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Anthocyanins from Chinese bayberry extract activate transcription factor Nrf2 in beta cells and negatively regulate oxidative stress-induced autophagy Bo Zhang, Miranbieke Buya, Wenjie Qin, Chongde Sun, Haolei Cai, Qiuping Xie, Bing Xu, and Yulian Wu J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/jf4012399 • Publication Date (Web): 11 Aug 2013 Downloaded from http://pubs.acs.org on September 4, 2013

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Journal of Agricultural and Food Chemistry

Title page

Anthocyanins from Chinese bayberry extract activate transcription factor Nrf2 in beta cells and negatively regulate oxidative stress-induced autophagy †﹟ ﹟ ﹟ Bo Zhang , Miranbieke Buya† , Wenjie Qin† , Chongde Sun‡, Haolei Cai†, Qiuping Xie†, Bing Xu†, Yulian Wu*,†

†Department of Surgery (Key Laboratory of Cancer Prevention and Intervention, China National Ministry of Education), Second Affiliated Hospital, School of Medicine, Zhejiang University. No.88 Jiefang Road, Hangzhou, Zhejiang Province, 310009, P.R.China ‡Laboratory of Fruit Quality Biology/The State Agriculture Ministry Laboratory of Horticultural Plant Growth, Development and Quality improvement, Zhejiang University, Huajiachi Campus, Hangzhou, Zhejiang Province, 310029, P.R. China ﹟ These authors contribute to this work equally. * Address correspondence and reprint requests to Yulian Wu, M.D., Ph.D. (Tel: 86-0571-87784604; Fax: 86-0571-87784604; E-mail: [email protected])

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Abstract

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Islet replacement is the promising cure for insulin-dependent diabetes, but is

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limited by a massive early cell death following transplantation. Overburden oxidative

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stress is one of the major factors causing cell damage. We have shown previously that

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anthocyanins in Chinese bayberry extract protected beta cells (INS-1) from hydrogen

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peroxide

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transplantation partially through heme oxygenase-1 (HO-1) upregulation. In the

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present study, we observed that H2O2 stimulation induced autophagy in beta cells.

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Inhibition of autophagy increased cell viability and decreased cell death.

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Anthocyanins pretreatment attenuated oxidative stress-mediated autophagic cell death.

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Anthocyanins activated antioxidant transcription factor Nrf2 in INS-1 cells, and

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Nrf2/HO-1 negatively regulated autophagy process. Furthermore, we here

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demonstrated that autophagy also took place in beta-cell grafts during the early

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post-transplantation phase. Beta cells pretreated with anthocyanins displayed

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decreased extent of autophagy after transplantation. Taken together, these findings

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further supported the conclusion that anthocyanins could serve as a protective agent of

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beta cells and suggested that autophagy might play a role in beta cells during

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transplantation.

(H2O2)-induced

apoptosis

and

decreased

grafts’ apoptosis

after

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Keywords: Anthocyanins; Nrf2; Heme oxygenase-1; Islet transplantation;

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Autophagy; Oxidative stress.

22 23 24 25


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Introduction

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Islet transplantation is currently the effective treatment of type 1 diabetes that

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achieves insulin-independence. However, the grafts will experience a progressive

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deterioration of cellular viability and function overtime, leading to a large proportion

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of subjects returning to insulin dependence several years later (1). This outcome is

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mainly attributed to (1) early graft damage during islet isolation, preservation and

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early post-transplantation phase. During this stage, hypoxia, interrupted blood supply,

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ischemia/reperfusion injury and non-specific inflammation are the main causes of

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beta-cell death, with the highest percentage of islet graft loss and dysfunction

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occurring just days after transplantation; (2) late phase loss. Immunoreactions play

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major roles in this stage, which are capable of inflicting beta-cell damage and

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impairing beta-cell function. Because of reduced antioxidant defenses, beta cells are

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increasingly vulnerable to oxidative/nitrosative damage arising during these stages (2).

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Increasing antioxidant capacity to render islets more resistance to free radical damage

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is theoretically a promising strategy to delay cell failure. Antioxidant treatment is

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reported to possess functions of decreasing free radical-induced beta cell damage in

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several animal and human studies (3).

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Autophagy is the major cellular pathway for the degradation of long-lived

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proteins and cytoplasmic organelles in animal cells(4). During autophagy, cytoplasmic

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materials are sequestered into double-membrane vesicles, ‘autophagosome’, which

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then fuse with lysosomes to form autolysosomes, where degradation of cellular

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structures occurs (5, 6). Autophagy could be stimulated by a change of environmental

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conditions such as nutrient deprivation. Hypoxia, ischemia/reperfusion and excessive

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free radicals, which are also the stress stimulations during islet transplantation as

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mentioned above, are considered as the inducers of autophagy in certain



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circumstances. However, up to now, limited information has been reported about

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whether autophagy appears in beta cells during transplantation procedure.

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Chinese bayberry is one of six Myrica species native to China and possesses

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various biological activities including notable radical-scavenging ability(7-9). The

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fruit extract is rich in anthocyanins, and cyanidin-3-O-glucoside (C3G) is identified as

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a major anthocyanin component (9, 10). We previously showed that anthocyanins

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pretreatment protected pancreatic beta cells (rat insulinoma cell line INS-1) against

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hydrogen peroxide (H2O2)-induced necrosis and apoptosis via HO-1 induction, and

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decreased graft apoptosis after transplantation(11) . In the present study, we explore

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the occurrence of autophagy in beta cells during oxidative stress stimulation in vitro

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and at early post-transplantation phase, and further investigate the protective effect of

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anthocyanin with the emphasis on its impact on autophagy.

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Materials and methods

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Chinese bayberry fruit extract preparation

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Chinese bayberry fruits were obtained from Yuyao County, Zhejiang Province,

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China. The fruit was then ground and extracted with extract solvent (methanol

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acidified with 0.05% HCl) as described previously(11). After being extracted for 3

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times and dried by evaporation at low temperature, the extract was dissolved in 80%

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ethanol solvent (4.5% formic acid) to remove impurities such as sugar. After being

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dried by evaporation again, anthocyanins were dissolved in double-distilled water,

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and underwent further cryo-concentration. HPLC analysis of the extract was carried

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out (10), and the content of C3G was calculated. HPLC analysis demonstrated that

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C3G accounted for 95% of total anthocyanins with a dominant peak at 520nm. The

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concentration of C3G was 2.77 mmol/L (1.245 mg/ml), determined by peak area


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measurement with pure C3G as standard sample (FigureS1). Since C3G is the major

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component, the usage dose of anthocyanins was standardized by the concentration of

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C3G..

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Cell culture conditions, reagents and plasmid

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Rat pancreatic beta-cell line INS-1 were grown in RPMI-1640 supplemented

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with 10% fetal bovine serum at 37 ℃ in a humidified atmosphere (5% CO2 and 95%

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air). MTT [3-(4,5-dimethythiazol-2-yl)-2,5-diphenyl tetrazolium bromide], Trypan

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blue dye, 3-Methylamphetamine (3-MA), monodansylcadaverine (MDC, a specific

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autophagic vacuole marker) and Hoechst 33258 (H33258) were the products of Sigma

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Chemical Co. (St. Louis, MO, USA). The commercially available in situ cell

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apoptosis detection kit (used for TUNEL test) was purchased from Promega

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(Madison,WI,USA). Annexin V-FITC/PI apoptosis detection kit was obtained from

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Abcam Public Limited Company (Abcam, Cambridge, MA, USA). LipofectamineTM

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2000 transfection reagent was obtained from Invitrogen (Carlsbad,CA, USA).

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GFP-mRFP-LC3 plasmid was kindly provided by Prof. T. Yoshimori (Osaka

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University, Osaka, Japan). The pLNCX2-HO-1 vector carrying the coding sequence

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of the rat HO-1 gene and INS-1 cells which were transfected with pLNCX2-HO-1

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stably overexpressing HO-1 (INS-1/HO1) were established in our previous study(11) .

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Cell viability assay

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Cell viability was determined using MTT dyes as previously described(12). 5

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×103 INS-1 cells per well were seeded in 96-well plates, after 1mM H2O2 stimulation

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for 2 hours, 20 µL of MTT solution was added to each well. After 4 hours’ incubation,

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the supernatant was removed and 150 µL of DMSO was added to each well. The

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plates were shaken for 10 minutes, and Optical Density was measured by an ELISA

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reader (BIO-Tek ELx800, Winooski, VT, USA) at a wavelength of 570 nm. Cell



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viability was also measured using the trypan blue dye exclusion test as previously

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described(11). After trypan blue dye was added to treated INS-1 cells for 5 minutes,

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the number of staining cells (unviable cells) were counted and calculated as a percent

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of total number of cells.

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Flow cytometric detection of apoptosis

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Cellular apoptosis was detected quantitatively by flow cytometry (FCM) as

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previously described(12). After treatment, 1×106 INS-1 cells were suspended in 1 mL

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1×PBS and centrifuged (1200 rpm×5 minutes), then the supernatant was removed and

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cells were resuspended in 500 µL 1×Binding Buffer. 10 µL Annexin V-FITC and 5 µL

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PI were added and kept in darkness with incubation of 15 minutes. The cellular

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apoptosis was assessed by flow cytometry using the Cell Quest program (Becton

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Dickinson, San Jose, CA, USA).

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Transmission electron microscope (TEM)

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After experimental manipulations, cells or tissues were fixed in 2.5% glutaraldehyde

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and 1% osmic acid for 1h respectively, followed by 4% uranyl acetate for 30 minutes

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and gradient ethanol dehydration. At last, the fixed samples were infiltrated with

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absolute acetone and the final resin mixture. Then the materials were embedded in

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Epon 812, cut with Ultracut UCT ultramicrotome (Leica, Bensheim, Germany), and

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photographed by electron microscopy (JEM-1230).

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Protein extraction and western blot analysis

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Whole cell lysates were obtained by resuspending cells in a cell lysis buffer

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containing a protease inhibitor cocktail as previously described(12). Nuclear and

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cytoplasmic extracts were prepared using commercial NE-PER Nuclear and

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Cytoplasmic Extraction Reagents (Pierce Biotechnology, USA; Cat: 78833). Protein

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concentration was determined using the BCA protein assay kit (Pierce Biotechnology,


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USA). Protein samples (equal amounts) were separated by SDS-PAGE (sodium

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dodecylsulfate-polyacrylamide) and transferred to PVDF membranes, which were

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then blocked with 5% non-fat milk, and probed overnight with primary antibody at

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4℃. After undergoing washes for 3 times, the membranes were incubated with

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secondary antibodies. The blots were visualized by enhanced chemiluminescence.

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Primary antibodies against beclin-1(BECN1), Nrf2 and actin were purchased from

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Santa Cruz Biotechnology (Santa Cruz, CA, USA). Anti-LC3 antibody was obtained

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from MBL International (MBL, Nagoya, Japan). Antibody against HO-1 was

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purchased from Cell Signaling Technology, Inc. (Beverly, MA,USA). Caspase-3 and

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Lamin A/C antibodies were obtained from Abcam Public Limited Company (Abcam,

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Cambridge, MA,USA).

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Electrophoretic Mobility Shift Assay (EMSA)

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To determine whether anthocyanins increase Nrf2 DNA-binding activity, we

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implemented electrophoretic mobility shift assay (EMSA) using the Viagene EMSA

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kit (Viagene Biotech Inc., China) following the manufacturer’s protocol. Briefly,

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equal amounts of nuclear protein (5 µg) were incubated with the biotin-labeled Nrf2

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oligonucleotide and poly dI:dC for 20 min at room temperature in binding reaction

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buffer. The DNA-protein complexes were resolved on a 6.5% polyacrylamide gel

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pre-electrophoresed in 0.25×TBE solution at 120V for 1h. Then the gel was

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transferred on to positively charged nylon membrane. The transferred DNA was

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cross-linked to the membrane and detected with horseradish peroxidase-conjugated

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streptavidin.

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Monodansylcadaverine (MDC) staining and Nrf2 immunofluorescence

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INS-1 cells were grown on glass coverslips. After being treated with the

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indicated agents, cells were incubated with 0.05mM MDC for 20 minutes at 37°C.



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Cells were washed 2 times with PBS and observed under fluorescence microscope.

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For immunofluorescence detection of Nrf2, treated cells were fixed with 4%

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paraformaldehyde (PFA) for 30 minutes at room temperature. After treatment with

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Triton X-100 (0.1%) for 10 minutes and normal goat serum for 30min at room

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temperature, anti-Nrf2 antibody (Santa Cruz, CA,USA; 1:200 in 2%BSA) was added

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and incubated overnight at 4°C. After an additional incubation for 1 hour at room

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temperature with Alexor 488-conjugated goat-anti-rabbit antibody (Molecular Probe,

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Invitrogen, USA) (1:200 in PBS), the slides were mounted and observed under

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fluorescence microscope.

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Plasmid transfection and RNA interference

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For detection of autophagosomes, INS-1 cells were transfected with

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GFP-mRFP-LC3 plasmid using LipofectamineTM 2000 reagent, and the transfected

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cells were treated with H2O2 for 2 hours and observed under fluorescence microscope.

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For RNA interference studies, the target sequence for HO-1-specific small

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interference RNA (siRNA) was 5’- CCGUGGCAGUGGGAAUUUAUGCCAU-3’,

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BECN1 siRNA sequence was5’- GUCCCUGACAGACAAAUCU-3’, both of which

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and the control siRNA (no silencing) were synthesized by GenePharma Co. (Shanghai,

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China). Nrf2 siRNA was purchased from Santa Cruz Biotechnology (Santa Cruz, CA,

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USA; Cat: sc-156128). Transfection was performed using LipofectamineTM 2000

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reagent according to the manual instructions.

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Animal studies

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ICR mice of 8 weeks in age were used as the recipients in beta-cell

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transplantation experiment and the experimental procedures were described in our

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previous study(11). Pretreated INS-1 cells (2×106 cells with 1µM anthocyanins for 24

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hours) and untreated INS-1 cells (2×106 cells) were transplanted under renal capsules


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of ICR mice respectively. 72 hours after transplantation, beta cell grafts with a part of

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kidneys were harvested and fixed with 4% PFA, embedded in paraffin for

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immunohistological analysis. Immunohistological staining for LC3, BECN1, and

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TUNEL staining were performed according to the manual instructions. Some samples

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were fixed in glutaraldehyde for further transmission electronic microscopic analysis

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as described above. All experiments were carried out in accordance with the ethical

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guidelines of Animal Experimentation Committee in our institute.

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Statistical analysis

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When necessary, data were expressed as mean±SD. Statistical analysis was

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performed using SPSS version 13.0. One-way ANOVA with Bonferroni’s post hoc

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test and Student’s t test were used to evaluate statistical significance and p

Anthocyanins from Chinese Bayberry Extract ... - ACS Publications

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