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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    https://doi.org/10.1007/s11684-018-0655-7
RESEARCH ARTICLE |
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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Abstract

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.
 URL:  
http://academic.hep.com.cn/fmd/EN/10.1007/s11684-018-0655-7
http://academic.hep.com.cn/fmd/EN/Y2018/V12/I6/697
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.
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