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The Leaves of Persimmon (Diospyros kaki Thunb.) Ameliorate Nmethyl-N-nitrosourea (MNU)-Induced Retinal Degeneration in Mice Kyung-A Kim, Suk Woo Kang, Hong Ryul Ahn, Youngwoo Song, Sung Jae Yang, and Sang Hoon Jung J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.5b02578 • Publication Date (Web): 11 Aug 2015 Downloaded from http://pubs.acs.org on August 17, 2015

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The Leaves of Persimmon (Diospyros kaki Thunb.) Ameliorate N-methyl-N-nitrosourea (MNU)-Induced Retinal Degeneration in Mice Kyung-A Kim,†,§ Suk Woo Kang,† Hong Ryul Ahn,† Youngwoo Song,† Sung Jae Yang,‡ and Sang Hoon Jung†,§*

†

Natural Products Research Center, Korea Institute of Science and Technology (KIST),

Gangneung, Korea ‡

Department of Ophthalmology, University of Ulsan, Gangneung Asan Hospital, Gangneung,

Korea §

Department of Biological Chemistry, University of Science and Technology (UST), Daejeon,

Korea.


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ABSTRACT

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The purpose of the study was to investigate the protective effects of the ethanol extract of

3

Diospyros kaki (EEDK) Persimmon leaves to study N-methyl-N-nitrosourea (MNU)-induced

4

retinal degeneration in mice. EEDK was orally administered after MNU-injection. Retinal

5

layer thicknesses were significantly increased in the EEDK-treated group, compared with the

6

MNU-treated group. The outer nuclear layer was preserved in the retinas of EEDK-treated

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mice. Moreover, EEDK treatment reduced the MNU-dependent up-regulation of glial

8

fibrillary acidic protein (GFAP) and nestin expression in Müller and astrocyte cells. EEDK

9

treatment also inhibited MNU-dependent down-regulation of rhodopsin expression.

10

Quercetin exposure significantly attenuated the negative effects of H2O2 in R28 cells,

11

suggesting that quercetin can act in an anti-oxidative capacity. Thus, EEDK may be

12

considered as an agent for treating or preventing degenerative retinal diseases, such as

13

retinitis pigmentosa and age-related macular degeneration.

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Keywords

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Diospyros kaki; N-methyl-N-nitrosourea; persimmon; photoreceptor; retinal degeneration

17 18



19

INTRODUCTION

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Retinitis pigmentosa and age-related macular degeneration (AMD) are leading

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worldwide causes of retinal degeneration and blindness.1, 2 Several important risk factors for

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retinal degeneration have been identified, and substantial evidence indicates that retinal

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degeneration is associated with reactive oxygen species (ROS).3 In the signaling mechanism

24

of vision, light is converted into electronic signals via retinal photoreceptors, a process that

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requires a large supply of oxygen.4 Oxygen is continuously consumed during the visual

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process; therefore, retinas are highly exposed to ROS, such as superoxide anions, hydroxyl

27

radicals, and hydrogen peroxide. Although anti-oxidative defenses exist to protect the retina

28

from ROS, the excessive accumulation of ROS causes photoreceptor cell death and,

29

subsequently, retinal damage and visual loss.5 This model is supported by epidemiological

30

studies wherein patients with retinal degeneration exhibit high ROS levels and low levels of

31

anti-oxidative proteins, including glutathione, superoxide dismutase, and catalase, compared

32

to healthy controls.6 These findings explain how antioxidants may protect against retinal

33

degeneration, and it has been reported in epidemiological studies that the consumption of

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foods containing high concentrations of antioxidants are associated with a reduced risk for

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developing retinal degeneration.7, 8 Therefore, the consumption of antioxidants from natural

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foods or nutritional supplements can be helpful in intervention against retinal degeneration.

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N-methyl-N-nitrosourea (MNU) is known to be a potent alkylating agent that interacts

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with DNA, and leads to the loss of retinal photoreceptor cells.9, 10 Although the underlying

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mechanism has not been clearly elucidated, Prater et al. reported that MNU induced the

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production of ROS via protein alkylation, which promoted apoptosis.11

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It has also been reported that NADPH oxidase plays a critical role in the generation of

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the oxidative stress, which was shown to promote cone cell death in retinitis pigmentosa,

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using a genetic model (rd1 mice).12

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Persimmon (Diospyros kaki Thunb.) belongs to the Ebenaceae and is widely cultivated

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in Korea, China, Japan, and Eastern Asia. The leaves of D. kaki are rich in bioactive

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compounds, such as polyphenols, flavonoids, and vitamins13 and it has been reported that

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these compounds, which contain gallic acid derivatives and glucose units linked together via

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glycosidic bonds, have potent radical-scavenging and anti-oxidative activities.14,

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compounds have also been used for medicinal purposes as remedies to treat a wide variety of

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conditions, including neuronal injury and neurodegeneration, due to their anti-oxidative

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properties.16, 17

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Such

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Antioxidants have been shown to have beneficial effects in human eyes.18, 19 The goal of

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this study was therefore to determine whether D. kaki has protective effects on MNU-induced

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retinal degeneration.

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MATERIALS AND METHODS

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Chemicals

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Anti-nestin and anti-rhodopsin antibodies were purchased from Novus Biologicals

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(Littleton, CA, USA). Anti-glial fibrillary acidic protein (GFAP), anti-superoxide dismutase

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(SOD)-3, and anti-chicken IgG antibodies were purchased from Millipore (Billerica, MA,

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USA). Anti-SOD-1, anti-SOD-2, anti-mouse IgG, anti-rabbit IgG, and anti-sheep IgG

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antibodies were purchased from Santa Cruz (Santa Cruz, CA, USA). Anti-GAPDH and

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glutathione peroxidase (GPx-1) antibodies were purchased from Cell Signaling Technology

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(Beverly, MA, USA). Alexa 488-conjugated anti-sheep, Alexa 594-conjugated anti-mouse



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and Alexa 633-conjugated anti-sheep antibodies were purchased from Invitrogen (Carlsbad,

67

CA, USA). A blocking solution was purchased from Dako (Santa Clara, CA, USA), and

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mounting medium was purchased from Vector Laboratories (Burlingame, CA, USA). Zoletil

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and Rumpun were purchased from Virbac Laboratories (Fort Worth, TX, USA) and Bayer

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(Newbury, UK), respectively, and used as anesthesia. All other chemicals and reagents were

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purchased from Sigma-Aldrich (St. Louis, MO, USA).

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Plant materials and sample preparation

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D. kaki was collected in Gangneung, Gangwon Province of Korea in August 2013. A

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voucher specimen (D-521) was deposited at the KIST Gangneung Institute. Eight hundred

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grams of the dried D. kaki leaves were extracted 3 times with 7 L of ethanol at room

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temperature for 3 h in an ultrasonic cleaning bath (model RK 158s, Bandelin, Germany) and

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filtered through Whatman No. 1 filter paper. The combined filtrate was concentrated to

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dryness by rotary evaporation at 40 °C to obtain 33 g (yield: 4.13%) of the ethanol extract of

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D. kaki leaves (EEDK). HPLC-PDA-MS analysis was applied using a Thermo Accela HPLC

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system and LCQ FLEET ion trap mass spectrometer (Thermo Fisher Scientific Inc., SanJose,

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CA, USA) for compound determinations in EEDK. The mobile phase consisted of 0.1%

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formic acid in water (Solvent A) and 0.1% formic acid in acetonitrile (Solvent B), which was

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used with a YMC-Triart C18 column (3-µm particle size, 150 mm × 4.6 mm I.D., YMC Co.,

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Kyoto, Japan). A linear gradient (from 10 to 60% solvent B for 30 min) at a flow rate of 1

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mL/min was used at 254 nm. The mass spectrometer conditions were as follows: negative ion

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mode; mass range, m/z 200−800; capillary voltage, 10.0 V; capillary temperature, 350 °C.

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The NMR spectra were recorded on a Varian 500 MHz NMR system (Varian, Palo Alto, CA,


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USA) at 500 MHz for 1H NMR and 125 MHz for 13C NMR in DMSO-d6. Compounds 1–9

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were purified by semi-preparative HPLC.

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Animals

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Six-week-old male C57BL/6J mice weighing 20–25 g (Central Lab. Animal Inc., Seoul,

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Korea) were housed at 23 ± 0.5 °C and 10% humidity, with a 12-h light-dark cycle. All

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animals were acclimated for at least 1 week, caged in groups of 5 or less, and fed with a diet

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of animal chow and water ad libitum.

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All animal studies were performed in a pathogen-free barrier zone at the KIST

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Gangneung Institute in accordance with the procedure outlined in the ARVO Statement for

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the Use of Animals in Ophthalmic and Vision Research. Procedures used in this study were

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approved by the Animal Care and Use Committee of the KIST Gangneung Institute (No.

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2014-011)

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MNU was used as a negative insult for retinal degeneration and was induced by single

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intraperitoneal (IP) injection of 50 mg/kg MNU (50 mg/kg body weight) in C57BL/6J mice.20

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After the injection of MNU, EEDK was orally administrated at doses of 10, 50, or 100

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mg/(kg·day) for 4 weeks. Animals were examined at 1 and 4 weeks after the injection of

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MNU. Eyes were dissected and examined either for histological or western blot analysis.

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Fixed eyes were cut into 4-µm slices and were then studied by staining with hematoxylin and

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eosin (H&E) or by immunofluorescence.

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Spectral-domain optical coherence tomography (SD-OCT)

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SD-OCT imaging was performed at 1 and 4 weeks after 50-mg/kg MNU treatment. The

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animals were anaesthetized using a mixture of Zoletil (1.6 µg/g, Verbac Laboratories, USA)

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and Rumpun (0.05 µL/g, Bayer, UK). Anesthetized animals were placed in front of the



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Spectral OCT system (Heidelberg Engineering, Germany) and analyzed as described

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previously.21

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Hematoxylin & eosin (H&E) staining

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H&E staining of the retina slides was performed using a standard procedure. Briefly, the

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retina slides were treated with hematoxylin buffer (0.1% hematoxylin and 10% ammonium)

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at room temperature. The slides were then washed 3 times with distilled water and dipped in

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1% eosin Y solution. Next, the slides were washed twice with 95% alcohol and mounted.

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Histological analysis was evaluated under a CKX41 inverted phase contrast microscope

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(Olympus, Tokyo, Japan).

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Western blot analysis

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Proteins were separated on 10% or 12% SDS-PAGE gels and then transferred onto

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polyvinylidene difluoride (PVDF) membranes. The membranes were blocked at room

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temperature in 5% skim milk before overnight incubation at 4 °C with antibodies (diluted as

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indicated in PBS) against nestin (1:1000), GFAP (1:1000), GAPDH (1:2000), rhodopsin

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(1:1000), SOD-1 (1:1000), SOD-2 (1:1000), SOD-3 1 (1:1000), or GPx-1 (1:1000). Binding

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of the primary antibodies was detected following incubation with appropriate horseradish

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peroxidase-conjugated IgG secondary antibodies (diluted 1:5000) for 2 h at room temperature.

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The membranes were developed using an enhanced chemiluminescence detection kit

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(Thermo Scientific, Waltham, MA, USA) and measured by densitometry using an LAS-4000

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image reader and Multi Gauge 3.1 software (Fujifilm, Tokyo, Japan). GAPDH expression

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was detected as a loading control.

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Immunofluorescence staining


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Paraffin-embedded retinal sections were deparaffinized and blocked in normal goat serum

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overnight at 4 °C. Subsequently, the sections were incubated with specific primary antibodies

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against nestin (diluted 1:200), GFAP (diluted 1:200), and rhodopsin (diluted 1:200) overnight

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at 4 °C. The sections were washed 3 times with PBST buffer (8 g/L NaCl, 0.2 g/L KCl, 1.44

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g/L, Na2HPO4, 0.24 g/L, NaH2HPO4, and 0.1% Triton X-100) and incubated with matching

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secondary antibodies (Alexa 488-conjugated anti-sheep, Alexa 594-conjugated anti-mouse,

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and Alexa 633-conjugated anti-chicken antibodies; diluted 1:500) for 2 h at room temperature

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in the dark. The sections were washed with PBST, mounted, and covered with a glass

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coverslip. Staining images were examined using a Leica TCS SP5 confocal microscope

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system (Leica, Wetzlar, Germany).

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Cell culture

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Immortalized rat retinal precursor cells (R28 cells) were purchased from Kerafast

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(Kerafast, Inc., MA, USA). R28 cells were grown in 75-cm2 culture flasks in low-glucose

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Dulbecco's Modified Eagle's Medium (DMEM, HyClone, Logan, UT, USA) supplemented

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with 10% (v/v) heat inactivated fetal bovine serum (FBS; HyClone, Logan, UT, USA) and

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100 U/mL penicillin/streptomycin (HyClone, Logan, UT, USA). Cultures were maintained at

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37 °C in a humidified atmosphere containing 5% CO2. The cells were passaged twice a week.

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R28 cells were pretreated with the indicated compounds for 1 h after 300 µM hydrogen

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peroxide (H2O2) was added to cultures for 24 h.

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Cell viability

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R28 cell viabilities were determined by performing 3-(4,5-dimethylthiazol-2-yl)-2,5-

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diphenyltetrazolium bromide (MTT) assays. Briefly, MTT assays were performed by treating

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cells with MTT solution (0.5 mg/mL final concentration) for 1 h at 37 °C. After a 1-h



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incubation, the blue formazan crystals that formed in intact cells were solubilized with

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DMSO, and absorbance values at 570 and 590 nm were measured with a microplate reader

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(BioTek Instruments, Winooski, VT, USA). Results are expressed as the percent of MTT

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reduction.

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Assessment of ROS production

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To quantify intracellular ROS production, we used DCFH-DA as a probe for free radicals

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22

. The R28 cells were seeded at a density of 5.0 × 103 cells per well into 96-well plates and

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incubated in a humidified atmosphere of 95% air and 5% CO2 at 37 °C. Twenty-four hours

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later, the cell culture medium was replaced with DMEM containing 1% FBS. Superoxide

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radicals effectively oxidize nonfluorescent dichlorofluorescein (DCFH) to fluorescent

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dichlorofluorescein (DCF). The cells were loaded with the radical probe DCFH-DA (10 µM)

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through incubation for 20 min at 37 °C. Then, the cell culture medium was replaced to

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remove the excess probe. To generate radical species, we added KO2 at 1 mM (O2·−) to the

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radical probe-loading medium. Fluorescence was then measured after various durations,

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using excitation and emission wavelengths of 485 and 535 nm, respectively (Luminescence

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Spectrometer LS50B, Perkin-Elmer Ltd., England).

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Statistical analysis

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Experimental values were analyzed by Kruskal–Wallis and Mann–Whitney tests using

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the Bonferroni correction and are presented as mean ± standard deviation. A result of p < 0.05

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was considered to be statistically significant. Quantification of western blotting results was

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performed using Fujifilm Multi Gauge software, version 3.0 (Tokyo, Japan). All statistical

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analyses were performed using SPSS, version 16.0 (IBM Corporation, Armonk, NY).

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RESULTS AND DISCUSSION

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The chromatogram of EEDK showed 9 major peaks (Figure 1). Peak 1 was identified as p-

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salicylic acid by mass (Figure 1) and NMR spectra. The major NMR peaks detected by 1H-

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NMR were 12.33 (1H, s, OH-7), 10.22 (1H, s, OH-4), 7.77 (2H, d, J = 8.7 Hz, H-2, 6), and

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6.80 (2H, d, J = 8.7 Hz, H-3, 5), while the major peaks detected with 13C-NMR were 167.6

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(C-7), 162.0 (C-4), 132.0 (C-2, 6), 121.9 (C-1), and 115.6 (C-3, 5). Peaks 2–9 were identified

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by MS as quercetin, kaempferol, or their glycoside, galactoside, or galloylated derivatives

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(Figure 1). The chromatograms and spectral data of Peaks 2–9 were consistent with results

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from a previous study. 23

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The concentrations of peaks 1–9 in EEDK were measured as 0.8 ± 0.02 mg/g (1), 4.8 ±

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0.08 mg/g (2), 10.6 ± 0.18 mg/g (3), 12.7 ± 1.24 mg/g (4), 9.4 ± 0.15 mg/g (5), 17.3 ± 0.27

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mg/g (6), 17.2 ± 0.23 mg/g (7), 2.1 ± 0.03 mg/g (8) and 5.0 ± 0.08 mg/g (9).

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In order to observe retinal degeneration caused by MNU treatment, we performed H&E

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staining of retinal tissues, as shown in Figure 2a and 2b. The total thickness of the retinal

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layer and outer nuclear layer significantly decreased in the mouse group treated with MNU

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alone (Figure 2). However, the retinas of C57BL/6J mice administrated EEDK were

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protected against MNU-induced retinal degeneration (Figure 2a and 2b). During the

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observation period, the inner nuclear layer and ganglion cell layer appeared to be unaffected

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except for in MNU-treated group at 4 weeks post-MNU treatment. Our results also revealed

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that EEDK administration protected the outer retina against MNU-induced retinal

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degeneration. These results are consistent with those from a previous study demonstrating

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that MNU affects the outer layer by causing a reduction in the number of photoreceptors in

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the retina,24 and demonstrate protective effects of EEDK against retinal degeneration.

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We observed further morphological changes in the retinas of live mice via SD-OCT scanning, as shown in Figure 3. 25



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SD-OCT is a non-invasive scanning technique that has been widely adopted clinically to

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assess retinal disorders involving changes in retinal thickness, structural changes, and edema,

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among others. SD-OCT enables accurate analysis because it can provide cross-sectional

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images of retinas with high resolution. Recently, OCT has been used in many studies to

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evaluate changes occurring during retinal degeneration in small animal-model studies. 21

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Figure 3a shows an OCT image of a normal retina (Figure 3a). The thickness of outer

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retinal layer was slightly reduced in the MNU-treated group at 1 week, compared to the

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control group (Figure 3b). After 4 weeks, significant differences were found in retinal

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thickness between the control- and MNU-treated groups (Figure 3b). However, MNU-

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induced retinal thinning was significantly attenuated by pre-treatment with EEDK (Figure 3b).

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The reference drug Lutein also showed a protective effect against MNU-induced retinal

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thinning (Figure 3c).

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Western blot analysis was conducted to investigate the effects of EEDK treatment on

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nestin, GFAP, and rhodopsin expression in mouse retinal tissues after MNU-induced retinal

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degeneration. Nestin, a type-VI intermediate filament protein is expressed in stem cells and

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progenitor cells, as well as the related Müller cells that differentiate in the central nerve

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system.26 The observed configuration of Müller cells and astrocytes has demonstrated that

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these glial cells help maintain the structural integrity of the retina.

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expression is often observed during late-stage pathology of neurodegenerative diseases.

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As shown in Figure 4, nestin protein expression was considerably increased in the MNU-

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treated group at 1 week compared to the control group, but nestin expression was not

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increased at 4 weeks post-MNU treatment. This result may have been due to low number of

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cells preserved in the whole retina.

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27

Up-regulated nestin 28, 29

Importantly, the elevation in nectin protein expression was significantly attenuated by EEDK administration (Figure 4a and 4b).


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The results of recent studies have indicated that GFAP, a type-III intermediate filament

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protein, is present in astrocytes and Müller cells in the retina. Increased GFAP expression is a

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reliable biomarker of retinal degeneration.

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time-dependent manner after mice were treated with MNU alone, compared to the control

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group; this up-regulation was inhibited by EEDK administration (Figure 4a and 4b). The dose

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of 50 mg/kg EEDK showed a significant inhibition, compared with the MNU-treated group.

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30

GFAP protein expression was up-regulated in a

Rhodopsin is a photosensitive pigment in the rod-containing outer retinal layer and plays 24

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a key role in the visual system.

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protein was down-regulated in a time-dependent manner compare to the control group.

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However, rhodopsin protein expression was maintained by EEDK administration (Figure 4a

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and 4b), and the photoreceptor tissue layer was well preserved by EEDK treatment. These

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results demonstrated that the protective effects of EEDK treatment on retinal degeneration

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might be related to the expression of retinal factors including nestin, GFAP, and rhodopsin

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during MNU-induced retinal degeneration.

In the MNU-treated group, expression of the rhodopsin

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To further evaluate the protective effects of EEDK on retinal degeneration, we analyzed

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nestin, GFAP, and rhodopsin expression by immunofluorescence staining, as shown in Figure

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4c and 4d. Immunofluorescence staining revealed that the expression levels of nestin and

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GFAP, which are related to early signs of gliosis, were significantly increased in the MNU-

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treated group compared with the control group (Figure 4c and 4d). However, the 50 and 10

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mg/kg EEDK-treated groups showed decreased nestin and GFAP protein expression

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compared with that of the MNU-treated group.

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The loss of photoreceptor cells was evaluated by immunofluorescence staining for

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rhodopsin. The expression of rhodopsin was considerably decreased in the MNU-treated

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group relative to that observed in the control group (Figure 4c and 4d). In contrast, rhodopsin

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protein expression was preserved in the 50 and 10 mg/kg EEDK-treated groups (Figure 4c



264

and 4d). As shown in Figures 2, 3, and 4, the 100 mg/kg EEDK-treated groups showed no

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significant differences compared with the MNU-treated group. Thus, administration of EEDK

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can potentially confer a protective benefit, but a high dose may elicit retinotoxicity.

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As observed by western blot analysis, immunofluorescence staining demonstrated the

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capacity of EEDK to inhibit retinal degeneration by down-regulating nestin and GFAP

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expression, and by up-regulating rhodopsin expression.

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Cell viability assays were performed to confirm the protective effects of compounds 1–9

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(Figure 5). As shown in Figure 5a, only quercetin (10 µM; compound 8) significantly

272

inhibited H2O2-induced cell death (~65%) among compounds 1–9. The inhibition of H2O2-

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induced cell death by quercetin occurred in a dose-dependent manner (Figure 5b).

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Recently, Keiichi et al. reported that the administration of exogenous antioxidants such

275

as α-tocopherol, ascorbic acid, Mn (III) tetrakis (4-benzoic acid) porphyrin, and α-lipoic acid

276

protected against oxidative damages in an rd1 mouse model. 31 Quercetin is known to possess

277

antioxidant and free radical-scavenging activity. 32

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DCFH-DA was used as a radical probe to quantify intracellular ROS production. The

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intracellular ROS levels caused by O2·− were increased relative to the control by up to 360%.

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However, pre-treating cells with EEDK and quercetin decreased the ROS levels in a dose-

281

dependent manner (Figure 6a and 6b).

282

The expression levels of antioxidant proteins such as SOD 1–3 and Gpx-1 induced by

283

MNU in mouse retina were evaluated by western blot analysis. Our results showed that SOD-

284

1, SOD-3, and Gpx-1 expression levels were significantly inhibited by EEDK treatment

285

(Figure 6c).

286

Endogenous antioxidant enzymes, including SODs, GPx, and catalase, served to reduce

287

the oxidative stress of photoreceptors in retinas. Previous findings have shown that

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endogenous antioxidant enzymes up-regulated under oxidative stress conditions decreased


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ROS production and prevented cone cell death in several retinitis pigmentosa models. 33, 34 Therefore, the protective effects of EEDK in MNU-induced retinal degeneration may be due to its direct or indirect anti-oxidative properties.

292

In summary, our results showed that EEDK has protective effects against oxidative

293

stress-induced cell death in vitro and considerably alleviated MNU-induced retinal

294

degeneration in vivo. In particular, compound 8 (quercetin) could possibly be the acting

295

compound due to anti-oxidative capacity. Quercetin glycosides, Compounds 2–4, did not

296

show significant activity. However, it was known that flavonoid mono-glucosides such as

297

quercetin-3-O-β-glucoside are hydrolyzed by lactase phlorizin hydrolase

298

glucosidase

299

that compounds 2–4 (quercetin-3-O-β-galactoside, quercetin-3-O-β-glucoside, quercetin-3-O-

300

β-2″galloylglucoside) may be absorbed from the small intestine of humans as aglycone forms

301

to exhibit anti-oxidative activity. Collectively, our findings indicate that EEDK may be useful

302

as a potential agent for the prevention and treatment of retinal degeneration, such as with

303

retinitis pigmentosa and AMD.

36, 37

35

and cytosolic β-

to be absorbed in the small intestine as aglycone forms. This demonstrates

304 305

ABBREVIATIONS USED

306

AMD, age related macular degeneration; CAT, catalase; CNS, central nerve system; EEDK,

307

ethanol extract of Diospyros kaki; ELM, external limiting membrane; GFAP, glial fibrillary

308

acidic protein; GCL, ganglion cell layer; GSH, glutathione; GPx, glutathione peroxidase; INL,

309

inner nuclear layer; IP, intraperitoneal; IPL, inner nuclear layer; MNU, N-methyl-N-

310

nitrosourea; ONL, outer nuclear layer; RD-1, retinal degeneration-1 mice; RNFL/GC, retinal

311

nerve fiber layer/ganglion cell layer; ROS, reactive oxygen species; RPE, retinal pigment



312

epithelium; SD-OCT, spectral-domain optical coherence tomography; SOD, superoxide

313

dismutase

314

AUTHOR INFORMATION

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Corresponding Authors

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*Telephone: +82-33-650-3653. Fax: +82-33-650-3679. E-mail: [email protected].

317

Funding

318

This work was financially supported by an intramural grant (2Z04381) from the Korea

319

Institute of Science and Technology (KIST), Republic of Korea.

320

Notes

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The authors declare no competing financial interest.

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(2) Fletcher, A. E., Free radicals, antioxidants and eye diseases: evidence from

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440

Figure legends

441

Figure 1. (a) HPLC-UV chromatogram of EEDK. Retention time for compound 1: 7.665 min;

442

compound 2: 11.350 min; compound 3: 11.514 min; compound 4: 11.828 min; compound 5:

443

12.441 min; compound 6: 12.889 min; compound 7: 13.305 min; compound 8: 17.918 min;

444

compound 9: 21.222 min. (b) MS spectra of major peaks (1–9). (c) Identified compounds.

445 446

Figure 2. Histological evaluation in hematoxylin and eosin (H&E) staining. Panel (a) shows

447

retinal cross sections of control (A1, A2), MNU-treated (B1, B2), MNU plus EEDK (100

448

mg/kg)-treated (C1, C2), MNU plus EEDK (50 mg/kg)-treated (D1, D2), and MNU plus

449

EEDK (10 mg/kg)-treated (E1, E2) mice at 1 and 4 weeks, with or without MNU and/or

450

EEDK treatment. Panel (b) shows total retinal thicknesses and outer layer thicknesses at the

451

indicated time points. Scale bar = 50 µm. Experimental values are expressed as mean ±

452

S.E.M. (**) p < 0.01, (***) p < 0.001 from 3 independent experiments (n = 9 mice per group).

453 454

Figure 3. Evaluation of retinal thicknesses by optical coherence tomography. (a) SD-OCT

455

cross-sectional image of a control retina. The following retinal layers were labeled:

456

RNFL/GC, retinal nerve fiber layer/ganglion cell layer; IPL, inner plexiform layer; INL,

457

inner nuclear layer; OPL, outer plexiform layer; ONL, outer nuclear layer; ELM, external

458

limiting membrane; RPE, retinal pigment epithelium. (b) Representative images were

459

obtained from the experimental animal retina at 1 and 4 weeks after, with or without MNU

460

and/or EEDK treatment; control (A1, A2), MNU-treated (B1, B2), MNU plus EEDK (100

461

mg/kg)-treated (C1, C2), MNU plus EEDK (50 mg/kg)-treated (D1, D2), and MNU plus

462

EEDK (10 mg/kg)-treated (E1, E2) (c) Lutein plus vitamin A supplement was used as


Page 22 of 33

Page 23 of 33


463

reference drug (Bright lutein, Ildong Pharmaceutical, Korea). Reference drug group was

464

received lutein plus vitamin A supplement (0.2%/kg/day). Scale bar = 50 µm.

465 466

Figure 4. (a) Western blot analysis showing the effects of EEDK treatment on protein-

467

expression levels of nestin (Müller cell marker; ~220–240 kDa), glial fibrillary-acidic protein

468

(GFAP; Müller cell and astrocyte marker; 51 kDa), rhodopsin (photoreceptor marker; 40

469

kDa), and GAPDH (loading control; 37 kDa) in C57BL/6J mice treated with 50 mg/kg MNU,

470

at 1 and 4 weeks. (b) Quantification of relative protein levels. (c, d) Representative

471

immunostaining images were stained anti-netin (purple), anti-rhodopsin (green), and anti-

472

GFAP (red) in the retina (Nuclei were stained with DAPI) and obtained by confocal

473

microscopy (original magnification, 630×). Retinal cross section from control, MNU-treated,

474

MNU plus EEDK (100 mg/kg)-treated, MNU plus EEDK (50 mg/kg)-treated, and MNU plus

475

EEDK (10 mg/kg)-treated mice at 1 and 4 weeks, with or without MNU and/or EEDK

476

treatment. Scale bar = 50 µm. Experimental values are expressed as mean ± S.E.M. (*) p

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