Soybean-Derived Glycine-Arginine Dipeptide Administration Promotes

1. Factor Expression in the Mouse Brain. 2. 3. Ayano Shimizu. †. , Takakazu Mitani. ‡ .... 50 synaptic plasticity. Among these, brain-derived neur...
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Bioactive Constituents, Metabolites, and Functions

Soybean-Derived Glycine-Arginine Dipeptide Administration Promotes Neurotrophic Factor Expression in the Mouse Brain Ayano Shimizu, Takakazu Mitani, Sachi Tanaka, Hiroshi Fujii, Motohiro Maebuchi, Yusuke Amiya, Mitsuru Tanaka, Toshiro Matsui, Soichiro Nakamura, and Shigeru Katayama J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b01581 • Publication Date (Web): 09 Jul 2018 Downloaded from http://pubs.acs.org on July 10, 2018

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Journal of Agricultural and Food Chemistry

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Soybean-Derived Glycine-Arginine Dipeptide Administration Promotes Neurotrophic

2

Factor Expression in the Mouse Brain

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Ayano Shimizu†, Takakazu Mitani‡, Sachi Tanaka†, Hiroshi Fujii‡, Motohiro Maebuchi§,

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Yusuke Amiya∥, Mitsuru Tanaka∥, Toshiro Matsui∥, Soichiro Nakamura†, and Shigeru

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Katayama*†‡

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8304 Minamiminowa Kamiina, Nagano 399-4598, Japan

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Department of Agriculture, Graduate School of Science and Technology, Shinshu University,

Interdisciplinary Cluster for Cutting Edge Research (ICCER), Shinshu University, 8304

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Minamiminowa Kamiina, Nagano 399-4598, Japan

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§

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Ibaraki 300-2497, Japan

13

14



Research Institute for Creating the Future, Fuji Oil Holdings Inc., 4-3, Kinunodai, Tsukuba,

Division of Bioresources and Bioenvironmental Sciences, Faculty of Agriculture, Graduate

School, Kyushu University, Hakozaki, Higashi-ku, Fukuoka 812-8581, Japan

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Corresponding author:

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Shigeru Katayama, Ph.D.

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Telephone/FAX: +81-265-77-1603. E-mail: [email protected]

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ABSTRACT

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Brain-derived neurotrophic factor (BDNF), a member of the neurotrophin family, plays an

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important role in cognitive abilities, including memory and learning. We have demonstrated that

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soybean protein hydrolysate (SPH) diet suppresses age-related cognitive decline, via the

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upregulation of BDNF in a mouse model of senescence. Our purpose was to identify novel

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bioactive peptides in SPH, which enhance BDNF expression. We treated mouse primary

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astrocytes with SPH, as well as with its positively-charged chromatographic fraction. Significant

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increases in the expression of BDNF were observed in the treatment with positively-charged

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fraction of SPH. Among the synthesized peptides, the dipeptide glycine-arginine (GR) increased

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BDNF expression in vitro, and TNBS-LC-TOF-MS analysis showed the presence of GR in the

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SPH. Furthermore, its administration in vivo increased the expression of BDNF in the cerebral

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cortex, and the number of neurons in hippocampus and cerebral cortex. These data indicate that

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GR might promote neurogenesis by upregulating BDNF levels.

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Key words: astrocytes, BDNF, soybean protein hydrolysate, glycine-arginine dipeptide,

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neurogenesis

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INTRODUCTION

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Aging is an inevitable biological process which constitutes the progressive decline of

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physical and functional tissue capacities. Brain aging is defined as a progressive loss of

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neurophysiological functions, which is often accompanied by age-related neurodegeneration.

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Particularly, cognitive impairment and dementia, including Alzheimer's disease (AD), have

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emerged as major debilitating illnesses associated with old age.1,2. In recent years, there has been

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an increasing interest in natural dietary bioactive compounds with potential neuroprotective

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properties, in order to prevent cognitive impairment and dementia.3 Omega-3 (ω-3)

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polyunsaturated fatty acids (PUFAs), and especially docosahexaenoic acid (22:6,n-3), mainly

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present in fish oil, play an important role in normal brain development and cognitive function.4

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Some studies in animal models and humans suggest that dietary ω-3 PUFA intake can slow down

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cognitive decline and attenuate the physiological brain disabilities.5 On the other hand, botanical

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polyphenols have been reported to attenuate mood disorders and cognitive impairment,6,7 and

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some flavan-3-ols, including anthocyanidins, such as catechin and epicatechin, have been shown

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to selectively reach the brain and accumulate in the form of metabolites.8

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Neurotrophins have emerged as a group of powerful molecular mediators in central

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synaptic plasticity. Among these, brain-derived neurotrophic factor (BDNF) and neurotrophin-3

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(NT-3) are considered as key players in the neurobiological mechanisms of learning and

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memory.9 Thus, the therapeutic modulation of BDNF and NT-3 levels is a promising treatment

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strategy for neurological and psychiatric disorders, in which BDNF levels are dysregulated. 3 ACS Paragon Plus Environment

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Moderate exercise has been demonstrated to increase BDNF levels in the plasma.10 Maejima et

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al. reported that moderate exercise contributes in the protection against cognitive function

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decline in the elderly, through the up-regulation of BDNF expression in the hippocampus.11

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Food-derived peptides have various functions in human health. Recently, functional

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peptides with brain-health benefits have been reported. Yamada et al. demonstrated that the

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enzymatic digestion products of β-lactoglobulin, a major component of bovine milk whey,

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exhibit anxiolytic-like activity in mouse behavioral experiments.12 We have previously reported

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that oral administration of soybean protein hydrolysates (SPH) for 6 months results in

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suppression of the age-related cognitive decline in senescence-accelerated prone 8 (SAMP8)

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mice, and in upregulation of BDNF and NT-3 expression in the brain of SPH-fed mice.13

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However, the bioactive peptides in SPH remain unknown. In this study, we identified the

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bioactive peptides in SPH, which exhibited a BDNF-enhancing effect. We further investigated

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the in vivo effects of the oral and intraperitoneal administration of SPH, as well as of a specific

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bioactive peptide, in healthy mice.

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MATERIALS AND METHODS

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Chemicals

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SPH composed mainly of di- and tripeptides was obtained from Fuji Oil Co. Ltd. (Osaka,

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Japan). The synthesized peptides KGRKG, KGRK, GRKG, KRG, KGR, GRK, RKG, KG, GR,

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and RK were obtained from GenScript Japan (Tokyo, Japan). 4 ACS Paragon Plus Environment

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Isolation of aromatic and positively-charged peptide fraction from SPH

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For the preparation of aromatic peptides, 20 % (w/w) SPH solution (pH 7.0) was prepared,

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by adding 19 volumes of 95 % ethanol, and kept at -20 °C for 30 min. After centrifugation at

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5,000 rpm for 10 min, the supernatant, containing the aromatic peptides, was collected. For the

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preparation of positively-charged peptides, SPH was dissolved in 10 mM ammonium formate

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(pH 8.0) and loaded onto a DEAE-Sepharose Fast Flow column (GE Healthcare, Wauwatosa, WI,

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USA). The flow-through fraction, containing the positively-charged peptides, was collected .

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Animals

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Eight-week-old male and ten-week-old female C57BL/6J mice were purchased from

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Charles River Laboratories Japan, Inc., (Kanagawa, Japan) and mated to obtain embryos.

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Pregnant females were sacrificed and embryos were collected at embryonic day 18 for primary

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cultures. For in vivo experiments, six-week-old male C57BL/6L mice were also obtained from

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Charles River Laboratories Japan, Inc. The mice were provided with free access to a

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commercially-available pellet diet (MF; Oriental Yeast Co., Tokyo, Japan) and water. They were

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acclimatized for one week before the start of the study. The animal room was maintained at

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constant temperature (22 ± 2°C) and humidity (55 ± 10%), with a 12-h light/dark cycle. All

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experiments were performed according to the institutional guidelines for animal experimentation

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at Shinshu University (Animal ethics, No. 270076 and 270077). 5 ACS Paragon Plus Environment

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Primary astrocyte cultures

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Astrocyte cultures were prepared from brains of mouse embryos. After removing the

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uterus, brains were immediately dissected from each embryo, immersed in Dulbecco's modified

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Eagle's medium/nutrient mixture F12 (DMEM/F12; Gibco/Invitrogen, Carlsbad, CA, USA), and

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treated with 0.25 % trypsin (Wako, Tokyo, Japan) in phosphate buffered saline (PBS), containing

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0.01 % DNase I (Worthington Biochem, Freehold, NJ, USA), at 37 °C for 10 min. After

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treatment, the cell suspension was centrifuged at 1500 rpm for 5 min and the supernatant was

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discarded. The cells were resuspended in DMEM/F12, containing 10 ng/mL G-5 supplement

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(Invitrogen, Carlsbad, CA, USA) and 10 % fetal bovine serum (Biowest, Nouaille, France), by

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gentle trituration with a pipette and the cell suspension was filtered through a cell strainer to

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remove undigested tissue. The cells were seeded at 2.0×106 cells in 75-cm2 flasks and incubated

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in a humidified atmosphere of 95% air and 5% CO2, at 37°C. Seven days later, astrocytes were

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harvested with trypsin and seeded at 2.0 × 105 cells in 60-mm dishes for real-time PCR, ELISA,

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and western blotting analyses. After four days of incubation, cells were treated with 1 mg/mL

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SPH or with 1 or 10 µM synthesized peptides for 6 or 24 h, and collected for qPCR or western

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blotting. The cultured cells were confirmed to be astrocytes by immunostaining with an anti-glial

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fibrillary acidic protein (GFAP) antibody.

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Luciferase reporter assay for CREB phosphorylation 6 ACS Paragon Plus Environment

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Primary astrocytes were cultured at a density of 2.5 × 106 cells/well in a 48-well plate and

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transiently transfected with the reporter vectors pCRE-Luc14 and pRL-SV40 (control reporter

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vector; Promega, Madison, WI, USA), by using Lipofectamine 2000, for 24 h. After the

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transfection medium was replaced with fresh medium, the cells were incubated with SPH at a

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concentration of 0.5 or 1 mg/mL for 4 h. The transfection efficiency was normalized to that of

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pRL-SV40. Firefly and Renilla luciferase activities were measured using the Dual Luciferase

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reporter assay kit and the GloMax 20/20 Luminometer (Promega). The relative light unit units

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were normalized to the constitutively active Renilla luminescence of the same sample.

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Real-time quantitative PCR (qPCR)

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Total RNA was prepared using RNAiso Plus (TaKaRa Bio, Shiga, Japan). RNA was used

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to reverse transcription using the ReverTra Ace real-time qPCR kit (Toyobo, Osaka, Japan).

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Real-time qPCR was carried out using a Kapa SYBR Fast qPCR kit (Kapa Biosystems, Woburn,

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MA, USA). Quantification of relative gene expression was performed by using the Thermal

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Cycler Dice real time system software (Takara Bio). β-actin was used as an internal standard to

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normalize the amount of total RNA present in each reaction mix, and the relative expression in

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each experimental group was expressed as the fold change compared to a control sample, using

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the

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5'-GCGGCAGATAAAAAGACTGC-3' (forward) and 5'-CTTATGAATCGCCAGCCAAT-3'

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(reverse), and for β-actin, 5’-CACTATTGGCAACGACAAGCGGTTC-3’ (forward) and

comparative

∆∆Cq

method.

The

primer

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sequences

for

BDNF

were

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5’-ACTTGCGGTGCACGATGGAG-3’ (reverse).

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Western blot analysis

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Astrocytes were collected using a cell scraper and homogenized in RIPA lysis buffer

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(Santa Cruz Biotechnology, Santa Cruz, CA, USA) on ice. Cell lysates were centrifuged at

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12,000 g for 10 min at 4°C, and the supernatants were collected. The total protein concentration

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was determined using the BCA assay with bovine serum albumin (BSA) as the standard. Proteins

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(15 µg) were separated by electrophoresis, on 15 % polyacrylamide gels, and transferred to

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polyvinylidene difluoride membranes (Clear Blot Membrane-P; ATTO, Tokyo, Japan). After

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blocking with 3 % BSA in Tris-buffered saline buffer, containing 0.1 % Tween 20, for 60 min at

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room temperature, the membranes were incubated with primary antibodies against BDNF

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(1:1,000, Abcam, Cambridge, MA, USA) and β-actin (1:5,000, Santa Cruz Biotechnology)

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overnight at 4°C or for 1 h at room temperature, respectively. The membranes were then

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incubated with the secondary anti-rabbit HRP-conjugated (1:5,000, Santa Cruz Biotechnology)

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and anti-mouse HRP-conjugated antibodies (1:10,000, Santa Cruz Biotechnology) for 60 min at

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room temperature, for the detection of BDNF and β-actin, respectively. Chemiluminescence

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detection was performed using the Pierce Western blotting substrate (Thermo Scientific,

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Rockford, IL, USA) and AE-9300 Ez-Capture (ATTO). proBDNF and mature BDNF (mBDNF)

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were detected as a band of 35 kDa and 28 kDa, respectively.

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Quantitative assay of peptides in SPH by LC-TOF-MS

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Target peptides in SPH were assayed by a liquid chromatography time-of-flight mass

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spectrometry (LC-TOF-MS) in combination with 2,4,6-trinitrobenzen sulfonate (TNBS)

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derivatization technique.15 The quantification of peptides was performed according to our

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reported standard additon method16, because the method could compensate unexpected

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reduction of MS detection by interferring matrix without any use of appropriate internal

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standard or expensive stable isotope labelling technique. Briefly, target peptides were spiked

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into SPH solution (1 mg/mL in 0.1 M borate buffer, pH 8.0) with the final cncentration of 0, 4,

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8, and 16 µg/mL. The spiked solutions (40 µL) were subjected to TNBS derivatization reaction

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by adding 10 µL of 150 mM TNBS solution in 0.1 M borate buffer (pH 8.0) at 30 °C for 30 min.

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Fifty µL of 0.2 % formic acid were, then, added to 50 µL of the resultant mixture, and an

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aliquot (10 µL) was injected to LC-TOF-MS.

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Chromatographic separation was carried on a Waters Biosuite C18 column (2.1 × 150 mm,

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3-µm pore size) (Waters, Milford, MA, USA). A linear gradient elution of methanol (0–100%),

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containing 0.1% formic acid, at a flow rate of 0.2 mL/min, was performed at 40°C. Electrospray

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ionization (ESI)-TOF-MS analysis was carried out in positive ESI mode, and the mass-detection

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range was set at 100-1,000 m/z. The conditions of the ESI source were as follows: drying gas

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(N2) flow rate = 8.0 L/min; drying gas temperature = 200 °C; nebulizing gas pressure = 1.6 bar;

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capillary voltage = 3,800 V. All data acquisition and analyses were performed by using a Bruker

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data analysis 3.2 software. To ensure optimal conditions for the analyses, we calibrated the 9 ACS Paragon Plus Environment

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detector using sodium formate clusters (10 mM sodium formate in water:acetonitrile (1:1, v/v)).

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The calibration solution was injected at the beginning of the run and all spectra were calibrated

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prior to characterization. The width was set at 0.05 m/z for the mono-isotopic isolation of the

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target ions: trinitrophenyl (TNP)-KGR and TNP-GRK = 571.222 m/z; TNP-GR = 443.127 m/z;

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TNP-RK = 514.201 m/z.

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In vivo experiments

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For oral administration, 7-week-old male C57BL/6J mice were treated with saline, GR

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dipeptide (15 or 100 mg/kg, once a day), or SPH (100 mg/kg, once a day) for 14 days. For

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intraperitoneal administration, mice were treated with saline, GR dipeptide (15 mg/kg, once a

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day), or SPH (100 mg/kg, once a day) for five days. Twenty-four hours after administration, the

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mice were sacrificed, and brains were collected, and processed for western blot analysis.

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Immunostaining

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The collected brain tissues were fixed with 30 % paraformaldehyde and then embedded in

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paraffin. The sections (6 µm) were blocked in PBS containing 10 % goat serum and 0.1 % Tween

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20 and were incubated with a rabbit anti-NeuN antibody (Abcam) diluted in 1:750. After

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washing six times with 0.1 % BSA in PBS, the sections were incubated with secondary antibody

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(Alexa Fluor 488 goat anti-rabbit IgG, Abcam) diluted 1:1,000, washed six times with 0.1 %

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BSA in PBS, and then were mounted in a mounting medium with 4',6-diamidino-2-phenylindole 10 ACS Paragon Plus Environment

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(DAPI) (ImmunoSelect Antifading Mounting Medium; Dianova, Hamburg, Germany). Images

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were taken using an EVOS fl fluorescence microscope (Advanced Microscopy Group, Bothell,

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WA, USA).

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Statistical analysis

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Data are presented as mean ± standard deviation (SD). Data on BDNF mRNA expression

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in primary astrocytes were analyzed using Student’s t-test. For all other comparisons, one- or

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two-way ANOVA followed by Dunnett’s post hoc test was used. Differences with p 0.988. Moreover, we found that GR dipeptide was

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successfully detected and quantified in the SPH used in this study (2.02 mg/g of SPH, Figure S1),

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whereas other peptides (KGR, GRK, and RK) failed to be detected within the present MS

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conditions, suggesting that SPH contains at least GR dipeptide at >0.2 wt% level.

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Effects of GR and SPH administration on BDNF expression in the mouse brain

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To examine the effects of GR administration in vivo, we measured BDNF expression

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levels in the brain after oral administration for 14 days. In the hippocampus, there was a trend for

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increased proBDNF levels after treatment with 15 mg/kg/day of GR, but it was not statistically

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significant (Figure 4A). Neither GR nor SPH treatment changed mBDNF expression in the

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hippocampus. On the other hand, a significant increase in proBDNF expression was observed in

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the cerebral cortex of mice treated with GR at 100 mg/kg/day, whereas mBDNF expression

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remained unaffected (Figure 4B). 13 ACS Paragon Plus Environment

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We next investigated the effects of the intraperitoneal administration of GR (15

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mg/kg/day) and SPH (100 mg/kg/day). In the hippocampus, GR treatment increased proBDNF

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expression, whereas no changes in mBDNF expression level were observed by either treatment

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(Figures 5A). In contrast, in the cerebral cortex, significant increases in the expression of both

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proBDNF and mBDNF were observed in the GR- and SPH-treated mice (Figure 5B). These

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results suggest that GR administration enhances BDNF levels in the brain, and especially in the

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cerebral cortex; this effect was stronger after intraperitoneal than oral GR administration.

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We further examined whether intraperitoneally-administered SPH can promote

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neurogenesis, a process that includes the proliferation, survival, and differentiation of neurons.

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For this purpose, we used the neuron-specific nuclear protein NeuN, as a mature neuronal marker.

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The number of NeuN positive cells was higher in GR- than saline-treated mice, in both the

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dentate gyrus of the hippocampus (Figure 6A) and the cerebral cortex (Figure 6B), thus

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suggesting that GR treatment might positively affect neurogenesis.

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DISUCSSION

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In this work, we demonstrated that the positively-charged peptides GRK and GR, induce

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proBDNF expression in primary astrocytes, while GR administration in vivo enhances BDNF

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expression in the brain. According to the LC-TOF-MS results, only the GR, but not the GRK

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peptide, was detected in the SPH. It is possible that during SPH preparation, the enzymatic

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treatment might have resulted in the cleavage of GRK to GR. To clarify the in vivo effect and 14 ACS Paragon Plus Environment

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metabolism of the GR dipeptide, we compared the effects of the oral to those of the

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intraperitoneal administration. The most significant increase was observed in proBDNF

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expression in the cerebral cortex of mice intraperitoneally-treated with GR for five consecutive

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days. However, a significant, but lower, increase was also observed in the cortex of mice

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orally-administered with GR. This discrepancy between the oral and intraperitoneal

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administration might be due to the potential cleavage of GR by enzymes of the digestive tract.

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No significant changes were observed after oral administration of SPH, suggesting that GR is a

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more potent agent than SPH. Our previous work has demonstrated that long-term administration

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of SPH enhances BDNF and NT-3 expression in the brain and suppresses the cognitive decline

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observed in SAMP8 mice.13 However, the effects of oral GR administration in SAMP8 mice still

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remain unclear and require further studies.

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BDNF is known to bind to TrkB and thus activate the phosphatidylinositol-3 kinase

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signaling pathway and the release of neurotransmitters for presynaptic sites.18,19 Previous studies

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have reported that the cyclic pentapeptide cyclo(-D-Pro-Ala-Lys-Arg-) was designed to act as an

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effective BDNF-like agonist. Massa et al.20 also demonstrated that subregion b of the second

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loop (Ser-Lys-Gly-Gln-Leu) of BDNF is involved in TrkB activation. Furthermore,

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Cardenas-Aguayo et al.17 demonstrated that tetrapeptides, such as Ac-Ile-Lys-Arg-Gly-CONH2

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and Ac-Ser-Lys-Lys-Arg-CONH2, which mimic different active regions of BDNF, activate the

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TrkB receptor and induce BDNF expression. These reports suggest that positively-charged amino

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acids in BDNF, such as lysine or arginine, might play important roles in its binding to TrkB. In 15 ACS Paragon Plus Environment

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our study, we show that the positively-charged fraction of SPH increases BDNF expression in

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primary astrocytes. GR was identified as one of bioactive peptides promoting BDNF expression

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from the SPH; however, the GR content in SPH was estimated at around 0.2 wt% level,

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suggesting that there must be still potential peptides in the positively-charged fraction of SPH.

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Here we focused on the the amino acid sequence of KGRKG and did not investigated the

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sequence combination except that. Therefore, there might be a possibility that oligopeptides

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containing GR sequence exert a potent enhancing effect of BDNF expression.

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In general, it is considered difficult for orally-administered peptides to reach the brain, as

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they are easily digested to single amino acids; however, some bioactive peptides found in SPH

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appear to directly contribute to BDNF regulation in the brain. Indeed, we found that both oral

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and intraperitoneal administration of SPH and of its bioactive peptide, GR, increase BDNF levels

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in the brain. However, it is still unknown whether these bioactive peptides cross the blood brain

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barrier and reach different brain areas. Further investigation of the peptide distribution in the

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brain will be necessary for the clarification of the underlying molecular mechanisms.

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BDNF exerts an important role in the formation of appropriate synaptic connections, both

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during development and during learning and memory in adults.21 It is well known that BDNF

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protein is secreted as proBDNF, which is then cleaved extracellularly via the tissue plasminogen

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activator/plasmin proteolytic cascade.22 Similarly, using western blotting, we detected BDNF in

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the cell lysate mainly as proBDNF. We measured the secreted BDNF in the cell culture medium

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by ELISA; however, BDNF levels were lower in the medium than the cell lysate (data not 16 ACS Paragon Plus Environment

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shown). Since astrocytes are known to closely interact with neurons, a co-culture assay of

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astrocytes and neurons will be necessary to examine the effects of SPH and its bioactive peptides

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on BDNF secretion.

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Several studies have demonstrated that orally administrated phytochemicals exhibit

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positive effects on brain functions. For example, the oral administration of quercetin, a natural

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flavonoid, increases the proliferation of mouse neural stem cells in the dentate gyrus, while

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quercetin-3-o-glucuronide, one of the major quercetin metabolites, activates the Akt/cyclin D1

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and BDNF signaling pathways.23 Further, oral administration of 7,8-dihydroxyflavone, a potent

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TrkB agonist, prevents the deposition of amyloid beta (Aβ) and inhibits the synaptic loss seen in

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the hippocampus of a transgenic mouse model of AD, by activating TrkB signaling.24

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Concerning the beneficial effects of soybean-derived bioactive compounds on brain function,

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isoflavone is considered as a potent neuromodulating agent. Oral administration of soybean

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isoflavone in rats suppresses the Aβ1-42-induced neuronal damage, via activation of the

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N-methyl-D-aspartate receptor signaling pathway.25 However, Chatterjee et al. reported that

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genistein, an isoflavone abundantly found in soybean, enhances the accumulation of the Aβ

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peptide, by increasing the expression of the amyloid precursor protein.26 Therefore, the

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beneficial effect of soybean isoflavone against AD is still questionable. In this study, we

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demonstrated that bioactive peptides of soybean ingredients are good candidates as

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neuromodulating agents, which might contribute to the suppression of Aβ1-42-induced neuronal

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damage. Hence, further studies on the potential beneficial effects of soybean peptides against AD 17 ACS Paragon Plus Environment

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are required, with a special focus on the effects of the positively-charged fraction or the GR

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dipeptide, identified in this study.

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In conclusion, we demonstrated that the GR dipeptide contained in SPH enhances BDNF

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expression both in vitro, in primary astrocytes, and in vivo, in the brain, and especially in the

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cerebral cortex. These findings provide useful insight on the biochemical processes by which soy

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proteins and peptides may promote good health in the elderly.

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ACKNOWLEDGMENTS

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This study was supported by the Ministry of Education, Culture, Sports, Science, and

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Technology of Japan KAKENHI (Grant No. 15K07426); the Ministry of Agriculture, Forestry,

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and Fisheries of Japan (Integration research for agriculture and interdisciplinary fields); and Fuji

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Oil Holdings Inc. (Osaka, Japan).

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Notes

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The authors declare no competing financial interest.

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References

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Fusco, D.; Colloca, G.; Monaco, M. R. L.; Cesari, M. Effects of antioxidant

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Maturitas. 2007, 58, 327-39.

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FIGURE CAPTIONS

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Figure 1. Effect of treatment with soybean protein hydrolysate (SPH) on brain-derived

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neurotrophic factor (BDNF) expression in primary cultured astrocytes. (A) Western blot

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analysis and quantification of proBDNF and mBDNF expression levels in the astrocyte lysates

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treated with SPH (0.5 or 1 mg/mL). (B) Relative luciferase activity, reflecting CREB

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phosphorylation levels, in primary astrocytes treated with different concentrations of SPH.

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Values are expressed as mean ± SD, n = 3. Data were analyzed by one-way ANOVA followed by

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Dunnett’s post hoc tests. *p