Resveratrol reduces intracellular reactive oxygen species levels by inducing autophagy through the AMPK-mTOR pathway

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

Introduction

The overproduction of intracellular oxidative stress has been implicated in the pathogenesis of cardiovascular diseases of metabolic syndrome. Long-term excessive reactive oxygen species (ROS) provokes lipid peroxidation, mitochondrial damage, or protein misfolding, leading to inflammation and cell apoptosis, which contribute to the pathophysiology of endothelial injury, an initial event to the development of vascular diseases [¹–³]. Thus, elimination of excessive ROS is critical for preventing and treating diabetic vascular disease.

Resveratrol, a dietary polyphenol, is a promising candidate for managing metabolic syndrome and preventing cardiovascular complications [⁴,⁵]. It has potent anti-inflammation, anti-aging, and anti-thrombotic roles [⁶–⁸]. Resveratrol has direct vascular protective effect and decreases diabetic cardiovascular complications in clinical trials [⁹,¹⁰]. However, the molecular mechanisms underlying the protective effect of resveratrol remain poorly understood.

Autophagy, an adaptive lysosome-dependent process with the removal of impaired cellular organelle, is a key regulator of cellular homeostasis [¹¹]. Accumulating evidence has shown that basal autophagy plays an indispensable role in protecting cells from ROS-induced cytotoxicity [²,¹²]. Thus, the induction of an autophagic response may exert a protective effect against the cytotoxic actions of metabolic stress in endothelial cells.

Resveratrol is reported to activate autophagy [¹³]. The AMP-activated protein kinase (AMPK) pathway, as a major metabolic energy gauge, is responsible for regulating eNOS phosphorylation, nitric oxide production, mitochondrial metabolism, inflammation, and angiogenesis [¹⁴–¹⁷]. Activated AMPK pathway inhibits mTOR, a serine/threonine protein kinase [¹⁸]. Emerging evidence has suggested that the AMPK-mTOR pathway can inhibit fibrosis and inflammation and reverse hyperglycemia-induced endothelial dysfunction [¹⁹,²⁰]. However, the role of the AMPK-mTOR pathway in resveratrol-mediated autophagy under metabolic stress remains unclear.

Therefore, we investigated whether resveratrol can repress intracellular ROS level and further explored the underlying mechanisms involved.

Materials and methods

Cell culture

Human aortic endothelial cells (HAECs) were obtained from ScienCell and were grown with ECM supplemented with 10% FBS and 1% penicillin/streptomycin. HAECs were transfected by specific siRNAs and subjected to resveratrol, palmitic acid (PA), AICAR, bafilomycin A1 (Baf), or Compound C for further study.

Preparation of BSA-free fatty acid conjugates

Saturated PA was conjugated to low-endotoxin, FFA-free bovine serum albumin (BSA) as described in a previous study [²¹]. PA stock solution (200 mmol/L) was prepared with ethanol and then diluted into concentrations of 1–5 mmol/L by 20% BSA. The final concentration of BSA was 1% in all PA media.

Detection of intracellular ROS levels

ROS-sensitive fluorescence detector 5-(and-6)-chloromethyl-2′,7′-dichlorodihydrofluorescein diacetate acetyl ester (CM-H2DCFDA) (Sigma-Aldrich, Saint Louis, MO) and the specific superoxide probe dihydroethidium (DHE; Invitrogen, Carlsbad, CA) were applied to detect intracellular ROS as previously described [²¹]. Confluent HAECs were subjected to non-serum medium with 5 µmol/L CM-HB2BDCFDA or 2 µmol/L DHE for 30 min and then rinsed with PBS thrice. Images were observed using a fluorescence microscope.

Flow cytometry

Cells were digested and resuspended into a single cell suspension after stimulation with the indicated agents described above. The cell suspension was incubated with CM-H2DCFDA in the dark for 30 min at 37 °C. Treated HAECs were then rinsed with PBS and resuspended with FACS buffer. The ROS levels in single cells was then immediately determined via flow cytometry (by 530 nm).

Evaluation of SOD levels

SOD levels were assayed using superoxide dismutase (SOD) kit (Beyotime, Changsha, China) in accordance with the manufacturer’s protocols. All data were calculated from three independently repeated experiments.

Transfection of cells with siRNA

Specific siRNAs against AMPK or Atg5 were obtained from Santa Cruz Biotechnology (CA, USA). siRNA transfections in HAECs were achieved using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) in accordance with the manufacturer’s protocol. Silencer Negative scrambled siRNA (Santa Cruz Biotechnology, Santa Cruz, CA, USA) was applied as a negative control parallelly. After transfection, HAECs were incubated with PA and/or resveratrol for the following experiments.

Western blot

The cell protein was lysed in lysis buffer. After separation by SDS-PAGE, the protein extracts were electrophoresed onto PVDF membranes. The membranes were blocked by 5% skimmed milk for an hour, immunoblotted with primary antibodies overnight, and then washed with washing buffer three times. After the corresponding HRP-conjugated secondary antibody incubation for an hour, the bands were developed with enhanced Western Chemiluminescence HRP Systems (Millipore, Darmstadt, Germany). The gray density of the band was quantified using the Gel-Pro Analyzer software.

Animal study

Fifty male four-week-old C57BL/6 mice received a 16-week standard chow diet (N = 10) and high-fat diet (HFD) feeding (N = 40) (D12492, New Brunswick, NJ, USA). The HFD mice were divided into four groups: resveratrol treatment (20 mg/kg/day), chloroquine treatment (CQ, 10 mg/kg/day), combined resveratrol (20 mg/kg/day) and chloroquine (CQ, 10 mg/kg/day) treatment, or treatment with an equal amount of normal saline. The aortas of the mice were rinsed with PBS, harvested, and then lysed in lysis buffer for protein extraction.

Statistical analysis

The variables are shown as mean±SD. The significance of variation was analyzed using one-way ANOVA by SPSS 13.0 statistical software. The significances were considered as two-tailed P<0.05.

Results

Resveratrol decreased PA-induced ROS in HAECs

To address the role of resveratrol in PA-induced ROS, we first detected ROS levels via CM-H2DCFDA staining. HAECs were incubated with different doses of resveratrol with or without PA. As shown in Fig. 1A, PA dramatically stimulated ROS production in HAECs, which is consistent with a previous study [²²]. CM-H2DCFDA staining and flow cytometry revealed that resveratrol dose-dependently abolished PA-induced ROS overproduction (Fig. 1B and 1C). Resveratrol attenuated the PA-induced decrease in SOD activity and increase in superoxide formation (Fig. 1D and 1E). Resveratrol also counteracted the suppressive role of PA in eNOS phosphorylation and decreased the expression of iNOS (Fig. 1F). Our findings indicate that resveratrol can effectively alleviate excessive ROS production and improve endothelial cell dysfunction induced by PA.

Resveratrol activated autophagy in HAECs

We explored the mechanisms driving the protective effect of resveratrol against ROS. Considering that autophagy is a cellular protective process with degradation of damaged organelles under metabolic stress [²³], we tested whether resveratrol can regulate autophagy in endothelial cells. Although a low concentration of PA increased autophagy flux, a high concentration of PA decreased autophagy significantly (Fig. 2A). Importantly, resveratrol greatly increased the LC3II/LC3I expression ratio and decreased p62 expression in a dose-dependent manner (Fig. 2B). These findings were also confirmed by the immunostaining of LC3 (Fig. 2C). Lysosomal inhibitor Baf significantly promoted the expression of p62 and LC3II/LC3I ratio, but enhanced turnover of LC3-II was found under co-treatment with resveratrol and Baf (Fig. 2D). These data indicate that resveratrol can induce autophagy.

Autophagy-mediated resveratrol-induced reduction in ROS

To further investigate whether the resveratrol-induced ROS reduction is mediated by autophagy, we used Atg5 siRNA transfection to inhibit autophagy. Transfected cells were incubated with resveratrol and/or PA. As shown in Fig. 3A, silencing Atg5 with siRNA reversed the reduction of ROS levels by resveratrol. Autophagy inhibitor 3-MA also abolished the beneficial effect of resveratrol on ROS levels (Fig. 3B). These data indicate that autophagy mediates the resveratrol-induced decrease in ROS.

Activation of the AMPK pathway induced protective autophagy and improved endothelial dysfunction

We investigated the possible pathway responsible for the resveratrol-induced autophagy. As indicated in Fig. 4A and 4B, AICAR, the specific AMPK pathway activator, dramatically increased the ratio of LC3II/LC3I in the presence of PA. AMPK siRNA also suppressed LC3II/LC3I ratio while increasing p62 expression. AMPK siRNA significantly decreased eNOS phosphorylation with or without PA (Fig. 4C), suggesting that activation of the AMPK pathway can induce autophagy and improve endothelial dysfunction.

AMPK-mTOR pathway was involved in resveratrol-induced autophagy

We investigated the pathway responsible for resveratrol-induced autophagy. As shown in Fig. 5A, resveratrol induced AMPK phosphorylation but suppressed the phosphorylation of mTOR in a dose-dependent manner, suggesting that this pathway was activated by resveratrol. Compound C, a specific AMPK inhibitor, reduced the basal and resveratrol-induced autophagy flux, as indicated by a significant reduction in LC3-II formation but an increased level of p62 expression (Fig. 5B), indicating that the AMPK-mTOR pathway contributes to resveratrol-induced autophagy regulation.

Resveratrol activated autophagy and decreased ROS in vivo

Finally, we investigated whether resveratrol could regulate autophagy and decrease ROS in vivo. C57BL/6 mice received chow diet or HFD and then were treated with normal saline, resveratrol, or CQ. The autophagy marker protein levels in the aortas were detected. We found that resveratrol increased the LC3II/LC3I ratio and decreased p62 expression in vivo, supporting the in vitro findings (Fig. 6A). Increased LC3 synthesis or reduced LC3 degradation could result in the elevation of LC3II/LC3I ratio; thus, lysosome inhibitor CQ was used to evaluate autophagic flux. Increased LC3II/LC3I ratio and p62 were found after CQ treatment in HFD mice, and these effects were enlarged with the co-treatment of resveratrol and CQ (Fig. 6B). These results suggest that RSV activates autophagy and subsequently inhibits ROS in the aortic endothelial cells of HFD mice.

Discussion

In the present study, we found that resveratrol inhibits intracellular ROS production by inducing autophagy via the AMPK-mTOR pathway. The proposed mechanisms of the resveratrol-induced reduction in intracellular ROS are shown in Fig. 6C.

Oxidative stress, characterized by an imbalance between oxygen/nitrogen radical production and antioxidant defense, is the key causal factor for diabetic vascular events [²⁴,²⁵]. Elimination of intracellular oxidative damage may provide therapeutic benefit for cardiovascular complications.

Resveratrol, a dietary polyphenolic compound, exerts cardioprotective effects against metabolic stress-induced injury [²⁶]. Numerous mechanisms, including alleviation of oxidative stress, reducing inflammation and amelioration of fibrosis, have been verified to explore the cardiovascular protective role of resveratrol [²⁷]. In the present study, resveratrol dramatically decreased PA-induced ROS levels in HAECs, which demonstrates that resveratrol may be an important therapeutic approach for cardiovascular complication prevention.

We also explored the underlying mechanisms involved in the decrease of ROS levels by resveratrol. Emerging evidence has shown that resveratrol can reduce intracellular ROS by inhibiting protein kinase C or activating NADPH oxidase [²⁸]. Autophagy plays key role in many cellular processes, including aging, survival, starvation, infection, or oxidative stress [²]. The best well-known indicators for autophagy are LC3 and p62. The LC3II/LC3I ratio elevates as autophagosomes are induced, whereas the increased expression of p62 suggests decreased autophagic flux [²⁹]. In the present study, resveratrol promoted autophagic flux, which mediated the antioxidant effect of resveratrol. Therefore, the induction of autophagy, which subsequently inhibits intracellular ROS and attenuates endothelial dysfunction, may represent a novel mechanism for the vascular protection of resveratrol.

The AMPK pathway is reportedly involved in the protective effect of resveratrol [³⁰,³¹]. AMPK, a key cellular energy sensor, is the upstream target for mTOR and negatively regulates mTOR. Many studies revealed that the AMPK-mTOR pathway can normalize ROS production by regulating mitochondrial biogenesis [³¹]. The present study suggests that the AMPK-mTOR pathway reduces intracellular ROS levels to mediate resveratrol-induced autophagy, thereby providing a novel insight into the activity of resveratrol. The AMPK-mTOR pathway also prevents endothelial apoptosis and inhibits vascular inflammation [³²,³³]. Thus, the upregulation of the AMPK-mTOR pathway by resveratrol may provide therapeutic benefits for cardiovascular diseases in metabolic syndrome. Additional detailed studies are needed to explore which mTOR protein complex is involved in autophagy regulation. Detailed studies with ROS inhibitors, such as NAC, are desirable to investigate further the effect of RSV on ROS in vivo.

Conclusions

Taken together, the results of this study suggest that resveratrol dramatically reduces ROS production and ameliorates endothelial dysfunction by inducing autophagic flux through the AMPK-mTOR pathway, which mediates resveratrol’s cardioprotective effects. Thus, resveratrol may hold promising therapeutic potential in preventing cardiovascular events of metabolic syndrome.

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