, Das UN, Eynard AR. Beneficial action of resveratrol: how and why? Nutrition 2016; 32(2): 174–178 pmid: 26706021
10 Novelle MG, Wahl D, Diéguez C, Bernier M, de Cabo R. Resveratrol supplementation: where are we now and where should we go? Ageing Res Rev 2015; 21: 1–15 pmid: 25625901
11 Antonioli M, Di Rienzo M, Piacentini M, Fimia GM. Emerging mechanisms in initiating and terminating autophagy. Trends Biochem Sci 2017; 42(1): 28–41 pmid: 27765496
12 Kiffin R, Bandyopadhyay U, Cuervo AM. Oxidative stress and autophagy. Antioxid Redox Signal 2006; 8(1-2): 152–162 pmid: 16487049
13 Gu J, Hu W, Song ZP, Chen YG, Zhang DD, Wang CQ. Resveratrol-induced autophagy promotes survival and attenuates doxorubicin-induced cardiotoxicity. Int Immunopharmacol 2016; 32: 1–7 pmid: 26774212
14 Nagata D, Mogi M, Walsh K. AMP-activated protein kinase (AMPK) signaling in endothelial cells is essential for angiogenesis in response to hypoxic stress. J Biol Chem 2003; 278(33): 31000–31006 pmid: 12788940
15 He C, Li H, Viollet B, Zou MH, Xie Z. AMPK suppresses vascular inflammation in vivo by inhibiting signal transducer and activator of transcription-1. Diabetes 2015; 64(12): 4285–4297 pmid: 25858560
16 Youn JY, Wang T, Cai H. An ezrin/calpain/PI3K/AMPK/eNOSs1179 signaling cascade mediating VEGF-dependent endothelial nitric oxide production. Circ Res 2009; 104(1): 50–59 pmid: 19038867
17 Zou MH, Hou XY, Shi CM, Nagata D, Walsh K, Cohen RA. Modulation by peroxynitrite of Akt- and AMP-activated kinase-dependent Ser1179 phosphorylation of endothelial nitric oxide synthase. J Biol Chem 2002; 277(36): 32552–32557 pmid: 12107173
18 Gwinn DM, Shackelford DB, Egan DF, Mihaylova MM, Mery A, Vasquez DS, Turk BE, Shaw RJ. AMPK phosphorylation of raptor mediates a metabolic checkpoint. Mol Cell 2008; 30(2): 214–226 pmid: 18439900
19 Zhang L, Wei J, Ren L, Zhang J, Wang J, Jing L, Yang M, Yu Y, Sun Z, Zhou X. Endosulfan induces autophagy and endothelial dysfunction via the AMPK/mTOR signaling pathway triggered by oxidative stress. Environ Pollut 2017; 220(Pt B): 843–852 pmid: 27814983
20 Jo HK, Kim GW, Jeong KJ, Kim DY, Chung SH. Eugenol ameliorates hepatic steatosis and fibrosis by down-regulating SREBP1 gene expression via AMPK-mTOR-p70S6K signaling pathway. Biol Pharm Bull 2014; 37(8): 1341–1351 pmid: 25087956
21 Li XN, Song J, Zhang L, LeMaire SA, Hou X, Zhang C, Coselli JS, Chen L, Wang XL, Zhang Y, Shen YH. Activation of the AMPK-FOXO3 pathway reduces fatty acid-induced increase in intracellular reactive oxygen species by upregulating thioredoxin. Diabetes 2009; 58(10): 2246–2257 pmid: 19592618
22 Koshkin V, Wang X, Scherer PE, Chan CB, Wheeler MB. Mitochondrial functional state in clonal pancreatic β-cells exposed to free fatty acids. J Biol Chem 2003; 278(22): 19709–19715 pmid: 12642585
23 Lee Y, Lee HY, Gustafsson AB. Regulation of autophagy by metabolic and stress signaling pathways in the heart. J Cardiovasc Pharmacol 2012; 60(2): 118–124 pmid: 22472907
24 Elnakish MT, Hassanain HH, Janssen PM, Angelos MG, Khan M. Emerging role of oxidative stress in metabolic syndrome and cardiovascular diseases: important role of Rac/NADPH oxidase. J Pathol 2013; 231(3): 290–300 pmid: 24037780
25 Hutcheson R, Rocic P. The metabolic syndrome, oxidative stress, environment, and cardiovascular disease: the great exploration. Exp Diabetes Res 2012; 2012: 271028 pmid: 22829804.
26 Bradamante S, Barenghi L, Villa A. Cardiovascular protective effects of resveratrol. Cardiovasc Drug Rev 2004; 22(3): 169–188 pmid: 15492766
27 Hao HD, He LR. Mechanisms of cardiovascular protection by resveratrol. J Med Food 2004; 7(3): 290–298 pmid: 15383221
28 Xia N, Förstermann U, Li H. Resveratrol and endothelial nitric oxide. Molecules 2014; 19(10): 16102–16121 pmid: 25302702
29 Klionsky DJ, Abdelmohsen K, Abe A, Abedin MJ, Abeliovich H, Acevedo Arozena A, et al.. Guidelines for the use and interpretation of assays for monitoring autophagy (3rd edition). Autophagy 2016; 12(1): 1–222 pmid: 26799652
30 Gu J, Hu W, Song ZP, Chen YG, Zhang DD, Wang CQ. Resveratrol-induced autophagy promotes survival and attenuates doxorubicin-induced cardiotoxicity. Int Immunopharmacol 2016; 32: 1–7 pmid: 26774212
31 Wu SB, Wu YT, Wu TP, Wei YH. Role of AMPK-mediated adaptive responses in human cells with mitochondrial dysfunction to oxidative stress. Biochim Biophys Acta 2014; 1840(4): 1331–1344 pmid: 24513455
32 Fan X, Wang J, Hou J, Lin C, Bensoussan A, Chang D, Liu J, Wang B. Berberine alleviates ox-LDL induced inflammatory factors by up-regulation of autophagy via AMPK/mTOR signaling pathway. J Transl Med 2015; 13:92 pmid: 25884210
33 Zheng XT, Wu ZH, Wei Y, Dai JJ, Yu GF, Yuan F, Ye LC. Induction of autophagy by salidroside through the AMPK-mTOR pathway protects vascular endothelial cells from oxidative stress-induced apoptosis. Mol Cell Biochem 2017; 425(1-2): 125–138 pmid: 27848074
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