Myricetin ameliorates scopolamine-induced memory impairment in mice via inhibiting acetylcholinesterase and down-regulating brain iron

Accepted Manuscript Myricetin ameliorates scopolamine-induced memory impairment in mice via inhibiting acetylcholinesterase and down-regulating brain iron Beiyun Wang, Yuan Zhong, Chengjie Gao, Jingbo Li PII:

S0006-291X(17)31175-0

DOI:

10.1016/j.bbrc.2017.06.045

Reference:

YBBRC 37951

To appear in:

Biochemical and Biophysical Research Communications

Received Date: 8 June 2017 Accepted Date: 11 June 2017

Please cite this article as: B. Wang, Y. Zhong, C. Gao, J. Li, Myricetin ameliorates scopolamineinduced memory impairment in mice via inhibiting acetylcholinesterase and down-regulating brain iron, Biochemical and Biophysical Research Communications (2017), doi: 10.1016/j.bbrc.2017.06.045. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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Myricetin ameliorates scopolamine-induced memory impairment in

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mice via inhibiting acetylcholinesterase and down-regulating brain

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iron

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Beiyun Wang1*, Yuan Zhong1, Chengjie Gao1 , Jingbo Li2

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1. Department of Gerontology, Shanghai Sixth People’s Hospital Affiliated to

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Shanghai Jiaotong University, Shanghai, 200233,China

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2. Department of Cardiology, Shanghai Sixth People’s Hospital Affiliated to Shanghai Jiaotong University, Shanghai, 200233, China

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* Correspondence: Beiyun

Wang, Department of Gerontology, Shanghai Sixth

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People’s Hospital Affiliated to Shanghai Jiaotong University, No.600 Yishan Road,

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Shanghai 200233, China. Tel: +86 64369181; Fax: +86 64369181; Email:

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[email protected]

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Abstract

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The aim of our study was to investigate to investigate the effect of myricetin on

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Alzheimer’s disease (AD) and its underlying mechanisms. In our study, Myricetin

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effectively attenuated Fe2+-induced cell death in SH-SY5Y cells in vitro. In a mouse

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model of AD， myricetin treatment significantly reversed scopolamine-induced

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cognitive

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acetylcholinesterase(AChE) and down-regulating brain iron. Furthermore, Myricetin

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treatment reduced oxidative damage and increased antioxidant enzymes activity in

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mice. Interestingly, the effect of myricetin was largely abolished by high iron diet.

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Therefore we suggested that treatment with myricetin attenuated cognitive deficits in

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mice via inhibiting AChE and brain iron regulation. In addition, myricetin reduce iron

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contents may via inhibiting transferrin receptor 1(TrR1) expression. In conclusion,

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accumulated data demonstrates that myricetin is a potential multifunctional drug for

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AD.

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Keywords: Myricetin, Alzheimer’s disease, acetylcholinesterase, Iron reducer

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1. Introduction

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Alzheimer’s disease (AD) is an age-related degenerative brain disorder characterized

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by a progressive worsening in cognitive function and memory. In accordance with the

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Alzheimer's Association, the patient of this disease will reach to 114 million by

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2050[1], which would clearly impose huge economical burden to the social security

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system and patient’s family [2]. However, there are no effective drugs to alter the

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course of AD, only slow the progression modestly via using symptomatic agents, such

deriving

from

a

novel

action

of

inhibiting

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deficits

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ACCEPTED MANUSCRIPT as cholinesterase inhibitors [3]. The etiology of AD is complicated, aging is

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considered to be the primary dangerous factor, other predisposing factors including

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cholinergic deficit, oxidative stress and iron overloading in brain have also been

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confirmed. Iron metabolism plays a critical role in the human body and excess iron

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produces reactive oxygen species (ROS), which severely damage tissues. Moreover,

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there is a growing body of evidence indicated that iron accumulation was

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age-related[4]. Many studies indicated that the misregulation of iron in the brain

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would cause many neurodegenerative diseases, such as AD, Parkinson’s and

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Huntington’s diseases [5,6]. Recent studies revealed that iron concentration in the

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brain of AD patients notably elevated compared to healthy controls [7,8]. The

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excessive deposition of iron is considered to induce oxidative stress [9]:

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Fe3++HO-+OH.

Fe2++H2O2

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The toxicity of superoxide anion (O2-) and hydrogen peroxide (H2O2) arises from their

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iron dependent conversion into the extremely reactive hydroxyl radical (OH )

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(Haber-Weiss reaction) that causes severe damage to membranes, proteins, and

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DNA[10]. In recent studies, considerable evidences have accrued demonstrating that

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the brain of AD patients is under increased oxidative stress and this may have a role in

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the pathogenesis of neuron degeneration and death in this disorder [11,12,13]. Due to

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the crucial role of acetylcholine in the pathogenesis of AD and the great contribution

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of iron-mediated free radicals to its development, application of acetylcholinesterase

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inhibitors and iron chelators treatment maybe a helpful therapeutic strategy for AD.

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Myricetin, a common natural flavonoid, presented abundantly in our diet, 3

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including vegetables, red wine, tea and berries[14,15]. A growing body of studies

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have demonstrated that myricetin has various kinds of biological effects, such as

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anticancer[16], anti-inflammatory[17] and antioxidant[18]. Recent studies indicated

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that

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[19,20].Although the neuroprotective effect of myricetin appear to be attribute to its

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antioxidant property, the underlying mechanism of myricetin in cognitive impairment

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is still unclear. In our study, young mice were treated with scopolamine to induce

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amnesia and myricetin was treated to investigate for its effects on improving these

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deficits.

has

neuroprotective

effect

at

physiological

concentration

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2. Materials and methods

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2.1 Cell culture

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myricetin

Human SH-SY5Y cells (China Centre for Type Culture Collection, Wuhan,

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China) were cultured in high-glucose Dulbecco’s modified Eagle’s medium (DMEM),

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supplemented with 10% fetal bovine serum (Zhejiang Tianhang Biological

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Technology Co, Ltd), at 37 °C in 5% CO2. For all cell experiments, SH-SY5Y cells

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were plated on 96-well plates with a density of 1×104 cells per well.

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2.2 Cell viability assay

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24h after cell seeding, cells were treated with FeSO4 (200 µM) for another 2h at

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37℃. After that, the culture medium was replaced by fresh culture medium, and the

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cells were growth in fresh culture medium for 24h [21]. Various concentrations

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myricetin (0.016~4 µM) had been incubated with cells for 2 h prior to treatment with 4

ACCEPTED MANUSCRIPT Fe2+. Cell viability was measured using MTT (3- （ 4,5-dimethyl-2-thiazolyl)

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-2,5-diphenyl-2-H-tetrazolium-bromide）assay. In brief, 10 µL of MTT (5 mg/ml) was

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added to each well and incubated for 4h at 37℃.After abolishing the medium, 150 µL

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dimethylsulphoxide (DMSO) was added to each well. The OD at 490 nm was

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measured in a microplate reader.

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2.3 Animals care and drug application

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Kunming (KM) mice (weighing 25±2g, half male and female) were obtained

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from Wuhan University Laboratory Animal Center. The mice were housed under a

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12h light/dark cycle at 25±2°C and 60±10% humidity and fed standard laboratory

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chow and water ad libitum. Animal study followed ARRIVE (Animal Research:

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Reporting In Vivo Experiments) guidelines and was approved by The Institutional

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Animal Care and Use Committee (IACUC), Wuhan University Center for Animal

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Experiment, Wuhan, China (AUP No. S201411012I)

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After acclimatized for 5 days, KM mice were randomly divided into 5 groups

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(N=12). Myricetin group was divided into low dose group (25mg/kg/Day), high dose

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group (50mg/kg/Day) and high dose group (50mg/kg/Day) supplied with a high iron

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diet (HID) (FeSO4, 75mg/kg/Day). Myricetin and FeSO4 were administered via

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gavage. The age-matched KM mice were used as the normal controls. Memory

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impairment was induced by intraperitoneal injection of scopolamine (0.2 mg/kg/Day)

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for 6 days except the normal group.

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2.4 Morris water maze test

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A spatial memory and learning test was performed as described previously with 5

ACCEPTED MANUSCRIPT some modification [22]. The circular pool (160 cm in diameter, 55 cm in height) was

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filled with water (20 ± 1°C), and nonfat milk was added to water. The pool was

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divided into four quadrants. A white platform (diameter in 10cm) was placed at the

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centre in one of four quadrants of pool and hidden 1cm below the water surface.

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During training trial sessions, mice were given four acquisition trials per day for 5

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days. If the moue found the platform in 60s, the mouse was allowed to stay on the

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platform for 15s. If the mouse couldn’t found the platform in 60s, it was placed on the

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platform for 15s. The probe test was performed following the 24 hours of last day of

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training. The mice were allowed to probe in the pool for 60s after the platform was

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removed. In our experiment, we recorded the platform-site crossovers, the path length

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and the time spent in the target quadrant.

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2.5 Ex vivo AChE activity assay

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After the Morris water maze (MWM) test, all mice were euthanized under

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anesthesia. The hippocampus was separated and homogenized in cold phosphate

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buffer saline (PBS). The homogenates were centrifuged for 10 min at 12000g at 4℃.

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The supernatants were used to AChE activity assay. The AChE activity assay and

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their protein amount were determined using their respective assay kits (Jiancheng

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Bioengineering Institute, Nanjing, JS, China). AChE activity per protein amount of

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the homogenate supernatant (mg) was calculated.

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2.7 In vitro AChE activity Assay.

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AChE activity (from Electrophorus electricus, purchased from Sigma) was determined

according

to

the

method 6

described

previously

with

slight

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S-Acetylthiocholine iodide (ATCh ,25 µL), 3 mM 5,5'-Dithiobis-(2-nitrobenzoic

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acid)(DTNB,125 µL), 50mM Tris-HCl (50 µL, pH 8.0), and test agents (25 µL) was

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preincubated in a microplate for 10 min., then AChE (0.25 U/mL, 25 µL) was added

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to the mixture to trigger the recation, and scanned for 10 min at 405 nm in a

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microplate reader. Enzyme activity was indicated as a percentage of the activity when

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treated with vehicle instead of test agents.

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2.7 Measurement of acetylcholine(ACh) content in hippocampus

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The homogenate supernatant of hippocampus also used to determine the content acetylcholine

using

acetylcholine

assay

kit

according

to

manufacturers’

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recommendations (Jiancheng Bioengineering Institute, Nanjing, JS, China).

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2.8 The severity of oxidative stress of hippocampus

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The level of oxidative stress of hippocampus was assessed by level

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malondialdehyde (MDA) level and activities of antioxidant enzymes including

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Superoxide Dismutase (SOD), glutathione peroxidase (GPx) and catalase (CAT). The

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supernatant samples of hippocampus were prepared according to the protocol of assay

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kits. These assays were measured using their respective assay kits (Jiancheng

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Bioengineering Institute, Nanjing, JS, China).

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2.9 Brain iron measurement

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The iron level in brain was measured using elemental analyzer. Briefly, the

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whole brain was separated and homogenized with Hepes buffer (1:10, wt/vol). the

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homogenate was digested with ultra-pure nitric acid at 50℃ for 24 hours. The 7

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digestive solutions were used to measure iron level after diluted with ultra-pure water.

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2.10 Fe(II) chelation assays Fe(II) chelation capacity of myricetin was evaluated using the ferrozine assay

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described previously[24]. The Fe(II)-ferrozine complex have the maximum

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absorbance at 560 nm, which was used to determine the free Fe(II) concentration.

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Myricetin solution (50 µL) mixing with Fe(II) solution (50 µL, 25 µM) stored for 30

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min at room temperature. A solution of ferrozine (50 µL, 1 mM) was added and the

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absorbance at 560 nm was measured. Standard curve was obtained by recording the

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absorbance at 560 nm of Fe(II) solutions containing ferrozine. Ethylenediamine

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tetraacetic acid (ETDA) was used to positive control. The capacity of Fe(II) chelation

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was calculated as follows (mol/g) : Y=（C0-C）×V / M; Where C0, is initial Fe(II)

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concentration; C, is free Fe(II) concentration after complex formation; V, is reaction

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volume; M, is the mass of test agents.

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2.11 Western blot assay

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To understand the mechanism of myricetin, western blot was conducted as

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previously described [25]. In brief, equal amount of brain sample proteins were

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electrophoresed using SDS-PAGE. To prevent the nonspecific binding, the

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membranes were blocked with 5% nonfat milk at room temperature for 2 h, and

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subsequently with primary antibodies overnight at 4 °C, including TfR1 (1:1000,

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Proteintech Group, Inc., Chicago, USA) and -actin (1 : 500, Proteintech Group, Inc.,

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Chicago, USA). Following, the membranes were also incubated with horseradish

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peroxidase-conjugated secondary antibodies. The expressions of proteins were

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measured using ECL detection reagents (Amersham Pharmacia Biotech Inc.,

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Piscataway, NJ, USA) and quantified by Image pro plus (IPP) software.

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2.12 Statistical analysis The data represented as the mean ± SEM; The data of escape latencies during the

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training in Morris water maze task were analyzed with two-way analysis of variance

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(ANOVA). Statistical evaluation of other results was performed using one-way

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analysis and p < 0.05 was considered as a significant differences.

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3、 、Results

3.1 Myricetin reduced Fe2+-induced injury in SH-SY5Y cells We examined the neuroprotective effects of myricetin using Fe2+-induced injury

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in SH-SY5Y cells and the MTT assay was used to measure cell viability. In the model

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of Fe2+-induced injury, myricetin (4, 1, 0.25, 0.063, 0.016 µM) significantly increased

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the cell viability in a concentration-dependent manner, compared to the model (Fig.

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2A).

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3.1 Myricetin improved the memory impairment in mice

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We investigated the therapeutic effects of myricetin against scopolamine-induced

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memory impairment in mice in the MWM test. The results showed that from the

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second day on, the escape latencies of control group were prolonged significantly (P <

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0.05), compared with the model group. Myricetin (25 mg/kg) group dramatically

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reduce the escape latencies on days 3 and 4 (P < 0.05) (Fig. 1A). Moreover, myricetin

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treatment group (50 mg/kg) significantly shorten the escape latency time from the 9

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mg/kg) group showed no significant difference compared with the model group,

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whereas significantly increased escape latency time compared with the myricetin

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group (50 mg/kg) (Fig. 1B). In the probe trial, the swimming time spent in the target

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quadrant and the platform crossings were significantly increased after myricetin

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administration (25 or 50 mg/kg), compared with the model group (P < 0.05, P < 0.01,

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respectively). In accordance with above discovery, HID + myricetin (50 mg/kg) group

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revealed no significant difference with the model group and decreased significantly,

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in comparison with myricetin (50 mg/kg) group, in the time spent in the target

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quadrant and the platform crossings (Fig. 1C and D).

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3.2 Effect of myricetin against AChE activity In vitro and in vivo To explore the underlying anti-AD mechanism of myricetin, it was tested at

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different concentrations for AChE activity in vitro. Results showed that myricetin

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potently inhibited AChE activity (IC50=58.9 µM), in a dose-dependent manner. In vivo,

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we also measured AChE activity in the hippocampus in mice. Our results showed that

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the model group remarkably increased AChE activity in the hippocampus, whereas

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these effects were reversed significantly after myricetin (50mg/kg) or HID +

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myricetin (50mg/kg) supplementation.

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3.3 Myricetin enhanced acetylcholine content in hippocampus

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Cholinergic hypofunction characterizes in AD patients[26] and AChE inhibitors

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are known to attenuate scopolamine-induced amnesia[27]. Therefore we examined

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the effect of myricetin on acetylcholine content in the hippocampus. Scopolamine 10

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comparison to normal group, while this effect was significantly reversed after

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treatment with myricetin (50 mg/kg or 25 mg/kg) or HID + myricetin (50 mg/kg).

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Furthermore, myricetin (50 mg/kg) group and HID+ myricetin (50 mg/kg) group had

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no significant differences.

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3.4 Myricetin ameliorated oxidative stress in hippocampus

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Our results showed that myricetin (50 mg/kg) group significantly decreased

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MDA level and increased antioxidant enzymes activities, such as SOD, GPx and CAT,

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compared with the model group. Whereas there was no significant difference between

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HID + myricetin (50 mg/kg) group and the model group (Fig. 3).

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3.5 Myricetin decreased iron level in brain.

We also examined the iron contents in brain of mice. Our datas showed that the

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iron contents in brain of the model group were higher than that of the normal group

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(P<0.05), and myricetin (50mg/kg) treatment significantly reduced the iron contents

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in brain as compared to the model group. The iron contents in brain of HID+

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myricetin (50mg/kg) group is higher than that of Myricetin(50mg/kg) group, and had

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no significant difference, as compared to the model group (Fig. 4B).

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3.6 Fe(II) chelation

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The ability of the myricetin to chelate Fe(II) was measured using the ferrozine

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assay and there existed a good linear relation between the absorbance at 560 nm and

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free Fe(II) concentration (r2 > 0.99). In Fe(II) chelation assays, myricetin was

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observed to be an excellent Fe(II)chelating agent, and stronger than the positive 11

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control compound EDTA.

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3.7 Myricetin significantly inhibited expression of TrR1 To understand the mechanisms involved in the reduction in iron contents induced

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by myricetin, we examined the expression of TrR1 in brain, an important iron uptake

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proteins. We found that myricetin at the dose of 50 mg per kg exerts the greatest

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protective effect on the mice. Myricetin was administered with this dosage in the

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western blot assay. The expression of TfR1 in scopolamine-induced mice was found

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to be significantly higher than that in normal mice. Myricetin supplementation

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significantly inhibited TrR1 expression compared to model group.

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4. Discussion

In our study, we found for the first time that myricetin treatment was able to

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reverse the spatial learning and memory impairments in scopolamine-induced

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dementia mice, and the effect of myricetin was almost abolished by high levels iron in

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the diet. Interestingly, our novel data showed that myricetin(50 mg/kg) and HID +

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myricetin(50 mg/kg) treatment both remarkably decreased AChE activity and

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increased ACh content in hippocampus. Myricetin(50 mg/kg) treatment could

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attenuate oxidative damage, whereas HID + myricetin(50 mg/kg) treatment was not

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able to reduce oxidative damage in hippocampus in mice. Moreover myricetin(50

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mg/kg) treatment significantly reduce iron level whereas the iron level of HID +

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myricetin(50 mg/kg) group had no significant difference, compared to model group.

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These results suggested that high levels of iron in the diet abolish the effect of

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myricetin may mainly due to the misregulation of iron in the brain which tends to

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cause oxidative stress, a vital pathogenesis mechanism of AD. Multiple researches have indicated that iron level increased in many brain region

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with age [28,29], especially occurs in the cortex and hippocampus. Previous studies

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have shown that iron is one of the most important element for health, for it is a

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component of a large of number oxidases and oxygenases. However, iron-overloaded

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is considered to the most one of factors to generate oxidative stress[30]. Iron have the

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ability to catalyze superoxide anion (O2-) and hydrogen peroxide (H2O2) turn into the

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exceedingly reactive hydroxyl radical (OH ) and , which could cause serious injury to

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membranes, proteins, and DNA[31]. Iron accumulation can lead to neuronal cell death

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because it can cause oxidative stress[32].

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Iron-loading commonly causes oxidative damage via catalysting excess reactive

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oxygen species (ROS) reacting with biomolecules. In our results, myricetin(50 mg/kg)

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treatment dramatically alleviated intracellular oxidative stress through increasing

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antioxidant enzymes (including SOD and GPx and CAT) activities and decreasing

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MDA level, one of markers of lipid peroxidation. However, high iron diet abolished

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these effects of myricetin, which suggested iron-loading had the ability to induce

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oxidative stress. In Fe(II) chelation assay, data revealed that myricetin was a strong

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Fe(II)chelating agent. To understand why myricetin could decrease iron level in the

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brain, we also explored the effects of myricetin on TrR1 expression, which indicated

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that myricetin reduce iron contents may via inhibiting TrR1 expression. Furthermore,

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in our cell assay, preincubation of SH-SY5Y cells with myricetin could prevent

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ACCEPTED MANUSCRIPT cytotoxicity induced by Fe2+. Therefore, we can speculate that myricetin plays a role

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in improving the ability of learning and memory in mice through attenuating oxidative

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damage induced by iron. Multiple articles have demonstrated that cholinergic

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neurodegeneration is the common existence in AD patients [33], thus AChE inhibition

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had an important role in curing AD. Moreover, in vitro, myricetin inhibited AChE

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activity in a dose-dependent manner (IC50=58.9µM). These findings agreed with the

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behavioral

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scopolamine-induced learning and memory impairments

together

showed

that

myricetin

could

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and

alleviate

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In our present study, the novel data indicated that myricetin treatment

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significantly reduced iron level in brain in mice. Thus myricetin not only serves as a

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selective AChE inhibition, but also may be an iron reducer. In conclusion, we

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confirmed that myricetin potently ameliorated memory deficits in mice, and mainly

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through inhibiting AChE activity and reducing oxidative stress via chelating iron ion,

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and myricetin could be a promising dual target drug for AD.

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Conflicts of interest

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The authors have declared no conflict of interest.

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Fig.1 Effect of myricetin on the spatial learning and memory impairments on the

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MWM. (A) The escape latencies during 5 consecutive days of training on the MWM.

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(B) The escape latencies at 5th day of training on the MWM. (C) The swimming time

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spent in the target quadrant during the MWM probe test. (D) The crossings into the

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former location of the submerged platform during the MWM probe test. All data were

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expressed as mean ± SEM. ##P < 0.05 versus normal; *P < 0.05 versus model, **P <

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Fig.3 (A) Effect of myricetin on AChE activity in vitro. Inhibition is expressed as

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percent inhibition of enzyme activity. IC50 = 58.9µM. (B) Effect of myricetin on

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AChE activity in hippocampus. (C) Neuroprotective effect of myricetin on 17

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Fe2+-induced injury in SH-SY5Y cells. (D) The ability of myricetin to chelate to

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Fe(II). All data were expressed as mean ± SEM. ##P < 0.05 versus normal; *P < 0.05

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versus model, **P < 0.01 versus model.

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Fig. 5 Effect of myricetin on antioxidant enzymes and lipid peroxidation. (A) SOD

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activity, (B) CAT activity, (C) GPx activity, (D) MDA content. All data were

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expressed as mean ± SEM. ##P < 0.05 versus normal; *P < 0.05 versus model, **P <

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Fig.4 (A) Effect of myricetin on ACh content in hippocampus. (B) Effect of myricetin

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on iron level in brain. (C) Effect of myricetin on TrR1 expression in brain. All data

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were expressed as mean ± SEM.

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**P < 0.01 versus model.

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P < 0.05 versus normal; *P < 0.05 versus model,

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