Effects of Sesaminol Feeding on Brain AÎ² Accumulation in a

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Effects of sesaminol feeding on brain A# accumulation in a senescence-accelerated mouse-prone 8, SAMP8 Shigeru Katayama, Haruka Sugiyama, Shoko Kushimoto, Yusuke Uchiyama, Masato Hirano, and Soichiro Nakamura J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.6b01237 • Publication Date (Web): 27 May 2016 Downloaded from http://pubs.acs.org on May 31, 2016

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Journal of Agricultural and Food Chemistry

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Effects of sesaminol feeding on brain Aβ β accumulation in a

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senescence-accelerated mouse-prone 8, SAMP8

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Shigeru Katayama,† Haruka Sugiyama,† Shoko Kushimoto,† Yusuke Uchiyama,§

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Masato Hirano,§ and Soichiro Nakamura*,†

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†

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Minamiminowa, Kamiina, Nagano 399-4598, Japan

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§

Department of Bioscience and Biotechnology, Shinshu University, 8304

Takemoto Oil & Fat Co., Ltd. 11 Hamacho, Gamagori, Aichi 443-0036, Japan

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*

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Soichiro Nakamura, PhD

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Department of Bioscience and Biotechnology, Shinshu University, 8304

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Minamiminowa, Kamiina, Nagano 399-4598, Japan

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Tel&FAX: +81-265-77-1609

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e-mail: [email protected]

Corresponding author:

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ACS Paragon Plus Environment


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Abstract

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Alzheimer’s disease (AD) is characterized by the progressive accumulation of

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extracellular beta-amyloid (Aβ) aggregates. Recently, the senescence-accelerated

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mouse P8 (SAMP8) model was highlighted as useful model of age-related AD.

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Therefore, we used the SAMP8 mouse to investigate the preventive effects of sesame

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lignans on the onset of AD-like pathology. In preliminary in vitro studies, sesaminol

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showed the greatest inhibitory effect on Aβ oligomerization and fibril formation

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relative to sesamin, sesamolin, and sesaminol-triglucoside. Hence, sesaminol was

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selected for further evaluation in vivo. In SAMP8 mice, feed-thorough sesaminol

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(0.05% w/w in standard chow) administered over a 16-week period reduced brain Aβ

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accumulation and decreased serum 8-OHdG, an indicator of oxidative stress.

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Furthermore, sesaminol administration increased the gene and protein expression of

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ADAM10, which is a protease centrally involved in the non-amyloidogenic processing

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of amyloid precursor protein. Taken together, these data suggest that long-term

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consumption of sesaminol may inhibit the accumulation of pathogenic Aβ in the brain.

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Keywords: Aβ, ADAM10, brain, SAMP8, sesaminol

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Introduction

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Alzheimer’s disease (AD) is the most common form of dementia and is

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characterized by cognitive decline and memory impairments that are severe enough to

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interfere with normal activities of daily living.1,

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accumulation of beta-amyloid (Aβ) in extracellular neuritic plaques as well as the

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hyperphosphorylation and aggregation of tau protein in intracellular neurofibrillary

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tangles. The accumulation of these proteins has subtle effects on synaptic efficacy and

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eventually leads to neuronal cell death and the progression of cognitive impairments.3

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Although many researchers have screened for anti-amyloidal agents among naturally

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occurring compounds, an effective and safe preventive therapy for dementia has not

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yet been developed.

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AD is associated with the

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The senescence-accelerated mouse (SAM) model of accelerated aging was

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initially established by Takeda et al.4 and has since been widely utilized for the study

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of aging. SAM prone 8 (SAMP8) mice show spontaneous age-related behavioral

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disorders, cognitive deficits, hair loss, and a shortened life span. Recently, it was

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shown that the SAMP8 mouse also exhibits age-related AD pathology, including

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alterations in secretase-mediated amyloid precursor protein (APP) processing,5,

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Aβ plaque accumulation,7 and hyperphosphorylated tau aggregation.8 Accordingly, the

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SAMP8 mouse has recently been used as an model of age-related AD onset in 3


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preclinical studies.9, 10

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In Asian countries, sesame (Sesamum indicum L.) has attracted significant

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attention for its potential health benefits. Sesame oil is rich in linoleic and oleic acids

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and has a high content of lignans including sesamin, sesaminol, sesamolinol, and

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sesaminol glucosides.11 Sesame lignans have been reported to have health beneficial

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effects on serum cholesterol, blood pressure, and liver function.12, 13 In the present

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study, we investigated the ability of sesame lignans to inhibit Aβ aggregation in vitro

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and to alleviate age-related oxidative stress, neuroinflammation, amyloidogenic Aβ

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processing, and Aβ accumulation in vivo.

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Materials and Methods

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Materials

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Sesamin, sesaminol, sesamolin, and sesaminol-triglucoside were supplied from

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Takemoto Oil & Fat Co., Ltd. (Aichi, Japan). Human Aβ1-42 was purchased from

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Peptide Institute Inc. (Osaka, Japan). Malondialdehyde (MDA) was purchased from

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Wako Pure Chemical Industries Ltd (Osaka, Japan), and 2-thiobarbituric acid (TBA)

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was purchased from Sigma-Aldrich Japan (Tokyo, Japan). All other chemicals were of

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analytical reagent grade.

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Preparation of Aβ β fibrils

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Human Aβ1-42 was dissolved in 0.1% ammonia solution to yield at a final

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concentration of 250 µM, separated into aliquots, and then stored at −80 °C until use.

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To prepare amyloid fibrils, aliquots of Aβ were diluted to a concentration of 25 µM in

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50 mM phosphate buffer (pH 7.5) containing 100 mM NaCl. The Aβ solution was then

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sealed in a microcentrifuge tube and incubated at 37 °C for 24 h in the presence or

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absence of test compounds (25 µM).

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ThT fluorescence assay

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Amyloid fibril formation was monitored using a Thioflavin T (ThT) assay.

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Briefly, 6 µL of each sample was dissolved in ethanol and added to 1.2 mL of ThT

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solution containing 50 mM glycine-NaOH buffer (pH 8.5). The mixture was briefly

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vortexed and fluorescence was quantified using a FP-6200 spectrofluorometer (JASCO,

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Tokyo, Japan). The excitation and emission wavelengths were 446 nm and 490 nm,

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respectively. The fluorescence signal of each sample was measured 4 times, averaged,

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and the fluorescence value of a ThT blank was subtracted from the result. Ethanol was

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used as a control.

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Electron microscopy

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Transmission electron microscopy (TEM) was used to characterize the structural

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morphology of Aβ fibrils in the presence or absence of test compounds. After

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incubation, 5 µL of each amyloid sample was spotted onto a collodion-coated 400

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mesh copper grid (Nisshin EM Co., Tokyo, Japan). The grids were negatively stained

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with 1% phosphotungstic acid and visualized with a JEM-1400 microscope (JEOL,

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Tokyo, Japan) operated with an accelerating voltage of 80 kV.

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Immuno-dot blotting

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After incubation, 5 µL of each amyloid sample was spotted onto a nitrocellulose

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membrane and dried at room temperature for 30 min. The membrane was then blocked

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at room temperature for 60 min with 3% (w/v) bovine serum albumin (BSA) in

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phosphate buffered saline (PBS) and subsequently washed 3 times with PBS

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containing 0.05% (w/v) Tween-20 (PBST). Next, the membrane was incubated with

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rabbit anti-oligomer antibody A11 (1:2500 dilution; Invitrogen, Camarillo, CA) for 60

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min at room temperature, washed 3 times with PBST, and incubated with anti-rabbit

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IgG-horseradish peroxidase secondary antibody (1:5000 dilution; Invitrogen) for 60

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min at 37 °C. Finally, the membrane was washed with PBST and developed in Pierce

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Western Blotting Substrate (Pierce, Rockford, IL) for 1 min at room temperature.

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Immunoreactive proteins were visualized using an AE-9300 Ez-Capture system (ATTO,

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Tokyo, Japan) using enhanced chemiluminescence.

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Animals

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Male SAMP8 mice aged 15 weeks were purchased from Japan SLC, Inc.

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(Shizuoka, Japan) and allowed to acclimate for 1 week before the experimental period.

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All mice were individually housed in a facility with controlled temperature (20-23 °C)

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and humidity (40-70%) on a 12-h light/dark cycle (light on at 8:00 am). All

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experiments were conducted in accordance with the institutional guidelines established

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by Shinshu University for the care and use of laboratory animals.

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Animal protocol

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Sixteen-week-old SAMP8 mice were divided into a control group and a

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sesaminol group (n = 8). The control group was fed a normal diet of standard chow

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(MF, Oriental Yeast, Tokyo, Japan) and the sesaminol group was fed a diet of standard

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chow containing 0.05% (w/w) freeze-dried sesaminol powder. Food and tap water

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were available ad libitum for a 16-week treatment period. Food intake and body weight

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were measured once per week. After 16 weeks, mice were tested in the Morris water

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maze and then sacrificed for blood and tissue harvesting. Brain and liver tissues were

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quickly excised on ice, weighed, and placed into RNA Later (Sigma) for RNA isolation,

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RIPA lysis buffer (Santa Cruz Biotechnology, Santa Cruz, CA) for Western blotting, or

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PBS containing 5 mM BHT for MDA measurement. Tissues were stored at −80 °C

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until use.

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Soluble Aβ β enzyme-linked immunosorbent assay (ELISA) Soluble Aβ was quantified in brain tissue samples using the ELISA method.

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Briefly, whole brain tissue was homogenized in 70% formic acid and the homogenates

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were centrifuged at 100,000g for 60 min at 4 °C. The resulting supernatants were

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neutralized with 1 M Tris and soluble Aβ was measured using a human Aβ1-42 ELISA

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kit (Wako Pure Chemicals) according to manufacturer specifications.

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Determination of serum 8-hydroxydeoxyguanosine (8-OHdG)

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Serum was filtered with an Amicon Ultra-10,000 NMWL centrifuge tube

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(Millipore, Bedford, MA) to remove any high molecular weight impurities that might

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have interfered with spectrophotometric measurement. Serum 8-OHdG was measured

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using a competitive ELISA kit (8-OHdG Check, Nikken Seil, Shizuoka, Japan)

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according to manufacturer specifications.

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Thiobarbituric acid reactive substances (TBARS) assay

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To assess lipid peroxidation, a TBARS assay was performed on brain tissue

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homogenates according to the method of Ohkawa et al.14 with modifications. Briefly,

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100 µl of 8.3% (w/v) tissue homogenate was combined with 0.2 mL of 8.1% sodium

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dodecyl sulfate, followed by the addition of 1.5 mL of 20% acetic acid (pH 3.5) and

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1.5mL of 0.8% TBA. The reaction mixture was vortexed and subsequently incubated at

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90 °C for 1 h. After cooling on ice, 5 mL of a butanol:pyrimidine mixture (15:1) was

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added and the resultant mixture was centrifuged at 1,000g for 15 min. The absorbance

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of the upper layer of the supernatant was measured at 532 nm with a UV-1700

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spectrophotometer (Shimadzu, Kyoto, Japan). MDA was used as standard and the

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results were expressed as µmol of MDA/g of protein measured by the Bradford

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method.

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Morris water maze testing

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Spatial learning and memory was evaluated using the Morris water maze.15 A

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circular plastic pool (diameter, 120 cm; height, 50 cm) was filled with water and the

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water temperature was maintained at 25 ± 1 °C. An escape platform (diameter, 11 cm)

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was hidden 1 cm below the surface of the water in a fixed location. Mice were trained

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to locate the platform in training trials 3 times per day for 4 consecutive days. Each

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trial lasted a maximum of 60 s and the inter-trial interval was approximately 30 s.

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When mice successfully located the platform, they were allowed to remain on the

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platform for 10 s; otherwise, mice that were unable to locate the platform within 60 s

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were placed on the platform for 10 s at the end of trial. One day after the last training

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trial, mice were subjected to a 60 s probe trial in which the platform was removed from

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the pool. Latency to cross the former location of the platform was recorded by an

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overhead camera and the ANY-maze video tracking system (Stoelting Co., Wood Dale,

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IL).

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Quantitative real-time PCR

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Total RNA was extracted from whole brain tissues using the RNAiso Plus kit

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(TaKaRa Bio, Shiga, Japan) according to manufacturer specifications. cDNA was

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synthesized from 0.25 mg of total RNA using a reverse transcription system (High

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capacity RNA-to-cDNA kit, Applied Biosystems). Quantitative real-time PCR was

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performed using the StepOne real-time PCR System (Applied Biosystems) with the

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KAPA SYBR FAST Universal qPCR kit (Kapa Biosystems, Cape Town, South Africa).

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The expression level of each gene was determined using the comparative Ct method

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and normalized to β-actin as an internal standard. The PCR reaction consisted of one

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cycle at 95 °C for 3 min, 40 cycles (95 °C for 3 s and 60 °C for 20 s), and a final

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dissociation cycle (95 °C for 15 s). The primer sequences used in this study are shown

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in Table 1.

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Western blotting

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Whole tissue extracts were homogenized in RIPA lysis buffer and subsequently

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centrifuged at 13,500g for 10 min at 4 °C. Tissue supernatants were collected and

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protein concentrations were determined using the DC protein assay kit (Bio-Rad,

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Hercules, CA) with BSA as a standard. Protein samples (25 µg) were then

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electrophoresed on 15% polyacrylamide gels and electroblotted onto polyvinylidene

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fluoride (PVDF) membranes (Clear Blot Membrane-P; ATTO). PVDF membranes

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were next incubated with antibodies against ADAM10 (1:5000 dilution; Enzo Life

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Sciences, Farmingdale, NY), BACE1 (1:100 dilution; Enzo Life Sciences), or β-actin

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(1:250 dilution; Enzo Life Sciences), followed by incubation with an anti-rabbit IgG

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horseradish peroxidase-conjugated secondary antibody (1:2500 dilution; Anaspec, San

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Jose, CA). Chemiluminescence detection was performed using Pierce Western blotting

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substrate (Thermo Scientific, Rockford, IL) and the AE-9300 Ez-Capture System

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(ATTO).

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Statistical analysis Data are presented as mean ± SE. Statistical analyses were performed by Student’s t-test. A level of p

Effects of Sesaminol Feeding on Brain AÎ² Accumulation in a

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