Journal Pre-proof

Quercetin protects against cerebral ischemia/reperfusion and

oxygen glucose deprivation/Reoxygenation neurotoxicity

Ya-Yu Wang, Cheng-Yi Chang, Shih-Yi Lin, Jiaan-Der Wang,

Chih-Cheng Wu, Wen-Ying Chen, Yu-Hsiang Kuan, Su-Lan Liao,
Wen-Yi Wang, Chun-Jung Chen

PII: S0955-2863(20)30468-X
DOI: https://doi.org/10.1016/j.jnutbio.2020.108436
Reference: JNB 108436

To appear in: The Journal of Nutritional Biochemistry

Received date: 28 May 2019

Revised date: 27 March 2020
Accepted date: 20 May 2020

Please cite this article as: Y.-Y. Wang, C.-Y. Chang, S.-Y. Lin, et al., Quercetin protects
against cerebral ischemia/reperfusion and oxygen glucose deprivation/Reoxygenation
neurotoxicity, The Journal of Nutritional Biochemistry (2020), https://doi.org/10.1016/
j.jnutbio.2020.108436

This is a PDF file of an article that has undergone enhancements after acceptance, such
as the addition of a cover page and metadata, and formatting for readability, but it is
not yet the definitive version of record. This version will undergo additional copyediting,
typesetting and review before it is published in its final form, but we are providing this
version to give early visibility of the article. 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.

© 2020 Published by Elsevier.

 Journal Pre-proof

Quercetin Protects against Cerebral Ischemia/Reperfusion and Oxygen Glucose

Deprivation/Reoxygenation Neurotoxicity

Ya-Yu Wang1,2#, Cheng-Yi Chang3#, Shih-Yi Lin2,4, Jiaan-Der Wang5,6, Chih-Cheng Wu7,8,9,

Wen-Ying Chen10, Yu-Hsiang Kuan11, Su-Lan Liao12, Wen-Yi Wang13, Chun-Jung Chen12,14*

1
Department of Family Medicine, Taichung Veterans General Hospital, Taichung City, Taiwan;

yywang@vghtc.gov.tw

f
oo
2
Institute of Clinical Medicine, National Yang Ming University, Taipei City, Taiwan;

yywang@vghtc.gov.tw (Y.-Y.W); sylin@vghtc.gov.tw (S.-Y.L)

3
pr
Department of Surgery, Feng Yuan Hospital, Taichung City, Taiwan; c.y.chang.ns@gmail.com
e-
4
Center for Geriatrics and Gerontology, Taichung Veterans General Hospital, Taichung City, Taiwan;
Pr

sylin@vghtc.gov.tw
5
Children’s Medical Center, Taichung Veterans General Hospital, Taichung City, Taiwan;
al

wangjiaander@gmail.com
rn

6
Department of Industrial Engineering and Enterprise Information, Tunghai University, Taichung
u

City, Taiwan; wangjiaander@gmail.com

Jo

7
Department of Anesthesiology, Taichung Veterans General Hospital, Taichung City, Taiwan;

chihcheng.wu@gmail.com
8
Department of Financial Engineering, Providence University, Taichung City, Taiwan;

chihcheng.wu@gmail.com
9
Department of Data Science and Big Data Analytics, Providence University, Taichung City, Taiwan;

chihcheng.wu@gmail.com
10
Department of Veterinary Medicine, National Chung-Hsing University, Taichung City, Taiwan;

wychen@dragon.nchu.edu.tw

1
 Journal Pre-proof

11
Department of Pharmacology, Chung Shan Medical University, Taichung City, Taiwan;

kuanyh001@gmail.com
12
Department of Medical Research, Taichung Veterans General Hospital, Taichung City, Taiwan;

slliao@vghtc.gov.tw (S.-L.L), cjchen@vghtc.gov.tw (C.-J.C)

13
Department of Nursing, Hung-Kuang University, Taichung City, Taiwan;

walice@sunrise.hk.edu.tw
14
Department of Medical Laboratory Science and Biotechnology, China Medical University,

Taichung City, Taiwan; cjchen@vghtc.gov.tw

f
oo
Running head: Neuroprotective effect of quercetin
pr
e-
#Ya-Yu Wang and Cheng-Yi Chang contributed equally to this study.
Pr

*Corresponding author: Chun-Jung Chen; Department of Medical Research, Taichung Veterans

al

General Hospital; No. 1650, Sec. 4, Taiwan Boulevard, Taichung City 407, Taiwan
rn

Phone: (886)-4-2359-2525 ext. 4022; Fax: (886)-4-2359-2705; E-mail: cjchen@vghtc.gov.tw

u
Jo

2
 Journal Pre-proof

Abstract

Beyong nutrition effect, quercetin is applicated as a complement or an alternative for promoting

human health and treating diseases. However, its complicated neuroprotective mechanisms have not

yet been fully elucidated. This study provides evidence of an alternative target for quercetin, and

sheds light on the mechanisms of its neuroprotection against cerebral ischemia/reperfusion (I/R)

injury in Sprague-Dawley rats. Oral pretreatment using quercetin has alleviated cerebral I/R-induced

neurological deficits, brain infarction, blood-brain barrier disruption, oxidative stress, TNF- and

f
IL-1 mRNA expression, along with apoptotic caspase 3 activity. The neuroprotective, anti-oxidative,

oo
anti-inflammatory, and anti-apoptotic effects of quercetin were replicated in rat hippocampal slice

pr
cultures and neuron/glia cultures which suffered from oxygen-glucose deprivation and reoxygenation

(OGDR). Biochemical studies revealed a reduction of extracellular signal-regulated kinase (ERK)

e-
and Akt phosphorylation, along with an increase in protein tyrosine and serine/threonine phosphatase
Pr

activity in cerebral I/R rat cortical tissues and OGDR hippocampal slice and neuron/glia cultures.

Quercetin alleviated the changes in ERK/Akt phosphorylation and protein phosphatase activities.
al

Inhibition of ERK or Akt alone was enough to cause apoptotic cell death and cytotoxicity in
rn

hippocampal slice cultures and neuron/glia cultures, while activators of ERK or Akt alleviated
u

OGDR-induced cytotoxicity. Taken together, our results demonstrate that quercetin alleviated the
Jo

increment of protein tyrosine and serine/threonine phosphatase activity, along with the reduction of

ERK and Akt phosphorylation, which may play pivotal roles in the expansion of brain injury after

cerebral I/R.

Keywords: Flavonoids; Neuroprotection; Phosphatase; Stroke

3
 Journal Pre-proof

1. Introduction

Cerebral ischemia or stroke is a consequential injury arising from the interruption of cerebral

blood circulation. It is a common cause of death and long-term disability in patients who have

suffered from the conditions. The early recanalization of circulation using a recombinant tissue

plasminogen activator is the clinical treatment of choice. However, only a minor number of patients

benefit from such an early reperfusion strategy [1]. The homeostasis and function of the brain relies

heavily upon the coordination amongst neurotransmission, bioenergetics, ion mobilization, and

cell-cell interplay. The insufficiency of oxygen and glucose supply due to cerebral ischemia causes a

f
oo
rapid onset of bioenergetic failure, which initiates a series of biochemical changes resulting in the

first wave of neuronal dysfunction and destruction. Although early reperfusion is a pivotal step
pr
towards restoring post-ischemic circulation, the boost of large amounts of oxygen and glucose
e-
oppositely augments and initiates a second wave of brain injury [2,3]. Oxidative stress and
Pr

inflammation represent the key players, and substantially contribute to post-ischemic brain injury

involving the strengthening of pro-damage mechanisms and subsiding pro-survival mechanisms [4,5].
al

Therefore, the resolution of oxidative stress and inflammation, along with a better understanding of
rn

pro-damage and pro-survival mechanisms, are theoretically pivotal towards offering insights into the
u

pathogenesis of ischemic brain injury, and the need to develop potentially preventive and/or
Jo

therapeutic strategies.

Plants, vegetables, and fruits are rich in bioactive compounds which offer health promoting

effects. The extracts and ingredients of medicinal plants possess antioxidant and anti-inflammatory

properties, while exhibiting therapeutic potential in a variety of models involving ischemic brain

injury [6-9]. Quercetin, a natural occurring flavonoid, possesses several biological activities

including anti-oxidation and anti-inflammation, while its neuroprotection has been demonstrated in

both cell and rodent models of ischemia. Toll-like receptors, NADPH oxidase, hypoxia-inducible

factor-1 (HIF-1), NF-E2-related factor (Nrf2), heme oxygenase-1 (HO-1), mitogen-activated

4
 Journal Pre-proof

protein kinase (MAPK), Akt, -enolase, antioxidant enzymes, BDNF, and NF-B are all proposed

targets of quercetin, with its glucoside form contributing to neuroprotection [10-19]. While a wide

range of studies have demonstrated the beneficial effects of quercetin in human healthy and diseased

models, the precise molecular and biochemical bases underlying its neuroprotection remain to be

fully investigated.

The central nervous system consists of a vast array of cell population. In biomedical research,

organotypic hippocampal slice cultures and primary neuron/glia cultures contain pivotal

parenchymal neurons, astrocytes, and microglia, which represent ex vivo and in vitro models for the

f
oo
study of neurological diseases, respectively [20,21]. To mimic in vivo ischemic insult,

pr
oxygen-glucose deprivation (OGD) is established in an in vitro model of ischemia [14,22]. Given

that the use of medicinal plants is an emerging prescription as a complement, along with being an
e-
alternative for promoting human health and treating diseases, the neuroprotective mechanisms of
Pr

quercetin are extremely complex and have yet to be fully elucidated. To extend the scope of

quercetin’s neuroprotective studies, we therefore explored the capacity of quercetin to mediate

al

neuroprotection in models of cerebral ischemia/reperfusion (I/R), hippocampal slice cultures, and

rn

neuron/glia cultures, and subsequently questioned whether it did; in order to determine the shared
u

intracellular targets related to its neuroprotection.

Jo

2. Materials and Methods

2.1. Cerebral I/R. All the procedures involving animal studies strictly adhered to the Institute’s

guidelines and had been approved by the Animal Experimental Committee of Taichung Veterans

General Hospital (IACUC No. La-98642, Oct. 14, 2009). Male Sprague-Dawley rats weighing

between 250-300 g were randomly allocated into two groups (n = 18 per group), with each receiving

a daily saline vehicle or quercetin (25 mg/kg) administration over a span of 21 days by oral gavage.

Rats were anesthetized with isoflurane (2-4%) prior to the surgical process. A midline cervical

5
 Journal Pre-proof

incision on the ventral side was first performed to expose the bilateral common carotid arteries

(CCAs). A craniectomy slightly anterior to the right foramen ovale was performed with an aim to

exposing the middle cerebral artery (MCA) without destroying the zygomatic arch. Transient focal

cerebral ischemia was produced by occluding the two CCAs and the right MCA for 90 min.,

followed by reperfusion in accordance with our previous study [23]. A daily administration of

quercetin continued until the death of the animal. All rats were then evenly divided into 3 groups (n =

6 per group) for further analyses; including neurological evaluation, brain infarction, Evans blue

extravasation, and biochemical analyses 3 days after surgery. Two additional groups (n = 6 per group)

f
oo
of animals receiving sham operations, all procedures performed were the same as above, but no

arterial ligation was performed.

pr
e-
2.2. Quantification of ischemic infarction. Rats were euthanized with isoflurane (2-4%)
Pr

anesthesia prior to decapitation. A serial coronal section of brain was cut at 2-mm intervals from the

frontal pole, while the first to seventh coronal slices were immersed in a phosphate-buffered saline
al

(PBS) solution containing 2% triphenyltetrazolium chloride (TTC) at 37°C for 30 min. Then, the
rn

sections were fixed in 10% phosphate-buffered formalin for 45 min [23]. The infarct areas were
u

measured with a computer image analysis system (IS1000; Alpha Innotech Corporation).
Jo

2.3. Evans blue extravasation assay. Evans blue (4%, 1 ml/kg) was injected via the tail vein 3

h prior to being sacrificed. Rats were perfused with heparinized saline solution, and the ipsilateral

and contralateral cortices were isolated. The isolated cortical tissues were homogenized with PBS

and precipitated with trichloroacetic acid (TCA) (100%). The contents of Evans blue in the

supernatants were measured (absorbance at 620 nm), according to a previously reported method [24].

2.4. Neurological evaluation. A modified six-point neurological deficit severity scale was

6
 Journal Pre-proof

applied for the evaluation of sensorimotor performance by technicians blinded to the treatment, as

described previously [24]. Neurological evaluation was done before animal sacrifice, as follows: 0,

no neurological deficit; 1, difficulty in fully extending the left forepaw; 2, unable to extend the left

forepaw; 3, mild circling to the left; 4, severe cycling to the left; and 5, falling to the left.

2.5. Behavioral observations. The spontaneous locomotion and motor coordination were

evaluated in an Open field test and Rotarod test, respectively [23]. Animals were placed at the

apparatus (30 cm in length and 30 cm in width) for a period of 30 min and the travel distance and

f
oo
moving time were recorded. The Rotarod performance test was evaluated by placing the rats on the

rotarod cylinder with speed increasing from 4 rpm to 40 rpm within 5 min, and the time for which
pr
the animals remained on the rotarod was measured. The mean duration on the device was recorded
e-
with three retarod measurements before surgery as baseline. Data are presented as percentage of
Pr

mean duration on the rotarod compared with each basal control.

al

2.6. Hippocampal slice cultures. The brains of both male and female Sprague-Dawley rats (n =
rn

20) were removed at postnatal days 5-7 after decapitation. The hippocampi were aseptically
u

dissected and slices (300 m) were prepared using a tissue chopper. Slices were then transferred to
Jo

sterile culture inserts (4 slices per insert), and placed in 6-well plates containing Minimum Essential

Medium (MEM) 50% (v/v), Hank’s Balanced Salt Solution (HBSS) 25% (v/v), horse serum 25%

(v/v), glucose 5 mg/ml, glutamine 1 mM, KCl 5 mM, fungizone 1.5%, penicillin 1%, and

streptomycin 1%, pH=7.2. Cultures were maintained at a temperature of 37C in an atmosphere of

5% CO2/95% air in 95% humidity for 10-12 days, with the media changed every other day,

according to a reported method after certain modifications [21,22].

2.7. Neuron/glia cultures. Neuron/glia cultures were prepared from the cerebral cortices of

7
 Journal Pre-proof

postnatal day 1 Sprague-Dawley rats (n = 20) in accordance with a previously reported method [20].

The dissociated cells obtained from the dissected cortical tissues were briefly plated onto

poly-D-lysine-coated plates and maintained in MEM containing 10% Fetal Bovine Serum (FBS) and

10% horse serum for 10-12 days in vitro.

2.8. Oxygen-glucose deprivation and reoxygenation (OGDR). Hippocampal slices and

neuron/glia cultures were maintained in MEM without glucose, and placed in an incubator with 1%

O2, 5% CO2, and 94% N2 for 2 or 3 h in accordance with the reported methods, including minor

f
oo
modifications [14,22]. The cultures were then supplemented with 5.5 mM glucose and transferred to

an incubator with 5% CO2/95% air for an additional 24 h. Paralleling cultures placed in an incubator
pr
with 5% CO2/95% air were referred to normoxia control. During the experimental periods, media
e-
contained quercetin at concentration levels of 0 and 10 M.
Pr

2.9. Propidine Iodide (PI) fluorescence detection. At the end of the experiments, the
al

hippocampal slice cultures were incubated with PI (10 g/ml) for 30 min and rinsed with a fresh
rn

medium according to the reported methods, including modifications [21,22]. Cultures were first
u

observed under a light microscope and then an epifluorescence microscope (Ex 530 nm and Em 580
Jo

nm). All images were taken with a fixed exposure time, and the fluorescence intensity of the

combined images was measured using Image Studio Lite Ver 5.2 software. The measured signals

were normalized by the surface areas of hippocampal slices.

2.10. Caspase 3 activity assay. Proteins were extracted from contralateral and ipsilateral cortical

tissues, hippocampal slice cultures, and neuron/glia cultures, prior to being subjected to enzymatic

measurement of caspase 3 activity using a commercial fluorometric protease assay kit, upon

following the instructions provided by the manufacturer (BioVision, Mountain View, CA).

8
 Journal Pre-proof

2.11. Measurement of lipid peroxidation. Contralateral and ipsilateral cortical tissues,

hippocampal slice cultures, and neuron/glia cultures were homogenized and subjected to the

measurement of lipid peroxidation using a Thiobarbituric Acid Reactive Substances (TBARS) assay

kit (ZeptoMetrix, Buffalo, NY), according to the manufacturer’s instructions. Data of the

measurements were calculated and expressed as Malondialdehyde (MDA) equivalents.

2.12. RNA isolation and quantitative real-time reverse transcriptase polymerase chain

f
oo
reaction (RT-PCR). Total cellular RNAs were extracted from the contralateral and ipsilateral

cortical tissues, hippocampal slice cultures, and neuron/glia cultures using a TriZol RNA isolation
pr
reagent (Invitrogen, Carlsbad, CA) and subjected to cDNA synthesis. PCR reaction was performed
e-
on ABI StepOneTM (Applied Biosystems, Foster City, CA), with the levels of gene expression
Pr

calculated by the ΔΔCT method. PCR primers were as follows: tumor necrosis factor- (TNF-),

5’-AAATGGGCTCCCTCTCATCAGTTC and 5’-TCTGCTTGGTGGTTTGCTACGAC;

al

interleukin-1 (IL-1), 5’-CACCTCTCAAGCAGAGCACAG and

rn

5’-GGGTTCCATGGTGAAGTCAAC; and -actin, 5’-AAGTCCCTCACCCTCCCAAAAG and

u

5’-AAGCAATGCTGTCACCTTCCC.
Jo

2.13. Immunocytochemical staining. Neuron/glia cultures were fixed with 4%

paraformaldehyde, permeabilized with 0.1% Triton X-100, and incubated with antibodies against

Microtube-associated Protein 2 (MAP-2, Transduction Laboratories, Lexington, KY), Glial

Fibrillary Acidic Protein (GFAP, Santa Cruz Biotechnology, Santa Cruz, CA), or CD68 (Santa Cruz

Biotechnology, Santa Cruz, CA), followed by horseradish peroxidase conjugated IgG. The

immunoreactive signals were developed with 3, 3’-Diaminobenzidine (DAB) and observed using a

light microscope.

9
 Journal Pre-proof

2.14. Cytotoxicity assessment. Cell damage was measured by the efflux of Lactate

Dehydrogenase (LDH) using a PierceTM LDH Cytotoxicity Assay Kit (Thermo Fisher Scientific,

Waltham, MA) in accordance with the manufacturer’s instructions. The cytotoxicity index was

indicated using the ratio of released LDH and total LDH.

2.15. Western blot. Proteins were extracted from the contralateral and ipsilateral cortical tissues,

hippocampal slice cultures, and neuron/glia cultures using Tissue Protein Extraction Reagents

f
oo
(T-PER, Pierce Biotechnology, Rockford, IL). Equal amounts of proteins were loaded and resolved

through SDS-polyacrylamide gel electrophoresis and transferred onto PVDF membranes. The
pr
membranes were then incubated with antibodies against phosphorylated and non-phosphorylated
e-
forms of extracellular signal-regulated kinase (ERK) and Akt (Santa Cruz Biotechnology, Santa Cruz,
Pr

CA) followed by horseradish peroxidase-labeled IgG. The membranes were developed using

enhanced chemiluminescence detection reagents. All measured protein levels were quantified
al

through densitometry.
u rn

2.16. Phosphatase assay. Contralateral and ipsilateral cortical tissues, hippocampal slice
Jo

cultures, and neuron/glia cultures were homogenized and subjected to enzymatic measurement of

serine/threonine and tyrosine phosphatase activities using a serine/threonine phosphatase assay kit

and a tyrosine phosphatase assay kit (Molecular Probes Inc., Eugene, OR), respectively, upon

following the instructions provided by the manufacturers.

2.17. Statistical analysis. The data are expressed as mean values ± standard deviation.

Statistical analysis was carried out using one-way Analysis Of Variance (ANOVA), followed by

10
 Journal Pre-proof

Dunnett’s test to assess the statistical significance between the treated and untreated groups. A level

of p < 0.05 was considered statistically significant.

3. Results

3.1. Quercetin alleviated cerebral I/R brain injury. To substantiate the neuroprotective

effects of quercetin against cerebral I/R brain injury, the parameters of neurobehaviors, brain

infarction, blood-brain barrier (BBB) permeability, apoptotic caspase 3 activity, oxidative stress, and

proinflammatory cytokine expression were all determined. Cerebral I/R rats developed neurological

f
oo
abnormalities in neurological deficits (Fig. 1A), motor coordination (Fig. 1B), and locomotion (Figs.

1C and 1D), along with brain infarctions (Figs. 2A and 2B), disrupted BBB integrity through
pr
parenchymal Evans blue extravasation (Fig. 2C), an increased caspase 3 activity (Fig. 2D), elevated
e-
lipid peroxidation product MDA content (Fig. 2E), and upregulated TNF- and IL-1 mRNA
Pr

expression (Fig. 2F) in the ipsilateral cortex. Quercetin treatment attenuated the assayed parameters

in cerebral I/R rats (Figs. 1 and 2). These findings suggest that quercetin exhibits a beneficial effect
al

against cerebral I/R brain injury.

u rn

3.2. Quercetin alleviated OGDR cytotoxicity in hippocampal slice cultures. To evaluate cell
Jo

death and cytotoxicity in experimental hippocampal slice cultures, the uptake of PI into injured cells

and the consequences of red fluorescence were photographed and quantified. Hippocampal slice

cultures were subjected to OGD for 2 h in the presence of quercetin (0 and 10 M), along with the

subsequent restoration of glucose and oxygen for 24 h. Paralleling cultures receiving the same

manipulation, except for OGD, were referred to normoxia control. Representative photographs of

phase and PI fluorescence amongst the groups are shown in Figure 3A. In normalizing by surface

areas, OGDR increased PI fluorescence in hippocampal slice cultures, with the increment being

alleviated by quercetin (Fig. 3B). OGDR-increased PI fluorescence was accompanied by elevated

11
 Journal Pre-proof

caspase 3 activity (Fig. 3C), MDA content (Fig. 3D), and TNF- and IL-1 mRNA expression (Fig.

3E). The presence of quercetin alleviated the aforementioned changes in the OGDR hippocampal

slice cultures (Figs. 3A-3E). Therefore, quercetin has an inhibitory effect on OGDR-induced cell

death and cytotoxicity in hippocampal slice cultures.

3.3. Quercetin alleviated OGDR cytotoxicity in neuron/glia cultures. The neuroprotective

effects of quercetin were further verified through the use of rat primary neuron/glia cultures.

Cultured neuron/glia consisted of 30-40% neurons, 40-50% astrocytes, and 10-15% microglia, as

f
oo
demonstrated by the immunoreactivity of MAP-2, GFAP, and CD68, respectively (Fig. 4A).

Neuron/glia cultures were subjected to OGD for 3 h in the presence of quercetin (0 and 10 M),
pr
and subsequent restoration of glucose and oxygen for 24 h. Concurrent treatments without OGD
e-
were under normoxia control. OGDR caused LDH efflux (Fig. 4B), caspase 3 activation (Fig. 4C),
Pr

MDA production (Fig. 4D), and TNF- and IL-1 mRNA expression (Fig. 4E) in neuron/glia

cultures. Quercetin alleviated OGDR-induced changes in LDH efflux (Fig. 4B), caspase 3
al

activation (Fig. 4C), MDA production (Fig. 4D), and TNF- and IL-1 mRNA expression (Fig.
rn

4E). These findings indicate that quercetin offers a protective effect on OGDR-induced cell death
u

and cytotoxicity in neuron/glia cultures.

Jo

3.4. Quercetin alleviated the reduction of ERK and Akt phosphorylation. Among the

intracellular signaling molecules, ERK and Akt each perform pivotal roles in cell survival, while

having neuroprotective effects [15,17,25]. To explore whether the neuroprotective effects of

quercetin may involve ERK and Akt signaling, the levels of total and phosphorylated ERK and Akt

were examined in models of in vivo, ex vivo, and in vitro studies. A reduction of both ERK and Akt

phosphorylation had been observed in ipsilateral cortical tissues of cerebral I/R rats (Figs. 5A and

5B), OGDR hippocampal slice cultures (Figs. 5C and 5D), and OGDR neuron/glia cultures (Figs. 5E

12
 Journal Pre-proof

and 5F). This would imply that the reduction of ERK and Akt phosphorylation was thus alleviated by

quercetin (Figs. 5A-5F). To further confirm the importance of ERK and Akt signaling in cell death

and cytotoxicity, the effects of pharmacological activators and inhibitors within ERK and Akt were

elucidated in hippocampal slice and neuron/glia cultures. Epidermal Growth Factor (EGF) and

insulin alleviated OGDR-induced PI fluorescence (Fig. 6A) and caspase 3 activity (Fig. 6B) in

hippocampal slice cultures as well as LDH efflux (Fig. 6C) and caspase 3 activity (Fig. 6D) in

neuron/glia cultures. The additions of U0126 and LY294002 subsequently increased both PI

fluorescence (Fig. 6E) and caspase 3 activity (Fig. 6F) in hippocampal slice cultures. Their cytotoxic

f
oo
(Fig. 6G) and apoptotic (Fig. 6H) effects were also noted in neuron/glia cultures. These results

suggest that ERK and Akt signaling play substantial roles in cell survival and are potential targets of

quercetin’s neuroprotection.
pr
e-
Pr

3.5. Quercetin alleviated increase of phosphatase activity. The phosphorus group in ERK and

Akt is identified at amino acid residue Thr202/Tyr204 and Ser473, respectively (Santa Cruz
al

Biotechnology, Santa Cruz, CA). The level of ERK and Akt phosphorylation is governed by the
rn

action balance between phosphatases and kinases. Through the enzymatic assays, ipsilateral cortical
u

tissues (Fig. 7A), OGDR hippocampal slice cultures (Fig. 7B), and OGDR neuron/glia cultures (Fig.
Jo

7C) each expressed a higher activity of protein tyrosine phosphatase and serine/threonine

phosphatase. The increment of protein phosphatase activities was alleviated by quercetin (Figs.

7A-7C). These findings suggest that the preservation of ERK and Akt phosphorylation by quercetin

may be partially attributed to the alleviation of protein phosphatase activities.

4. Discussion

Cerebral ischemia causes early onset brain injury, and progressively expands to the penumbra

areas. Prolonged ischemia and recanalization-associated reperfusion are common causes for the

13
 Journal Pre-proof

second wave or delayed type of brain injury. There are multiple, interrelated mechanisms which

cause progressive post-ischemic brain injury. Oxidative stress, inflammation, and apoptosis not only

have fundamental roles in the pathophysiology of cerebral ischemia, but are also considered to be

risk or trigger factors for stroke. A growing body of evidence has shown that the inhibition of

oxidative stress, inflammation, or apoptosis may provide effective, preventive, or therapeutic

intervention to help reduce cerebral ischemic injury [7,26,27]. In our study we have further

demonstrated the neuroprotective effects of quercetin against brain injury in cerebral I/R rats, OGDR

hippocampal slice cultures, and OGDR neuron/glia cultures. Through the use of distinct

f
oo
experimental models, the neuroprotective effects of quercetin were accompanied by the alleviation of

oxidative stress, inflammation, and apoptosis. Biochemical studies subsequently revealed a reduction
pr
of ERK and Akt phosphorylation, along with an increase in protein tyrosine and serine/threonine
e-
phosphatase activity during stressed conditions, and reversal effects due to quercetin. The inhibition
Pr

of ERK or Akt on its own was enough to cause apoptotic cell death and cytotoxicity in hippocampal

slice cultures and neuron/glia cultures. Taken as a whole, our results have demonstrated that
al

quercetin alleviated the increment of protein tyrosine and serine/threonine phosphatase activity,
rn

along with the reduction of ERK and Akt phosphorylation, which may play pivotal roles in the
u

expansion of brain injury after cerebral I/R. Despite its documented beneficial effects, the underlying
Jo

neuroprotective mechanisms and therapeutic effectiveness of quercetin against cerebral I/R remain

an important issue of interest which requires further investigation.

The brain is a metabolically active organ with a high demand for both oxygen and glucose.

Once it encounters ischemia, the insufficiency of bioenergetic supply impairs energy-guided pump

and cellular activities, which in turn initiates a series of biochemical and molecular events involving

cell survival and damage to its machinery. The intricate processes surrounding cell damaging

mechanisms contribute substantially towards the pathogenesis of cerebral I/R proceeding through

necrosis or apoptosis, and the augmentation of disease progression through concomitant changes,

14
 Journal Pre-proof

such as oxidative stress and inflammation. Therefore, cell damaging mechanisms are of clinical

importance for therapeutical intervention. The inhibition of oxidative stress, inflammation, and

apoptosis provides neuroprotective effects against cerebral I/R injury [7,26,27]. Quercetin possesses

both antioxidant and anti-inflammatory effects, while also offering beneficial effects against

oxidative and inflammatory diseases, including cerebral I/R. Evidence indicates that the scavenging

of free radicals, inhibition of free radical generation, and induction of Nrf2/HO-1 expression are all

proposed action mechanisms regarding quercetin’s antioxidant effects. The anti-inflammatory effect

of quercetin is mediated by the suppression of TLRs, MAPKs, and NF-B signaling

f
oo
[11,12,14-18,28,29]. In consistence with the aforementioned references, our findings have also

pr
demonstrated an inhibitory effect which quercetin has on oxidative stress, inflammation, and

apoptosis against neurodegenerative diseases. Although there was a lack of deep mechanistic study,
e-
quercetin’s universal or common effect on oxidative stress, inflammation, and apoptosis is
Pr

highlighted by current studies based on data from the three distinct models involving cerebral I/R

rats, OGDR hippocampal slice cultures, and neuron/glia cultures.

al

Cell survival mechanisms are adaptive, protective, and defensive systems which minimize and
rn

restrain the expansion of injury and promote regeneration. Among the cell survival mechanisms, the
u

master signaling molecules ERK and Akt could converge diverse signals to turn on gene expression
Jo

or phosphorylate cellular molecules which are critical to cell survival. Cyclin D1, -catenin, and

Bcl-2 family proteins are all representative molecules under the control of ERK and Akt and crucial

to cell survival [15,17,25,30]. However, ERK and Akt may also possess damaging potential, as they

are able to activate NF-B, and stimulate both TNF- and IL-1 expression [28]. It has been

demonstrated that the signaling of ERK and Akt can be activated during the ischemia period and

early phase of reperfusion (~2 h of reperfusion), eventually contributing to brain damage [31].

Additionally, OGDR-induced cell death in cortical neurons is associated with ERK

hyperphosphorylation [18]. Despite these detrimental findings, most studies indicate that the

15
 Journal Pre-proof

activation of ERK and Akt is neuroprotective against cerebral I/R injury [25,32-36]. The

enhancement of Akt signaling has been detected in the action of quercetin against cerebral I/R and

OGDR injury [14,15,17]. We identified that cell death and cytotoxicity in cerebral I/R rats with a

3-day reperfusion, OGDR hippocampal slice cultures, and OGDR neuron/glia cultures were

accompanied by a reduction of ERK and Akt phosphorylation. The neuroprotective actions of

quercetin were associated with elevated ERK and Akt phosphorylation. Parallel studies have shown

apoptotic cell death in hippocampal slice and neuron/glia cultures after the exposure of U0126 and

LY294002 and protection against OGDR apoptosis by each corresponding activator, EGF and insulin.

f
oo
Thus, cerebral I/R- and OGDR-injury may involve a decline of ERK- and Akt-related cell survival

mechanisms, with the preservation of ERK and Akt signaling being an explanation for quercetin’s

neuroprotective effects.
pr
e-
The homeostatic regulation of ERK and Akt phosphorylation is governed by the balance
Pr

between upstream kinases and phosphatases. Apart from free radicals, the activation of the

Epidermal Growth Factor Receptor (EGFR) is one way to increase ERK and Akt phosphorylation
al

during the early phase of cerebral I/R [31,37,38]. The inhibition of protein phosphatase activity
rn

represents an alternative way to maintain the status of ERK and Akt phosphorylation. It has been
u

reported that the broad-spectrum protein tyrosine phosphatase inhibitor, protein tyrosine phosphatase
Jo

1B inhibitor, and protein tyrosine phosphatase non-receptor type 9 microRNA all provide

neuroprotective effects against cerebral I/R injury, with concurrent activation of ERK and Akt

signaling [33,35,36,39]. Protein phosphatase 2A however, negatively regulates the ERK protective

signaling in cerebral I/R [40]. Cerebral I/R rats, OGDR hippocampal slice cultures, and OGDR

neuron/glia cultures each expressed elevated protein tyrosine phosphatase and serine/threonine

phosphatase activity, with the increments being alleviated by quercetin. Quercetin possesses the

ability to inhibit SHP2 phosphatase, along with PP2C expression and activity [41,42]. Our previous

study also demonstrated the inhibitory effect of quercetin against protein tyrosine phosphatase and

16
 Journal Pre-proof

serine/threonine phosphatase activity in microglia cells [28]. In this study, we discovered that

cerebral I/R- and OGDR-injury were strongly correlated with the reduction of ERK/Akt

phosphorylation, and the elevation of protein tyrosine and serine/threonine phosphatase activity.

Combining the aforementioned relevant studies together with our findings, ERK- and Akt-related

survival mechanisms are of importance in restraining the expansion of brain injury, with protein

tyrosine phosphatases and serine/threonine phosphatases representing crucial targets for therapeutic

intervention. The neuroprotective effects of quercetin may be partially mediated by the preservation

of ERK- and Akt-related survival mechanisms through the alleviation of both protein tyrosine and

f
oo
serine/threonine phosphatase activity. However, the specific phosphatase molecules involved were

not identified in the current study.

pr
In this study, one item should be noted. Both a 3 and 21 consecutive day oral quercetin
e-
pre-administration doses of 10 and 25 mg/kg were tested in the pilot study. The dosages and
Pr

administration of quercetin in in vivo, ex vivo, and in vitro were assessed based on our preliminary

tests and our previously reported studies [28,29]. In cerebral I/R rats, unlike the 21 consecutive day
al

study, an oral administration of quercetin for 3 consecutive days prior to cerebral I/R failed to
rn

provide significant neuroprotection (data not shown). Although quercetin is a flavonoid well-known
u

for its multiple health benefits, its water insolubility and low oral bioavailability limit its translational
Jo

utility. To overcome these obstacles, Ghosh et al. [12] have developed nanoencapsulated quercetin, in

order to help improve its bioavailability and neuroprotection. Thus, its lack of neuroprotective effect

in a short course of treatment may be due to its low oral bioavailability.

Quercetin offers multiple medicinal benefits. This study provides evidence of an alternative

target for quercetin, and sheds light on the mechanisms of its neuroprotection. In summary, we have

shown that quercetin exhibits neuroprotective, anti-oxidative, anti-inflammatory, and anti-apoptotic

effects in rats subjected to cerebral I/R, and that these effects were related to the preservation of ERK

and Akt phosphorylation through the alleviation of protein tyrosine and serine/threonine phosphatase

17
 Journal Pre-proof

activity. The neuroprotective consequences and biochemical changes caused by quercetin were

replicated in both hippocampal slice cultures and neuron/glia cultures which suffered from OGDR.

The novel effects of quercetin on the preservation of ERK and Akt signaling, along with the

alleviation of protein tyrosine and serine/threonine phosphatase activity, provide an alternative

strategy for the prevention of brain injury expansion, while also combating neurodegenerative

disorders such as stroke. Due to the fact that the dynamic regulation of ERK and Akt

phosphorylation is multifactorial, further studies are required in order to better determine the precise

neuroprotective and action signaling mechanisms of quercetin.

f
oo
Acknowledgments
pr
This study was supported by grants from both Taichung Veterans General Hospital and
e-
Hung-Kuang University (TCVGH-997318D, TCVGH-HK1028003 respectively), Ministry of
Pr

Science and Technology (MOST 105-2314-B-075A-004-MY3, MOST 105-2314-B-075A-006-MY3),

Feng Yuan Hospital, and the Ministry of Health and Welfare Hospital, Taiwan.
al
rn

Conflict of interest statement

u

The authors declare that they have no conflicts of interest.

Jo

18
 Journal Pre-proof

References

[1] Xiong Y, Manwani B, Fisher M. Management of acute ischemic stroke. Am J Med

2019;132:286-91.

[2] Jin R, Yang G, Li G. Inflammatory mechanisms in ischemic stroke: Role of inflammatory cells.

J Leukoc Biol 2010;87:779-89.

[3] Moxon-Emre L, Schlichter LC. Evolution of inflammation and white matter injury in a model

of transient focal ischemia. J Neuropathol Exp Neurol 2010;69:1-15.

[4] Enzmann G, Kargaran S, Engelhardt B. Ischemia-reperfusion injury in stroke: Impact of the

f
oo
brain barriers and brain immune privilege on neutrophil function. Ther Adv Neurol Disord

2018;11:1756286418794184.
pr
[5] Shekhar S, Cunningham MW, Pabbidi MR, Wang S, Booz GW, Fan F. Targeting vascular
e-
inflammation in ischemic stroke: Recent developments on novel immunomodulatory
Pr

approaches. Eur J Pharmacol 2018;833:531-44.

[6] Du S, Deng Y, Yuan H, Sun Y. Safflower yellow B protects brain against cerebral ischemia
al

reperfusion injury through AMPK/NF-kB pathway. Evid Based Complement Alternat Med
rn

2019;2019:7219740.
u

[7] El Khashab IH, Abdelsalam RM, Elbrairy AI, Attia AS. Chrysin attenuates global cerebral
Jo

ischemic reperfusion injury via suppression of oxidative stress, inflammation and apoptosis.

Biomed Pharmacother 2019;112:108619.

[8] Kao TK, Chang CY, Ou YC, Chen WY, Kuan YH, Pan HC, Liao SL, Li GZ, Chen CJ.

Tetramethylpyrazine reduces cellular inflammatory response following permanent focal cerebral

ischemia in rats. Exp Neurol 2013;247:188-201.

[9] Zhou F, Wang M, Ju J, Wang Y, Liu Z, Zhao X, Yan Y, Yan S, Luo X, Fang Y. Schizandrin A

protects against cerebral ischemia-reperfusion injury by suppressing inflammation and oxidative

stress and regulating the AMPK/Nrf2 pathway regulation. Am J Transl Res 2019;11:199-209.

19
 Journal Pre-proof

[10] Chen BH, Park JH, Ahn JH, Cho JH, Kim IH, Lee JC, Won MH, Lee CH, Hwang IK, Kim JD,

Kang IJ, Cho JH, Shin BN, Kim YH, Lee YL, Park SM. Pretreated quercetin protects gerbil

hippocampal CA1 pyramidal neurons from transient cerebral ischemic injury by increasing the

expression of antioxidant enzymes. Neural Regen Res 2017;12:220-7.

[11] Dai Y, Zhang H, Zhang J, Yan M. Isoquercetin attenuates oxidative stress and neuronal

apoptosis after ischemia/reperfusion injury via Nrf2-mediated inhibition of the

NOX4/ROS/NF-κB pathway. Chem Biol Interact 2018;284:32-40.

[12] Ghosh A, Sarkar S, Mandal AK, Das N. Neuroprotective role of nanoencapsulated quercetin in

f
oo
combating ischemia-reperfusion induced neuronal damage in young and aged rats. PLoS One

2013;8:e57735.
pr
[13] Jeon SJ, Kim MO, Ali-Shah F, Koh PO. Quercetin attenuates the injury-induced reduction of
e-
γ-enolase expression in a middle cerebral artery occlusion animal model. Lab Anim Res
Pr

2017;33:308-14.

[14] Lee YJ, Bernstock JD, Nagaraja N, Ko B, Hallenbeck JM. Global SUMOylation facilitates the
al

multimodal neuroprotection afforded by quercetin against the deleterious effects of

rn

oxygen/glucose deprivation and the restoration of oxygen/glucose. J Neurochem

u

2016;138:101-16.
Jo

[15] Lei X, Chao H, Zhang Z, Lv J, Li S, Wei H, Xue R, Li F, Li Z. Neuroprotective effects of

quercetin in a mouse model of brain ischemic/reperfusion injury via anti-apoptotic mechanisms

based on the Akt pathway. Mol Med Rep 2015;12:3688-96.

[16] Park DJ, Shah FA, Koh PO. Quercetin attenuates neuronal cells damage in a middle cerebral

artery occlusion animal model. J Vet Med Sci 2018;80:676-83.

[17] Pei B, Yang M, Qi X, Shen X, Chen X, Zhang F. Quercetin ameliorates

ischemia/reperfusion-induced cognitive deficits by inhibiting ASK1/JNK3/caspase-3 by

enhancing the Akt signaling pathway. Biochem Biophys Res Commun 2016;478:199-205.

20
 Journal Pre-proof

[18] Wang CP, Li JL, Zhang LZ, Zhang XC, Yu S, Liang XM, Ding F, Wang ZW. Isoquercetin

protects cortical neurons from oxygen-glucose deprivation-reperfusion induced injury via

suppression of TLR4-NF-B signal pathway. Neurochem Int 2013;63:741-9.

[19] Yao RQ, Qi DS, Yu HL, Liu J, Yang LH, Wu XX. Quercetin attenuates cell apoptosis in focal

cerebral ischemia rat brain via activation of BDNF-TrkB-PI3K/Akt signaling pathway.

Neurochem Res 2012;37:2777-86.

[20] Chen WY, Chang CY, Li JR, Wang JD, Wu CC, Kuan YH, Liao SL, Wang WY, Chen CJ.

Anti-inflammatory and neuroprotective effects of fungal immunomodulatory protein involving

f
oo
microglial inhibition. Int J Mol Sci 2018;19: E3678.

pr
[21] Lutz JA, Carter M, Fields L, Barron S, Littleton JM. The dietary flavonoid rhamnetin inhibits

both inflammation and excitotoxicity during ethanol withdrawal in rat organotypic hippocampal
e-
slice cultures. Alcohol Clin Exp Res 2015;39:2345-53.
Pr

[22] Patiño P, Parada E, Farré-Alins V, Molz S, Cacabelos R, Marco-Contelles J. Melatonin protects

against oxygen and glucose deprivation by decreasing extracellular glutamate and Nox-derived
al

ROS in rat hippocampal slices. Neurotoxicology 2016;57:61-8.

rn

[23] Kao TK, Ou YC, Kuo JS, Chen WY, Liao SL, Wu CW, Chen CJ, Ling NN, Zhang YH, Peng
u

WH. Neuroprotection by tetramethylpyrazine against ischemic brain injury in rats. Neurochem

Jo

Int 2006;48:166-76.

[24] Chang CY, Kuan YH, Li JR, Chen WY, Ou YC, Pan HC, Liao SL, Raung SL, Chang CJ, Chen

CJ. Docosahexaenoic acid reduces cellular inflammatory response following permanent focal

cerebral ischemia in rats. J Nutr Biochem 2013;24:2127-37.

[25] Pan HC, Kao TK, Ou YC, Yang DY, Yen YJ, Wang CC, Chuang YH, Liao SL, Raung SL, Wu

CW, Chiang AN, Chen CJ. Protective effect of docosahexaenoic acid against brain injury in

ischemic rats. J Nutr Biochem 2009;28:715-25.

[26] Gao XJ, Xie GN, Liu L, Fu ZJ, Zhang ZW, Teng LZ. Sesamol attenuates oxidative stress,

21
 Journal Pre-proof

apoptosis and inflammation in focal cerebral ischemia/reperfusion injury. Exp Ther Med

2017;14:841-7.

[27] Wen Z, Hou W, Wu W, Zhao Y, Dong X, Bai X, Peng L, Song L. 6'-O-Galloylpaeoniflorin

attenuates cerebral ischemia reperfusion-induced neuroinflammation and oxidative stress via

PI3K/Akt/Nrf2 activation. Oxid Med Cell Longev 2018;2018:8678267.

[28] Kao TK, Ou YC, Raung SL, Lai CY, Liao SL, Chen CJ. Inhibition of nitric oxide production by

quercetin in endotoxin/cytokine-stimulated microglia. Life Sci 2010;86:315-21.

[29] Lin SY, Wang YY, Chen WY, Chuang YH, Pan PH, Chen CJ. Beneficial effect of quercetin on

f
oo
cholestatic liver injury. J Nutr Biochem 2014;25:1183-95.

[30] Wang WY, Twu CW, Liu YC, Lin HH, Chen CJ, Lin JC. Fibronectin promotes nasopharyngeal
pr
cancer cell motility and proliferation. Biomed Pharmacother 2019;109:1772-84.
e-
[31] Zhou J, Du T, Li B, Rong Y, Verkhratsky A, Peng L. Crosstalk between MAPK/ERK and
Pr

PI3K/AKT signal pathways during brain ischemia/reperfusion. ASN Neuro

2015;7:1759091415602463.
al

[32] Cen J, Zhao N, Huang WW, Liu L, Xie YY, Gan Y, Wang CJ, Ji BS. Polyamine analogue QMA
rn

attenuated ischemic injury in MCAO rats via ERK and Akt activated Nrf2/HO-1 signaling
u

pathway. Eur J Pharmacol 2019;844:165-74.

Jo

[33] Hasegawa Y, Hamada J, Morioka M, Yano S, Kawano T, Kai Y, Fukunaga K, Ushio Y.

Neuroprotective effect of postischemic administration of sodium orthovanadate in rats with

transient middle cerebral artery occlusion. J Cereb Blood Flow Metab 2003;23:1040-51.

[34] Liu R, Tang JC, Pan MX, Zhuang Y, Zhang Y, Liao HB, Zhao D, Lei Y, Lei RX, Wang S, Liu

AC, Qin XP, Chen J, Zhang ZF, Wan Q. ERK 1/2 activation mediates the neuroprotective effect

of BpV(pic) in focal cerebral ischemia-reperfusion injury. Neurochem Res 2018;43:1424-38.

[35] Qu M, Pan J, Wang L, Zhou P, Song Y, Wang S, Jiang L, Geng J, Zhang Z, Wang Y, Tang Y,

Yang GY. MicroRNA-126 regulates angiogenesis and neurogenesis in a mouse model of focal

22
 Journal Pre-proof

cerebral ischemia. Mol Ther Nucleic Acids 2019;16:15-25.

[36] Sun M, Shinoda Y, Fukunaga K. KY-226 protects blood-brain barrier function through the

Akt/FoxO1 signaling pathway in brain ischemia. Neuroscience 2019;399:89-102.

[37] Liao SL, Ou YC, Lin SY, Kao TK, Pan HC, Chang CY, Lai CY, Lu HC, Wang WY, Chen CJ.

Signaling cascades mediate astrocyte death induced by zinc. Toxicol Lett 2011;204:108-117.

[38] Zhang G, He J, Ye X, Zhu J, Hu X, Shen M, Ma Y, Mao Z, Song H, Chen F. β-Thujaplicin

induces autophagic cell death, apoptosis, and cell cycle arrest through ROS-mediated Akt and

p38/ERK MAPK signaling in human hepatocellular carcinoma. Cell Death Dis 2019;10:255.

f
oo
[39] Sun M, Izumi H, Shinoda Y, Fukunaga K. Neuroprotective effects of protein tyrosine

phosphatase 1B inhibitor on cerebral ischemia/reperfusion in mice. Brain Res 2019;1694:1-12.

pr
[40] Zhao J, Wu HW, Chen YJ, Tian HP, Li LX, Han X, Guo J. Protein phosphatase 2A-negative
e-
regulation of the protective signaling pathway of Ca2+/CaM-dependent ERK activation in
Pr

cerebral ischemia. J Neurosci Res 2008;86:2733-45.

[41] Igbe I, Shen XF, Jiao W, Qiang Z, Deng T, Li S, Liu WL, Liu HW, Zhang GL, Wang F. Dietary
al

quercetin potentiates the antiproliferative effect of interferon-α in hepatocellular carcinoma cells

rn

through activation of JAK/STAT pathway signaling by inhibition of SHP2 phosphatase.

u

Oncotarget 2017;8:113734-48.
Jo

[42] Lu J, Wu DM, Zheng YL, Hu B, Zhang ZF, Shan Q, Zheng ZH, Liu CM, Wang YJ. Quercetin

activates AMP-activated protein kinase by reducing PP2C expression protecting old mouse

brain against high cholesterol-induced neurotoxicity. J Pathol 2010;222:199-212.

23
 Journal Pre-proof

Figure legends

Figure 1. Quercetin alleviated cerebral I/R-induced deficit in behaviors. Rats given a saline vehicle

or quercetin (25 mg/kg) pretreatment were subjected to 90-min cerebral ischemia, followed by 3-day

reperfusion. Neurological deficits were evaluated by neurological score (A). The motor performance

was assessed by a Rotarod test (B). The spontaneous locomotion including travel distance (C) and

moving time (D) was measured by Open field test. *p < 0.05 vs. the Sham vehicle group and #p <

0.05 vs. the I/R vehicle group, n = 6.

f
oo
Figure 2. Quercetin alleviated cerebral I/R brain injury. Rats given a saline vehicle or quercetin (25

mg/kg) pretreatment were subjected to 90-min cerebral ischemia, followed by 3-day reperfusion.
pr
Representative photographs show the histological examination of brain infarction by TTC staining
e-
(A). The average percentage of infarction volume in the ipsilateral hemisphere is depicted (B). The
Pr

contents of Evans blue in contralateral and ipsilateral cortical tissues were measured by an Evans

blue extravasation assay (C). Proteins were extracted from the contralateral and ipsilateral cortical
al

tissues and subjected to an enzymatic assay of caspase 3 activity (D). Total homogenates (ipsilateral
rn

and contralateral cortical tissues) were subjected to TBARS assay for the measurement of MDA
u

content (E). Total cellular RNAs were isolated from the ipsilateral and contralateral cortical tissues
Jo

and subjected to real time RT-PCR analysis for the measurement of TNF-, IL-1, and -actin

mRNA (F). The contents in the contralateral tissues of the vehicle groups were defined as 100%

(Panels D and F). *p < 0.05 vs. the contralateral tissues of the vehicle groups and #p < 0.05 vs. the

ipsilateral tissues of the vehicle groups, n = 6.

Figure 3. Quercetin alleviated OGDR injury in hippocampal slice cultures. Rat hippocampal slice

cultures were subjected to normoxia control or oxygen and glucose deprivation for 2 hours in the

presence of quercetin (0 and 10 M), followed by glucose supplement and reoxygenation for 24

24
 Journal Pre-proof

hours. Representative photographs show phase and PI fluorescence obtained after OGDR (A). The PI

fluorescence intensity is depicted (B). Proteins were extracted and subjected to enzymatic assay of

caspase 3 activity (C). Total homogenates were subjected to TBARS assay for the measurement of

MDA content (D). Total cellular RNAs were isolated and subjected to real time RT-PCR analysis for

the measurement of TNF-, IL-1, and -actin mRNA (E). The contents in normoxia control without

the quercetin groups were defined as 100% (Panels B, C, and E). *p < 0.05 vs. normoxia control

without the quercetin groups and #p < 0.05 vs. OGDR without the quercetin groups, n = 5.

f
oo
Figure 4. Quercetin alleviated OGDR injury in neuron/glia cultures. Neuron/glia cultures were

pr
subjected to normoxia control or oxygen and glucose deprivation for 3 hours in the presence of

quercetin (0 and 10 M) and followed by glucose supplement and reoxygenation for 24 hours.
e-
Representative photomicrographs show MAP-2-, GFAP-, and CD68-immunoreactive cells in
Pr

neuron/glia cultures (A). Cell damage was evaluated by the LDH efflux assay (B). Proteins were

extracted and subjected to enzymatic assay of caspase 3 activity (C). Total homogenates were
al

subjected to TBARS assay for the measurement of MDA content (D). Total cellular RNAs were
rn

isolated and subjected to real time RT-PCR analysis for the measurement of TNF-, IL-1, and
u

-actin mRNA (E). The contents in normoxia control without the quercetin groups were defined as
Jo

100% (Panels C and E). *p < 0.05 vs. normoxia control without the quercetin groups and #p < 0.05

vs. OGDR without the quercetin groups, n = 5. Scale bar = 50 m.

Figure 5. Quercetin alleviated reduction of ERK and Akt phosphorylation. Rats given a saline vehicle

or quercetin (25 mg/kg) pretreatment were subjected to 90-min cerebral ischemia and followed by

3-day reperfusion. Proteins were extracted from the contralateral and ipsilateral cortical tissues of

ischemic rats and subjected to Western blot analysis with indicated antibodies (A). Quantitative data

are shown (B). Rat hippocampal slice cultures were subjected to normoxia control or oxygen and

25
 Journal Pre-proof

glucose deprivation for 2 hours in the presence of quercetin (0 and 10 M), followed by glucose

supplement and reoxygenation for 24 hours. Proteins were extracted and subjected to Western blot

analysis with indicated antibodies (C). Quantitative data are shown (D). Neuron/glia cultures were

subjected to normoxia control or oxygen and glucose deprivation for 3 hours in the presence of

quercetin (0 and 10 M), followed by glucose supplement and reoxygenation for 24 hours. Proteins

were extracted and subjected to Western blot analysis with indicated antibodies (E). Quantitative data

are shown (F). The contents in the contralateral tissues of the vehicle groups and normoxia without

f
quercetin groups were defined as 100% (Panels B, D, and F). *p < 0.05 vs. the contralateral tissues

oo
of vehicle groups or normoxia without quercetin groups and #p < 0.05 vs. the ipsilateral tissues of

pr
vehicle groups or OGDR without quercetin groups, n = 4-6.
e-
Figure 6. ERK and Akt participated in cell survival. Rat hippocampal slice cultures were subjected to
Pr

normoxia control or oxygen and glucose deprivation for 2 hours in the presence of insulin (0 and 10

g/ml) or EGF (0 and 250 ng/ml), followed by glucose supplement and reoxygenation for 24 hours.
al

Proteins were extracted and subjected to Western blot analysis with indicated antibodies (A).
rn

Quantitative data are shown (B). Neuron/glia cultures were subjected to normoxia control or oxygen
u

and glucose deprivation for 3 hours in the presence of insulin (0 and 10 g/ml) or EGF (0 and 250
Jo

ng/ml), followed by glucose supplement and reoxygenation for 24 hours. Proteins were extracted and

subjected to Western blot analysis with indicated antibodies (C). Quantitative data are shown (D).

Hippocampal slice cultures were treated with vehicle, U0126 (20 M), or LY294002 (20 M) for 24

hours. The PI fluorescence intensity is depicted (E). Proteins were extracted and subjected to

enzymatic assay of caspase 3 activity (F). Neuron/glia cultures were treated with vehicle, U0126 (20

M), or LY294002 (20 M) for 24 hours. Cell damage was evaluated by the LDH efflux assay (G).

Proteins were extracted and subjected to enzymatic assay of caspase 3 activity (H). The contents in

the normoxia without quercetin groups and vehicle groups were defined as 100% (Panels B, D, F,

26
 Journal Pre-proof

and H). *p < 0.05 vs. the normoxia without quercetin groups or vehicle groups and #p < 0.05 vs. the

OGDR without quercetin groups, n = 4-6.

Figure 7. Quercetin alleviated phosphatase activity. Rats given a saline vehicle or quercetin (25

mg/kg) pretreatment were subjected to 90-min cerebral ischemia, followed by 3-day reperfusion.

Proteins were extracted and subjected to enzymatic assay of protein tyrosine phosphatase and

serine/threonine phosphatase activities (A). Rat hippocampal slice cultures were subjected to

normoxia control or oxygen and glucose deprivation for 2 hours in the presence of quercetin (0 and

f
oo
10 M), followed by a glucose supplement and reoxygenation for 24 hours. Proteins were extracted

pr
and subjected to enzymatic assay of protein tyrosine phosphatase and serine/threonine phosphatase

activities (B). Neuron/glia cultures were subjected to normoxia control or oxygen and glucose
e-
deprivation for 3 hours in the presence of quercetin (0 and 10 M), followed by a glucose
Pr

supplement and reoxygenation for 24 hours. Proteins were extracted and subjected to enzymatic

assay of protein tyrosine phosphatase and serine/threonine phosphatase activities (C). The contents in
al

the contralateral tissues of the vehicle groups and normoxia without quercetin groups were defined as
rn

100% (Panels A-C). *p < 0.05 vs. the contralateral tissues of the vehicle groups or normoxia without
u

quercetin groups and #p < 0.05 vs. the ipsilateral tissues of the vehicle groups or OGDR without
Jo

quercetin groups, n = 4-6.

27
 Journal Pre-proof

Author Statement:

Conceptualization, Y.-Y.W., C.-Y.C. and C.-J.C.

Methodology, S.-L.L., J.-D.W. and C.-C.W.

Validation, W.-Y.C. and Y.-H.K.

Formal analysis, C.-C.W.

Investigation, S.-L.L., J.-D.W., C.-C.W., W.-Y.C., Y.-H.K., S.-L.L. and W.-Y.W.

Data curation, S.-L.L. and W.-Y.W.

Writing-original draft preparation, Y.-Y.W. and C.-Y.C.

f
oo
Writing-review and editing, C.-J.C.

Supervision, C.-J.C.

Funding acquisition, Y.-Y.W., C.-Y.C. and C.-J.C.

pr
e-
Pr

All authors have read and agreed to the published version of the manuscript.
al
u rn
Jo

28
 Journal Pre-proof

Highlights

1. Quercetin protected rat brain against cerebral ischemia/reperfusion injury.

2. Quercetin protected hippocampal slice cultures against OGDR injury.

3. Quercetin protected neuron/glia cultures against OGDR injury.

4. Quercetin alleviated reduction of ERK and Akt phosphorylation.

5. Quercetin alleviated increase of protein phosphatase activity.

f
oo
pr
e-
Pr
al
u rn
Jo

29
Figure 1
Figure 2
Figure 3
Figure 4
Figure 5
Figure 6
Figure 7