Alcohol, Estres y Antocianinas

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Protection of the Developing Brain with Anthocyanins

Against Ethanol-Induced Oxidative

Stress and Neurodegeneration

Shahid Ali Shah &Gwang Ho Yoon &Myeong Ok Kim

Received: 25 April 2014 /Accepted: 23 June 2014# Springer Science+Business Media New York 2014

Abstract Oxidative stress has been implicated in the patho-

physiology of several neurodegenerative disorders. Numerous

studies have reported that ethanol exposure produces reactiveoxygen species (ROS), one of the best-known molecular

mechanisms of ethanol neurotoxicity. We recently reported

gamma-aminobutyric acid B1 receptor (GABAB1R)-depen-

dent protection by anthocyanins against ethanol-induced apo-

ptosis in prenatal hippocampal neurons. Here, we examined

the effect of anthocyanin neuroprotection against ethanol in

the hippocampus of the postnatal day-7 rat brain. After 4 h of

ethanol administration, either alone or together with anthocy-

anin, the expression of glutamate receptors (-amino-3-hy-

droxy-5-methyl-4-isoxazolepropionic acid receptors

(AMPARs)), intracellular signaling molecules, and various

synaptic, inflammatory, and apoptotic markers was evaluated.

The results suggest that anthocyanins significantly reversed

the ethanol-induced inhibition of glutamatergic neurotrans-

mission, synaptic dysfunction, GABAB1R activation, and

neuronal apoptosis by stimulating the phosphatidylinositol-

4,5-bisphosphate 3-kinase (PI3K)/v-akt murine thymoma vi-

ral oncogene (Akt)/glycogen synthase kinase 3 beta (GSK3)

pathway in the hippocampus of postnatal rat brain. Anthocya-

nins also inhibited the ethanol-activated expression of phos-

phorylated c-Jun N terminal kinase (p-JNK), phospho-nuclear

factor kappa B (p-NF-B), cyclooxygenase 2 (COX-2), as well

as attenuating neuronal apoptosis in the hippocampal CA1,

CA3 and DG regions of the developing rat brain. Furthermore,

anthocyanins increased cell viability, attenuated ethanol-induced

PI3K-dependent ROS production, cytotoxicity, and caspase-3/7

activation in vitro. In conclusion, these results suggest that

anthocyanins are beneficial against ethanol abuse during brain

development.

Keywords Apoptosis. Hippocampus . Intracellular

signaling .NF-B. COX-2

Introduction

A single exposure to ethanol lasting a few hours in rodents

during development results in significant neuronal loss

throughout the forebrain [13], and maternal exposure to

ethanol in humans causes many neuronal dysfunctions, in-

cluding cognition and behavioral abnormalities, broadly la-

beled as fetal alcohol syndrome (FAS) [4]. A study by

Jevtovic-Todorovic shows that serious damage and neuronal

loss during development may continue even when these ani-

mals are mature [3]. The hippocampus in the developing brain

is one of the most sensitive to ethanol, and hippocampal tissue

abnormalities disrupt cell density, inhibit presynaptic gluta-

mate release, affect glutamate binding, and ultimately cause

memory, learning, and behavioral impairments [58]. Al-

though the exact mechanisms leading to neuronal loss in the

developing brain after ethanol exposure are not fully under-

stood, existing data suggest that ethanol mediates its effect

through blockade of the NMDA receptor and inhibition of

phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K) and its

downstream signaling molecules, including suppression of v-

akt murine thymoma viral oncogene (Akt) and activation of

GSK3 [911].

Among the numerous mechanisms proposed for ethanol

neurotoxicity, oxidative stress is the most predominant [12,

13]. Because ethanol can cross the blood-brain barrier very

easily and impact the action of catalase in the brain, alcohol

dehydrogenase enzymes acting on ethanol result in the pro-

duction of reactive oxygen species (ROS), which damage

S. A. Shah :G. H. Yoon : M. O. Kim (*)

Department of Biology and Applied Life Science, College of Natural

Sciences, Gyeongsang National University, Jinju 660-701, Republic

of Korea

e-mail: [email protected]

Mol Neurobiol

DOI 10.1007/s12035-014-8805-7


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neuronal cells [13, 14]. Owing to its significant usage of

oxygen and small number of antioxidative enzymes, the

CNS is particularly vulnerable to oxidative stress [15].

Large-scale therapeutic efforts are needed to overcome the

deleterious effects of ethanol and ROS production on the

developing brain.

One logical approach is to target the ethanol-induced ROS

and oxidative stress. Natural antioxidants extracted fromplants and fruits are attractive candidates for scavenging

ROS and alleviating ethanol neurotoxicity due to their safety

and tolerance by oral administration. Anthocyanins are a class

of flavonoids consisting of water-soluble natural pigments that

are primarily distributed in various foods, including beans,

fruits, and vegetables. Anthocyanins have demonstrated pro-

tection against oxidative stress, inflammation, and cancer, as

well as other beneficial effects [16]. A number of studies

suggest that anthocyanins are highly beneficial against ethanol

toxicity [17]. Anthocyanins extracted from different sources

have shown neuroprotection; anthocyanins are the main phe-

nolic compounds of red wine [18], and the work of Assuncaoshowed that anthocyanins from red wine reduced ethanol-

induced lipid peroxidation, glutathione, antioxidant enzymes,

and improved spatial memory in the hippocampus of the adult

rat brain [19]. Recently, our group reported that anthocyanins

from the black bean protect against ethanol-induced neuronal

apoptosis via gamma-aminobutyric acid B1 receptors

(GABAB1 Rs) in prenatal hippocampal neurons [20]. There-

fore, in the present study, we also examined a possible neuro-

protective effect of black bean anthocyanins against ethanol-

induced ROS, inflammation, and neuronal apoptosis in the

hippocampus of a postnatal day-7 rat brain.

Materials and Methods

Animals and Drug Treatment

Sprague-Dawley postnatal day-7 (P7) rat pups with an aver-

age body weight of 18 g (n=5 animals/group) were used.

Ethanol (5 g/kg) in saline solution was administered by a

single subcutaneous injection. Anthocyanins (100 mg/kg)

were administered as a cotreatment 30 min after ethanol,

whereas the control group was treated with saline. The ani-

mals were sacrificed 424 h after injection [21]. All experi-

mental procedures were approved by the local animal ethics

committee of the Division of Applied Life Sciences, Depart-

ment of Biology, Gyeongsang National University, South

Korea.

Western Blot Analysis

For Western blotting, animals were sacrificed after 4 h [21] of

drug treatment, and their brains were removed immediately.

The hippocampus was carefully dissected out, and the tissue

was frozen on dry ice. After homogenization in 0.2 M

phosphate-buffered saline (PBS) plus a protease inhibitor

cocktail, the protein concentration was analyzed by using a

Bio-Rad protein assay solution. An equal amount of protein

(30 g per sample) was loaded on a 1015 % SDS-PAGE gel

under reducing conditions and transferred to a polyvinylidene

difluoride (PVDF) membrane (Santa Cruz Biotechnology,S a n ta Cru z , CA, US A). P re s ta in e d p ro te in la d d e r

(GangNam-STAIN, iNtRon Biotechnology, Inc., Republic

of Korea) covering a broad range of molecular weights (10

245 kDa) was run in parallel and used to determine the

molecular weights of the detected proteins. Membranes were

blocked using 5 % (w/v) skim milk to minimize the risk of

nonspecific binding. A wide range of antibodies was used to

detect different proteins, including rabbit-derived anti-actin,

anti-B cell CLL/lymphoma 2 (Bcl-2), anti-Bcl-2-associated X

protein (Bax), anti-cyclooxygenase 2 (COX-2), and anti-

caspase-3, goat-derived anti-cytochrome c and anti-glycogen

synthase kinase 3 beta (GSK3) (Ser9), and mouse-derivedanti-poly(ADP-ribose) polymerase 1 (PARP-1) polyclonal an-

tibodies (Santa Cruz Biotechnology, Santa Cruz, CA, USA).

We also used rabbit-derived anti-phospho-nuclear factor kap-

pa B (p-NF-B), anti-phospho-cAMP responsive element

binding protein (CREB) (Ser133

), anti-phospho-PI3K (Y458

/

Y199) anti-calmodulin-dependent protein kinase type II

(CaMKII), anti-phospho-Akt (Ser473

), anti-phospho-JNK

(Thr183/Tyr185), anti--amino-3-hydroxy-5-methyl-4-

isoxazolepropionic acid (AMPA), anti-phospho-AMPA

(Ser845), and anti-Synaptophysin from Cell Signaling Tech-

nology, Inc. After using membrane-derived secondary anti-

bodies, ECL (Amersham Pharmacia Biotech, Uppsala, Swe-

den) detection reagent was used for visualization, according to

the manufacturers instructions. The X-ray films were

scanned, and the optical densities of the bands were analyzed

by densitometry using the computer-based Sigma Gel pro-

gram, version 1.0 (SPSS, Chicago, IL, USA).

Tissue Collection and Sample Preparation

For morphological studies of the brain tissues, animals were

sacrificed following 24 h [21] of drug treatment. An equal

number of animals was maintained in each group (five per

group), and a transcordial perfusion with 4 % ice-cold para-

formaldehyde and 1 PBS was performed. Postfixing was

performed in 4 % paraformaldehyde overnight, after which

the brains were transferred to a 20 % sucrose solution until

they sank to the bottom of the tube. Prior to processing 16-m

sections in the coronal planes using a Leica cryostat (CM

3050C, Germany), the brains were frozen using O.C.T. com-

pound (A.O. USA). Sections were thaw-mounted on ProbeOn

Plus charged slides (Fisher).

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Extraction of Anthocyanins

Anthocyanins were extracted from Korean black soybeans

provided by the agriculture research facility of GNU, as

previously described [20]. Briefly, anthocyanins were extract-

ed three times from 1,500 g black soybeans with 1,500 ml

95 % methanol (1 % HCl) for 72 h in the dark at room

temperature. The solution was concentrated to a volume of150 ml in a rotary evaporator and loaded onto a XAD-7

column. Distilled water was used to elute the column until a

soft, red-colored eluate appeared. After discarding the eluate,

ethyl acetate (EA) was added to the column until the extract

color changed to purple except for 1 cm above the bottom of

the column. A solution of 95 % methanol (1 % HCl) was

passed through the column until the extract changed to a red

color. The filtrate was collected and concentrated to a volume

of 100 ml in a rotary evaporator and passed through 0.45-m

pore size filter. This filtrate was then loaded onto a Sephadex

column and eluted using a solution containing 50 % methanol,

50 % distilled water, and 1 % HCl until 8001,000 ml of thered color filtrate was obtained. The red-colored filtrate was

completely dried using a rotary evaporator, and the resulting

anthocyanin powder was stored at20 C until use.

Fluoro-Jade B Staining

Fluoro-Jade B staining was performed according to the sug-

gested protocol (Millipore, USA, cat# AG310, lot#2159662).

Brain tissue slides were air-dried overnight. Initially, the slides

were immersed in a solution of 1 % sodium hydroxide and

80 % ethanol for 5 min, then 70 % alcohol for 2 min, and

followed by 2 min in distilled water. The slides were trans-

ferred to a solution of 0.06 % potassium permanganate for

10 min and then rinsed with distilled water. Next, the slides

were immersed in a solution of 0.1 % acetic acid and 0.01 %

Fluoro-Jade B for 20 min. The slides were then rinsed with

distilled water and allowed to dry for 10 min. Glass coverslips

were mounted on the glass slides using mounting medium.

Images were captured using an FITC filter on a confocal laser

scanning microscope (FV 1000, Olympus, Japan).

Immunofluorescence

Tissue-containing slides were washed two times for 15 min in

0.01 M PBS, after which proteinase K solution was added to

the tissue and incubated for 5 min at 37 C. Then, the tissues

were incubated for 90 min in a blocking solution containing

normal swine/rabbit serum and 0.3 % Triton X-100 in PBS.

Primary antibodies p-Akt, COX-2, p-GSK3, and phosphor-

ylated c-Jun N terminal kinase (p-JNK) (all 1:100 in PBS)

were applied alternatively at 4 C overnight. Subsequently,

secondary antibodies (Dako, TRITC and FITC, Santa Cruz,

1:50 in PBS) were applied at room temperature for 90 min.

The slides were twice washed with PBS for 5 min. For double

staining, incubations were performed in parallel. Glass cover-

slips were mounted on the glass slides using mounting medi-

um. Images were captured using a confocal microscope

(FluoView FV1000 Olympus, Japan).

ApoTox-Glo Triplex Assay

An ApoTox-Glo Triplex Assay was performed to assess the

viability, cytotoxicity, and caspase-3/7 activation within a

single assay well, as previously described [22]. This assay

consists of two parts. The first part of the assay simultaneously

measures two protease activities as markers of cell viability

and cytotoxicity. Mouse hippocampal neuronal HT22 cells, a

generous gift from Prof. Koh (Gyeongsang National Univer-

sity) [23] were cultured in 96-well assay plates at a density of

2104 cells. Each well contained a final volume of 200-l

Dulbeccos modified Eagles medium (DMEM) containing

10 % fetal bovine serum (FBS) and 1 % penicillin/

streptomycin. After 48-h incubation at 37 C in a humidified5 % O2 incubator, the cells were treated with ethanol

(100 M) and anthocyanins (0.1 mg/ml) for a total of

20 min. For the assay, 20 l of the viability/cytotoxicity

re a g e n t c o n ta in in g b o th th e g ly c y l-ph e n y la lan y l-

aminofluorocoumarin (GF-AFC) substrate and the bis-

alanyl-alanyl-phenylalanyl-rhodamine 110 (bis-AAF-R110)

substrate was added to all wells, briefly mixed using orbital

shaking (500rpm for 30s),and incubated for 1 h at37 C. The

fluorescence was measured at two wavelengths: 400/505 nm

(viability) and 485/520 nm (cytotoxicity). The GF-AFC sub-

strate enters live cells and is cleaved by a live-cell protease to

release AFC. The bis-AAF-R110 substrate does not enter live

cells; rather, it is cleaved by a dead-cell protease to release

R110. The live-cell protease activity is restricted to intact

viable cells and measured using a fluorogenic, cell-

permeant, and pep tide sub str ate (GF-A FC). A sec ond

fluorogenic, cell-impairment peptide substrate (bis-AAF-

R110) is used to measure the dead-cell protease activity re-

leased from cells that have lost membrane integrity.

The second part of the assay uses a luminogenic caspase-3

substrate containing the tetrapeptide sequence DEVD in a

reagent to measure caspase activity, luciferase activity, and

cell lysis. The Caspase-Glo3/7 reagent was added (100 l) to

all wells and briefly mixed using orbital shaking (500 rpm for

30 s). After incubation for 30 min at room temperature, the

luminescence was measured to determine caspase activation.

Oxidative Stress (ROS) Detection

Mouse hippocampal cell lines (HT22) were cultured in 96-

well plates. Each well contained a final volume of 200 l

DM E M c o n ta in in g 1 0 % F BS a n d 1 % p e n ic il l in /

streptomycin. After 48-h incubation at 37 C in a humidified

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5 % CO2 incubator, the cells were treated with ethanol

(100 M), ethanol plus anthocyanins (100 M+0.1 mg/ml),

ethanol plus LY294002 (100 M+20M), and ethanol plus

LY294002 plus anthocyanins (100 M+20 M+0.1 mg/ml)

for a total of 20 min. The PI3K inhibitor LY294002 was added

to the cells 5 min before ethanol and anthocyanin treatment.

2,7-Dichlorofluorescin diacetate (DCFDA) 600 M dis-

solved in DMSO/PBS was added to each well and incubatedfor 30 min. Plates were read in ApoTox-Glo (Promega) at

488/530 nm.

Data and Statistical Analysis

Western blot results were scanned and analyzed by densitom-

etry through the computer-based Sigma Gel System (SPSS

Inc., Chicago, IL). Density values were expressed as the mean

SEM. The ImageJ software was used to analyze the integrat-

ed density. One-way analysis of variance (ANOVA) used to

determine significant differences, followed by Studentsttest.

Pvalues less than 0.05 (P


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c to the cytosol was inhibited. This cytochrome c re-

lease is responsible for the activation of a caspase

cascade that includes caspase-9 and caspase-3. Ethanol

administration to postnatal rat pups induces the activa-

tion of caspase-9, which in turn activates caspase-3.

However, cotreatment with anthocyanins showed a sig-

nificant reduction in the expression of activated caspase-

9 and caspase-3 in the hippocampus of postnatal rat

pups. Once caspase-3 is activated, it facilitates the acti-

vation of PARP-1, which induces DNA damage. Fol-

lowing ethanol treatment, a marked increase in the ex-

pre ssi on of PARP-1 was obs erv ed, whe reas Korean

black bean anthocyanins completely inhibited the acti-

v a tio n o f PARP-1 in th e p o stn a ta l h ipp o ca mpu s

(Fig. 3a).

The extent of neuronal apoptosis was examined morpho-

logically after 24 h of ethanol, with or without anthocyanin

administration, using Fluoro-Jade B (FJB), which is re-

ported to be one of the most important neuronal apo-

ptotic markers. Figure 3b clearly shows that ethanol is

primarily responsible for inducing neuronal apoptosis in

the hippocampal CA1, CA3, and DG regions of postna-

tal day-7 rat brains. By contrast, anthocyanin treatment

diminished the number of Fluoro-Jade B-positive cells,

suggesting that anthocyanins decreased neuroapoptosis

caused by ethanol exposure (Fig. 3b).

Anthocyanins Stimulate the PI3K/Akt/GSK3 Intracellular

Signaling Pathway to Protect the Developing Brain

Hippocampus Against Ethanol

After analyzing the anti-apoptotic and anti-inflammatory

effects of anthocyanins against ethanol in the develop-

ing brain, we set out to study the exact mechanism by

which anthocyanin provides neuroprotection in the de-

veloping rat brain. We were interested in the involve-

ment of PI3K pathway and its downstream signaling

molecules as reported earlier that ethanol impairs insulin

signaling and inhibits PI3K activity in the neurons of

postnatal day 7 rat pups [10]. Western blotting was used

to investigate the possible involvement of PI3K and its

downstream cellular signaling pathway. Our results sug-

gest that a single administration of ethanol caused a

significant inhibition of the cellular levels of phospho-

PI3K, phospho-Akt (Ser473), and phospho-GSK-3

(Ser9). However, Fig. 4a shows that anthocyanins re-

versed the ethanol-induced changes by increasing the

expression of phosphorylated PI3K, Akt (Ser473), and

GSK3 (Ser9) proteins in the hippocampus of postnatal

rat brains.

The expression of Akt (Ser473) and GSK3 (Ser9)

proteins was also inve stigate d morp holo gica lly usin g

immunofluorescence. These immunostaining results are

PKA

GABAB1R

AMPAR

C E E+A A

-Actin

p-AMPAR

Synaptophysin

p-CREB

CaMKII

C E E+A A

(a)

(b)

-Actin

Fig. 1 The effect of anthocyanins

on ethanol-induced expression

changes in GABAB1R and

AMPARs and various synaptic

protein markers in the

hippocampus of developing rat

brain. Shown are representative

Western blots ofa GABAB1R and

AMPARs andb synaptic protein

marker levels, including p-AMPA(Ser845), p-CREB, CaMKII, and

Synaptophysin, in the

hippocampus of 7-day-old rat

brain (n=5 rats/group) 4 h after

ethanol and anthocyanin

treatment. The graphs represent

the ratio of the proteins

normalized to-actin

(*#P


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p-NF-kB

C E E+A A

COX-2

(a)

(b)

p-JNK

-Actin

DAPI/Mergep-JNK COX-2 Merge

C

E

E+A

CA1

Fig. 2 Anthocyanins reduced ethanol-induced expression of various

inflammatory markers in the developing rat brain. Western blot analysis

ofap-JNK, p-NF-B, and COX-2 expression levels in the hippocampus

of the developing rat brain (n=5 animals/group) exposed to ethanol and

anthocyanins. The histograms containing relative intensity of proteins to

-actin (*#P


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consistent with our Western blot results and show that

ethanol is primarily responsible for the downregulation

of p-Akt (Ser473) and p-GSK-3 (Ser9) signaling pro-

teins in the hippocampal DG, CA1, and CA3 regions of

th e d e ve lo pin g b rain (F ig. 4b d) . H ow e ve r, t h e

anthocyanin-treated groups exhibited an opposite effect,

with the reversal of the changes induced by ethanol in

the expression of p-Akt (Ser473) and p-GSK-3 (Ser9)

in these three regions of the hippocampus of the devel-

oping rat brain (Fig. 4bd).

Anthocyanins Reduced Ethanol-Induced Neurotoxicity

and PI3K-Dependent ROS In Vitro

To further understand the role of anthocyanins in neuropro-

tection against ethanol in vitro, we measured cell viability/

cytotoxicity and apoptosis (apoptotic marker caspase-3/7)

using an ApoTox-Glo assay on mouse hippocampal HT22

neuronal cells. Our in vitro results show that treatment with

ethanol resulted in a significant reduction in the number

of viable HT22 cells, whereas cell toxicity and the

activation of caspase-3/7 increased significantly com-

pared with the control. By contrast, treatment with an-

thocyanins significantly reduced the effects of ethanol,

thereby increasing cell viability and decreasing cytotox-

icity and caspase-3/7 activation, suggesting that the an-

thocyanins reduced ethanol-induced neurotoxicity

(Fig. 5ac).

To understand how the antioxidant effect of anthocy-

anins against the ethanol-induced PI3K signaling path-

way mediated oxidative stress, we quantitatively mea-

s u red re ac tive o x yg e n s p ec ie s (ROS) p rod u ctio n

DAPI/Merge

DG

p-JNK COX-2 Merge

C

E

E+A

CA3CA3

(c)

Fig. 2 (continued)

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(b)

(a)

Bax

Bcl-2

C E E+A A

Cytochrome c

Cleaved Casp-3

PARP-1

Caspase-9

-Actin

Fig. 3 The anti-apoptotic effect

of anthocyanins against ethanol in

the hippocampus of the

developing rat brain. Western

blots of the various apoptotic

protein markersa Bax, Bcl-2,

Bax/Bcl-2, cytochrome c, and

caspase-9 and caspase-3 levels in

the hippocampus of postnatal

day-7 rat brain after 4 h ofcotreatment with ethanol and

anthocyanins. Thegraphs contain

their relative intensity of proteins

to-actin (*#P


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p-Akt (Ser473)

p-GSK3 (Ser9)

C E E+A A

Actin

p-PI3K

C

E

E+A

DAPI/Mergep-AKT p-GSK3 Merge

(b)

(a)

DG

DG

DG

Fig. 4 Anthocyanins stimulates the PI3K/Akt/GSK3 intracellular signal-

ing pathway against ethanol in the hippocampus of developing rat brain.

Western blots ofap-PI3K, p-Akt, and p-GSK3proteins in the hippocam-

pus of 7-day-old rat brain after 4 h of ethanol and anthocyanin administra-

tion. The graphs contain their relative intensity of proteins to -actin

(*#p


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in vitro. Exposure to ethanol induced a significant pro-

duction of oxidative stress by increasing ROS levels

compared with control cells; however, anthocyanins

m a r ke d l y r e d uc e d R O S l e v el s , i n d ic a t in g t h a t

anthocyanins are a potent antioxidant. We also used a

PI3K inhibitor (LY249002) to confirm that ethanol-

induced ROS is PI3K-dependent. Our results show that

the PI3K inhibitor almost completely abolished the

C

E

E+A

DAPI/Mergep-AKT Mergep-GSK3

CA1

CA1

CA1

(c)

CA3

CA3

CA3

C

E

E+A

DAPI/Mergep-AKT Mergep-GSK3

(d)

Fig. 4 (continued)

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ability of ethanol to induce oxidative stress, suggesting

that ethanol-induced ROS production is PI3K pathway-

dependent (Fig. 5d).

Discussion

In this study, we demonstrated that Korean black bean antho-

cyanins are able to reverse ethanol-induced glutamatergic

neurotransmission inhibition, reduction in synaptic proteins,

GABAB1 receptor activation, neuroinflammation, and ROS

production, as well as ameliorate neuronal apoptosis via the

PI3K/Akt/GSK3pathway (Fig.6) in the hippocampus of 7-

day-old rat brain.

Developmental exposure to alcohol structurally damages

the brain by increasing the densities of white and gray matter,

and affected individuals are reported to display behavioral and

many other disabilities [2528]. Neuroprotection against eth-

anol toxicity through flavonoids has been reported in several

studies. Flavonoid-containing diet has been shown to reduce

ethanol-induced damage to the brain and liver [29], and

another study also showed that flavonoids prevented

ethanol-induced apoptosis in vitro [30]. In comparison to

other flavonoids, anthocyanins have been highly valued be-

cause of their high antioxidant capabilities and formation of

electron delocalization and resonating structures [31,32]. In

this study, a natural antioxidant anthocyanin extracted from

Korean black bean was used to protect the developing brain

from the deleterious effects of ethanol. Our findings suggest

that anthocyanins not only reversed the ethanol-induced in-

crease in the Bax/Bcl-2 ratio and inhibited cytochrome c

release but also reduced the expression of activated caspases,

particularly caspase-3, thereby reducing DNA damage and

neuronal apoptosis due to the anti-apoptotic effect against

ethanol in the hippocampus of the developing rat brain. We

conclude from the above findings that natural antioxidants

such as anthocyanins are able to block ethanol-induced

neurotoxicity.

Anthocyanins are potent antioxidants that play a key role in

neuroprotection against ethanol-induced ROS production [31,

32]. Our observation that ethanol induced the dephosphoryla-

tion of phospho-PI3K and its downstream signaling molecules

in the brains of postnatal day-7 rats is also consistent with the

Fig. 5 Anthocyanins reversed ethanol-induced cell viability decrease,

cytotoxicity, caspase-3/7 activation, and ROS production in vitro. Repre-

sentative histograms for %age ofa cell viability, b cytotoxicity, and c

caspase-3/7 activation in mouse hippocampal HT22 cells with or without

ethanol, ethanol, and anthocyanins in vitro. The cells were cultured in 96-

well plates and then treated with ethanol and anthocyanins for 24 h. An

ApoTox-Glo Triplex Assay was performed as described in the Mate-

rials and Methods section. d The %age histogram shown for ROS in

HT22 cells after treatment with or without ethanol, ethanol, and antho-

cyanins with or without LY294002 (as an inhibitor of PI3K). Significant

differences were determined using a one-way analysis of variance

(ANOVA) followed by Studentsttest. Significance=P


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observation that the inhibition of these signaling cascades

contributes to ethanol-induced neurodegeneration and the

neuropathology associated with fetal alcohol exposure [9,

10]. Another study suggests that ethanol mediates its

neurotoxic effects via glycogen synthase kinase 3 beta

(GSK3), a serine/threonine kinase [3335]. Here, we

h a ve s h own th at a n th o cy an ins n o t o n ly re ve rse d

ethanol-induced inhibition of p-PI3K but also abolished

PI3K-mediated ROS generation. Similarly, anthocyanins

also antagonize ethanol-induced neuronal degeneration

by stimulating p-Akt Ser473 and p-GSK3 Ser9 activity

in the hippocampus of postnatal rat brains.

In the current study, our results suggest that ethanol admin-

istration induced the activation of the phosphorylated JNK

pathway and inflammatory markers such as NF-B and

COX2 and treatment with anthocyanins significantly inhibited

the activated NF-B and COX2 in the hippocampus of the

postnatal day-7 rat brains. Previous studies also demonstrated

that ethanol is responsible for inducing inflammation in the

brain [24]. For instance, a study by Crews et al. showed that

chronic ethanol treatment induces inflammation through the

induction of NF-B[36]. Similarly, the work of Izumi et al.

demonstrated that rats exposed to ethanol once on postnatal

day 7 showed significant neuronal apoptosis throughout the

forebrain after 24 h [37]. Compared with the adult brain, the

developing brain is more vulnerable to neurotoxic insults

because it lacks sufficient antioxidant enzymatic activity [38,

39]. Additionally, hippocampal and cerebellar regions of the

brain are very sensitive to oxidative stress due to the low levels

of vitamin E, which acts as a natural endogenous antioxidant,

and vitamin E has been shown to reverse ethanol-induced

activation of NF-B in the cortex and hippocampus of post-

natal rat brains and is therefore considered a neuroprotective

compound [40].

During the development of the CNS, several types of

events take place, including neuronal proliferation, migration,

differentiation, and synapse formation. Some of these events

begin prenatally, but following birth, neuronal plasticity and

remodeling increase at a rapid rate [41]. AMPA and NMDA

receptor transmission increases in synapses within the first

2 weeks of the postnatal period [4244]. Both of these recep-

tors are important for synaptic transmission and plasticity,

depending upon their biophysical and pharmacological prop-

erties. AMPAR function and glutamate release in the hippo-

campal CA1 neurons have been reported to be particularly

sensitive to ethanol [45], and it is now well established that

ethanol induces the hyperactivation of GABA receptors and

inhibits NMDA glutamate receptors [1, 2]. Previous studies

also support our current findings that ethanol induced the

activation of GABAB1 receptors and the inhibition of

ionotropic AMPARs, and ethanol also caused a reduction in

the expression of synaptic proteins and disturbed the normal

functioning of the developing brain. Anthocyanins diminished

the deleterious effect of ethanol and increased the expression

ROS

p-CREB CaMKII

p-AMPAR

p-JNK

NF-kB

COX2

PI3K

p-Akt

GSK3

Bax/Bcl-2

Cyt-c

Caspase-9

PARP-1

Caspase-3 Inflammation

Ethanol

AMPARGABAB1R

Neurodegeneration

Anthocyanins

Anthocyanins

Fig. 6 The schematic

representation of anthocyanin

neuroprotection against ethanol-

induced ROS production,

apoptosis, and inflammation in

the hippocampus of the

developing rat brain. The diagram

shows how ethanol induces its

toxic effects by inhibiting

glutamatergic neurotransmission,neuroinflammation, generation of

reactive oxygen species (ROS),

and neuroapoptosis via the

PI3K/Akt/GSK3pathway in the

hippocampus of the developing

rat brain. It also shows that

anthocyanins protect the postnatal

brain against ethanol toxicity

Mol Neurobiol


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of synapse-related proteins, suggesting that they may be ben-

eficial to neuronal synapses.

In conclusion, our findings suggest that the hippocampus is

a plausible target for the location of anthocyanin protective

effects against ethanol-induced hippocampal damage. These

findings are of clinical relevance and provide opportunities for

the development of nutraceutical therapies for treating neuro-

degenerative diseases.

Acknowledgments This research was supported by the Pioneer Re-

search Center Program through the National Research Foundation of

Korea funded by the Ministry of Science, ICT and Future Planning

(2012-0009521).

Conflict of Interest The authors declare no conflicts of interest.

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