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: mokim@gsnu.ac.kr
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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