J Mol Neurosci (2014) 53:617–625
DOI 10.1007/s12031-014-0228-4
Conditioned Media of Choroid Plexus Epithelial Cells Induces
Nrf2-Activated Phase II Antioxidant Response Proteins
and Suppresses Oxidative Stress-Induced Apoptosis in PC12 Cells
Abbas Aliaghaei & Fariba Khodagholi &
Abolhassan Ahmadiani
Received: 1 November 2013 / Accepted: 1 January 2014 / Published online: 4 February 2014
# Springer Science+Business Media New York 2014
Abstract Based on the critical role of the choroid plexus (CP)
in detoxification processes in the central nervous system
(CNS), herein we investigated the effect of choroid plexus
epithelial cells conditioned media (CPECs-CM) under oxidative conditions. CPECs were isolated from rat brains, cultured,
and the conditioned media were collected. Then pheochromocytoma neuron-like cells (PC12) were treated simultaneously
with CPECs-CM and H2O2 as the oxidative stressor. Next, the
effect of CPECs-CM on neurite outgrowth and cell differentiation in the presence of H2O2 was determined. Our results
showed that CPECs-CM improved the expansion of neurites
and differentiation in PC12 cells under oxidative stress conditions. Changes in apoptotic factors, nuclear factor erythroid
2-related factor 2 (Nrf2) and γ-glutamylcysteine synthetase as
the highlighted pathway in the antioxidant defense system
were determined by western blot. Also, the activity of antioxidant enzymes and lipid peroxidation level were determined.
CPECs-CM-treated PC12 cells could survive after exposure
to H2O2 by reduction of caspase-3 cleavage and Bax level and
elevation of anti-apoptotic factor Bcl2. Our data also revealed
that Nrf2 activation, and consequently its downstream protein
Electronic supplementary material The online version of this article
(doi:10.1007/s12031-014-0228-4) contains supplementary material,
which is available to authorized users.
A. Aliaghaei : F. Khodagholi : A. Ahmadiani
NeuroBiology Research Center, Shahid Beheshti University of
Medical Sciences, Tehran, Iran
A. Aliaghaei : F. Khodagholi : A. Ahmadiani (*)
Neuroscience Research Center, Shahid Beheshti University of
Medical Sciences, Tehran, Iran
e-mail: [email protected]
A. Ahmadiani
Department of Pharmacology, Medical Faculty, University of
Malaya, Kuala Lumpur, Malaysia
levels, increased in the presence of CPECs-CM. Based on our
data, we can conclude that CPECs-CM protects PC12 cells
against oxidative stress and apoptosis. It seems that CPECs
secrete antioxidative agents and neurotrophic factors that have
a role in the health of the CNS.
Keywords Choroid plexus epithelial cells . Nrf2 . Condition
media . Oxidative stress . Apoptosis
Abbreviations
ANOVA
Analysis of variance
BBB
Blood–brain barrier
BDNF
Brain derived neurotrophic factor
CNS
Central nervous system
CP
Choroid plexus
CPECs
Choroid plexus epithelial cells
CPECsChoroid plexus epithelial cells conditioned
CM
media
CSF
Cerebrospinal fluid
DMEM
Dulbecco’s Modified Eagle’s media
FGF2
Fibroblast growth factor 2
γ-GCS
γ-glutamylcysteine synthetase
GDNF
Glial derived neurotrophic factor
GSH
Glutathione
MDA
Malondialdehyde
NGF
Nerve growth factor
Nrf2
Nuclear erythroid 2-related factor 2
NT3-4
Neurotrophin 3-4
PMS
Phenazine methosulfate
ROS
Reactive oxygen species
SOD
Superoxide dismutase
TBA
Tribarbituric acid
TTR
Transthyretin
VEGF
Vascular endothelial growth factor
618
Introduction
Among the specialized structures of the brain, the choroid
plexus (CP) has a key role in maintenance of central nervous
system (CNS) homeostasis (Johanson et al. 2004). Constituent
cells of CP are derived from the ependyma and possess
epithelial properties. The well-delineated function of choroid
plexus epithelial cells (CPECs) is the production of cerebrospinal fluid (CSF) (Speake et al. 2001; Dohrmann 1970).
Also, CPECs contribute to detoxification procedures in the
brain, stabilization of the extracellular environment of neurons
and contribute to repair of the brain after traumatic injuries
(Emerich et al. 2004). CPECs synthesize and secrete numerous neurotrophic factors with therapeutic potentials such as
nerve growth factor (NGF), brain-derived neurotrophic factor
(BDNF), neurotrophin 3-4 (NT3-4), vascular endothelial
growth factor (VEGF) and fibroblast growth factor 2 (FGF2)
(Fuxe et al. 1996; Timmusk et al. 1995; Marti and Risau
1998). Several studies have indicated the critical role of these
agents in health, expansion and growth of neuronal processes
(Itoh et al. 2011; Takaku et al. 2013; Evans et al. 2009; Jin
et al. 2006; Kim et al. 2003).
Oxidative stress is a well-known event in the pathogenesis of
neurodegenerative diseases that results from accumulation of
free radicals and increase in the formation of reactive oxygen
species (ROS) (Sies 1997). The neuroprotective role of CP
against oxidative stress has been well established (Mesquita
et al. 2012; Xiang et al. 2012). Accordingly, investigations have
focused on antioxidant pathways which can be triggered by CP
in the oxidative stress-induced models in vitro and in vivo (Yon
et al. 2011; Turgut et al. 2007). In addition, induction of nuclear
factor erythroid 2-related factor 2 (Nrf2) by CP in the mouse
brain has been reported by D’Angelo and colleagues
(D’Angelo et al. 2012). Nrf2-driven gene products contain a
broad spectrum of enzymes and proteins participating in antioxidant defense systems such as enzymes involved in reduced
glutathione (GSH) synthesis (γ-glutamylcysteine synthetase,
γ-GCS) (Kaspar et al. 2009; Ma 2013). Furthermore, it has
Fig. 1 Choroid plexus tissue was isolated from rat brain and cultured.
After the cells reached an appropriate density, immunocytochemistry
(ICC) was done. For this purpose, cells were fixed and incubated with
primary antibody against transthyretin (TTR). Then cells were incubated
J Mol Neurosci (2014) 53:617–625
been shown that CP elevates the activity of antioxidant enzymes such as superoxide dismutase (SOD) and catalase, located downstream of Nrf2 (Fubini and Hubbard 2003). In the
blood–brain barrier (BBB), Nrf2 prevents the loss of endothelial cells and tight junction proteins following traumatic brain
injury (Zhao et al. 2007). Therefore, we chose the Nrf2/ARE
system to evaluate whether antioxidant systems are induced by
CPECs-CM. In the present study, we investigated the changes
in antioxidant enzyme activities, the Nrf2/ARE pathway and
apoptosis markers in the presence of CPECs-CM against oxidative stress in pheochromocytoma neuron-like cells (PC12).
Materials and Methods
Materials
Dulbecco’s Modified Eagle’s Media (DMEM/F12) was obtained from Invitrogen (Invitrogen, Carlsbad, CA, USA).
Antibodies against transthyretin (TTR) and goat anti-rabbit
IgG conjugated FITC and diamidino-2-phenylindole (DAPI)
were purchased from Santa Cruz Biotechnology (Santa Cruz,
CA, USA). RNAX plus was purchased from Qiagen (Qiagen
Ltd, Crawley, UK). Antibodies against Bax, Bcl-2, caspase-3
were purchased from Cell Signaling Technology (Beverly,
MA, USA). Nrf2 and γ-GCS antibodies were purchased from
ABCAM (Cambridge, UK). The electrochemiluminescence
kit (ECL) was purchased from Amersham Bioscience
(Piscataway, NJ, USA). Polyvinylidene difluoride membrane
(PVDF) was obtained from Millipore (Billerica, MA, USA),
and other materials were purchased from Sigma Aldrich (St.
Louis, MO, USA).
Isolation and Culture of CPECs
Under approval by the animal care committee of Shahid
Beheshti University of Medical Sciences and in accordance
with the Guide for the Care and Use of Laboratory Animals
with secondary antibody conjugated with FITC. a The round shape cells
appeared 3 days after culture. b Two week later, the cells reached the
maximum confluence. c ICC showed that CPECs are immunopositive for
TTR. The scale bar is 200 μm
J Mol Neurosci (2014) 53:617–625
(National Institutes of Health Publication No. 80-23, revised
1996), adult male albino Wistar rats (150-200 g) were decapitated under deep anesthesia and the brains were removed. After
washing with phosphate buffered saline (PBS), coronal sections
were made and the choroid plexus tissue was removed from the
ventricles, and then rinsed by PBS. Tissues were then incubated
with trypsin solution 0.25 % for 20 min at 37 ° C. Afterwards,
fetal bovine serum (FBS) was added and centrifuged. The pellet
was transferred to a culture media containing DMEM/F12, FBS
(10 %) and antibiotic. To prevent fibroblastic contamination,
we used cytosine arabinoside (20 μM). After 48 h, to remove
the debris and red blood cells, the culture media was changed.
For collecting the conditioned media of CPECs, CPECs were
cultured in serum-free medium. After 48 h, medium was collected, filtered and kept at -20 °C for further uses.
Immunocytofluorescence
CPECs were cultured in a 24-well plate and fixed by 4 %
paraformaldehyde (PFA). After washing with PBS, cells were
permeabilized with Triton Χ-100. Then, cells were incubated
with goat normal serum followed by an overnight incubation
with the primary antibody against TTR at 4 °C. The fluorescent secondary antibody was applied after washing with PBS.
For visualization of the nuclei, cells were stained by DAPI.
Preparations were examined under fluorescence microscope
(Olympus, IX 71, Japan).
619
streptomycin (1 %). Then, PC12 cells were treated with
CPECs-CM (4:1 ratio of CPECs-CM to DMEM/F12 medium)
and simultaneously exposed to H2O2 (150 μM) for 24 and 48 h.
Examination of PC12 Cell Morphology
PC12 cells were seeded in 6-well plates. For morphological
analysis, random images were acquired from each well, taking
20 images per well. A minimum of 50 cells per treatment were
quantified. After co-administration of CPECs-CM and H2O2,
four criteria were assessed: neurite length, cell body area,
neurite width and percentage of bipolar neurons. Neurite
length was defined as the sum of lengths of all primary
branches and their associated twigs. The area of a cell body
except its branches was defined as cell body area. To calculate
neurite width, cell body area was divided by neurite length.
Data analysis was done by using the Cell^A software (Frankel
et al. 2009).
MTT Assay
PC12 cells were cultured in 96-well plates. After treatment,
3-(4, 5-Dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) was added to each well and incubated for 4 h.
Then supernatant was removed and dark blue crystals of
formazan were dissolved in dimethyl sulfoxide. Absorption
of the suspension was read at 630 nm, and the measurements
were reported as percentage of control.
PC12 Cell Culture and Treatment
DAPI Staining
PC12 cells were obtained from the Pasteur Institute (Tehran,
Iran). The cells were cultured in DMEM/F12 media supplemented with 5 % FBS, 10 % horse serum, and penicillin and
Fig. 2 Effect of CPECs-CM on
the PC12 cells morphology. a
Neurite length. b Cell body area. c
Neurite width. d Percentage of
bipolar neurons. * P<0.05
significant different with H2O2
exposed groups; # P<0.05
significant different with control
group
For DAPI nuclear staining, PC12 cells were fixed by PFA 4 %
and then permeabilized by Triton Χ100. Cells were then
620
incubated with DAPI (600 nM) for 2 min. After washing with
PBS, stained cells visualized under fluorescent microscopy
(Park et al. 2007).
Western Blot
After treatment, cells were lysed in lysis buffer containing a
protease inhibitor. Protein content was determined based on
Bradford's technique (Bradford 1976). Twenty microgram
proteins were loaded on 12 % SDS-page gel, electrophoresed and then transferred to PVDF. The membrane was
incubated with blocking solution for 75 min. Afterwards,
the blots were incubated with primary antibodies at 4 °C.
Then, blots were washed and incubated with secondary
antibody for 90 min. Immunoreactive polypeptides were
detected using ECL reagents and autoradiography. The
resulting bands were quantified by densitometry using
Image J software.
J Mol Neurosci (2014) 53:617–625
Fig. 4 The evaluation of apoptosis in PC12 cells after treatment with„
CPECs-CM and H2O2 concomitantly. a Fluorescence microscope
analysis of cells stained with DAPI. Treatment of PC12 cells by H2O2
and or CPECs-CM, cells were fixed stained with DAPI and analyzed for
morphological characteristic associated with apoptosis. b Western blot
analysis of apoptosis markers. Blots were probed with anti-Bax, anti-Bcl2
and anti-caspase-3 antibodies and reprobed with β-actin antibodies (c).
The densities of Bax, Bcl-2 and β-actin bands were measured and the ratio
of Bax/Bcl-2 was reported. d The densities of cleaved caspase-3 and βactin bands were measured and ratio of cleaved caspase-3/β-actin was
reported. * P<0.05 significant difference between treated groups and
H2O2 groups; # P<0.05 significant difference between the control group
and H2O2 group
nitro-5-thiobenzoic acid, which was measured at 412 nm.
GSH concentrations were expressed as mol/mg protein
(Ellman 1959).
Measurement of Malondialdehyde (MDA) Content
The activity of SOD was measured based on the inhibition
of formation of amino-blue tetrazolium formazan in combination with nicotinamide adenine dinucleotide (NADH),
phenazine methosulfate (PMS) and nitro blue tetrazolium
(NBT). Cell lysate, PMS (186 μM), NBT (300 μM), NADH
(750 μM) and sodium pyrophosphate buffer (pH 8.3,
0.052 M) were combined and incubated at 30 °C for 90 s.
Absorbance of resulting color was measured at 560 nm
(Kakkar et al. 1984).
This method was performed based on optic absorbance of
MDA with the double heating procedure. The purple color
in this method results from the reaction of tribarbituric acid
(TBA) with MDA. Briefly, 200 μg of proteins of cell lysis
were mixed with tricholoroacetic acid (10 % w/v) solution and
placed in boiling water for 15 min. Reaction tubes were
centrifuged at 3000 rpm for 10 min. The resulting supernatant
was transferred to another tube containing TBA (0.67 % w/v).
The tubes were again put in boiling water for 15 min, and
optic absorption was measured at 532 nm wavelength (Draper
and Hadley 1990).
Measurement of GSH Content
Data Analysis
Reduced glutathione (GSH) reduces with 5-5 -dithiobis (2nitrobenzoic acid) (DTNB) to form the colored product 2-
All data are represented as the mean ± S.E.M. Comparison
between groups was made by one-way analysis of variance
(ANOVA) followed by Tukey’s multiple comparison test to
analyze the difference. The statistical significances were
achieved when P<0.05.
SOD Activity Measurement
Results
CPECs Have Epithelial Feature In Vitro and Express TTR
Fig. 3 Effect of CPECs-CM on cell viability. CPECs-CM and H2O2 was
added to PC12 cells at the same time. After 24 and 48 h, cell viability was
determined by MTT assay. Viability was calculated as the percentage of
living cells in treated cultures compared to control cultures. * P<0.05
significant different from H2O2 groups; # P<0.05 significant different
from control group
Three days after CPECs culture, round cells were detected in
the flasks (Fig. 1a). Two weeks later, the cells covered the
whole surface of the flasks. Cells were polygonal and had
epithelial appearance (Fig. 1b). Based on our immunocytochemistry results, the cells were immunopositive for TTR that
is a marker for CPECs (Herbert et al. 1986) (Fig. 1c). RT-PCR
analysis showed that CPECs express mRNA of TTR. Our
results also showed that CPECs express mRNAs of BDNF,
GDNF, NGF, FGF2 and VEGF (supplementary Fig.).
J Mol Neurosci (2014) 53:617–625
621
622
CPECs-CM Prevented PC12 Cells Neurite Outgrowth
Disruption Induced by H2O2
In the next step, we evaluated criteria of neurite outgrowth and
differentiation in PC12 cells treated with CPECs-CM and H2O2
simultaneously. Based on our data, in cells exposed to CPECsCM and H2O2, neurite length increased significantly compared
to cells incubated only with H2O2. Neurite length in the groups
that received H2O2 and CPECs-CM simultaneously, after 24
and 48 h, was two- and sevenfold higher than H2O2-exposed
cells, respectively (Fig. 2a). As shown in Fig. 2b, cell body area
increased in the present of H2O2 (4.8- and 5.8-fold 24 and 48 h
after exposure to H2O2 compared to control cells, respectively).
While, in CPECS-CM treated cells exposed to H2O2 the area of
cell body was reduced (1.2- and 1.3-fold 24 and 48 h, respectively, after exposure to H2O2 compared to H2O2 only treated
cells). In addition, calculation of neurite width showed significant decrease in the groups receiving CPECs-CM and H2O2
after 24 and 48 h relative to the stress (H2O2 treated) group.
These values were 7.4-fold for H2O2 group after 24- and 37.9fold after 48 h and for groups treated with CPECs-CM and
H2O2 simultaneously were 2.4- and 9.9-fold compared to the
stress group after 24 and 48 h, respectively (Fig. 2c). As a
marker of undifferentiation, percentage of bipolar neurons also
increased in H2O2 exposed cells (1.8-fold at 24 h and 3.9-fold at
48 h compared to intact cells). While percentage of bipolar
neurons significantly decreased in groups treated with CPECsCM and H2O2 compared to stress groups (34.5 % and 41.2 %
decrease at 24 and 48 h, respectively), the number of multipolar
cells increased in the presence of CPECs-CM as well (Fig. 2d).
CPECs-CM Increased PC12 Cells Viability in the Presence
of H2O2
To assess the effect of CPECs-CM on PC12 cells, viability of
cells treated with CPECs-CM and H2O2 were determined by the
MTT assay. As shown in Fig. 3, MTT assay revealed that cell
viability 24 and 48 h after the presence of CPECs-CM and H2O2
increased significantly compared to the cells incubated only
with H2O2. In the group treated with CPECs-CM and H2O2
concomitantly, these value increased 1.9- and 1.4-fold after 24
and 48 h relative to the H2O2 treated groups, respectively.
CPECs-CM Reduced Apoptosis in PC12 Cells in Oxidative
Stress Condition
To determine the ability of CPECs-CM to suppress apoptosis
in PC12 cells that were exposed to H2O2, we measured the
level of three factors Bax, Bcl-2 and caspase-3 through western blotting. The ratio of pro-apoptotic factor Bax to antiapoptotic factor Bcl-2 has been reported to be correlated to
initiation of a cascade which leads to apoptosis (Salakou et al.
2007). Based on our results, Bax: Bcl-2 ratio in cells exposed
J Mol Neurosci (2014) 53:617–625
concurrently to CPECs-CM and H2O2, reduced notably compared to cells that incubated only with H2O2 (Fig. 4c).
Our data also revealed that cleavage of caspase-3 significantly reduced when cells co-incubated with H2O2 and CPECs-CM
compared to H2O2 exposed cells. Cleavage of caspase-3 significantly increased in the presence of H2O2 relative to control cells
(Fig. 4d). These results were confirmed by morphological analysis, DAPI staining as well. The apoptotic cells show bright blue
nuclei while healthy cells have a dark blue nucleus. As shown in
Fig. 4a, the number of cells with bright blue nuclei significantly
increased in the presence of H2O2. On the other hand, incubation
of PC12 cells with CPECs-CM along with H2O2 revealed
reduction of apoptotic cells after 24 h.
CPECs-CM Increased Nrf2 and γ-GCS Levels
in the Oxidative Stress Condition
Nrf2 is a transcription factor that responds to ROS by activating phase II detoxification enzymes (Pi et al. 2010). Our
results showed that the level of Nrf2 in the presence of H2O2
decreased 64 % and 42.3 % after 24 and 48 h, respectively,
compared to the control (Fig. 5b), while the level of Nrf2 in
cells co-incubated with H2O2 and CPECs-CM increased remarkably compared to H2O2 exposed cells.
The same behavior was observed for γ-GCS as one of the
downstream factors of Nrf2 (Fig. 5c). These results show the
ability of CPECs-CM to keep the Nrf2 pathway in the active
form.
CPECs-CM Increased SOD Activity in the Oxidative Stress
Condition
SOD catalyses the dismutation of superoxide into hydrogen
peroxide and oxygen. As shown in Table 1, SOD activity in
CPECs-CM and H2O2 exposed cells after 24 and 48 h were
1.7- and 1.5-fold higher than the H2O2 treated group (Table 1).
Glutathione (GSH) Level Increased by CPECs-CM
in the Presence of H2O2
GSH is synthesized from amino acids L-glutamate and Lcysteine by action of γ-GCS (Liu et al. 2001). As shown in
Table. 1, GSH content reduced in H2O2 exposed cells compared to the control cells. Our data also showed that in the
presence of both CPECs-CM and H2O2, GSH content increased 1.4- and 1.5-fold after 24 and 48 h compared to
H2O2 exposed cells (Table 1).
CPECs-CM Decreased Lipid Peroxidation Induced
by Oxidative Stress
Previous studies have shown that MDA increases after induction of oxidative stress (Horakova et al. 2003; Kowalczuk and
J Mol Neurosci (2014) 53:617–625
623
Table 1 Effect of CPECs-CM on lipid peroxidation, GSH level and SOD
activity in the presence of H2O2 in PC12 cells
Treatment
MDA (nmol/mg
protein)
GSH (μmol/mg
protein)
SOD (U/mg
protein)
Control
CPECs-CM
H2O2 (24 h)
H2O2 +CPECsCM(24 h)
H2O2 (48 h)
H2O2 +CPECsCM(48 h)
0.61±0.01
0.63±0.003
0.73±0.003###
0.64±0.002***
6.51±0.38
5.87±0.37
5.08±0.33#
6.92±0.18*
41.7±1.18
41.1±0.62
23.31±0.52###
39.08±1.41***
0.67±0.009###
0.58±0.005***
4.3±0.32##
6.32±0.11**
18.28±0.14###
27.67±0.5***
* P<0.05 significant difference from H2O2 groups
#
Fig. 5 Nrf2 and γ-GCS levels in PC12 cells after treatment with CPECsCM and/or H2O2. Western blot probed with anti-Nrf2, anti-γ-GCS antibodies and reprobed with β-actin antibodies. a Bands of Nrf2, γ-GCS
were detected in all of groups. b The densities of Nrf2 and β-actin bands
were measured and the ratio of Nrf2/β-actin was reported. c The densities
of γ-GCS and β-actin bands were measured and the ratio of γ-GCS/βactin ratio was reported. * P<0.05 significant difference between treated
groups and H2O2 groups; # P<0.05 significant difference between control
groups and H2O2 groups
Stryjecka-Zimmer 2002). Based on our data, lipid peroxidation was 1.2-fold higher than the control cells 24 h after
induction of stress. In the CPECs-CM treated group, lipid
peroxidation showed 87 % reduction compared to stressed
group 24 h after stress induction. This decrease was 86.2 %
after 48 h (Table 1).
Discussion
In the present study, we demonstrated that CPECs-CM attenuates apoptosis and activates the Nrf2/ARE pathway. We used
H2O2 as the oxidative inducer. H2O2 is an attractive candidate
for signaling because it is relatively stable and long-lived
compared to other free radicals (Stone and Yang 2006). As
P<0.05 significant difference from control groups
one of the first observable changes in neurons, we determined
the effect of CPECs-CM on morphological criteria in the
oxidative situation. It has been observed that CPECs-CM
promotes neurite outgrowth in cultured spinal dorsal root
neurons (Chakraborty et al. 2000). Based on our results from
the calculation of other parameters, CPECs-CM not only
increased length of neurons but also decreased cell body area,
average of neurite width and percentage of bipolar neurons.
We can presume that these effects of CPECs-CM on neurite
outgrowth depend on the secretion of extracellular components. It has been shown that CPECs produce extracellular
matrix such as laminin and fibronectin that provide a scaffold
for spreading and expansion of shrinkage neurite-induced by
H2O2 (Zhou 1990; Peraldi-Roux et al. 1990). Previous study
showed CPECs express and secrete TTR that promoted expansion of neurite in vitro and in vivo (Fleming et al. 2007).
So, we can discuss that contents of CPECs-CM trigger intracellular signaling in PC12 cells in order to improve spreading
out of neuronal branches. In addition, secretion of intracellular
matrix components by CPECs-CM provide suitable basis for
expansion of neurite.
A well-known consequence of oxidative stress is overproduction of ROS that attack protein and DNA structures. ROS
is involved in several intracellular pathways that sooner or
later lead to apoptosis. Several studies reported the ability of
CPECs-CM to promote neuronal survival (Matsumoto et al.
2010; Watanabe et al. 2005; Borlongan et al. 2004a). In this
regard, our data showed that the CPECs-CM reduced the level
of apoptotic factors Bax and caspase-3 in PC12 cells following induction of oxidative stress by H2O2. In this manner,
level of Bcl2 as an anti-apoptotic factor increased to protect
cells against death. This effect has been attributed to the ability
of secreted materials of the choroid plexus. Borlongan et al.
have shown that neuronal cells survive and grow in CPECsCM as well as serum-containing medium. They reported that
CPECs-CM contains proteins and growth factors for survival
and growth of neuronal cells (Borlongan et al. 2004b).
624
Neuroprotective role of Nrf2 has been reported in preservation of BBB function after traumatic injury (Zhao et al.
2007). It is tempting to speculate that CPECs have good
potentiality to recruit Nrf2 and enhance antioxidant defense,
even in pathological conditions. To further investigate whether CPECs secret any neuroprotective and Nrf2-inducing
agents in their condition media, we measured the level of
Nrf2 and its downstream factor in PC12 cells exposed to
CPECs-CM and H2O2. Our data suggest that the presence of
CPECs-CM significantly increased Nrf2 stabilization 24 and
48 h after oxidative insult which is consistent with decreased
apoptosis and increased cell survival. Our data also showed
that incubation of stressed PC12 cells with CPECs-CM increases γ-GCS levels. Several studies have shown that there is
a positive feedback loop between growth factor level and Nrf2
expression (Mimura et al. 2011; Kweider et al. 2011). Our data
showed that CPECs can express mRNA of VEGF and BDNF.
It seems that growth factors such as VEGF and BDNF have
significant roles in stabilization and overexpression of Nrf2, in
which VEGF and BDNF have been shown to increase Nrf2
activation and its target gene expression (Kweider et al. 2011;
Li et al. 2013). In oxidative stress conditions, Nrf2 translocates into nucleus and activates ARE-mediated factors such as
γ-GCS that lead to detoxification of chemicals and ROS and
prevention of free radical generation. Taken together, we can
suggest that CPECs-CM contains a series of protective factors
that modulate the endogenous cellular defense against detrimental effects of oxidative stress.
As the first line of defense against oxidative agents, antioxidant enzymes immediately are activated in the presence of
stressors. These defensive enzymes directly/indirectly are located downstream of Nrf2 (Fubini and Hubbard 2003). As
mentioned above, CPECs-CM is able to induce the Nrf2/ARE
pathway. So, it will be predictable that content of antioxidant
enzymes increases in the presence of CPECs-CM. Not only
the level of antioxidant enzymes, but also their activity is
considered to be increased, because of the early role of these
enzymes in detoxification of oxidants. As our data showed, in
the presence of CPECs-CM and H2O2 there was an increase in
activity of SOD and content of GSH compared to the H2O2
exposed cells. Elevated activity of antioxidant enzymes ameliorates destructive effects of H2O2 by scavenging free radicals. It seems that CPECs-CM content protects neuronal cells
against oxidative stress via induction of the Nrf2/ARE pathway and increase of antioxidant enzymes activity.
In conclusion, activation of Nrf2 has been shown to combat
oxidative stress by induction of phase II detoxifying antioxidant proteins. As studies progress, it seems that Nrf2-inducers
are highlighted as a promising agent for future research in this
field. In this study, we showed that the application of CPECsCM could maintain cellular redox status by increasing SOD
activity and GSH content. So, it seems that CPECs cause
neuroprotection by proteins and factors that release into
J Mol Neurosci (2014) 53:617–625
CSF. Functions of many of these secreted proteins are not
yet fully understood. In our laboratory, we are now studying
the role of CPECs and their product, CSF, in CNS. We
presume that the CPECs can secrete many proteins in CSF
that seems to have role in health of nervous system.
Acknowledgments This work was supported by Shahid Beheshti University of Medical Sciences Research Funds. This project is part of the
PhD student thesis of A. Aliaghaei.
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