Visible Light-Induced Lipid Peroxidation of Unsaturated Fatty Acids in

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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.

1

ACS Paragon Plus Environment


1

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

25

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

26

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

27

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

28

(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,

32

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

39

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

40

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

41

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

42

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

43

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

44

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

3



45

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

46

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

47

reaction consequently leads to retinal pigment epithelial dysfunction and

48

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

49

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

50

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

51

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

52

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

53

increasing amount of attention as potential agents for improving retinal defense

54

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

55

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

56

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

57

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

58

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

60

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

61

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

62

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

63

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

64

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

65

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

66

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


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67

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

68

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

69

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

70

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

71

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

72

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

74

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

75

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

76

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

77

retinal damage.

78

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

79

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

80

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

81

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

82

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

83

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

84

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

85

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

86

light-induced retinal damage was checked in vivo.

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MATERIALS AND METHODS

88

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

89

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

91

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

92

use.

93

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

94

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

95

Oasis HLB cartridges used for isolating and purifying anthocyanins were purchased

96

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

97

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

98

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

99

high-performance liquid chromatography (HPLC) grade.

The ethanol and

100

hydrochloric acid were purchased from China National Pharmaceutical Industry

101

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

102

during extraction.

103

DHA,

AA,

OA,

methylsulfonic

acid,

1-Methyl-2-phenylindole,

104

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

105

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

106

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

107

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

108

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

109

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

110

chloride was purchased from China National Pharmaceutical Industry Corporation

111

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


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112

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

113

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

114

Institute of Biotechnology (Jiangsu, China).

115

Extraction and Fractionation. Polyphenol separation and purification from

116

blueberries were performed as previously described with several modifications.

117

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

118

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

119

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

120

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

121

supernate was filtered through a moderate speed 102 qualitative filter paper

122

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

123

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

124

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

125

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

126

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

127

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

128

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

129

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

130

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

131

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

132

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

133

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


9,25,26


134

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

135

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

136

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

137

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

138

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

139

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

140

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

141

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

142

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

143

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

144

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

145

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

146

kept at -20 °C until use.

147

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

148

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

150

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

151

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

152

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

153

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

154

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

155

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


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156

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

157

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

158

Determination of Total Anthocyanins. Total anthocyanin content was measured

159

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

160

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

161

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

162

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

163

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

164

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

165

which was calculated as follows:

166

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

167

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

168

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

169

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

170

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

171

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

172

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

173

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

174

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

175

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

176

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

177

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



178

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

179

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

180

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

181

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

182

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

183

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

185

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

186

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

187

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

189

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

190

UFA-rich mediums were analyzed.

191

To evaluate the potential protective activities of blueberry polyphenols against

192

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

193

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

194

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

195

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

196

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

197

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

198

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

199

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


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200

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

201

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

202

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

203

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

204

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

205

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

206

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

207

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

208

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

209

fluorescence from each well was recorded by a multifunctional microplate reader

210

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

211

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

212

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

213

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

214

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

215

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

216

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

217

(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

221

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



Page 12 of 41

222

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

223

advance.

224

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

225

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

226

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

227

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

228

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

229

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

230

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

231

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

232

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

233

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

234

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

235

calculated according to the MDA standard curve.

236

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

238

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

239

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

241

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

242

atmosphere.

243

UFAs

Reagent

Cytotoxicity.

1:

1-methyl-2-phenylindole

Cell

viability

o

was

dissolved

in

a

C under a humidified 5% CO2

was

measured

12


by

MTT

Page 13 of 41


244

[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

246

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

247

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

248

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

249

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

250

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

251

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

257

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.

260

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

261

Biotechnology) according to the manufacturer’s instructions.

262

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.

264

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



266

MTT assay.

267

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

268

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

269

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

270

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

272

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

273

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

274

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

275

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

276

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

277

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

278

Whole blueberries and blueberry polyphenols were fed to animals by intragastric

279

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

280

morphological changes in retinas were observed.

281

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

285

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

286

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

287

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


Page 14 of 41

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288

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

289

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

290

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

291

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

292

run in triplicate. p

Visible Light-Induced Lipid Peroxidation of Unsaturated Fatty Acids in

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