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Neuropharmacology 67 (2013) 419e431

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Neuropharmacology

journal homepage: www.elsevier .com/locate/neuropharm

Quercetin protects against the Ab25e35-induced amnesic injury: Involvementof inactivation of RAGE-mediated pathway and conservation of the NVU

Rui Liu a, Tian-tai Zhang a, Dan Zhou a, Xiao-yu Bai a, Wei-ling Zhou a, Chao Huang a, Jun-ke Song a,Fang-rui Meng a,b, Cai-xia Wu a,c, Lin Li d, Guan-hua Du a,*

aBeijing Key Laboratory of Drug Target and Screening Research, Institute of Materia Medica, Chinese Academy of Medical Sciences & Peking Union Medical College,1 Xiannongtan Street, Beijing 100050, PR Chinab Pharmaceutical College, Liaoning University, Shenyang 110031, PR Chinac Shenyang Pharmaceutical University, Shenyang 110016, PR ChinadXuanwu Hospital Capital Medical University, Beijing 100053, PR China

a r t i c l e i n f o

Article history:Received 21 March 2012Received in revised form15 November 2012Accepted 20 November 2012

Keywords:Alzheimer’s diseaseAmyloid b peptideThe bloodebrain barrierThe neurovascular unitThe receptor for advanced glycation endproducts

* Corresponding author. Tel.: þ86 10 63165184.E-mail addresses: bessie0420@163.com, dugh@im

0028-3908/$ e see front matter � 2012 Elsevier Ltd.http://dx.doi.org/10.1016/j.neuropharm.2012.11.018

a b s t r a c t

Quercetin has demonstrated protective effects against Ab-induced toxicity on both neurons and endo-thelial cells. However, whether or not quercetin has an effect on the neurovascular coupling is unclear. Inthe present study,we aim to investigate the anti-amnesic effects of quercetin and to explore the underlyingmechanisms. Ab25e35 (10 nmol) was administrated to mice i.c.v. Quercetin was administrated orally for 8days after injection. Learning and memory behaviors were evaluated by measuring spontaneous alterna-tion in Morris Water Maze test and the step-through positive avoidance test. The regional cerebral bloodflowwas monitored before the Ab25e35 injection and on seven consecutive days after injection. Mice weresacrificed and cerebral cortices were isolated on the last day. The effects of quercetin on the neurovascularunit (NVU) integrity, microvascular function and cholinergic neuronal changes, and the modification ofsignaling pathways were tested. Our results demonstrate that quercetin treatment for Ab25e35-inducedamnesic mice improved the learning and memory capabilities and conferred robust neurovascularcoupling protection, involving maintenance of the NVU integrity, reduction of neurovascular oxidation,modulation of microvascular function, improvement of cholinergic system, and regulation of neuro-vascular RAGE signaling pathwayandERK/CREB/BDNFpathway. In conclusion, in Ab25e35-induced amnesicmice, optimal doses of quercetin administration were beneficial. Quercetin protected the NVU likelythrough reduction of oxidative damage, inactivation of RAGE-mediated pathway and preservation ofcholinergic neurons, offering an alternative medication for Alzheimer’s disease.

� 2012 Elsevier Ltd. All rights reserved.

1. Introduction

Quercetin, one of the most common flavonoids, is a strongantioxidant and free radical scavenger, found in many edible plants(Saponara et al., 2002; Chander et al., 2005). Quercetin has beenshown to display both anti-inflammatory and anti-ischemic effects,and can pass the bloodebrain barrier (BBB) and flux into brainregions (Youdim et al., 2004; Rogerio et al., 2007). Its anti-inflammatory activity may be due to the blockage of the trans-location of the nuclear transcription factor nuclear factor-kappa B(NF-kB) (Comalada et al., 2005) and the reduction of NO releaseinduced by lipopolysaccharide (LPS) (Cho et al., 2012), and itsneuroprotective effect is probably associated with the inhibition of

m.ac.cn (G.-h. Du).

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voltage-dependent sodium channels (Yao et al., 2010). Additionally,it has been thoroughly investigated for the ability to express anti-proliferative (Eberhardt et al., 2000), vasorelaxing (Appeldoornet al., 2009; Chen and Pace-Asciak, 1996), anti-platelet (Kim et al.,1998) and anti-oxidant properties (Kim et al., 2005) in varioussystems. In recent studies, investigators found that quercetin pro-tected a mouse hippocampal cell line from glutamate-inducedoxidative toxicity and lipid peroxidation by blocking reactiveoxygen species (ROS) production (Ishige et al., 2001), reversedD-galactose induced neurotoxicity in mice (Lu et al., 2006), andinhibited the formation of fibrillar Ab and destabilized fibrils inprimary cultured neurons (Ansari et al., 2009). Even though quer-cetin has potential effects on both neurons and endothelial cells, ithas been unclear whether there is a direct action of quercetinwithin the neurovascular unit (NVU).

The concept of the NVU arose from studies on the hypothalamicmedian eminence andwas referred to in earlywork as “neurohemal

R. Liu et al. / Neuropharmacology 67 (2013) 419e431420

organs” (Tweedle et al., 1988). The NVU comprises cerebral bloodvessels and surrounding astrocytes, neurons, and other supportingcells (e.g. perivascular microglia and pericytes) (Abbott, 2002;Iadecola, 2004). Overall, the concept of the NVU highlights thefunctional and pathological interactions between BBB function andneuronal activity (Abbott, 2004; Kalaria, 2009; Persidsky et al.,2006; Wilcock et al., 2009; Zlokovic, 2008a, 2008b).

In the NVU, the receptor for advanced glycation end products(RAGE) and low density lipoprotein receptor related protein-1(LRP-1) are essential in controlling Ab levels in the brain(Zlokovic et al., 2000). RAGE and LRP-1 maintain Ab levels in thebrain by controlling its transport across the BBB and play a role inimpaired vascular remodeling and cerebral blood flow dysregula-tion in the disease process. LRP-1 is a major clearance receptor forAb at the BBB, while RAGE binds different forms of Ab. The reactionsof RAGE with Ab at the luminal membrane of the BBB mediate there-entry of circulating Ab into the brain across the BBB followed byAb binding to neurons, NF-kB-dependent activation of endotheliumwith expression of proinflammatory cytokines, and secretion ofendothelin-1 resulting in cerebral blood flow (CBF) reductions(Zlokovic, 2008a). Further, ligation of RAGE in neurons and glialcells has been shown to activate multiple signaling pathways,including Ras-extracellular signal-regulated kinase 1/2 (ERK1/2)(Zill et al., 2001), Cdc42/Rac (Taguchi et al., 2000), stress-activatedprotein kinase/c-Jun-NH2-terminal kinase (SAPK/JNK) and p38MAP kinase pathways (p38 MAPK) (Li et al., 2004). These signalingpathways finally lead to neurotransmitter system dysfunctionand cholinergic system dysregulation. Therefore, neurovascularRAGE pathway is increasingly considered as a new therapeuticalapproach for Alzheimer’s disease (AD).

AD is a progressive neurodegenerative disorder characterized byneuropathological features such as amyloid plaques, neurofibrillarytangles and cholinergic neuronal loss (Coyle et al., 1983; Hendrie,1997). There are accompanying pathologies that likely contributeto the progression of the disease including neuroinflammation(Akiyama et al., 2000) and cerebrovascular involvement (Nicollet al., 2004). A variety of evidence suggests that the accumulationof Ab proteins, mainly the 40e42-amino-acid peptide fragment andalso Ab25e35 oligomers that are mediators in AD (Wilquet and DeStrooper, 2004; Ostrovskaya et al., 2007), mediate neurovasculardysfunction (Iadecola, 2004; Liu et al., 2011; Zlokovic, 2005),chronic neurodegenerative process, and amyloid deposition inbrain parenchyma and vessels (Han et al., 2008; Shin et al., 2007),all of which involve neurovascular coupling mechanisms and couldcontribute to pathologies in AD. As neuroprotectants are used assole treatment means, an effective therapy is absent at present.Current treatment for AD needs could be more effective with other

Fig. 1. Experimental procedure. (1) The MWM test began on the 3rd day of quercetin treatmquercetin treatment, a single probe trial was conducted. (2) The step-through positive avo(3) rCBF was monitored before the Ab25e35 injection and on seven consecutive days duringisolated on the 9th day. The anti-amnesic effects and mechanism of quercetin were tested.

complementary therapies. An efficacious therapy may need to offerwide cytoprotection to the NVU, instead of the traditional neuro-nocentric protection for AD.

In this study, we found that quercetin protects the NVU likelythrough reduction of oxidative damage, inactivation of RAGE-mediated pathway and preservation of cholinergic neurons, basedon multiple immunofluorescent and biological assays by cellularand molecular measures. This study might provide the basis ofa novel therapeutic management of AD.

2. Material and methods

2.1. Drugs

Ab25e35 were purchased from Sigma Chemical Company (St. Louis, MO, USA), thelatter dissolved in sterile saline at a concentration of 1 mM and aggregated byincubation at 37 �C for 7 days before use. Quercetin was extracted and isolated fromthe whole plant of Elsholtzia rugulosa Hemsl., which was collected in the YunnanProvince of China and identified in Chinese Academy of Medical Sciences. The HPLCpurity of the compound was over 98% (Liu et al., 2009a).

2.2. Experimental schedules

The experimental schedules are shown in Fig. 1. Male Kunming mice, 25e30 g,were provided by the Animal Breeding Center of the Chinese Academy of MedicalSciences. Mice were housed 5 per cage and acclimated to standard laboratoryconditions (12 h light, 12 h dark cycle) with free access to mouse chow and water.The animal breeding and experiments were conducted in accordance with institu-tional guidelines and ethics and approved by the Laboratories Institutional AnimalCare and Use Committee of Chinese Academy of Medical Sciences and Peking UnionMedical College.

Mice were anesthetized with 50 mg/kg sodium pentobarbital and placed ina stereotaxic instrument. The aggregated Ab25e35 was injected into the right lateralventricle with the following coordinates (Laursen and Belknap, 1986): �0.5 mmanterior/posterior, þ1.0 mm medial/lateral and �2.5 mm dorsal/ventral fromBregma (10 nmol in 3 ml of saline per injection). Sham animals were injected in anidentical manner with the same amount of sterile saline.

Therewere totally 114mice. They were divided into six groups the next day aftersterile saline or Ab25e35 injection: sham group, Ab25e35-treated group, quercetin5 mg/kg group, quercetin 10 mg/kg group, quercetin 20 mg/kg group, and quercetin40 mg/kg group (19 mice in each group). Quercetin was dissolved in distilled watercontaining 20% hydroxypropyl-beta-cyclodextrin at a concentration of 5 mg/ml, andwas administered to mice receiving Ab25e35 injection in treatment groups by oralgavage once a day continuously for 8 days. The sham group and Ab25e35-treatedgroup received oral gavage in the same manner using distilled water containing20% hydroxypropyl-beta-cyclodextrin without quercetin.

There are two parallel experiments. (1) The Morris Water Maze (MWM) trainingbegan on the 3rd day of quercetin treatment, and mice of each group were trainedfor five consecutive days; a single probe trial was conducted on the 9th day ofquercetin treatment. The step-through positive avoidance test was performed onthe 8th day and 9th day after the MWM test. After that, mice of each group weresacrificed and cerebral cortices were isolated on the 9th day. The effects of quercetinon NVU integrity, cholinergic neuronal changes, and the modification of BDNF,RAGE, LRP-1, ERK, CREB, p38MAPK and NF-kB p65 levels were tested. (2) Theregional cerebral blood flow (rCBF) was monitored before sterile saline or Ab25e35

ent, and mice of each group were trained for five consecutive days; on the 9th day ofidance test was performed on the 8th day and 9th day in parallel to the MWM test.quercetin treatment. (4) Mice of each group were sacrificed and cerebral cortices were

R. Liu et al. / Neuropharmacology 67 (2013) 419e431 421

injection and on seven consecutive days. After that, the effects of quercetin oncerebral oxidation were tested.

2.3. Morris water maze performance

The MWM task was performed (Morris, 1984) and a probe test was assigned(Song et al., 2006) in the standard methods reported. Briefly, spatial training beganon 3rd day of quercetin administration. Mice were trained for five consecutive days.In each trial, the time required to escape onto the hidden platform was recorded asescape latency. The mice were given a maximum of 60 s to find the hidden platform.If a mouse failed to find the platform within 60 s, the training was terminated,a maximum score of 60 s was assigned, and the mouse was manually guided to thehidden platform. On 9th day of quercetin administration, a single probe trial wasconducted. The platformwas removed and the mice were placed into the pool fromthe quadrant opposite to the training quadrant. In each probe trial, the mice wereallowed to swim for 60 s. The time the mouse staying in the platform quadrant andnumbers of crossings where the platform had been located were recorded. Thetreatments were continued during the water maze task. One hour before the MWMtest, mice were treated with vehicle or quercetin administrations.

2.4. Step-through positive avoidance test

The contextual long-term memory of the animals was assessed using the step-through passive avoidance procedure (Kim et al., 2008; Lu et al., 2011). Passiveavoidance tasks were carried out in identical illuminated and non-illuminated boxes(20 cm � 20 cm � 20 cm), separated by a guillotine door (5 cm � 5 cm). The illu-minated compartment contained a 50 W bulb and the floor of the non-illuminatedcompartment (20 cm � 20 cm � 20 cm) was composed of 2 mm stainless steel rodsspaced 1 cm apart. For the acquisition trial, mice were initially placed in the illu-minated compartment; the door between the two compartments was opened 5 slater. When the mice entered the non-illuminated compartment, an electrical footshock (0.30 mA) for 3 s was delivered through the stainless steel rods. The retentiontest was carried out 24 h after training. The time and numbers a mouse took to enterthe NON-illuminated compartment after opening the door were defined as latencyand error times respectively. Latency to enter the dark compartment was recordedup to 300 s to avoid ceiling effects.

2.5. Confocal microscopy

Brains of mice of each group were perfusion-fixed with 4% paraformaldehydeafter behavioral tests. Series of 8 mm thick coronal sections were cut throughtemporal cortices. Neurons, astrocytes and endothelial cells were stained with goatpolyclonal anti-MAP-2 antibody (1:400, Sigma), mouse monoclonal fibrillary acidicprotein (GFAP) antibody (1:800, Sigma) and CD34 antibody (1:400, Sigma),respectively. The secondary antibodies were Cy5, tetramethyl rhodamine iso-thiocyanate and FITC conjugated IgG for each antibody. Confocal images of sliceswere taken for the NVU image using excitation and emission wavelengths asfollows: Cy5 (excitation 650 nm, emission 670 nm), tetramethyl rhodamine iso-thiocyanate (excitation 585 nm, emission 590 nm) and FITC (excitation 488 nm,emission 530 nm). Fluorescent images from cortical slices were acquired and fluo-rescent intensity was calculated by Image Pro Plus.

2.6. rCBF measurement

A Laser-Doppler Flowmetry (LDF) was used to monitor the regional cerebralblood flow (rCBF) before the Ab25e35 injection and on seven consecutive days duringquercetin treatment, as described in previous reports (Liu et al., 2009b, 2011). Briefly,the rCBF was monitored before Ab25e35 or sterile saline injection to establish thebaseline value. The rCBF was monitored for eachmouse the next day after Ab25e35 orsterile saline injection every day. The values of rCBF were first expressed asa percentage of the baseline value and then calculated as a percentage of pre-Ab25e35 injection.

2.7. Tracer application and assessment of BBB permeability

The tracer lanthanum (lanthanum ionsw139 Da, effective hydrated radius 3.1�A)was used to assess the restoration of the BBB function by previous reported methods(Liu et al., 2009b, 2011). Briefly, mice were anaesthetized with 45 mg/kg sodiumpentobarbital, i.p. The heart was exposed and the left ventricle was perfused with0.9% saline, followed by perfusion with a fixative consisting of one part 4%lanthanum nitrate and two parts 6% glutaraldehyde in 0.1 M sodium cacodylate (pH7.40e7.50). At the end of brain perfusion, the prefrontal cerebral cortices wereisolated and cut into 1e2 mm3 pieces. These samples were kept for transmissionelectron microscopy (TEM) studies. Ultrathin sections (60 nm thick) wereobtained of selected blocs of cerebral regions. The ultrathin sections weremounted on copper grids (200 mesh) and double-contrasted with uranyl acetateand lead citrate for examination in a LEO 906 (Zeiss, Oberkochen, Germany) trans-mission electron microscope operated at 60 kV. Tracer leakage was also evaluated.The evaluation was performed by counting at least 10 randomly selected

microvessels (arterioles, venules and capillaries) in one ultrathin section (w1 mm2)of cortex per animal (n ¼ 4 mice � 10 vessels per mouse ¼ 40 vessels/group).Vascular leakage was evaluated by the criterion of whether or not there was leakageof lanthanum ions, and calculated as a percentage by dividing the number of affectedvessels by the total number of vessels examined in the sections.

2.8. Detection of oxyradicals using hydroethidine in situ

The production of oxidative stress was investigated by a previously describedmethod of in situ detection of oxidized hydroethidine (HEt) (Bindokas et al., 1996;Liu et al., 2011). HEt is diffusible into the CNS parenchyma after an intravenousinjection and is selectively oxidized to ethidium by superoxide anion but not byother ROS such as hydrogen peroxide, hydroxyl radical, or peroxynitrite. Eachmousewas anesthetized with 45 mg/kg sodium pentobarbital, i.p., and administeredintravenously through the right jugular vein 150 ml of HEt [1 mg/ml diluted inphosphate-buffered saline (PBS); Molecular Probes, Eugene, OR, USA]. One hourafter the HEt injection, mice were sacrificed and fixed with 4% paraformaldehyde bytranscardial perfusion. Series of 8 mm thick coronal sections were cut throughtemporal cortices. The sections were incubated with 2 mM Hoechst 33342 (DojindoLaboratory, Kumamoto, Japan) in PBS. Photomicrographs of slices were taken usingdouble exposures to oxidized HEt and Hoechst 33342. The percentage of oxidizedHEt-expressing cells, relative to the number of total cells stained with Hoechstnuclear staining, or the percentage increase in fluorescent intensity relative tobackground values, was separately calculated.

2.9. Acetylcholinesterase assay

Prefrontal cortex homogenate in a volume of 500 ml was mixed with 1% Triton X-100 (1% w/v in 0.03M sodium phosphate buffer, pH 7.0) and samples were centri-fuged at 100,000 g at 4 �C for 60 min in a Beckman Ultracentrifuge (LE 80, USA). Thesupernatant was collected and stored at 4 �C for acetylcholinesterase estimation(Ellman et al., 1961). The kinetic profile of enzyme activity was measured spectro-photometrically at 412 nm with an interval of 15 s. One unit of AChE activity wasdefined as the number of mmol of acetylthiocholine iodide hydrolyzed per minuteper milligram of protein. The specific activity of AChE is expressed in mmol/min/mgof protein.

2.10. ELISA analysis

Two site enzyme immunoassay system assay (ELISA) was used to detect brain-derived neurotrophic factor (BDNF) content and acetylcholine level in bilateralcerebral cortices as previously reported (Arancibia et al., 2007). Briefly, on the 9thday after Ab25e35 injection, each mousewas decapitated under ether anesthesia. Thecerebral cortices were quickly removed and cleaned with chilled saline on the ice.The tissues were homogenized with ultrasonication and centrifuged at 4 �C for10 min, 13,000 rpm. The supernatants were recovered and used for detection. Dataare represented as pg/g wet tissue and all assays were performed in duplicate.

2.11. Western blot analysis

The expression of RAGE, ERK1/2, CREB, p38MAPK and NF-kB p65 in cerebralcortex homogenate, and RAGE, LRP-1, ERK1/2 and NF-kB p65 in cerebral cortexmicrovessel homogenate were determined by western blot analyses.

Mice of each groupwere anaesthetized and sacrificed by decapitation. The brainswere quickly dissected, and the gray matter of the bilateral cerebral cortex wascarefully isolated. Part of the tissues was used to determine protein expression incerebral cortex homogenate. Others were homogenized in the microvessel isolationbuffer (147mMNaCl, 4mMKCl, 3mMCaCl2,1.2mMMgCl2, 5mMglucose and 15mMHEPES, pH 7.4). Then, the suspensionwas passed through a sieve of 220 mmpore size,followedbya sieve of 76mmpore size. Finally, thefiltratewas centrifugedat 5000 rpmfor 30 min at 4 �C. The proteins were extracted from the pellets by using lysis buffer(50mMTriseHCl, pH7.4. 20mMEDTA, 0.1% sodiumdodecyl sulfate,100mMNaCl,1%NP-40, 0.5% sodium deoxycholate, 50 mM sodium fluoride, 1 mM sodium orthova-nadate, 1 mM PMSF, 2 mM sodium pyrophosphate, 1 mg/ml pepstatin A, 100 mg/mlleupeptin and one protease inhibitor cocktail tablet/50 ml, Roche Biochemicals,Indianapolis, IN). NF-kB p65 expressionwas determined by western blot assessmentin cytoplasmic and nuclear extracts of cerebral tissues that were obtained usinga nuclear/cytoplasmic isolation kit (Pierce, Biotechnology, Inc., Rockford, IL, USA).

The antibodies used in this experiment were as follows: anti-RAGE (polyclonalantibody, 1:400, Sigma), anti-a-LRP-1 (polyclonal antibody, 1:400, Santa CruzBiotechnology, Santa Cruz, CA), anti-ERK (polyclonal antibody,1:500, Sigma), anti-p-ERK1/2 (polyclonal antibody, 1:500, Sigma), anti-p38 MAPK (polyclonal antibody,1:400, Sigma), anti-p-p38 MAPK (polyclonal antibody, 1:400, Sigma), anti-CREB(polyclonal antibody, 1:400, Sigma), anti-p-CREB (polyclonal antibody, 1:400,Sigma), anti-NF-kB p65 (polyclonal antibody, 1:400, Santa Cruz Biotechnology, SantaCruz, CA) and anti-b-actin (monoclonal antibody 1:2000, Sigma). Horseradishperoxidase (HRP)-conjugated secondary antibodies were purchased from ZSGB-Bio(China). Membranes were washed several times with TBST prior to incubation withHRP-conjugated secondary antibody (1:1000) for 45min at room temperature. After

R. Liu et al. / Neuropharmacology 67 (2013) 419e431422

subsequent washes in TBST, the protein bands were visualized using an ECL�detection kit (GE Healthcare) and exposure to X-ray films. Relative optical densitiesand areas of bands were quantified using an image densitometer. The densitometricplots of the results were normalized to the intensity of the actin band.

2.12. Statistical analysis

All data are represented as the mean� S.E.M. The P values of less than 0.05 wereconsidered statistically significant. Statistical analyses were performed on computerusing the SPSS software (Version 16.0; SPSS, Inc., Chicago, IL). Treatment differencesin the escape latency in MWM task and rCBF measurement were analyzed usingtwo-factor analysis of variance with repeated measures on one factor. Tukey’s posthoc test was used if the treatment was significant in ANOVA. The other studies wereanalyzed using one-way ANOVA followed by an appropriate post-hoc test to analyzethe difference.

3. Results

3.1. Effects of quercetin on learning and memory performance in theAb25e35-induced amnesia model in mice

Spatial learning was initiated on the 3rd day of quercetintreatment (day 1 for MWM test) and assessed by the time requiredto find the hidden platform (escape latency). Fig. 2A shows theresults of all mice during acquisition training. Repeated-measuresANOVA revealed a significant day effect on escape latency(F(4,336) ¼ 35.99, P < 0.001) within the groups, indicating that micein different groups showed different spatial learning capabilityduring the 5 day acquisition training. There is also a significanttreatment effect (F(5,84) ¼ 5.66; P < 0.001) on the escape latency,and subsequent comparisons further suggested that 20 mg/kg and40 mg/kg quercetin treatment reduced the escape latency incomparison to the Ab25e35 group (20 mg/kg, P < 0.01; 40 mg/kg,P < 0.05) across the 5 d training period, which demonstrated that20 mg/kg and 40 mg/kg quercetin was effective in attenuatingspatial learning deficits in Ab25e35-treated mice.

Probe trials were conducted on the 9th day of quercetin treat-ment (day 6 for MWM test) to assess the spatial memory. All themice spend the same amount of time (60 s) searching for theplatform, but the data suggested that the Ab25e35-treated mice

Fig. 2. Effects of quercetin on learning and memory capabilities in the Ab25e35-induced aComparison of latency to platform during 5 training days in Morris water maze. (B) Quercetintest. **P < 0.01 vs. sham, #P < 0.05 vs. Ab25e35. (C) Quercetin increased the numbers of crossivs. Ab25e35. (D) Quercetin increased the latency to enter the dark compartment in the step-thpresent the Mean � S.E.M. n ¼ 15 mice per group. (E) Quercetin decreased the error times

spent significantly less time searching for the platform in the targetquadrant and showed less numbers of crossings where the plat-form previously located relative to the sham mice (P < 0.01,P < 0.05, Fig. 2B and C). During the same amount of searching time,20 mg/kg quercetin-treated mice spent more time searching in thetarget quadrant, and the numbers of crossings where the platformlocated was also increased in this group (P < 0.05, Fig. 2B and C).These results demonstrated that quercetin partly improved spatialmemory capability against Ab25e35-induced toxicity.

The effect of quercetin onmemory performancewas also carriedout in the passive avoidance task. During the acquisition trial, nosignificant difference in step-through latency and error times wasobserved between groups (Fig. 2D and E). During the retentiontrials, the sham mice hesitated to re-enter the ILLUMINATEDcompartment, but the Ab25e35-treated mice re-entered the ILLU-MINATED compartment more frequently, and showed the shorterstep-through latency and more error times (P < 0.05, P < 0.01,Fig. 2D and E). In the quercetin-treated groups, mice showed thelonger step-through latency and less error times (P< 0.05, P< 0.01,Fig. 2D and E), which demonstrated that quercetin improvedmemory capability against Ab25e35-induced toxicity in a dose-dependent manner at 5 mg/kg, 10 mg/kg and 20 mg/kg.

In the behavioral tests, due to the significant effects from 5 mg/kg to 40 mg/kg among the tested doses of quercetin, the highestdose of 40 mg/kg was not as effective as the lower 20 mg/kg. Wechose 20 mg/kg to investigate the anti-amnesic mechanism of thiscompound.

3.2. Effects of quercetin on morphology changes of theneurovascular unit in the Ab25e35-induced amnesia model in mice

Fig. 3 shows the morphology changes of major components ofNVU examined under confocal microscopy. The normal capillary(green) exhibited the integrative junction, and peripheral astrocytes(red) and neurons (blue) did not exhibit any apparent signs of per-ivascular swelling or deficiency (Fig. 3A). However, in cortical slicesof Ab25e35-treated mice, microvessels were broken, astrocytesproliferatedand therewas extensiveneuron loss (P<0.05, P<0.001,

mnesia model in mice. Data present the Mean � S.E.M. n ¼ 15 mice per group. (A)increased the percentage of time the mouse staying in the target quadrant in the probengs where the platform has been located in the probe test. *P < 0.05 vs. sham, #P < 0.05rough passive avoidance test. *P < 0.05 vs. sham, #P < 0.05, ##P < 0.01 vs. Ab25e35. Datain the step-through passive avoidance test. **P < 0.01 vs. sham, #P < 0.05 vs. Ab25e35.

Fig. 3. Effects of quercetin on morphology of NVU in the Ab25e35-induced amnesia model in mice. Morphological changes of major components of the NVU were examined underconfocal microscopy. Neurons, astrocytes and microvessels were stained and appear as blue, red and green, respectively. There are representative photomicrographs of sham (A),Ab25e35 (B) and 20 mg/kg quercetin (C) groups. (D) Values of microvessel, astrocyte and neuron fluorescent intensity. Data present the Mean � S.E.M., n ¼ 3, *P < 0.05, ***P < 0.001vs. sham, #P < 0.05 vs. Ab25e35. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 4. Effects of quercetin on neurovascular oxidative damage in the Ab25e35-induced amnesia model in mice. Photomicrographs were taken using double exposures to oxidizedHEt (red) and Hoechst 33342 (blue). Oxidized HEt signals were localized in cytoplasm of cells (arrows). (A) Sham. (B) Ab25e35. (C) 20 mg/kg quercetin. (DeE) Values of the relativecytosolic oxidized HEt intensity and the percentage of ethidium-expressing cells relative to total Hoechst-stained cells. Data present as the Mean � S.E.M, n ¼ 4, **P < 0.01,***P < 0.001 vs. sham, #P < 0.05, ##P < 0.01 vs. Ab25e35. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

R. Liu et al. / Neuropharmacology 67 (2013) 419e431 423

Fig. 5. Effects of quercetin on regional cerebral blood flow in the Ab25e35-inducedamnesia model in mice. The rCBF value of Ab25e35-treated mice significantly declinedprogressively from day 3 to day 9. Data presented as the Mean � S.E.M, n ¼ 4,***P < 0.001 vs. sham, #P < 0.05, ##P < 0.01, ###P < 0.001 vs. Ab25e35.

R. Liu et al. / Neuropharmacology 67 (2013) 419e431424

Fig. 3B, D). 20 mg/kg quercetin protected the vessel integrity andprevented loss of surrounding neurons (P< 0.05, Fig. 3C, D), but wasnot able to reduce the Ab25e35-induced proliferation of astrocytes.

3.3. Effects of quercetin on neurovascular oxidative damage in theAb25e35-induced amnesia model in mice

Under double exposure of oxidized HEt (red) and Hoechstnuclear staining (blue), most oxidized HEt was localized in a rela-tively weak signal in cell cytosol of cells and neurovascular inter-face in the cortical slices of sham group (Fig. 4A, arrows). Butoxidized HEt signals in the cytosol were widely and intensivelydistributed in the Ab25e35-treated cortex (Fig. 4B). In addition,endothelial cells and neurovascular interface showed strongoxidized HEt signals after Ab25e35 injection (Fig. 4B, arrows). In the

Fig. 6. Effects of quercetin on cortical capillaries and the surrounding neuropil in the Ab25e3neuropil around. The distribution of lanthanum ions (arrows) indicates the integrity of theelectron microscopy. Each bar represents the mean number of affected vessels per grou(n ¼ 4 mice � 10 vessels per mouse ¼ 40 vessels/group). Data presented as the Mean � S.

20 mg/kg quercetin group, slices showed a significant decrease ofoxidized HEt signals in the cytosol and in the neurovascular inter-face (Fig. 4C, arrow); the relative cytosolic oxidized HEt intensityand the percentage of oxidized HEt-positive cells relative to totalHoechst-stained cells were decreased by 41% and 25% (P < 0.05,P < 0.01, Fig. 4D, E), respectively.

3.4. Effects of quercetin on rCBF in the Ab25e35-induced amnesiamodel in mice

Fig. 5 shows the values of all mice in rCBF measurement.Repeated-measures ANOVA revealed a significant day effect(F(7,63) ¼ 48.51, P < 0.001) within the groups and a significanttreatment effect (F(2,9) ¼ 235.60; P < 0.001) on rCBF values. Therewas also a significance in the interactions of treatment and days(F(14,63) ¼ 31.83, P < 0.001). Subsequent comparisons further sug-gested that the rCBF value of Ab25e35-treated mice was significantlydeclined progressively from day 3 (P < 0.001); treatment with20mg/kg quercetin for 8 days had a significant effect on rCBF values(P < 0.001), and the rCBF value of quercetin-treated mice showedsignificant changes compared with the Ab25e35-treated group fromday 6 (P < 0.05, P < 0.01, P < 0.001).

3.5. Effects of quercetin on cortical capillaries and the surroundingneuropil in the Ab25e35-induced amnesia model in mice

Ultrastructural observations by TEM in the cortex of mice of thesham group showed lanthanum ions confined into the vesselslumen and normality in the perivascular tissue (Fig. 6A, arrows).The normal endothelial cell layer showed distinct tight junctionsand underlying astrocytes did not exhibit any apparent signs ofperivascular edema (Fig. 6A). Neuron showed a smooth surface andits nuclei was not condensed. In vessels of Ab25e35-treated mice,cortex presented leakage of lanthanum ions from the lumen ofvessels and a robust increase in BBB permeability (Fig. 6B).Lanthanum ion was filling the endocytotic vesicles of endothelialcells (EC) and leaked out of basement membrane of endothelium.

5-induced amnesia model in mice. Photomicrographs show the luminal surface and theBBB. (A) Sham. (B) Ab25e35. (C, D) 20 mg/kg quercetin. (E) Counting by transmissionp by the total number of vessels counted in one section per mouse of each groupE.M, ***P < 0.001 vs. sham, #P < 0.05 vs. Ab25e35. EC, endothelial cells; As, astrocytes.

R. Liu et al. / Neuropharmacology 67 (2013) 419e431 425

Observations done by TEM revealed that in cortex a significantincrease of vessels with tracer extravasation (14.33-fold higher)occurred in Ab25e35-treated mice in comparison to sham group(P < 0.001, Fig. 6E). Water filled astrocytes (As) and extracellularspace, and adjacent degeneration of neuropil were clearly visible(Fig. 6B). Neurons became pyknotic (Fig. 6B). 20 mg/kg quercetintreatment alleviated the leakage of lanthanum ions from the lumenof vessels and protected BBB integrity (Fig. 6C, D). The number ofaffected vessels showed a significant decrease compared to theAb25e35-treated group (P < 0.05, Fig. 6E). Astrocytes did not showapparent signs of end-foot swelling after quercetin treatment.Neuronal pycnosis was relieved.

3.6. Effects of quercetin on expression of LRP-1, RAGE, ERK1/2 andNF-kB p65 of cerebral microvessels in the Ab25e35-induced amnesiamodel in mice

As shown in Fig. 7A, expression of a-LRP-1 was significantlydecreased with Ab25e35 treatment compared to expression in thesham group (P < 0.001); treatment with quercetin did not rescuethis effect.

RAGE expression was significantly increased by Ab25e35 treat-ment (P < 0.001, Fig. 7A). Phosphorylated ERK1/2 level was alsoincreased in Ab25e35-treatedmice while no significancewas seen intotal ERK1/2 expression (P < 0.01, Fig. 7B). The nuclear expression

Fig. 7. Effects of quercetin on cerebral microvascular expression of LRP-1, RAGE, ERK1/2 andimmunoblots for a-LRP-1 and RAGE and quantitative analysis of them in cerebral micro###P < 0.001 vs. Ab25e35. (B) Representative immunoblots for p-ERK1/2 and total ERK1/2 anMean � S.E.M, n ¼ 4, ***P < 0.001 vs. sham, ##P < 0.01 vs. Ab25e35. (C) Representative immuData present as the Mean � S.E.M, n ¼ 4, ***P < 0.001 vs. sham, ###P < 0.001 vs. Ab25e35.

of NF-kB p65 was significantly increased (P< 0.001, Fig. 7C), but theexpression of NF-kB p65 in cytoplasm was unchanged in Ab25e35-treated mice. Therefore, 20 mg/kg quercetin reduced the expres-sion of RAGE, suppressed phosphorylated ERK1/2 level anddecreased the ratio of NF-kB p65 in nuclei and in cytoplasm in thecerebral microvessels of Ab25e35-treated mice.

3.7. Effects of quercetin on ACh level and AChE activity of cerebralcortex in the Ab25e35-induced amnesia model in mice

Changes of cortical ACh level were detected in parallel to AChEactivity. As shown in Fig. 8, there was a significant decrease incortical ACh level in Ab25e35-treated group (P < 0.001); simulta-neously, AChE activity in the Ab25e35-treated group was signifi-cantly increased (P < 0.001). 20 mg/kg quercetin restored corticalACh levels and suppressed AChE activity in Ab25e35-treated mice(P < 0.01).

3.8. Effects of quercetin on expression of RAGE, p38 MAPK and ERK/CREB/BDNF pathway of cerebral cortex in the Ab25e35-inducedamnesia model in mice

As shown in Fig. 9A, in the Ab25e35-treated group, cerebral RAGEexpression was significantly increased (P < 0.001). According toFig. 9B, phosphorylated p38 MAPK expression was up-regulated by

NF-kB p65 activation in the Ab25e35-induced amnesia model in mice. (A) Representativevascular extracts. Data present as the Mean � S.E.M, n ¼ 4, ***P < 0.001 vs. sham,d quantitative analysis of them in cerebral microvascular extracts. Data present as thenoblot for NF-kB p65 and quantitative analysis in the cytoplasmic and nuclear extracts.

Fig. 8. Effects of quercetin on ACh level and AChE activity of cerebral cortex in the Ab25e35-induced amnesia model in mice. (A) Quercetin increases cortical ACh level in Ab25e35-treated mice. Data present as the Mean � S.E.M, n ¼ 4, ***P < 0.001 vs. sham, ##P < 0.01 vs. Ab25e35. (B) Quercetin decreases the AChE activity in the Ab25e35-treated mice. Datapresent as the Mean � S.E.M, n ¼ 4, ***P < 0.001 vs. sham, ##P < 0.01 vs. Ab25e35.

R. Liu et al. / Neuropharmacology 67 (2013) 419e431426

2.16 fold (P < 0.001), whereas total p38 MAPK expression wasunchanged comparedwith the shamgroup. In the samemanner, thenuclear expression of NF-kB p65 was increased, shown as a signifi-cant increased ratio of nuclear expression to cytoplasmic expression(P < 0.001). Oral administration of 20 mg/kg quercetin significantlydecreased the expression of RAGE and phosphorylated p38MAPK incerebral cytoplasm, and inhibited the nuclear expression of NF-kBp65 in the cerebral cortex of Ab25e35-treated mice (P < 0.05). Nosignificant changes of total p38 MAPK and cytoplasmic NF-kB p65expression were seen in the treatment group.

As shown in Fig. 10, our findings showed that cortical BDNFlevel, phosphorylated ERK1/2 and phosphorylated CREB expressionof cerebral cortex were suppressed in Ab25e35-treated mice(P < 0.01, P < 0.001). After 20 mg/kg quercetin treatment, coupledwith the elevation of BDNF level, enhanced phosphorylated ERK1/2and CREB expression were detected in the cerebral cortex(P < 0.05).

4. Discussion and conclusion

This study investigates the potential mechanisms of quercetinagainst Ab25e35-induced amnesiawith respect to the damage of theNVU. The results demonstrate that in Ab25e35-induced amnesicmice optimal doses of quercetin administration were beneficial,

Fig. 9. Effects of quercetin on cerebral cortex expression of RAGE, p38 MAPK and NF-kBimmunoblot for RAGE and quantitative analysis in cerebral cortex extracts. Data present as timmunoblots for p-p38 MAPK, total p38 MAPK and NF-kB p65 and quantitative analysis***P < 0.001 vs. sham, #P < 0.05 vs. Ab25e35.

which improved the learning and memory capabilities andconferred robust neurovascular coupling protection. This study hasthree prospective facts that are potentially beneficial for thetreatment of AD. These prospects are: (1) quercetin showedmultiple protective effects on cerebral cholinergic neurons inducedby Ab; (2) quercetin played a pivotal role in molecular cascades ofthe BBB against Ab-induced damage; (3) quercetin altered the NVUchanges and affected RAGE-mediated transduction.

4.1. Neuroprotection of quercetin against Ab-induced amnesia

Intracerebroventricular infusion of Ab25e35 was demonstratedto induce spatial learning and memory deficits in AD animalmodels (Nakdook et al., 2010; Pavia et al., 2000; Rovira et al., 2002;Sigurdsson et al., 1997). Herein, a single i.c.v. injection of Ab25e35peptides in mice could induce the significant learning andmemory impairment when compared to the sham group as shownin the results from behavioral performance. This confirmed thatcognitive impairment was induced by Ab25e35 peptides itself, notattributable to an i.c.v. injection.

Different brain area has been shown to be involved in a range oflearning and memory performance tasks. The prefrontal cortexparticipates in learning, memory, and other high-level cognitivetasks (Miller et al., 2002). Pyramidal neurons in the layers III and V

p65 activation in the Ab25e35-induced amnesia model in mice. (A) Representativehe Mean � S.E.M, n ¼ 4, ***P < 0.001 vs. sham, #P < 0.05 vs. Ab25e35. (B) Representativein the cytoplasmic and nuclear extracts. Data present as the Mean � S.E.M, n ¼ 4,

Fig. 10. Effects of quercetin on cerebral cortex BDNF/ERK/CREB pathway in the Ab25e35-induced amnesia model in mice. (A) Quercetin increases cerebral BDNF level of cortex inAb25e35-treated mice. Data present the Mean � S.E.M, n ¼ 4, **P < 0.01 vs. sham, #P < 0.05 vs. Ab25e35. (B) Representative immunoblots for p-ERK, total ERK, p-CREB and CREB andquantitative analysis of them in cerebral cortex extracts. Data present as the Mean � S.E.M, n ¼ 4, **P < 0.01, ***P < 0.001 vs. sham, #P < 0.05 vs. Ab25e35.

R. Liu et al. / Neuropharmacology 67 (2013) 419e431 427

of the cerebral cortex are in charge of the spatial memory andlearning both in human beings and in experimental animals (Block,1999; Lim et al., 2004; Zola-Morgan et al., 1986). Thus, long-termpreservation of neurons in vulnerable cerebral structures andfunctions are considered to be amajor end-point of neuroprotectivestrategies. Vulnerable cerebral structure and function and therelated molecular changes of NVU were selected for furtherinvestigation in this study.

Our results confirmed the protective effects of quercetin againstAb-induced toxicity. Quercetin, by oral gavage of 20 mg/kg/day and40 mg/kg/day, improved the spatial learning effectively across the5 d acquisition training period. In subsequent memory capabilitytrials, the Ab25e35-administerd mice after 20 mg/kg quercetintreatment showed a better performance.

Many studies indicate that quercetin showed neuroprotection,but some of its controversy also arises because of the potential riskfor neurotoxicity (Jung et al., 2010). This dispute may be owing totwo possible reasons. One explanation may lie in changes in theBBB permeability that seems to be present in disease models. Thereis an alteration of the BBB permeability in AD patients (Persidskyet al., 2006), which could be speculated that only people with theBBB disruption will benefit from antioxidant supplementation(Gilgun-Sherki et al., 2002). Another explanation is partly due tothe extensive quercetin metabolism by the intestinal and liver cells.A certain range of quercetin (as low as 1 mM) could significantlyreverse the neurotoxin-induced cell death under pathologicalconditions (Vauzour et al., 2008). Therefore, improper liposomalpreparations or higher BBB permeability conditions, by increasingthe amount of quercetin aglycone reaching the CNS parenchyma,may elevate the risk of neurotoxicity in CNS and impair the cellulardefense mechanisms.

We previously detected whether quercetin impairs learning andmemory capability in normal mice, and found that it did not showsignificant effects (oral gavage of the doses used in present study,data not shown here). This provided additional information thatquercetin did not have toxic effects in healthy animals. So its effects

on improving the learning and memory capabilities and its mech-anisms underlying neuroprotection under the following diseasedcondition are attributable to the therapeutic action of itself.

Quercetin has been suggested to inhibit Ab fibril formation andprotect HT-22 murine neuroblastoma cells from Ab25e35 oxidativeattack (Kim et al., 2005; Bastianetto and Quirion, 2002). Theprotection is correlated with the action on the expression of pro-and anti-apoptotic genes and the inhibition of heat-shock protein(HSP) 70 through enhancement of specific cleavage of poly (ADP-ribose) polymerase into apoptotic fragments (Bournival et al., 2009;Schroeter et al., 2001). In this study, oral administration of quercetinexerted protective effect in scavenging ROS in Ab25e35-inducedamnesic mice. Although the precise mechanism by which quer-cetin exerts its beneficial actions is unclear, effects that are associ-atedwith its antioxidant property for treatment reversing cognitivedeficits due to aging and Ab toxicity should not be ruled out.

In addition, quercetin restored AChE activity and ACh level inAb25e35-treated mice. Although the amyloid and the cholinergichypothesis of AD have been widely investigated, the pathwayslinking them are not clear to date. Evidence illustrated that thecholinergic alteration existed in APP transgenic mice, and that thecerebral b-amyloidosis caused significant cholinergic fiber loss inthe neocortex without any loss of cholinergic basal forebrainneurons (Boncristiano et al., 2002). Ab did not result in any changesin the mAChR ligand binding parameters, but in subtoxic concen-trations it impaired the mAChR activation of G-protein coupling bya free radical-mediated mechanism in cortical cultures (Kelly et al.,1996). Previous studies demonstrated that quercetin had actions onthe activity of some subsets of nicotinic acetylcholine receptors(Lee et al., 2011a, 2011b). Our results showed the restorative effectson cholinergic system of quercetin subjected to Ab toxicity. Asquercetin with beneficial antioxidant function has been illustratedin this study, it is in accordance with the protective effects oncerebral cholinergic neurons induced by Ab.

Next, in this study, quercetin also restored ERK/CREB/BDNFsignaling pathway in Ab25e35-induced amnesic mice. Learning and

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memory is controlled at the molecular level in the brain (Carew,1996). There are several major signal pathways participating in theprocess, one ofwhich ismitogen-activated protein kinase (MAPK). Inthe MAPK family, ERK1/2 converges to signal to CREB (Barco et al.,2006), the transcription factor, and then CREB binds to thepromoter regions of many genes associated with memory andsynaptic plasticity (Pham et al., 1999). BDNF is one of the manyeffectors of CREB regulation and participates in the learning andmemoryprocesses (Bekinschtein et al., 2007;Heldt et al., 2007; Tyleret al., 2002), and ERK/CREB/BDNF can be seriously suppressed bysublethal Ab treatment in cortical neuronal loss (Echeverria et al.,2007; Tong et al., 2004). In the present study, phosphorylation ofERK and CREB proteins were shown to be lowered by Ab25e35treatment compared to the levels observed in sham mice. Quer-cetin treatment rescued the Ab25e35-induced ERK1/2/CREB/BDNFsignal pathway, which is in good agreement with a previous studyindicating thatCREB/BDNFsignal is amolecular therapeuticpathwayin the treatment of double transgenic AD mice (Hou et al., 2010).Thus, quercetin also showed the effect on themodulationof synaptictransduction of ERK/CREB/BDNF in the cortical cholinergic system.

Here, taken together, although quercetin is well-known for theantioxidant effects, it possesses other possible neuroprotectivemolecular modulations independent of antioxidation against Ab-induced toxicity. According to a lot of reports, quercetin can exertprotection through multiple pathways, such as anti-oxidation(Arredondo et al., 2010; Sharma et al., 2012), anti-inflammation(Youdim et al., 2004; Sharma et al., 2007; Kao et al., 2010), anti-excitotoxicity (Silva et al., 2008), and the increase of BNDF and itstransduction (Hou et al., 2010). Our findings are consistent withprevious studies that quercetin protected neurons associated withthe antioxidant property, restoring cholinergic system, andpreserving ERK/CREB/BDNF signaling pathway for reversingcognitive deficits. Thus, combined with the antioxidant activity, wespeculate that quercetin-induced reversal of the impairment oflearning and memory performance induced by Ab25e35 might berelated to multiple pathways in the cortical cholinergic system.

4.2. The protective effects of quercetin on the BBB

Accumulation of toxic free radicals in the neurovascular inter-face plays a pivotal role in early molecular cascades of the BBBdisruption (Del Zoppo, 2010). Although not all of the Ab actionsseem to be oxidative in nature (Lovell and Markesbery, 2006),oxidative damages to both the cortical neurons and endothelialcells following Ab25e35 injection were demonstrated in the study.We also demonstrated a gradually decrease of rCBF after Ab25e35injection, accompanied with the serious leakage of lanthanumions from the lumen of vessels and the robust increase in BBBpermeability. These results are consistent with observations in ADpatients, where a reduction of CBF was demonstrated in the earlystages and a leaky BBB could potentially disrupt the CNS homeo-static environment.

Since the BBB is easily disrupted, a strategy with neuro-protective properties to reduce neurovascular oxidative injury andmaintain the integrity of BBB is recommended for the treatment ofbrain damage. Oral administration of quercetin firstly reducedoxidization in the neurovascular interface. Additionally, quercetintreatment was effective in preserving anatomical and functionalintegrity of the BBB permeability. Increase of rCBF was also ach-ieved. A relevant effect of quercetin by partial vasodilation throughactivation of vascular potassium channels might be attributed tothe changes of rCBF (Calderone et al., 2004).

Further, as the BBB is increasingly considered as a target of newtherapeutical approaches in AD, in addition to production of Ab,enhancing Ab clearance across the BBB, as well as protection of the

BBB from injury, are among the proposed new strategy for thetherapy of AD. Clearance of Ab out of the brain is mediated by theLRP-1 (Deane et al., 2003), while an increase in Ab influx to thebrain is due to increased expression of RAGE (Deane et al., 2003;Zlokovic, 2008a). These two transporters were identified in thisstudy. In good agreement with previous reports, LRP-1 expressionwas decreased while RAGE was in a contrary change. Oral admin-istration of quercetin mainly reduced RAGE expression in Ab25e35-induced amnesic mice. Ab-RAGE interaction is an especiallymediator in the phosphorylation state of MAPKs. Among thesubfamilies of MAPKs, ERK1/2 has been evidenced to play impor-tant roles in Ab/RAGE-induced toxicity (Sun et al., 2009) anddirectly lead to inflammation by activating NF-kB mediated secre-tion of proinflammatory cytokines that may reduce the BBBpatency (Deane et al., 2003). In this study, protection of quercetinon the BBB participated in the inhibition of the activation of RAGEsignaling, and the subsequent decreased expression of phosphor-ylated ERK1/2 and nuclear p65.

4.3. Potential mechanism of quercetin in affecting the cerebral NVU

The neurovascular unit is not only an obstacle to CNS drugdelivery but also a therapeutic target itself. In the present study,quercetin regulated the cerebral neurovascular unit mainly throughtwo principal pathways. One of the explanations is the regulation ofRAGE-mediated pathway both in microvessels and in cerebralparenchyma. Ab-RAGE interaction not only mediates re-entry of Abfrom blood to brain, but activates p38 and ERK1/2 MAPK pathwayswhich are responsible, at least in part, for Ab-mediated glial acti-vation and the induction of proinflammatorymediators in the brain(Fang et al., 2010; Liu et al., 2012). Based on our results, activation ofp38 MAPK and ERK1/2 was found as down-stream responses in theAb25e35-treated mice. In the microvessels, quercetin showed theeffects on the expression of RAGE, as well as the following changesof phosphorylation of ERK1/2 and nuclear expression of NF-kB p65in the same manner. In cerebral parenchyma, quercetin treatmentattenuated RAGE transduction, partially directly by inhibitingoxidative damage or indirectly by affecting cerebral parenchymalcells through decreasing p38MAPK-NF-kB p65 pathway (Fig. 11).Although there has been no direct experimental evidence indi-cating the role for RAGE in the NVU dysfunction relevant to ADpathology, we speculate that RAGE-mediated molecular signalingmay have a harmful effect on the NVU integrity and function, andthat regulation of RAGE might evoke a beneficial response to theamplification of neuroinflammation linked to neuronal perturba-tion. Then taken together, inhibition of the RAGE signaling pathwayinvolving suppression of phosphorylation of p38 MAPK and acti-vation of NF-kB p65 by quercetin relevant to the improvement ofthe NVU integrity and BBB function, might be attributed to thecellular coupling of cerebral neurovascular unit protection.

Another explanation is that modulation in the cholinergicneuronal function involving ACh level increases was simulta-neously accompanied by the regulation of microvascular functionthrough quercetin treatment. A hypothesis was advanced thatcerebral cholinergic fibers originating in the nucleus basalis ofMeynert and projecting to the small blood vessels in the cortexhave a vasodilator function (Roman and Kalaria, 2006; Sato et al.,2002; Whitehouse, 2004). The basalocortical pathway thatinvolves basal forebrain neurons located in the nucleus basalis andadjoining substantia innominata is partly in connection withcortical cerebral blood flow regulation. The results described hereprovide an indirect support for this hypothesis. Consistent with thespatial learning and memory improvement, the preservation inneuronal ERK-CREB-BDNF pathway is accompanied with theregulation of local microvessels by limited ACh released from

Fig. 11. Schematic diagram of potential mechanism of quercetin against Ab-induced neurotoxicity in AD brain. (1) Quercetin showed multiple protective effects on cerebral neuronsinduced by Ab25e35. (2) Quercetin played a role in molecular cascades in the BBB against Ab25e35-induced damage. (3) Quercetin showed the effects on the decrease of RAGEexpression both in the microvessels and in the parenchyma, as well as their related signal pathways.

R. Liu et al. / Neuropharmacology 67 (2013) 419e431 429

cerebral cholinergic fibers. These observations might provideevidence supporting one of complicated neurovascular couplingmechanisms during the major components of the NVU. In view ofthis, we suggest that quercetin-induced reversal of the impairmentof learning and memory performance induced by Ab25e35 might bepartly related to the preservation of cholinergic neuronal regulationof cortical cerebral blood flow.

In summary, with the experimental design and methodologyused in the present study, we suggest that quercetin-inducedreversal of the impairment of learning and memory performanceinduced by Ab25e35 might be partly related to the conservation ofcholinergic neuronal regulation of cortical cerebral blood flow andcerebral neurovascular RAGE signaling pathways. These findingssuggest that quercetin, a natural flavonoid, could offer an alterna-tive medication for AD.

Conflict of interest

There are no actual or potential conflicts of interest.

Acknowledgment

This work was supported by Major Scientific and TechnologicalSpecial Project for “Significant New Drugs Creation” (No.2009ZX09302-003), National Natural Science Foundation of China(No. 81102830, 81073120), Central Public Scientific Research Insti-tution Fundamental Project (2011CHX01), Peking Union MedicalCollege Youth Fundamental Project (2012J20), and Research Fundfor the Doctoral Program of Higher Education of China (No.20111106120023).

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