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DOI 10.1002/mnfr.201400161

RESEARCH ARTICLE

Delphinidin-3-glucoside protects human umbilical vein endothelial cells against oxidized low-density lipoprotein-induced injury by autophagy upregulation via the AMPK/SIRT1 signaling pathway Xin Jin1∗ , Mingliang Chen1∗ , Long Yi1 , Hui Chang1 , Ting Zhang1 , Li Wang1 , Wanqiang Ma2 , Xiaoli Peng1,3 , Yong Zhou1∗∗ and Mantian Mi1 1

Chongqing Key Laboratory of Nutrition and Food Safety, Research Center for Nutrition and Food Safety, Institute of Military Preventive Medicine, Third Military Medical University, Chongqing, P. R. China 2 PLA 210th Hospital, Dalian, P. R. China 3 School of Public Health, Chengdu Medical College, Chengdu, Sichuan, P. R. China Scope: Oxidized LDL (oxLDL) induced vascular endothelial cell injury is a key event in the pathogenesis of atherosclerosis (AS). In our previous studies, we showed that delphinidin-3glucoside (Dp), a natural anthocyanin, attenuated oxLDL-induced injury in human umbilical vein endothelial cells (HUVECs), indicating its potential role in preventing AS. However, the involved mechanism is not fully understood. Methods and results: Via methyl thiazolyl tetrazolium and flow cytometry assay, we found that Dp-attenuated oxLDL-induced cell viability decrease and apoptosis in HUVECs. Depending on confocal microscopy, transmission electron microscopy, and Western blot assay, we found that Dp-induced autophagy in HUVECs, whereas suppression of autophagy significantly abolished the protective role of Dp against oxLDL-induced endothelial cell injury. Furthermore, Dp upregulated sirtuin 1 (SIRT1) expression and SIRT1 knockdown notably suppressed Dpinduced autophagy in HUVECs. Dp also increased the expression of phosphorylated adenosine monophosphate-activated protein kinase, while adenosine monophosphate-activated protein kinase (AMPK) knockdown remarkably abolished Dp-induced SIRT1 expression and subsequent autophagy. Conclusion: Our data suggested that Dp protected HUVECs against oxLDL-induced injury by inducing autophagy via the adenosine monophosphate-activated protein kinase/SIRT1 signaling pathway. This new finding might shed light to the prevention and therapy of AS.

Received: March 6, 2014 Revised: April 28, 2014 Accepted: June 1, 2014

Keywords: Atherosclerosis / Autophagy / Delphinidin-3-glucoside / Endothelial cells / Injury

1 Correspondence: Dr. Mantian Mi, Chongqing Key Laboratory of Nutrition and Food Safety, Research Center for Nutrition and Food Safety, Institute of Military Preventive Medicine, Third Military Medical University, 30th Gaotanyan Main Street, Shapingba District, Chongqing 400038, P. R. China E-mail: [email protected] Fax: +86-2368752643 Abbreviations: 3-MA, 3-methyladenine; AMPK, adenosine monophosphate-activated protein kinase; AS, atherosclerosis; Atg, autophagy-related protein; Dp, delphinidin-3-glucoside; ERK1/2, extracellular signal-regulated kinase 1/2; FBS, fetal bovine serum; GFP-MAP1LC3B, green fluorescent proteinmicrotubule-associated protein 1 light chain 3 beta; HUVECs,  C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

Introduction

Atherosclerosis (AS) remains the most frequent cause of death worldwide with consequences of coronary artery and cerebrovascular diseases such as myocardial infarction or stroke [1]. Although it is a multifactorial disease, vascular human umbilical vein endothelial cells; MAP1LC3B2, microtubule-associated protein 1 light chain 3 beta-2; oxLDL, oxidized LDL; siRNA, small interfering RNA; SIRT1, sirtuin 1; SQSTM1, sequestosome 1; TEM, transmission electron microscopy ∗ These authors contributed ∗∗ Additional corresponding

equally to this work. author: Dr. Yong Zhou, E-mail: [email protected] www.mnf-journal.com

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endothelial dysfunction is a driving force in its initiation and development, and oxidized LDL (oxLDL) is a particularly important risk factor in this process [2, 3]. OxLDL induces vascular endothelial cell injury, and results in elimination of cells, enhanced vessel wall permeability, and increased coagulation activity, which induces atherosclerotic lesion rupture and subsequent clinical complications [4–6]. Therefore, inhibiting vascular endothelial cell injury caused by oxLDL might be a potential therapeutic strategy for AS prevention and therapy. Delphinidin-3-glucoside (Dp) as a member of natural anthocyanins is abundant in fruits, vegetables, and cereals [7]. A series of recent studies have shown the notable role of anthocyanins in attenuating endothelial dysfunction, implying the potential benefits of anthocyanins on AS [8–11]. Our previous studies showed that Dp protects against oxLDL-induced injury in human umbilical vein endothelial cells (HUVECs) [12, 13]; however, the precise mechanisms responsible for such effects are still not clear. Autophagy is a regulated cellular pathway involved in the turnover of cytoplasmic organelles and proteins through a lysosome-dependent degradation process [14]. A recent study showed that autophagy deficiency promoted AS [15], indicating its protective function. And autophagy can be regulated by several pathways, the most important one of which is the adenosine monophosphate-activated protein kinase/silent mating type information regulation 2 homolog 1 (AMPK/sirtuin 1 (SIRT1)) signaling pathway [16–18]. It is reported that resveratrol, another kind of natural polyphenol, regulates autophagy by activating the AMPK/SIRT1 signaling pathway in neuroblastoma and endothelial cells [19, 20]. Furthermore, Dp is reported to induce autophagy in breast cancer cells [21]. However, the effect of Dp on autophagic level in oxLDL-treated HUVECs is still unknown. In the current study, we attempted to explore the role of autophagy in it as well as the underlying mechanisms with an emphasis on the AMPK/SIRT1 signaling pathway. As previous studies have revealed that the morphological changes of cultured vascular endothelial cells associated with oxLDL toxicity are similar to those observed in the endothelium covering atherosclerotic lesions [22], in the present study, we first validated the protective function of Dp against oxLDL-induced injury in HUVECs. Then we focused on the effects of Dp on autophagy and the relative function in oxLDL-treated HUVECs. At last we investigated the mechanism by which Dp stimulated autophagy in oxLDL-treated HUVECs.

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of Cell Biology, National Institute for Basic Biology, Precursory Research for Embryonic Science and Technology, Okazaki, Japan). Cell culture media HyQ M199/EBSS (M199; SH30351.01) and fetal bovine serum (FBS, SH30370.03) was purchased from Hyclone Laboratories. The MitoTracker Red (MTR, M22425), LysoTracker Green (LTG, L7526) molecular probes, and LipofectamineTM 2000 transfection reagent (11668-019) were purchased from Invitrogen. DMSO (D2650) and 3-methyladenine (3-MA, M9281) were purchased from Sigma-Aldrich; Dp was purchased from Polyphenols Laboratories AS, Sandnes, Norway. Antibodies against MAP1LC3B (L7543) were obtained from Sigma-Aldrich. Phosphorylated AMPK (sc-33524) and AMPK (sc-74461) were obtained from Santa Cruz Biotechnology, whereas antibodies against SIRT1 (2028) and sequestosome 1 (SQSTM1, 5114) were obtained from Cell Signaling Technology, Inc. And antibody against Actin (TA-09) was obtained from Zhongshan Jinqiao Biotechnology Co.

2.2 Cell culture and treatment HUVECs were isolated by collagenase digestion from fresh umbilical cord veins obtained at normal deliveries in accordance with the ethical standards formulated in the Helsinki Declaration. HUVECs were cultured in M199 containing 10% FBS and 3.2 mM glutamine as previously described [13]. Cells were incubated with Dp for 2 h before exposure to oxLDL according to our previous study [13]. Cells were incubated with 5 mM of 3-MA for 1 h before the addition of Dp to block autophagy. The ethics review board at Third Military Medical University approved this study (approval SYD-2011-0001), and informed consent was obtained from all patients before participation.

2.3 OxLDL-treated HUVECs Native LDL and oxLDL were prepared as previously described [23]. Briefly, native human LDL was isolated from human plasma by sequential ultracentrifugation. LDL (2 mg/mL) was oxidized by exposure to 10 ␮M CuSO4 for 24 h at 37⬚C. The extent of oxidation was determined by measuring the amount of thiobarbituric acid-reactive substances. HUVECs were incubated with 100 ␮g/mL oxLDL for additional 24 h.

2.4 Flow cytometry assay for cell apoptosis

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Materials and methods

2.1 Antibodies and reagents The green fluorescent protein-microtubule-associated protein 1 light chain 3 beta (GFP-MAP1LC3B) plasmid was kindly provided by Dr. Tamotsu Yoshimori (Department  C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

Cells were harvested, washed three times with ice-cold PBS, and assessed for apoptosis using an annexin-V-FITC and propidium iodide double-staining kit (Clontech Laboratories, Inc., Mountain View, CA, USA) according to the manufacturer’s instructions. Cell apoptosis was analyzed on a FACScan flow cytometer (Becton Dickinson & Co., Franklin Lakes, NJ, USA). www.mnf-journal.com

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2.5 Methyl thiazolyl tetrazolium assay for cell viability HUVECs were seeded in a 96-well microplate at a density of 8000 cells/well and then pretreated with Dp for 2 h at a series of concentrations (50, 100, and 200 ␮M) followed by treatment with or without oxLDL (100 ␮g/mL) for another 24 h. The control group was treated with 0.2% DMSO. Subsequently, cells were incubated with 5 mg/mL of methyl thiazolyl tetrazolium (Sigma) for 4 h, and the blue formazan crystals of viable cells were dissolved in DMSO and then measured spectrophotometrically at 490 nm. The optical density value at 490 nm was reported as the percentage of cell viability in relation to the control group (set as 100%). 2.6 GFP-MAP1LC3B puncta formation assay Endothelial cells were transfected with plasmids expressing GFP-MAP1LC3B. After 24 h, cells were exposed to various indicated treatments. Then cells were washed with PBS, fixed by incubation for 20 min at 37⬚C in 4% paraformaldehyde, permeabilized with 0.1% v/v Triton X-100, and washed with PBS containing 2% FBS albumin. All steps were performed at room temperature. Thereafter, cells were immediately visualized by confocal microscopy. The number of GFP-MAP1LC3B-positive puncta in each cell was counted. 2.7 Transmission electron microscopy (TEM) TEM was used to detection of autophagosomes as described before [24]. Briefly, endothelial cells were collected and fixed in 2% paraformaldehyde and 0.1% glutaraldehyde in 0.1 M sodium cacodylate for 2 h, postfixed with 1% OsO4 for 1.5 h, washed, and stained for 1 h in 3% aqueous uranyl acetate. The samples were then washed again, dehydrated with graded alcohol, and embedded in Epon-Araldite resin (Canemco & Marivac, 034). Ultrathin sections were cut on a ultramicrotome (Reichert-Jung, Inc., Cambridge, UK), counterstained with 0.3% lead citrate, and examined on a transmission electron microscope (model no.: EM420; Koninklijke Philips Electronics N.V., Amsterdam, The Netherlands). 2.8 Confocal microscopy of lysosomes and mitochondria Endothelial cells were cultured overnight at a density of 16 000 cells per dish. Then cells were exposed to various treatments as specifically indicated. Thereafter, cells were washed with fresh medium for two times and loaded with MTR (50 nM) for 15 min in humidified air at 37˚C in M199 complete culture medium. Afterwards, the MTR-loaded endothelial cells were washed two times with fresh complete growth medium and incubated with LTG (50 nM) for 15min under identical conditions. After MTR and LTG had been loaded, cells were washed with PBS for three times and were measured by a  C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

Radiance 2000 laser scanning confocal microscope. Excitation of LTG at 504 nm was provided by a helium/neon laser, and fluorescence emission was measured through a 511 nm long pass barrier filter. Excitation of MTR at 581 nm was provided by an argon laser, and fluorescence emission was measured at through a 644 nm long pass barrier filter. Lysosomes (green) and mitochondria (red) were observed. And mitophagy (yellow) was evaluated by merging the lysosomal and mitochondrial images. 2.9 RNA interference Small interfering RNA (siRNA) targeting SIRT1 (human, sc-40986) and AMPK (human, sc-45312) were purchased from Santa Cruz Biotechnology along with control siRNA (sc-44230) and siRNA Transfection Reagent (sc-29528). Endothelial cells were transfected with 100 nM siRNA for 5– 7 h according to the manufacturer’s protocol. Then, the cells were switched into M199 medium and incubated for an additional 24 h. Where indicated, cells were treated with Dp (200 ␮M) for 2 h and then exposed to 100 ␮g/mL of oxLDL for another 4 h. Thereafter, cells were harvested and Western blot analysis was performed. 2.10 Western blot analysis Total cell lysate was analyzed by Western blot analysis as previously described [25]. Briefly, 30–50 ␮g of proteins were resolved by SDS-PAGE and then electroblotted onto polyvinylidene difluoride membranes for Western blot analysis. Blots were probed with 1:1000 diluted primary antibodies overnight at 4⬚C, followed by horseradish peroxidaseconjugated secondary antibodies (Thermo Scientific Lab Vision; 31340 and 31455). Then, the proteins were visualized by enhanced chemiluminescence exposure to X-ray film. Finally, the blots were scanned, and densitometric analysis was performed on the scanned images using Scion Image-Release Beta 4.02 software. 2.11 Statistical analysis Results are expressed as the mean ± standard error of the mean (SEM). Raw data were analyzed with SPSS 13.0 software (SPSS, Inc., Chicago, IL, USA) and each experiment was performed three times. Statistical analysis of between group comparisons was performed using t-test analysis. p-Values < 0.05 was considered statistically significant.

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Results

3.1 Dp attenuated oxLDL-induced injury in HUVECs First, we validated the protective function of Dp in oxLDLtreated HUVECs. By flow cytometry analysis, HUVECs apoptosis was significantly increased by oxLDL treatment, which www.mnf-journal.com

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ysis showed lysosomes were increased by Dp single treatment in HUVECs (Fig. 2G). Therefore, our data indicated that an autophagic response was stimulated by Dp single treatment in HUVECs. Moreover, similar results were found in Dp- and oxLDL-cotreated cells (Fig. 2C–G). Furthermore, we used 3-MA to inhibit Dp-induced autophagy. We found that Dp-induced MAP1LC3B2 expression was significantly decreased by 3-MA in oxLDL-treated HUVECs (Fig. 3A and B). And the GFP-MAP1LC3B puncta formation assay and confocal microscopy showed that Dpinduced increase of autophagosomes and lysosomes were also inhibited (Fig. 3C and D). Subsequently, Dp-induced decrease of apoptosis and increase of cell viability were remarkably blocked (Fig. 3E and F) in HUVECs. Therefore, our data showed that inhibiting autophagy markedly abolished the protective role of Dp in oxLDL-treated HUVECs. Figure 1. Dp-inhibited oxLDL-induced injury in HUVECs. HUVECs were preincubated with different concentrations (50, 100, and 200 ␮M) of Dp for 2 h followed by treatment with or without oxLDL (100 ␮g/mL) for another 24 h. The control group was treated with 0.2% DMSO. Cell apoptosis was measured by flow cytometry (A) and cell viability was detected by methyl thiazolyl tetrazolium assay (B). Values are presented as means ± SD (n = 3); b p < 0.01 versus the vehicle-treated control group; c p < 0.05, d p < 0.01 versus oxLDL-treated group.

was notably inhibited by Dp in a dose-dependent manner (Fig. 1A). Consistently, oxLDL-induced decreased cell viability was markedly suppressed by Dp (Fig. 1B). Together, these data suggested that Dp attenuated oxLDL-induced injury in HUVECs, which was consistent with our previous study [26]. In addition, Dp (50, 100, and 200 ␮M) itself had no effect on the viability and apoptosis of endothelial cells (Fig. 1A and B). 3.2 Dp attenuated oxLDL-induced injury through stimulating autophagy in HUVECs Previous studies showed that autophagy deficiency promoted AS [15], and Dp-induced autophagy in breast cancer cells [21], thus we speculated that Dp exerted its protective effect by stimulating autophagy in oxLDL-treated HUEVCs. We firstly examined the impact of Dp single treatment on autophagy in HUEVCs. Via Western blot analysis, microtubule-associated protein 1 light chain 3 beta-2 (MAP1LC3B2) protein, an accurate indicator of autophagy, was significantly upregulated and the measurement of SQSTM1 degradation revealed a gradual decrease in SQSTM1 protein levels by Dp single treatment (Fig. 2A and B). Then, the effects of Dp single treatment on autophagosome and lysosome formation were also tested. By TEM analysis, Dp single treatment increased autophagosomes in HUVECs (Fig. 2E). Meanwhile, GFP-MAP1LC3B puncta formation assay showed that Dp single-treated cells displayed a significant increase in the number of autophagic structures (GFP-MAP1LC3B dots) compared with the control cells (p < 0.01; Fig. 2F). In addition, confocal microscopy anal C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

3.3 Dp stimulated mitophagy in oxLDL-treated HUVECs As mitochondrial dysfunction plays important roles in oxLDL-induced apoptosis [27], we investigated whether Dp affected mitophagy in oxLDL-treated HUVECs. By merging lysosome and mitochondria staining of confocal microscopy, we found that Dp single treatment increased mitophagy in HUVECs (yellow dots; Fig. 4 A3 versus B3 ). And the same results were found in Dp- and oxLDL-cotreated cells (Fig. 4 C3 versus D3 ). Moreover, Dp-induced mitophagy was inhibited by 3-MA (Fig. 4 D3 versus E3 ). Therefore, the data indicated that Dp increased mitophagy in HUVECs, which might facilitate HUVECs to eliminate dysfunctional mitochondria.

3.4 Dp stimulated autophagy through activating SIRT1 expression in oxLDL-treated HUVECs Then we investigated the mechanism by which Dp stimulated autophagy in oxLDL-treated HUVECs. SIRT1 is an NAD+ dependent deacetylase and is an important regulator of autophagy [16]. By Western blot analysis, SIRT1 protein was significantly upregulated by Dp single treatment in a dosedependent manner in HUVECs (Fig. 5A and B). And the same results were found in Dp- and oxLDL-cotreated cells (Fig. 5C and D). Furthermore, SIRT1 knockdown was achieved by SIRT1 siRNA (siSIRT1) transfection. Western blot validation showed that SIRT1 protein in siSIRT1 cells was notably lower than that in the control cells. And Dp-induced overexpression of MAP1LC3B2 was significantly inhibited by SIRT1 knockdown in oxLDL-treated HUEVCs (Fig. 5E and F). Meanwhile, Dp-induced high levels of autophagosomes and lysosomes were also inhibited by SIRT1 knockdown in oxLDL-treated HUVECs (Fig. 5G). Moreover, Dp-induced mitophagy was also inhibited (Fig. 5H, c3 versus d3 ). Together, the results suggested that Dp stimulated autophagy through regulating SIRT1 in oxLDL-treated HUVECs. www.mnf-journal.com

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Figure 2. Dp-induced autophagy in oxLDL-treated HUVECs. (A) Dp increased MAP1LC3B2 formation and SQSTM1 degradation in HUVECs. Cells were treated with Dp at a series of concentrations (50, 100, and 200 ␮M) for 2 h and the expression of MAP1LC3B2 and SQSTM1 was detected by Western blot. (B) Bar charts show the quantification of endogenous MAP1LC3B2 and SQSTM1. (C) Cells were pretreated with Dp as described in (A) and then exposed to oxLDL (100 ␮g/mL) for another 24 h. The expression of MAP1LC3B2 and SQSTM1 was detected by Western blot. (D) Bar charts show the quantification of endogenous MAP1LC3B2 and SQSTM1. (E) HUVECs were pretreated with Dp (200 ␮M) for 2 h, followed by treatment with or without oxLDL (100 ␮g/mL) for an additional 24 h. Following fixation, cells were visualized by TEM. Arrows indicate the autophagosomes. (F) HUVECs were transfected with a plasmid expressing GFP-MAP1LC3B. After 24 h, the cells were pretreated with Dp (200 ␮M) for 2 h, followed by treatment with or without oxLDL (100 ␮g/mL) for an additional 24 h. Following fixation, cells were immediately visualized by confocal microscopy and the number of GFP-MAP1LC3B dots in each cell was counted. (G) HUVECs were treated as described in (E). Then, the cells were incubated with LTG (50 nM, 15 min, and 37⬚C) and visualized by confocal microscopy. The average LTG fluorescence was expressed as the mean fluorescence intensity using IPP 6.0 software. Values are expressed as the mean ± SD (n = 3). a p < 0.05, b p < 0.01 versus the vehicle-treated control group; c p < 0.05, d p < 0.01 versus oxLDL-treated group.

3.5 Dp upregulated SIRT1 expression by activating AMPK in oxLDL-treated HUVECs We then investigated how Dp regulated SIRT1 expression in oxLDL-treated HUVECs. AMPK is reported to enhance SIRT1 by increasing cellular NAD+ levels [17]. By Western  C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

blot analysis, phosphorylated AMPK proteins were significantly upregulated by Dp single treatment in HUVECs, however AMPK protein levels were not affected by Dp (Fig. 6A and B), indicating that AMPK was activated by Dp. And the same results were found in Dp- and oxLDL-cotreated cells (Fig. 6C and D). Then, we used AMPK siRNA (siAMPK) to www.mnf-journal.com

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Figure 3. Dp attenuated oxLDL-induced injury through stimulating autophagy in HUVECs. (A) Cells were treated with Dp (200 ␮M) in the presence or absence of 3-MA (5 mM) for 2 h, followed by treatment with oxLDL (100 ␮g/mL) for another 24 h. MAP1LC3B2 contents were detected by Western blot analysis. (B) Bar charts show the quantification of MAP1LC3B2. (C) HUVECs were transfected with a plasmid expressing GFP-MAP1LC3B. After 24 h, the cells were preincubated with Dp (200 ␮M) with or without 3-MA for 2 h. Then, cells were incubated with oxLDL (100 ␮g/mL) for an additional 24 h. Following fixation, cells were immediately visualized by confocal microscopy and the number of GFPMAP1LC3B dots in each cell was counted. (D) Cells were treated as described in (A). Then, the cells were incubated with LTG (50 nM, 15 min, and 37⬚C) and visualized by confocal microscopy. The average LTG fluorescence was expressed as the mean fluorescence intensity using IPP 6.0 software. Cells were treated as described in (A). Then, cell apoptosis was measured by flow cytometry (E) and cell viability was detected by methyl thiazolyl tetrazolium assay (F). Values are expressed as the mean ± SD (n = 3). b p < 0.01 versus the vehicle-treated control group; d p < 0.01 versus oxLDL-treated group; # p < 0.01 versus Dp- and oxLDLcotreated groups.

knockdown AMPK. Western blot validation showed AMPK protein in siAMPK cells was remarkably lower than that in the control cells. And Dp-induced upregulation of SIRT1 was notably inhibited by siAMPK in oxLDL-treated HUVECs (Fig. 6E and F). Meanwhile, Dp-induced upregulation of MAP1LC3B2

was also inhibited by siAMPK (Fig. 6E and F), indicating that Dp-induced autophagy was blocked by siAMPK in oxLDLtreated HUVECs. All together, our data suggested that Dp stimulated autophagy through the AMPK/SIRT1 signaling pathway in oxLDL-treated HUVECs.

Figure 4. Dp increased mitophagy in oxLDL-treated HUVECs. HUVECs were preincubated with Dp (200 ␮M) in the presence or absence for 2 h, followed by treatment with or without oxLDL (100 ␮g/mL) for another 24 h. Thereafter, cells were loaded with redfluorescing MTR (50 nM, 15 min, and 37˚C). Afterwards, the MTRloaded endothelial cells were incubated with green-fluorescing LTG (0.50 nM, 15 min, and 37˚C). Cells were visualized by confocal microscopy. As shown, lysosome was green and mitochondria were red. And mitophagy (yellow) was evaluated by merging them together.  C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

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Figure 5. Dp stimulated autophagy through activating SIRT1 expression in oxLDLtreated HUVECs. (A) Cells were pretreated with Dp at a series of concentrations (50, 100, and 200 ␮M) for 2 h. SIRT1 expression was detected by Western blot. (B) The bar graph shows the quantification of endogenous SIRT1. (C) Cells were pretreated with Dp with different concentrations followed by exposure to oxLDL (100 ␮g/mL) for an additional 24 h. SIRT1 expression was detected by Western blot. (D) The bar graph shows the quantification of endogenous SIRT1. (E) SIRT1 was knocked down by siSIRT1 as described in Section 2. At 24-h posttransfection, the cells were treated with Dp (200 ␮M) for 2 h and then incubated with oxLDL (100 ␮g/mL) for an additional 24 h. Cells were collected and lysed, then Western blot analysis was performed. (F) The bar graphs show the quantification of the indicated proteins. (G) Cells were transfected with a plasmid expressing GFP-MAP1LC3B and siSIRT1. After 24 h, the cells were treated with Dp (200 ␮M) for 2 h and then incubated with oxLDL (100 ␮g/mL) for an additional 24 h. Following fixation, the cells were immediately visualized by confocal microscopy. The number of GFP-MAP1LC3B dots in each cell was counted. (H) Cells were treated as described in (E). Thereafter, cells were loaded with red-fluorescing MTR (50 nM, 15 min, and 37˚C). Afterwards, the MTR-loaded endothelial cells were incubated with greenfluorescing LTG (0.50 nM, 15 min, and 37˚C). As shown, lysosome was green and mitochondria were red. And mitophagy (yellow) was investigated by merging them together. Cells were visualized by confocal microscopy. Values are expressed as the mean ± SD (n = 3). a p < 0.05, b p < 0.01 versus the vehicle-treated control group; c p < 0.05, d p < 0.01 versus oxLDL-treated group; # p < 0.01 versus Dp- and oxLDL-cotreated group.

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Discussion

AS is a serious health concern worldwide and oxLDL-induced vascular endothelial injury is a driving force in its initiation and development [4–6]. Recent studies have shown a protective role of Dp against oxLDL-induced injury [28], however, the relative mechanisms remain unclear. In the present  C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

study, we demonstrated that Dp attenuated oxLDL-induced injury by stimulating autophagy in HUVECs through the AMPK/SIRT1 signaling pathway (Fig. 7). To our knowledge, this is the first time to report Dp protected against oxLDLinduced injury through stimulating autophagy. Dp is a member of natural anthocyanins which are a class of compounds belonging to the larger flavonoids class. Due www.mnf-journal.com

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Mol. Nutr. Food Res. 2014, 58, 1941–1951 Figure 6. Dp upregulated SIRT1 expression by activating AMPK in oxLDL-treated HUVECs. (A) Cells were treated with Dp alone at a series of concentrations (50, 100, and 200 ␮M) for 2 h. The expression of AMPK and p-AMPK was measured by Western blot. (B) The bar graphs show the quantification of endogenous p-AMPK. (C) Cells were pretreated with Dp with different concentrations followed by exposure to oxLDL (10 ␮g/mL) for an additional 24 h. The expression of AMPK and p-AMPK was measured by Western blot. (D) The bar graphs show the quantification of endogenous p-AMPK. (E) AMPK was knocked down by siRNA transfection as described in Section 2. At 24-h posttransfection, the cells were pretreated with Dp (200 ␮M) for 2 h and then incubated with oxLDL (100 ␮g/mL) for an additional 24 h. The cells were collected, lysed, and the proteins were subjected to Western blot analysis. (F) The bar charts show the quantification of the indicated proteins. Values are expressed as the mean ± SD (n = 3). a p < 0.05, b p < 0.01 versus the vehicletreated control group; c p < 0.05, d p < 0.01 versus oxLDL-treated group; # p < 0.01 versus Dp- and oxLDL-cotreated group.

to the low bioavailability of anthocyanins in vivo, the serum concentration of unmetabolized anthocyanins generally peak in the sub- to low-micromolar range and the peak concentration reached in the first 30 min to 2 h [29–31]. However, the concentrations of Dp tested in vitro were almost much higher than that determined in the plasma. In our previous work, the effects of Dp with different concentrations (0.1–200 ␮M) on endothelial injury have been measured and we found that Dp can significantly attenuated oxLDL-induced endothelial damage at the concentration of >1 ␮M [32]. More interesting, we found that Dp can penetrate into endothelial cells via sodium-dependent glucose transporter 1, where it may attain an intracellular concentration higher than the initial extracellular concentration. It has been demonstrated that the vascular endothelium may extend for up to about 240 m2 , making it likely the largest tissue target of Dp [32, 33]. And our previous studies also showed that several anthocyanins with concentrations ranged from 5 to 200 ␮M could notably suppress oxLDL-induced endothelial injury [12, 13, 34, 35]. Furthermore, the concentrations of Dp tested in the current study were in line with other published data [36–39]. Thus, it could be concluded that the endothelium might be a tissue target of Dp. And the appropriate concentrations of Dp tested in endothelial cells in vitro were ranged from 1 to 200 ␮M, which might be attributed to the strong antioxidant capacity and instability of Dp in vitro, however, the precise reason remains unknown. Autophagy plays important roles in both cell protection and cell death. Adaptive autophagy promotes cell survival against injurious stimuli. However, uncontrolled upregula C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

Figure 7. Regulation of Dp-induced autophagy in endothelial cells. Dp attenuated oxLDL caused endothelial injury by inducing autophagy via the proposed signaling pathways: Dp activates AMPK thereby increasing SIRT1 expression, and ultimately inducing autophagy especially mitophagy.

tion of autophagy leads to cell death [40, 41]. In response to most forms of cellular stress, autophagy plays a cytoprotective role. Autophagy deficiency is reported to promote AS [15]. Herein, we found that Dp stimulated autophagy and blocking autophagy significantly inhibited the protective action of Dp in oxLDL-treated HUVECs. Both previous studies and our www.mnf-journal.com

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results show that during AS development, autophagy might act as a cytoprotective factor. It is reported that oxLDL activates autophagy leading to the degradation of oxLDL [42]. This indicates that autophagy is a cellular self-protective measure against oxLDL-induced injury. However, oxLDL-induced autophagy is not sufficient for cell survival. In the current study, we found that Dp amplified this self-protective measure and facilitated HUVECs escape from oxLDL-induced injury. Autophagy could protect endothelial cells against oxLDLinduced injury through several pathways. First, autophagy can lead to the degradation of oxLDL [42]. Second, oxLDL increases several proinflammatory factors production, such as reactive oxygen species, and then induces cell injury and subsequent cell death [28, 43]. Activation of autophagy eliminates these proinflammatory factors through a lysosome-dependent process and benefits cell survival. Third, mitochondrial dysfunction plays important roles in oxLDL-induced injury in endothelial cells [44, 45]. The dysfunctional mitochondria produce a serious of proapoptotic factors and activate intrinsic apoptosis, which is known as the mitochondrial apoptotic pathway [46, 47]. In this study, we found that Dp increased mitophagy in oxLDL-treated HUVECs, which might help cells to eliminate the dysfunctional mitochondria and thus block mitochondrial apoptotic pathway. This has been validated by our following study (data not shown). Then we further identified a potential mechanism of Dp stimulating autophagy in oxLDL-treated HUVECs. SIRT1 is an NAD+ -dependent deacetylase and several reports have shown that SIRT1 improves or retards age-related diseases such as cardiovascular disease by regulating a variety of cellular processes [48]. An SIRT1 knockout mice study shows SIRT1 is an important regulator of autophagy [49]. SIRT1 interacts with several essential components of the autophagy machinery, including autophagy-related protein (Atg) 5, 7, and 8 (Atg 5, Atg 7, and Atg 8), and directly deacetylates these components, and promotes autophagy. As SIRT1 exerts the above function in an NAD+ -dependent fashion, its activity is regulated by those proteins regulating cellular NAD+ level. Among them, one important protein is AMPK. It has been reported that AMPK phosphorylation regulates SIRT1 activity by increasing cellular NAD+ levels [50]. Herein, we found that Dp promoted AMPK phosphorylation in oxLDL-treated HUVECs. In addition, AMPK and SIRT1 knockdown significantly inhibited Dp-induced autophagy. Together, our data demonstrated that Dp stimulated autophagy through AMPKSIRT1 signaling pathway in oxLDL-treated HUVECs. Moreover, anthocyanins, a class of compounds belonging to the larger flavonoids class, have been considered as potential effective anti-AS agents. During the past decades, the underlying mechanisms of this effect have been widely studied. Mauray et al. [10] found that bilberry anthocyaninrich extract alters expression of genes related to AS development in aorta of apoE-deficient mice. And many studies have demonstrated that anthocyanins from blackberry extract ex C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

ert a protective effect against endothelial dysfunction and vascular failure through the antioxidant activity and the regulation of enzymes involved in the Nitric oxide (NO) synthesis. Researchers found that anthocyanins can enhance NO synthase expression and escalate NO production via an Srcextracellular signal-regulated kinase 1/2-Sp1 signaling pathway and enhance NO synthase activity by regulating its phosphorylation [51–53]. Recently, Dongliang Wang et al. claimed that gut microbiota metabolism of anthocyanins promotes reverse cholesterol transport in mice via repressing microRNA10b, suggesting a role of gut microbiota in anthocyanins’ anti-AS effect [54]. Our previous study has shown that anthocyanins can attenuate oxLDL-induced endothelial injury and the activity is closely related to their chemical structures among which Dp is the most effective one [12, 13]. And we also found that Dp attenuates oxLDL-induced endothelial injury by inhibiting the reactive oxygen species/p38MAPK/NFкB signaling pathway [13]. Last year, we have proved that Dp protects against oxLDL-induced mitochondrial dysfunction in endothelial cells via sodium-dependent glucose transporter 1 [32]. However, the exact molecular target of anthocyanins, Dp in particular, still needs to be fully elucidated. Herein, we demonstrated, for the first time, that Dp attenuated oxLDLinduced injury by stimulating autophagy in HUVECs through the AMPK/SIRT1 signaling pathway. Our results will provide an experimental evidence for future clinical applications of Dp or Dp-related products to prevent AS which giving new insights into the cardiovascular benefits of anthocyanins. However, there are still some limitations in this study, which require further explanation. First, our data was based on isolated HUVECs in vitro, as the more complex microenvironment in vivo, whether Dp stimulates autophagy in vivo need to be validated further, which is our main following work. Besides, it has been reported that Dp can affect apoptosis through other pathway in other cell types [55–57]. Whether Dp takes effects through other mechanisms in HUVECs also need to be explored. This work was supported by the General Program of National Natural Science Foundation of China (grant numbers: 81202202 and 81000133) and the Youth Innovation Foundation of the Third Military Medical University (2012XJQ06). The authors have declared no conflicts of interest.

5

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