Visible Light-Induced Lipid Peroxidation of Unsaturated Fatty Acids

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Visible Light-Induced Lipid Peroxidation of Unsaturated Fatty Acids in Retina and the Inhibitory Effects of Blueberry Polyphenols Yixiang Liu, Di Zhang, Jimei Hu, Guang-Ming Liu, Jun Chen, Lechang Sun, Zedong Jiang, Xichun Zhang, Qingchou Chen, and Baoping Ji J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.5b04341 • Publication Date (Web): 12 Oct 2015 Downloaded from http://pubs.acs.org on October 12, 2015

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Journal of Agricultural and Food Chemistry

Visible Light-Induced Lipid Peroxidation of Unsaturated Fatty Acids in Retina and the Inhibitory Effects of Blueberry Polyphenols Yixiang Liu,† Di Zhang,§ Jimei Hu,※ Guangming Liu,† Jun Chen,† Lechang Sun,† ※ Zedong Jiang,† Xichun Zhang,† Qingchou Chen,† and Baoping Ji*,※ †

College of Food and Biological Engineering, Jimei University, Xiamen, Fujian, PR

China. §

School of Food and Biological Engineering, Jiangsu University, Zhenjiang, Jiangsu,

PR China. ※

College of Food Science & Nutritional Engineering, China Agricultural University,

Beijing, PR China.

Running title: blueberry polyphenols protecting against visible light-induced retinal injury

*

Corresponding author: Email, [email protected]; Fax, +86-010-62347334.

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ABSTRACT: The lipid peroxidation of unsaturated fatty acids (UFAs) in retina not

2

only threatens visual cells but also affects the physiological health of retina. In this

3

work, the potential damages caused by daily visible light exposure on retinal UFAs

4

were evaluated via a simulated in vitro model. At the same time, benefits of dietary

5

supplementation of blueberries to the eyes were also assessed. After prolonged light

6

exposure, lipid peroxidation occurred for both docosahexaenoic and arachidonic acids

7

(DHA and AA, respectively). The oxidized UFAs presented obvious cytotoxicity and

8

significantly inhibited cell growth in retinal pigment epithelium cells. Among the

9

different blueberry polyphenol fractions, the flavonoid-rich fraction, in which

10

quercetin was discovered as the main component, was considerably better in

11

preventing visible light-induced DHA lipid peroxidation than the anthocyanin- and

12

phenolic acid-rich fractions. Then the retinal protective activity of blueberry

13

polyphenols against light-induced retinal injury was confirmed in vivo. Based on the

14

above results, inhibiting lipid peroxidation of UFAs in retina is proposed to be another

15

important function mechanism for antioxidants to nourish eyes.

16

KEYWORDS: Unsaturated fatty acids, retina, lipid peroxidation, blueberries,

17

polyphenols

18 19 20 21 22 2

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INTRODUCTION

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Unsaturated fatty acids (UFAs), which are abundant in the outer photoreceptor

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segments of the retina, perform important functions in retinal structure and functions.

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In human retina, the major UFAs in the outer photoreceptor segments include

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docosahexaenoic acid (DHA, 22:6n–3), oleic acid (OA, 18:1) and arachidonic acid

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(AA, 20:4n–6), and the levels of the three UFAs reached about 50%, 10%, and 8% of

29

the total fatty acids, respectively.1-3 Increasing evidences have shown that both DHA

30

and AA affect photoreceptor membrane function though altering permeability, fluidity,

31

thickness,

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oxidation-induced photoreceptor apoptosis and could promote rod and cone

33

development. DHA may also be involved in phototransduction and rhodopsin

34

regeneration.4-6

and

lipid

properties.4

Moreover,

DHA

alone

could

prevent

35

However, the negative effects of UFAs in vision health are beginning to attract

36

researcher attention, because an increasing number of people are currently suffering

37

from certain eye diseases. These eye diseases are due to overexposure to the light of

38

video terminals, such as computers, widescreen mobile phones and televisions, and

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are attributed to the lipid peroxidation of UFAs in the retina induced by excessive

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light radiation.7-9 This kind of photo-induced retinal injury presents physiological

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inevitability. Besides being rich in UFAs, the retina is where light is focused.

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Moreover, the retinal area is always under high oxygen tension because of its visual

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imaging function and active metabolism.10 Numerous present studies have revealed

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that when lipid peroxidation of retinal UFAs occurs, lipid peroxidation products, such

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as malondialdehyde (MDA), hydroxynonenal (HNE), and hydroxyhexenal (HHE), in

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turn react with cellular macromolecules (DNA, proteins, and lipids) in the retina. This

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reaction consequently leads to retinal pigment epithelial dysfunction and

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photoreceptor cell damage.11-13 As a result, new approaches for protecting retina from

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light-induced injury should be the focus of research. Studies should concentrate on

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both preventing lipid peroxidation of UFAs in retina and rescuing retinal pigment

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epithelium (RPE) and photoreceptor cells that have been damaged.

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As a preventive measure, specific antioxidants in the diet are receiving an

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increasing amount of attention as potential agents for improving retinal defense

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against oxidative stress. The Age-Related Eye Disease Study recently confirmed that

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increasing the body’s defenses against oxidative stress with specific antioxidants, such

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as lutein, vitamin A, and lycopene, can preserve vision in patients with macular

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degeneration and reduce the rate of disease progression.14 At present, certain

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polyphenolic compounds that are present in high concentrations in fruits, vegetables

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and other plant-derived foods have been highlighted to present eye benefits. Specific

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dietary flavonoids, such as fisetin, luteolin, quercetin, eriodictyol, baicalein, galangin,

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curcumin, and epigallocatechin gallate, are reportedly capable of protecting retinal

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cells from light- and oxidant stress-induced cell death by blocking the accumulation

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of reactive oxygen species.15,16 In recent years, the unique physiological activity of

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these antioxidants, especially of anthocyanins, in protecting or improving one’s vision

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has been noticed both by nutritionists and food scientists. As for conclusive evidences

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that confirm the visual benefits of anthocyanins, one milestone involves the detection 4

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of anthocyanins in the eye organ after animals, such as rabbits, rats, and pigs, were

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fed with anthocyanin-rich forage.17,18 The possible routes of action may be associated

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with the accelerated resynthesis of rhodopsin,8,19,20 modulation of retinal enzymatic

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activity,19,21 protection of retinal cells by antioxidation,22,23 and improved

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microcirculation.19 In our previous studies, we discovered that polyphenol-rich

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blueberries were efficient in ameliorating light-induced retinal damage in vivo.24 One

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of the protective routes that were clarified is that blueberry anthocyanins exhibit

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protective activities against aging and light-induced damage in RPE cells.9 However,

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as seen in the abovementioned analysis, blocking the lipid peroxidation of UFAs in

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the retina may be another important pathway for blueberries to prevent photo-induced

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retinal damage.

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Therefore, this work aims to further verify whether blueberry polyphenols can

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preserve the outer segments of photoreceptors when the retina is under an

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environment of excessive illumination. Three main fatty acids (DHA, AA, and OA) in

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the outer photoreceptor segments were selected to simulate visible light-induced lipid

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peroxidation in the retina. Then, the differences in cytotoxicity of the three oxidized

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fatty acids on RPE cells were assessed. Different fractions of blueberry polyphenols

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were subsequently evaluated for their potential to defend against lipid peroxidation in

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the retina. Finally, the protective effect of blueberry polyphenols on visible

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light-induced retinal damage was checked in vivo.

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MATERIALS AND METHODS

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Plant Materials and Sample Preparation. Fresh wild blueberries (Vacciniun spp.),

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grown from the Greater Hinggan Mountains in Northeast China, were supplied by the 5

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Science and Technology Bureau of Greater Hinggan Mountains district. For the

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long-term preservation, these fresh berries were freeze-dried and stored at -20 °C until

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use.

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Chemicals and Reagents. Amberlite XAD-7 used for purifying blueberry

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polyphenols was obtained from Sigma (Sydney, Australia). The Sephadex LH20 and

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Oasis HLB cartridges used for isolating and purifying anthocyanins were purchased

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from Amersham Biosciences AB (Uppsala, Sweden) and Waters (Milford, MA),

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respectively. Deionized water was produced using a Milli-Q unit (Millipore, Bedford,

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MA). The acetonitrile from Mallinckrodt Baker (Phillipsburg, NJ) was of

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high-performance liquid chromatography (HPLC) grade.

The ethanol and

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hydrochloric acid were purchased from China National Pharmaceutical Industry

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Corporation Ltd. (Shanghai, China). Analytical reagent-grade solvents were used

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during extraction.

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DHA,

AA,

OA,

methylsulfonic

acid,

1-Methyl-2-phenylindole,

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2′,7′-dichlorofluorescin diacetate (DCFH-DA), 1,1,3,3-Tetramethoxypropan (TMOP)

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and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) were

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purchased from Sigma Chemicals Co. (St. Louis, MO). Standard flavonoids such as

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resveratrol, myricetin, quercetin, rutin, and quercitrin were obtained from Chengdu

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Mansite Co. (Sichuan, China) Fetal bovine serum and Dulbecco’s modified

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Eagle’s/Ham’s F12 media were provided by Invitrogen Co. (Carlsbad, CA). Ferric

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chloride was purchased from China National Pharmaceutical Industry Corporation

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Ltd. Penicillin and streptomycin were obtained from Gibco Life Technologies (Grand 6

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Island, NY). Bovine serum albumin (BSA) was purchased from EMD Biosciences

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(La Jolla, CA). The lactic dehydrogenase (LDH) kit was purchased from Beyotime

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Institute of Biotechnology (Jiangsu, China).

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Extraction and Fractionation. Polyphenol separation and purification from

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blueberries were performed as previously described with several modifications.

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Ten grams of dry blueberries were immersed by 140 mL absolute methanol for 1 h in

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a 250–mL round–bottomed flask, and then homogenized using a homogenizer

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(XHF–D, Ningbo Science & Biotechnology Co., Ningbo, Zhejiang, China). The

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homogenized sample was further centrifuged for 10 min at 4,000 ×g, and the

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supernate was filtered through a moderate speed 102 qualitative filter paper

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(Hangzhou Special Paper Industry Co. Ltd., Hangzhou, Zhejiang, China). The above

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procedure was repeated to re-extract the residue. The two filtrates were combined and

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evaporated using a rotary evaporator at 40 °C and a vacuum pressure of 0.1 MPa. Part

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of the concentrated solution was loaded onto an Amberlite XAD-7 column, and the

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remaining part was lyophilized (crude extract) for further analysis. After 1 h, the XAD

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was washed with about 800 mL 1% (v/v) formic acid aqueous solution to remove

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non-polyphenolic compounds, after which the polyphenolics were eluted with about

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600 mL absolute methanol with 1% (v/v) formic acid. The eluent was concentrated at

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40 °C and lyophilized in vacuum using a freeze dryer (Four–ring Science Instrument

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Plant Beijing Co., Ltd., Beijing, China). Forty-eight hours later, a friable dark red

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powder was obtained. A 50–mg lyophilized sample (polyphenol mixture) was

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resolubilized in pH 7 phosphate buffer and applied to a Sephadex LH20 column. The 7

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column was first washed with a pH 7 phosphate buffer to remove phenolic acids, and

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then with 70% (v/v) methanol acidified with 10% (v/v) formic acid to elute

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anthocyanins and flavonoids. The phenolic acids removed, also called the phenolic

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acid-rich fraction, were collected and lyophilized for later use. The anthocyanin and

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flavonoid fraction was freeze–dried, resolubilized in 5% (v/v) formic acid in water,

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and then applied to an Oasis HLB cartridge. The cartridge was washed with 5% (v/v)

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formic acid, followed by ethyl acetate, and then with 10% (v/v) formic acid in

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methanol. The ethyl acetate eluted flavonoids, and this fraction was called the

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flavonoid-rich fraction after drying in a vacuum at 40 °C using a DZF-6021 vacuum

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drying oven (Hangzhou Lihui Environmental Testing Equipment Co. Ltd., Hangzhou,

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China). The anthocyanins were eluted with acidified methanol, and the eluents were

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freeze-dried and called anthocyanin-rich fraction. These extracts or fractions were

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kept at -20 °C until use.

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Determination of Total Phenolics. The total phenolic content was determined using

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the Folin–Ciocalteu assay as described by Singleton and Rossi,27 with some

149

modifications. Briefly, 1 mL of diluted samples (100 µg/mL) was mixed with 0.5 mL

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of 0.2 mol/L Folin–Ciocalteu reagent in 10 mL volumetric flasks. After 5 min, 1.5 mL

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of a 20% sodium carbonate solution was added. The volume was then increased to 10

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mL with distilled water and mixed thoroughly. After 30 min at room temperature, the

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absorbance was measured at 760 nm using a spectrophotometer (722S, Shanghai

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Precision & Science Instrument Co., Ltd., Shanghai, China), and the total phenolic

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content was calculated using the standard curve of gallic acid. Results were expressed 8

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as weight percentages, which were calculated as follows: gallic acid equivalent

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(GAE)/wet weight of extracts or fractions × 100.

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Determination of Total Anthocyanins. Total anthocyanin content was measured

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using a pH differential method described previously. 28,29 Two dilutions of the extracts

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were prepared, one for pH 1.0 using potassium chloride buffer and the other for pH

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4.5 using sodium acetate buffer. The samples were diluted to a final concentration of

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50 µg/mL. The absorbance was then measured at 515 nm with distilled water as a

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blank. The samples showed no haze or sediment; thus, correction at 700 nm was

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omitted. The total anthocyanin content was then determined using Lambert-Beer’s law,

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which was calculated as follows:

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Total anthocyanin content (mg/L) = (A × MW × DF × 103)/(ε × L)

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where A is the difference in the absorbance at 515 nm between pH 1.0 and 4.5, MW is

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449.2 [the molecular weight of cyanidin-3-glucoside (g/mol)], DF represents a

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dilution factor, and ε denotes the extinction coefficient of cyanidin-3-glucoside

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[26,900 L × mol-1 × cm-1, where L (path length) = 1 cm]. Results were expressed as

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weight percentages, which were calculated as follows: (cyanidin-3-glucoside

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equivalent/wet weight of extracts or fractions) × 100.

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HPLC Analysis. A Shimadzu LC-10 A Series high performance liquid

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chromatography (HPLC) system was used to analyze flavonoids in this experiment.

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The analytical column used was a 150 mm×4.6 mm i.d. Shimadzu Inertsil ODS-3 C18

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column (Shimadzu, Tokyo, Japan) maintained at 35 °C. The injection volume was 20

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µL, and elution solvent--(A) H2O with 1.0% acetic acid and (B) methyl cyanides with 9

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1.0% acetic acid were applied as follows: flow rate 1 mL/min, isocratic 15% B for 1

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min, from 15%–40% B over 30 min, from 40%–60% B over 5 min, from 60%–80% B

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over 2 min, isocratic 80% B for 3 min and from 80%–15% B over 5 min.

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Simulating Light-Induced Lipid Peroxidation of UFAs in Retina. UFAs was

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dissolved in ethanol before being added into serum-free F12 mediums and the final

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concentration of ethanol in the mediums was 0.1% (v:v). The UFAs-rich mediums

184

were transferred into a 24-well plate with the volume of 2 mL every well and then the

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plate was covered with its lid. To simulate the in vivo environment, the plate was

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placed into a cell culture incubator at 37 °C in a humidified 5% CO2 atmosphere.

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Next, the mediums were subjected to white light (420–800 nm) irradiation by an

188

integrated light-emitting diode (LED) lamp system designed by the authors.9 After the

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light illumination, the level of lipid peroxidation and cytotoxicity of the oxidized

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UFA-rich mediums were analyzed.

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To evaluate the potential protective activities of blueberry polyphenols against

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light-induced lipid peroxidation in retina, different fractions of blueberry polyphenols

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were added into the UFAs-rich mediums. The changes of both the lipid peroxidation

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free radicals and aldehydic products were observed until the light exposure ended.

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Measurement of Lipid Hydroperoxide (LOOH) level. Lipid peroxidation free

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radicals produced by UFAs were measured with a DCFH fluorescent probe.30 DCFH

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was prepared from DCFH–DA by basic hydrolysis according to the previous method.

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A 5 mg portion of DCFH–DA was dissolved in 10 mL absolute methanol as the stock

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solution, which was then stored under nitrogen at −20 °C and used within 2 months. A 10

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volume of 500 µL of DCFH–DA stock solution was mixed with 2 mL of 0.01 mol/L

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NaOH aqueous solution at 4 °C and then stored at 4 °C for 30 min in the dark. The

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reaction was neutralized with 2 mL of 0.01 mol/L HCl and diluted with pH 6.0

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phosphate-buffered saline (PBS). The final concentration of the working solution was

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5.0 µmol/L. After irradiation by visible light, cell culture media containing UFAs were

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transferred to a standard 96-well plate. In the plate, 150 µL of different culture

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medium per well was added, and 10 µL of the working solution was added as quickly

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as possible. To prevent air disturbance, we covered the plate with the lid and sealed

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the plate with adhesive tape. After incubation of the media for 1 h at 37 °C, the

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fluorescence from each well was recorded by a multifunctional microplate reader

210

(Molecular Devices Co.) at 522 nm emission and 498 nm excitation.

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Measurement of Active Carbonyl Compounds. The 1-methyl-2-phenylindole

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colorimetric method was employed to assay the levels of active carbonyl compounds

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of UFA rich media after light radiation.31,32 As for omega-3-polyunsaturated fatty

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acids, 4-hydroxy-2-hexenal (4-HHE) is the major product in the peroxidative

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decomposition of DHA. AA is the omega-6-polyunsaturated fatty acids, and its major

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lipid peroxidation products are malondialdehyde (MDA) and 4-hydroxy-2-nonenal

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(4-HNE). In the methanesulfonic acid reaction system, 4-HHE, 4-HNE, and MDA can

218

react with 1-methyl-2-phenylindole. The reaction molar ratio for the three aldehydic

219

products is 1:2, and the reaction end-products present the same characteristic

220

absorption spectrum at 586 nm. Therefore, MDA has been used as the standard

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material in calculating the contents of aldehydic products in DHA, AA, and OA 11

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systems. First, Reagent 1, Reagent 2, and MDA stock solution were prepared in

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advance.

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acetonitrile–methanol (v:v, 3:1) solution to a final concentration of 10 mmol/L.

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Reagent 2: Ferric chloride was dissolved in 37% methanesulfonic acid to a final

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concentration of 60 µmol/L. 10 mmol/L MDA stock solution: 1 mmol TMOP was

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dissolved in 100 mL of 1% (v:v) sulfuric acid aqueous solution, and the mixture was

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reacted for 2 h. To construct the MDA standard curve, the MDA stock solution was

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diluted to 0.5, 1.0, 5.0, 10.0, 20, 30, 50.0 µmol/L with serum-free F12 medium, and

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then 200 µL of the media were mixed with 650 µL of Reagent 1 and 150 µL of

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Reagent 2. The mixture was incubated for 40 min at 37 °C and detected at 586 nm.

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Based on the different absorbance values in different MDA concentrations, the MDA

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standard curve was constructed. For sample analysis, the MDA standard solution was

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replaced with oxidized UFA rich medium, and the level of aldehydic products was

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calculated according to the MDA standard curve.

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Cell Culture. A human RPE cell line, ARPE-19 (ATCC CRL–2302) (American Type

237

Culture Collection, Manassas, Virginia, USA), was used in the present study and

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cultured as previously described.9 Cell cultures were grown in Dulbecco’s modified

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Eagle’s/Ham’s F12 media (Invitrogen) supplemented with 10% fetal bovine serum

240

(Sigma–Aldrich), containing 1% antibiotic mixture of penicillin (100 U/mL) and

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streptomycin (100 mg/mL) (Invitrogen) at 37

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atmosphere.

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UFAs

Reagent

Cytotoxicity.

1:

1-methyl-2-phenylindole

Cell

viability

o

was

dissolved

in

a

C under a humidified 5% CO2

was

measured

12

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[3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide] (Sigma–Aldrich)

245

assay as previously described.9 RPE cells were seeded in 96-well plates

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(Corning–Costar, Corning, NY, USA) at a concentration of 5 × 105 cells/mL, and then

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allowed to attach after 48 h. The medium was then replaced with serum–free F12

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medium containing 0.01, 0.025, 0.05, 0.075, 0.1, and 0.5 mmol/L UFAs, respectively.

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Considering the hydrophobic nature of UFAs, they were firstly dissolved with slight

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ethanol before being added into the medium. Twenty–four hours later, the cell

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supernate was removed for lactate dehydrogenase (LDH) assay. About 200 µL of 0.5

252

mg/mL MTT serum–free F12 medium was added into each well of plates, after which

253

incubation was performed for 4 h. After removal of the MTT solution, 150 µL of

254

dimethyl sulfoxide (Sigma–Aldrich) was added, and the absorbance was measured at

255

570 nm using a plate reader (Molecular Devices Co., CA, USA). Results were

256

expressed as the percentage of viable cells with respect to untreated control cells. Cell

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viability (%) was calculated as follows: [(mean absorbance of the sample – reference

258

absorbance)/mean absorbance of the control] ×100. The cellular release of LDH

259

following UFAs exposure was used as a measure of cellular damage/integrity.

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Enzymatic activity was determined using an LDH kit (Beyotime Institute of

261

Biotechnology) according to the manufacturer’s instructions.

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Cell Viability Assay. After subjecting to light irradiation, the cell supernatants of RPE

263

cells with about 80% confluence were replaced by the oxidized UFAs-rich mediums.

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RPE cells were continuously incubated in this medium for 24 h under the same

265

normal condition described above and cell viability was finally analyzed using an 13

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MTT assay.

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Verification of the Retinal Benefits of Blueberry Polyphenols in vivo. The

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protective effects of blueberry polyphenols on light-induced retinal damage were

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further estimated in a rabbit model. Animal administration and light exposure were

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adopted according to our earlier study.24 All animals were handled according to the

271

Association for Research in Vision and Ophthalmology (ARVO) statement for Use of

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Animals in Ophthalmic and Vision Research. After 1 week adaptation period, the

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rabbits were randomly divided into the following four groups (n = 5 per group):

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control (no light exposure and vehicle administration), Group 1 (light exposure and

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vehicle administration), Group 2 (light exposure and administration of whole

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blueberries, 4.8 g/kg/day), and Group 3 (light exposure and administration of

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blueberry polyphenols, 31.2 mg/kg/day, amounting to 4.8 g of whole blueberries).

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Whole blueberries and blueberry polyphenols were fed to animals by intragastric

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administration for 4 consecutive weeks prior to light exposure. After light exposure,

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morphological changes in retinas were observed.

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Histological Analysis. After isolation of eyes and removal of cornea and lens, the

282

remaining ocular tissue was immersed for 2 h in 4% paraformaldehyde, followed by

283

cryoprotection in 30% sucrose in PBS (pH 7.4) at 4 °C. The eyes were then embedded

284

in paraffin and sliced into 12 µm-thick sections. Then the hematoxylin-eosin staining

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was employed and the retinal sections were analyzed under a light microscope

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(Chongqing Optical and Electrical Instrument Co. Ltd., Chongqing, China).

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Statistical Analysis. The statistical significance of the differences between the control 14

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and treatment groups was analyzed by one-way ANOVA using Origin version 8.0,

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followed by Tukey tests. A normality test showed that all the raw data had a normal

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distribution, and all groups were determined to have equal variance by a variance test.

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Data were expressed as the means ± SD of at least three individual experiments, each

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run in triplicate. p