Protective Effects of Blueberry Anthocyanins against H2O2-Induced

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Protective Effects of Blueberry Anthocyanins against H2O2Induced Oxidative Injury in Human Retinal Pigment Epithelial Cells Wuyang Huang, Han Wu, Dajing Li, Jiangfeng Song, Yadong Xiao, Chunquan Liu, Jianzhong Zhou, and Zhongquan Sui J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b06135 • Publication Date (Web): 02 Feb 2018 Downloaded from http://pubs.acs.org on February 4, 2018

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Journal of Agricultural and Food Chemistry

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Protective Effects of Blueberry Anthocyanins against H2O2-Induced Oxidative Injury in

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Human Retinal Pigment Epithelial Cells

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Wu-Yang Huang a,b,#, Han Wu a,#, Da-Jing Li a, Jiang-Feng Song a, Ya-Dong Xiao a,

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Chun-Quan Liu a, Jian-Zhong Zhou a,*, and Zhong-Quan Sui c,*

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a

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PR China, 210014

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b

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Agricultural Sciences, Nanjing 210014, China

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c

Institute of Farm Product Processing, Jiangsu Academy of Agricultural Sciences, Nanjing,

Jiangsu Key Laboratory for Horticultural Crop Genetic Improvement, Jiangsu Academy of

Department of Food Science and Engineering, Key Lab of Urban Agriculture (South), Bor S.

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Luh Food Safety Research Center, School of Agriculture and Biology, Shanghai Jiao Tong

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University, Shanghai, PR China, 200240

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Running title: Protective Effects of Blueberry Anthocyanins against H2O2 in ARPE-19

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#

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* Corresponding author.

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Tel.: 86-25-84392177; 86-21-34206613; Fax: 86-25-84391677; 86-21-34206613.

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E-mail address: [email protected] (J.Z. Zhou); [email protected] (Z.Q. Sui).

Both authors contributed equally to this work.

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ACS Paragon Plus Environment


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ABSTRACT

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Blueberry anthocyanins are considered to be protective for eye health due to their

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recognized antioxidant properties. In this study, blueberry anthocyanin extract (BAE),

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malvidin (Mv), malvidin-3-glucoside (Mv-3-glc), and malvidin-3-galactoside (Mv-3-gal)

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reduced H2O2-induced oxidative stress by decreasing the levels of reactive oxygen species

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and malondialdehyde and increasing the levels of superoxide dismutase, catalase, and

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glutathione peroxidase in human retinal pigment epithelial cells. BAE and anthocyanin

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standards enhanced cell viability from 63.69 ± 3.36% to 86.57 ± 6.92% (BAE), 115.72 ±

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23.41% (Mv), 98.15 ± 9.39% (Mv-3-glc), and 127.97 ± 20.09% (Mv-3-gal) and significantly

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inhibited cell apoptosis (all P < 0.01). Mitogen-activated protein kinase pathways, including

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ERK1/2 and p38, were involved in the bioactivities. In addition, the anthocyanins decreased

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the vascular endothelial cell growth factor levels and activated Akt signal pathways. These

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combined results supported the hypothesis that blueberry anthocyanins could inhibit the

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induction and progression of age-related macular degeneration (AMD) through antioxidant

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mechanisms.

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KEYWORDS: age-related macular degeneration, antioxidant, blueberry anthocyanins,

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oxidative stress, retinal pigment epithelial cells

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INTRODUCTION

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Age-related macular degeneration (AMD) is a major cause of vision loss in the elderly

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population worldwide.1 AMD primarily affects the retinal pigment epithelium (RPE), leading

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to dysfunction of the epithelial cells and degeneration of the photoreceptors, and AMD also

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results in the accumulation of extracellular deposits in the macula, incrassation of the Bruch’s

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membrane, and even neovascularization of the retina in the central region.2 The RPE is a

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monolayer of pigmented cells forming a part of the blood/retina barrier, which secretes a

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variety of growth factors to maintain the structural integrity of photoreceptors and capillary

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endothelium.3 The RPE is essential for visual function as the retinal pigment epithelial cells

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play important roles in inhibiting retinal edema, in blocking abnormal neovascularization, and

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in protecting the retina from oxidative stress, making it a valuable model to study the

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oxidative stress response in AMD.4

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Oxidative stress is a well-established factor that contributes to the pathogenesis of several

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age-related diseases, including AMD.5 Oxidative stress in the retina is aggravated by

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lipofuscin, which accumulates in the RPE with age.1 Hydrogen peroxide (H2O2) is generated

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in the RPE during the phagocytosis of the oxidized outer segment in the physiological process

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of photoreceptor renewal.6 Growing evidence suggests that RPE cells are vulnerable to

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oxidative stress, particularly the stress from reactive oxygen species (ROS), and the damage is

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thought to be an early event in AMD.7 Despite the potent antioxidant systems, including

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superoxide dismutase (SOD), catalase (CAT), and glutathione (GSH), that exist in the RPE

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cells, oxidative damaged bio-molecules (e.g., malondialdehyde, MDA, a bio-marker of lipid

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peroxidation) have also been identified in RPE cells.5 During early AMD, the RPE cells

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become progressively dysfunctional and finally die by apoptosis, because an inadequate

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cytoprotective response converts oxidative stress.8 Therefore, the protection of RPE cells

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against oxidative damage may be an effective strategy for the amelioration of early AMD.

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Numerous studies have reported the protective effects of dietary antioxidants on eye

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health. 9-12 For example, lutein and zeaxanthin limited retinal oxidative damage by absorbing

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blue light and/or quenching ROS.10 Anthocyanins exhibited protective effects on visual signal

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transduction and prevented age-related blindness by decreasing the oxidative burden in RPE

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cells.11 An age-related eye disease study demonstrated by the National Eye Institute of the

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USA in 2001 and 2013 proved that higher doses of dietary antioxidants play an important role

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in preventing catastrophic loss of vision in patients with high risk of AMD retinopathy.12

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Therefore, the use of dietary countermeasures, especially antioxidants, to prevent AMD

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progression has been a topic of increasing interest.

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Fruits and vegetables are good resources of dietary antioxidants, including polyphenols,

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flavonoids, carotenoids, vitamins, and minerals. In particular, anthocyanins, one class

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of flavonoids synthesized via the phenylpropanoid pathway, have an antioxidant role in plants

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against reactive oxygen species and great antioxidant capacity in vitro or in vivo.13,14

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Blueberries have the highest level of anthocyanins among 40 vegetables and fruits.15 Several

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scientists have reported that blueberry components (e.g., anthocyanins, polyphenols, and

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pterostilbene) could protect RPE cells against light-induced injury via the antioxidative,

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anti-angiogenic and anti-aging effects in vitro, and the retinal protective activity of blueberries

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were also confirmed in vivo.16-18 Malvidins glycosylated with hexose or pentose were the 4


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dominant anthocyanin components in blueberries, accounting for > 46% of the total

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anthocyanin content in wild Chinese blueberries.16 Our previous survey investigated and

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identified the polyphenol constituents of Brightwell rabbiteye blueberry (Vaccinium ashei) in

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Nanjing using HPLC-DAD-MSn and found that these fruits were all composed of delphinidin,

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cyanidin, petunidin, peonidin, and malvidin glycosides, where malvidin-3-glucoside

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(Mv-3-glc) and malvidin-3-galactoside (Mv-3-gal) were also the major components

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accounting for nearly 50% of the total anthocyanin extracts.19 Most previous studies focused

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on the chemical profiling of anthocyanins, in vitro and the in vivo antioxidant activity of the

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whole berries or phytochemical extracts. We have reported the antioxidant and

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anti-inflammatory properties of malvidin, Mv-3-glc, and Mv-3-gal in human umbilical vein

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endothelial cells.20,21 However, the protective effects of these anthocyanins on retinal

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epithelial cells exposed to H2O2 are still unknown. In this study, protective effects of

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blueberry anthocyanin extract, as well as malvidin and its glycosides (Mv-3-glc and Mv-3-gal)

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on H2O2-induced oxidative injury in human RPE cells were investigated to reveal the effects

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of these antioxidants on AMD.

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MATERIALS AND METHODS

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Chemicals. Trypsin and standards, including malvidin (Mv), malvidin-3-glucoside

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(Mv-3-glc), and malvidin-3-galactoside (Mv-3-gal), were obtained from Sigma Aldrich

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(Shanghai, China). The human retinal pigment epithelia cell line (ARPE-19) was obtained

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from Meixuan Biological Science and Technology, Ltd. (Shanghai, China). Dulbecco’s

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modified Eagle medium (DMEM) and fetal bovine serum (FBS) were obtained from

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Gibco/Invitrogen (Shanghai, China). Penicillin and streptomycin were bought from Life

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Technologies

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/propidium iodide (PI) Apoptosis Detection Kit was bought from Yeasen Biotechnology Co.,

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Ltd. (Shanghai, China). An MTT Cell Proliferation Kit, reactive oxygen species (ROS) Assay

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Kit, and BCA (bicinchoninic acid) Protein Assay Kit were bought from Beyotime Institute of

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Biotechnology

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glutathione peroxidase (GSH-PX), and superoxide dismutase (SOD) Activity Assay Kits,

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malondialdehyde (MDA), and vascular endothelial cell growth factor (VEGF) ELISA Kits

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were bought from Xinzhetianyou Biotechnology Co., Ltd. (Beijing, China). All the chemicals

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and reagents were analytical grade.

(Shanghai,

(Shanghai,

China).

China).

The

Annexin V-fluorescein isothiocyanate (FITC)

Andygene

human

caspase-3,

catalase

(CAT),

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Antibodies. Mouse monoclonal primary antibody against Bcl-2 and rabbit polyclonal

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primary antibodies against Akt, phospho-Akt, Bax, Erk1/2, p38, and cleaved-caspase-3 were

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obtained from Ruiying Biological Technology Co., Ltd. (Jiaozuo, Henan, China). Mouse

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monoclonal primary antibody against VEGF was bought from Abcam (Shanghai, China).

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Mouse monoclonal primary antibody to β-actin was bought from Sigma Aldrich (Shanghai,

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China). Mouse monoclonal primary antibody against p-p38 was purchased from Santa Cruz

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Biotechnology, Inc. (Santa Cruz, CA, USA), using 1:500 dilutions. All the other primary

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antibodies were used at 1:1000 dilutions. Rabbit polyclonal primary antibody against

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phospho-p4442 MAPK (Erk1/2) (Thr202Tyr204) and Goat anti-mouse/rabbit IgG

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HRP-linked secondary antibodies were purchased from Cell Signaling Technology Inc.

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(Shanghai, China). Secondary antibodies were used at 1:4000 dilutions.

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Extraction of Anthocyanins from Blueberries.19 Brightwell rabbiteye blueberry

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(Vaccinium ashei) was collected from the Nanjing countryside in July 2016 and stored at

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-18°C avoiding light. The blueberries were defrosted at room temperature. After beating at

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10,000 rpm for 30 s using a homogenizer, 250 g blueberries were soaked in 1000 mL of

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methanol containing 1% HCl solution for 24 h. The extract was collected and centrifuged at

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5000 × g for 15 min. After in vacuo evaporation of the solvent at 40°C, the residue was

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extracted with 1:1 (v/v) ethyl acetate three times. The crude anthocyanin extract (522.3 mg)

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was further purified with AB-8 macroporous resin and then dried using an Eyela FDU-1200

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freeze dryer (Tokyo Rikakikai, Japan) to get 166.2 mg blueberry anthocyanin extract (BAE)

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powder. The purity of the anthocyanins was determined by UV-Vis spectrophotometry using

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cyanidin as standard, and was calculated as high as 78.9%. Blueberry anthocyanin extract was

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characterized by HPLC-DAD to determine the exact amount of each compound in the extract.

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Malvidin glycosides were dominant anthocyanins in the blueberries, accounting for 47.9% of

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the total anthocyanin content, in which Mv-3-glc and Mv-3-gal were 17.2% and 22.6%,

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respectively (unpublished data).

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Retinal Pigment Epithelial Cell Culture and Treatment. ARPE-19 cells were cultured

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in DMEM supplemented with 10% FBS, 1% streptomycin and penicillin at 37°C in a 5% CO2

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humidified incubator.22 The cells were used for all the experiments at 80-90% confluence.

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ARPE-19 cells were grown in reduced serum medium for 4 h prior to the experiment. The

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cells were seeded in 6-well or 96-well plates at a concentration of 2×105 cells/mL and

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pretreated with 5 µg/mL of BAE, Mv, Mv-3-glc, or Mv-3-gal aqueous solution for 6 h. Then,

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the sample cells were stimulated with 800 µmol/L H2O2 for 2 h. A normal cell group without 7



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blueberry anthocyanin pretreatment and H2O2 stimulation was used as the control. An H2O2

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stimulation group without blueberry anthocyanin pretreatment was used as the oxidative

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model. The 6-well plates were used in apoptosis analysis, ELISA, and Western blotting. The

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96-well plates were used in the MTT and ROS assay.

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Cell Viability Detection. The cell viability was determined by the MTT method.5 The

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cells in 96-well plates were pretreated with 5 µg/mL BAE, Mv, Mv-3-glc, or Mv-3-gal for 6 h

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and stimulated with 800 µmol/L H2O2 for 2 h. The cells without anthocyanin and H2O2 were

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used as the blank control, while the cells stimulated with H2O2 only were used as the

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oxidative model. Each well was added with 10 µL MTT (5 mg/mL) and then continued to

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culture 4 h. Formazan crystals were dissolved by adding 100 µL DMSO (dimethyl sulfoxide)

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and shaking 10 min slowly. The absorbance was measured at 550 nm on a Synergy H4

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Multi-Mode Microplate Reader (BioTek Instruments, Inc. Winooski, VT, USA). The reader

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was controlled via Hyper Terminal Applet ELISA software. The cell viability was calculated

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with the following formula:

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Cell viability (%) = sample group OD value/blank group OD value ×100%

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Analysis of Apoptosis. ARPE-19 cells in 6-well plates with different treatments were

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collected, centrifuged, washed twice with phosphate buffered saline (PBS) and resuspended in

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200 µL 1 × Binding Buffer. After adding 5 µL of 0.1 mg/mL AnnexinV-FITC solution and 10

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µL of 20 µg/mL PI solution according to the instructions of AnnexinV-FITC/PI Apoptosis

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Detection Kit, the cells were incubated in the dark for 15 min at room temperature and

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400 µL of Binding buffer was added. Immediately, the result was analyzed with a Becton

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Dickinson (BD) FACS Calibur Flow Cytometer (BD Biosciences, San Jose, CA), and the

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percentage of apoptosis was calculated.

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Reactive Oxygen Species (ROS) Assay. A Dichloro-dihydro-fluorescein diacetate

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(DCFH-DA) Detection Kit was used to assess the ROS level in ARPE-19 cells. Briefly, the

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cells were suspended with reduced serum medium to reach a concentration of 2×105

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cells/mL and were seeded in 96-well plates with different treatments. After washing the cells

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with PBS, 10 µmol/L DCFH-DA was added to each well and left to react for 20 min at 37°C.

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The fluorescence intensity of the cells in each well were determined using a Synergy H4

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Multi-Mode Microplate Reader (BioTek Instruments, Inc. Winooski, VT, USA) with 488-P

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excitation and 525-P emission filters. The total fluorescence intensities of the cells in each

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well were noted, and the fluorescence intensity relative to the cell viability was calculated to

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reflect the real ROS generation level of the cells. The data was measured as fold increase over

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the control.

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ELISA Analysis. The supernatants in 6-well plates with different treatments were

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collected for ELISA analysis on extracellular proteins. The levels of CAT, GSH-PX, MDA,

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SOD, and VEGF were quantified using ELISA kits or enzyme activity assay kits. The total

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cell protein in each well was detected using the BCA Protein Assay Kit. The assay procedure

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was employed according to the kit protocol instructions. The absorbance of the resulting

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yellow color was measured at 450 nm on a Synergy H4 Multi-Mode Microplate Reader

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(BioTek Instruments, Inc. Winooski, VT, USA). The reader was controlled via Hyper

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Terminal Applet ELISA software.

9



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Western Blotting. The cells in 6-well plates with different treatments were prepared for

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Western blotting on intracellular proteins. Protein expression of pro-caspase-3, Bcl-2, Bax,

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Akt, p-Akt, Erk1/2, p-Erk1/2, p38, p-p38, and VEGF was analyzed for the ARPE-19 lysates.

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Data were normalized by re-probing the membrane with an antibody against β-actin, which

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was used as a loading control. Cell lysates from untreated cells were loaded on every gel, and

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the data were expressed as a fold increase over the corresponding control. The ratios of

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phosphorylated proteins to total proteins for Akt, Erk1/2, and p38 were also calculated.

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Statistical Analysis. All data presented are the mean value ± standard deviation (SD) of

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three or four independent experiments. Figures were obtained using GraphPad Prism Version

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5 (GraphPad Software, Inc., CA, USA). One-way analysis of variance (ANOVA) with

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Dunnett’s posttest was used for comparison with the control, and t-test with a two-tailed P

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value was performed to determine the significant differences between each of the two

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different columns (oxidative model group and sample group). Differences were considered

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significant with a P value < 0.05.

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RESULTS

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Protective Effects against H2O2-induced Cytotoxicity in ARPE-19 Cells. The

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oxidative model with 5 µg/mL anthocyanin pretreatment for 6 h and 800 µmol/L H2O2

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stimulation for 2 h was established according to our preliminary experiment by detecting

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ARPE-19 cells treated with different concentrations of BAE (1, 5 and 10 µg/mL) and H2O2 (0,

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10, 20, 40, 60, 80, 100, 200, 400, 800, 1600, and 3200 µmol/L) for various times (BAE: 6 and

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24 h; H2O2: 2, 6, and 24 h) using an MTT and ROS assay. Hydrogen peroxide significantly

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influenced the growth of ARPE-19 cells as 800 µmol/L H2O2 induced a 36.31% loss of cell 10


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viability (63.69 ± 3.36%, P < 0.001). Blueberry anthocyanins were previously determined as

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having no cytotoxicity on ARPE-19 cells (our unpublished data), and could even protect

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ARPE-19 cells against H2O2-induced cytotoxicity by increasing the cell viability.

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Pretreatment with 5 µg/mL BAE, Mv, Mv-3-glc, and Mv-3-gal significantly inhibited the loss

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of cell viability (P < 0.01 or P < 0.001). Their values were 86.57 ± 6.92%, 115.72 ± 23.41%,

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98.15 ± 9.39%, and 127.97 ± 20.09%, respectively (Figure 1), compared to the control group.

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Compared to the crude blueberry anthocyanin extract and the aglycone malvidin (P < 0.01),

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malvidin glycosides (Mv-3-glc and Mv-3-gal) exhibited more pronounced protective effects

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(P < 0.001).

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Protective Effects against H2O2-induced Cell Apoptosis in ARPE-19 Cells. The

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exposure to 800 µmol/L H2O2 led to a significantly higher rate of total apoptosis (combined

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the early and late apoptotic cells, 15.04 ± 2.92%) compared with the control cells (total

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apoptotic rate: 1.27 ± 0.33%; P < 0.01). The percentage of necrotic cells also increased from

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2.86 ± 0.03% (the control) to 7.64 ± 0.48% after adding 800 µM H2O2. Blueberry

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anthocyanins could protect ARPE-19 cells against H2O2-induced cell apoptosis and necrosis

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(P < 0.01). Pretreatment with 5 µg/mL BAE, Mv, Mv-3-glc, and Mv-3-gal inhibited 71.87 ±

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3.57%, 83.02 ± 5.32%, 81.27 ± 3.61%, and 83.38 ± 7.52% of H2O2-induced cell apoptosis,

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and the total apoptotic rates were 5.14 ± 0.68%, 3.61 ± 0.33%, 3.85 ± 0.73%, and 3.56 ±

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0.32% (P < 0.01 vs the oxidative model), respectively (Figure 2). The inhibitory rates of BAE,

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Mv, Mv-3-glc, and Mv-3-gal on necrotic cells induced by 800 µM H2O2 were 44.98 ± 1.39%,

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33.89 ± 0.68%, 42.47 ± 1.89%, and 28.66 ± 0.51%, respectively. The inhibitory effects on cell

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total apoptosis seemed be more pronounced than those on cell necrosis. Pure anthocyanin 11



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malvidin and its glycosides (Mv-3-glc and Mv-3-gal) seemed to attenuate more cell apoptosis

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compared with the crude blueberry anthocyanin extract since BAE had no effect on the early

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apoptotic cells.

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Protective Effects against H2O2-induced ROS productions in ARPE-19 Cells.

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ARPE-19 cells exposed to 800 µmol/L H2O2 had a significantly higher ROS level compared

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with the control cells (3.08 ± 0.30-fold; P < 0.001). Blueberry anthocyanins could protect

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ARPE-19 cells against H2O2-induced ROS. Pretreatment with 5 µg/mL BAE, Mv, Mv-3-glc,

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and Mv-3-gal significantly inhibited 67.11 ± 6.35%, 104.68 ± 4.23%, 88.52 ± 9.09%, and

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116.32 ± 10.52% of H2O2-induced ROS (all P < 0.001 vs the oxidative model), respectively

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(Figure 3). Pure anthocyanin malvidin and its glycosides (Mv-3-glc and Mv-3-gal) seemed to

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attenuate more ROS compared with the crude blueberry anthocyanin extract. Mv-3-gal

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exhibited the strongest antioxidant capacity with a ROS level even less than the control (0.66

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± 0.06-fold, P < 0.001 vs the control; P < 0.001 vs the oxidative model). The ROS level of

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Mv-treated cells (0.90 ± 0.04-fold, P < 0.05 vs the control; P < 0.001 vs the oxidative model)

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were also less than the control.

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Protective Effects against H2O2-induced MDA productions in ARPE-19 Cells.

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ARPE-19 cells exposed to 800 µmol/L H2O2 had a significantly higher MDA content

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compared with the control cells (1.85 ± 0.09-fold; P < 0.001). Pure anthocyanin malvidin and

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its glycosides (Mv-3-glc and Mv-3-gal) could significantly attenuate the MDA contents in

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ARPE-19 cells (P < 0.001 vs the oxidative model). The crude blueberry anthocyanin extract

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also decreased the MDA level but had no significant difference with the oxidative model. The

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inhibitory rates of BAE, Mv, Mv-3-glc, and Mv-3-gal on H2O2-induced MDA were 52.69 ± 12


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11.73%, 103.25 ± 16.36%, 123.16 ± 28.95%, and 47.14 ± 2.59%, respectively (Figure 4A).

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Mv-3-glc exhibited the strongest inhibitory effects.

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Effects on Antioxidant Enzymes SOD, CAT, and GSH-PX in ARPE-19

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Supernatants. After exposure to 800 µmol/L H2O2, the SOD activity decreased from 394.79

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± 34.67 to 296.63 ± 35.90 mU/L (0.75 ± 0.09-fold of the control; P < 0.05), and the CAT

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content decreased from 44.92 ± 3.47 to 16.43 ± 6.62 mU/L (0.36 ± 0.14-fold of the control; P

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< 0.001), but the GSH-PX content had no significant change. Blueberry anthocyanins could

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decrease the consumption of these antioxidant biomolecules to some extent, but the changes

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were not always significant. The inhibitory rates of BAE, Mv, Mv-3-glc, and Mv-3-gal were

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30.02 ± 2.32%, 100.41 ± 6.45%, 79.29 ± 4.91%, and 75.04 ± 15.79%, respectively, for

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H2O2-consumed SOD and 16.69 ± 1.75%, 72.75 ± 4.34%, 14.81 ± 1.11%, and 49.99 ±

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11.26%, respectively, for CAT (Figure 4B and C). The crude blueberry anthocyanin extract

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had no influence on SOD and CAT. Mv and Mv-3-glc could increase the SOD activity (P

Protective Effects of Blueberry Anthocyanins against H2O2-Induced

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