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ORIGINAL PAPER
Neuroprotective Effect of Salvia sahendica is Mediated
by Restoration of Mitochondrial Function and Inhibition
of Endoplasmic Reticulum Stress
Fatemeh Shaerzadeh •Shabnam Zeighamy Alamdary •
Mohammad Ali Esmaeili •Nazanin Namazi Sarvestani •
Fariba Khodagholi
Accepted: 7 July 2011 / Published online: 16 July 2011
ÓSpringer Science+Business Media, LLC 2011
Abstract Herein, we investigated the protective effect of
Salvia sahendica against H
2
O
2
-induced cell death in rat
pheochromocytoma (PC12) cells. Our data show that
S. sahendica blocks apoptosis pathway by inhibition of
cytochrome crelease from mitochondria and leakage of
calcium from endoplasmic reticulum. It also activates/
inactivates two members of Bcl-2 family, Bax and Bcl-2.
Bax inhibition and Bcl-2 activation suppress release of
cytochrome cfrom mitochondria that prevents cleavage of
caspase-3. Besides S. sahendica suppresses ER stress via
attenuation of intracellular levels of calcium. Suppression
of ER stress decreased calpain activation and subsequently
cleavage of caspase-12. Altogether, these results indicate
that S. sahendica protects PC12 cells treated with H
2
O
2
via
suppression of upstream factors of apoptosis pathway.
While oxidative stress is an early event in Alzheimer dis-
ease, it seems that S. sahendica prevents deleterious effects
of reactive oxygen species by stabilizing mitochondrial
membranes and inhibiting ER stress.
Keywords Apoptosis Oxidative stress ER stress
Mitochondrial dysfunction Salvia sahendica
Abbreviations
AbAmyloid-beta
AD Alzheimer’s disease
AO/EB Acridine orange/ethidium bromide
BSA Bovine serum albumin
DCF-DA 20,7
0-dichlorofluorescein diacetate
DMEM Dulbecco’s modified Eagle’s medium
ER Endoplasmic reticulum
HEPES 4-(2-hydroxyethyl)-piperazineethanesulfonic
acid
MMP mitochondrial membrane potential
MPT mitochondrial permeability transition
MPTPs mitochondrial permeability transition pores
NGF nerve growth factor
PARP Poly (ADP-ribose) polymerase
PBS phosphate buffered saline
PC12 pheochromocytoma cells
PMSF phenylmethylsulfonyl fluoride
Rh123 Rhodamine 123
ROS reactive oxygen species
Introduction
Age-related neurodegenerative diseases such as Alzhei-
mer’s disease (AD) are multifactorial in nature and involve
genetic, environmental and endogenous factors [1,2].
These diseases are commonly associated with increased
reactive oxygen species (ROS) production and abnormal
protein dynamics including the mitochondrial accumula-
tion of disease-specific proteins such as amyloid-beta (Ab)
in AD [3,4]. Uncontrolled production of oxygen radicals is
a common step in many models of apoptosis [5]. Since
mitochondria are the main cellular organelles that produce
ROS in physiological conditions [6], it seems that mito-
chondria may be the main source of ROS over-production
in pathological conditions of neurodegenerative diseases
F. Shaerzadeh S. Z. Alamdary N. N. Sarvestani
F. Khodagholi (&)
Neuroscience Research Center, Shahid Beheshti University
of Medical Sciences, Tehran, Iran
e-mail: khodagholi@sbmu.ac.ir
M. A. Esmaeili
Department of Biology, Medicinal Plants and Drugs Research
Institute, Shahid Beheshti University, G.C. Tehran, Iran
123
Neurochem Res (2011) 36:2216–2226
DOI 10.1007/s11064-011-0545-8
such as AD. Also, mitochondria are key regulators of
programmed cell death by apoptosis [7].
Endoplasmic reticulum (ER) is an organelle that regu-
lates the folding and assembly of secretory and membrane
proteins, lipid metabolism, the cellular response to stress,
and the homeostasis of intracellular calcium [8,9]. Certain
pathological stress conditions such as loss of the ER intra-
luminal oxidative environment and depletion of intracellu-
lar calcium stores can disrupt homeostasis in the ER. This
condition is referred to as ER stress [10,11]. Prolonged ER
stress induces apoptotic cell death and is linked to the
pathogenesis of some neurodegenerative disorders includ-
ing AD [12].
Salvia (sage) is the largest genus of the Lamiaceae
family and comprises about 900 species. Salvia have been
widely used all over the world because of their insecti-
cidal [13], antifungal [14], antioxidant [15–19], anticho-
linesterase [20] and neuroprotective [21] activities. In
addition, S. officinalis, a medicinal plant with memory
improving properties, has been used in traditional Chinese
medicine to treat AD patients [22]. About 58 Salvia spe-
cies have been identified in Iran, among them Salvia
sahendica is an aromatic, endemic perennial herb that
grows in the wild in Iran (Tabriz, East Azarbaijan province)
[23]. In our recent study, we revealed that S. sahendica can
protect PC12 cells from cell death and its presence could
regulate cellular redox status [16]. Considering that cur-
rently little information exists regarding protective effects
of S. sahendica as well as the involved mechanisms, in this
study, we investigated the antiapoptotic role of this plant
and provided some insight into its mechanism of action.
Experimental Procedure
Materials
Antibodies directed against caspase-3, Bax, Bcl-2, cyto-
chrome c, calpain-2, b-actin and Poly (ADP-ribose) poly-
merase (PARP) were obtained from Cell Signaling
Technology. Antibody directed against caspase-12 was
obtained from ABCAM. Fura 2/AM was obtained from
Invitrogen. All the other reagents, unless otherwise stated,
were from Sigma Aldrich (St. Louis, MO).
Plant Material
Aerial parts of S. sahendica were collected from Tabriz,
Iran (June 2004; Voucher herbarium specimen: MPH-848).
The plant aerial parts were air-dried, protected from direct
sunlight, and then powdered. The powder was kept in a
closed container in cold room. The powdered plant mate-
rial (50 g) was extracted four times with methanol at room
temperature overnight. The methanolic extract was com-
bined and concentrated under reduced pressure on a rotary
evaporator, after that, it was filtered and then lyophilized.
For cell treatment, lyophilized powder was suspended in
distilled water.
Cell Culture and PC12 Differentiation
Rat pheochromocytoma cells (PC12) obtained from Pasteur
Institute (Tehran, Iran) and maintained in Dulbecco’s
modified Eagle’s medium (DMEM) supplemented with
heat-inactivated 10% horse serum, 5% fetal bovine serum
and 1% antibiotic mixture comprising penicillin–strepto-
mycin (Gibco), in a humidified atmosphere of 5% CO
2
at
37°C. Growth medium was changed three times a week.
The cells were differentiated by treating with nerve growth
factor (NGF; 50 ng/ml) every other day for 6 days.
Treatment Condition
Differentiated PC12 cells were pretreated with different
concentrations (10, 25, 50 and 100 lg/ml) of methanolic
extract of S. sahendica for 24 h. The pretreated cells
were then incubated with H
2
O
2
(150 lM) for an additional
24 h.
Acridine Orange/Ethidium Bromide (AO/EB) Double
Staining
Apoptosis was determined morphologically after staining
the cells with AO/EB followed by fluorescence micros-
copy inspection. Briefly, PC12 cells were seeded in a
6-well plate and were treated with different concentra-
tions of extract (10, 25, 50 and 100 lg/ml). After 24 h,
150 lMH
2
O
2
was added as an oxidative agent. After
24 h incubation, the cells were harvested and washed
three times with phosphate buffered saline (PBS) and
were adjusted to a density of 1 910
6
cells/ml of PBS.
AO/EB solution (1:1 v/v) was added to the cell suspen-
sion in a final concentration of 100 lg/ml. Cellular
morphology was evaluated by fluorescence microscope
(Zeiss, Germany).
Isolation of Mitochondria from the PC12 Cells
Mitochondrial and cytosolic fractions were prepared as
described previously [24], with minor modifications.
Briefly, cells were harvested and re-suspended in buffer A
(10 mM 4-(2-hydroxyethyl)-piperazineethanesulfonic acid
(HEPES), 10 mM KCl, 1 mM EDTA, 1 mM EGTA,
68 mM sucrose, 220 mM mannitol, 0.1% Bovine serum
albumin (BSA)), supplemented with protease inhibitors
(1 mM phenylmethylsulfonyl fluoride (PMSF), 2 lg/ml
Neurochem Res (2011) 36:2216–2226 2217
123
aprotinin, and 0.1 mM leupeptin). After 30 min incuba-
tion on ice, cells were disrupted with a 27 gauge syringe
(25–40 strokes), and the homogenates were centrifuged at
200gfor 2 min to eliminate unbroken cells. The 500 ll
supernatants were then centrifuged at 7,000g for 15 min
to obtain the heavy membrane pellet enriched with
mitochondria. The resultant supernatants pre-cleared at
16,000g for 30 min were collected as the cytosolic frac-
tions. Mitochondrial fractions were subsequently solubi-
lized in TBSTDS (10 mM Tris, (pH 7.5), 150 mM NaCl,
1 mM EDTA, 1% Triton X-100, 0.5% sodium deoxy-
cholate, 0.5% SDS, 0.02% NaN
3
, 0.0004% NaF) supple-
mented with protease inhibitors. All manipulation steps
were carried out at 4°C. Fractions then were stored at
-70°C until used.
Measurement of the Mitochondrial Membrane Potential
(MMP)
MMP was estimated by a fluorescence assay with fluores-
cent dye Rhodamine 123 (Rh123) as described previously
[25]. After treatment, differentiated-PC12 cells were
incubated for 30 min at 37°C with PBS containing 5 lM
Rh123. After being washed with PBS, cells were trypsin-
ized at room temperature and suspended in PBS. The
fluorescence intensity was measured by the Varian Cary
Eclipse spectrofluorometer with excitation and emission
wavelengths of 485 and 530 nm, respectively.
Western Blot Analysis
Total proteins were electrophoresed in 12% SDS–PAGE
gels, transferred to polyvinylidene fluoride membranes and
probed with specific antibodies. Immunoreactive polypep-
tides were detected by chemiluminescence using Electro-
ChemiLuminescence reagents (Amersham Bioscience,
USA) and subsequent autoradiography. Quantification of
results was performed by densitometric scan of films. Data
analysis was done by Image J, measuring integrated density
of bands after background subtraction. Protein concentra-
tion was determined by Bradford method [26], using BSA
as a reference standard.
Measurement of Intracellular ROS
The fluorescent probe 20,7
0-dichlorofluorescein diacetate
(DCF-DA) was used to monitor intracellular accumulation
of ROS. For this purpose, the DCFH-DA solution
(10 lM) was added to the suspension of the cells
(1 910
6
/ml), the mixture was incubated at 37°C for 1 h.
Cells were then washed twice with PBS and finally, the
fluorescence intensity was measured by Varian Cary
Eclipse spectrofluorometer with excitation and emission
wavelengths of 485 and 530 nm, respectively.
Measurement of Intracellular Calcium level
After treatments, PC12 cells were collected and prepared
to generate a 0.5 ml cell suspension for each sample.
Fura-2/AM (final concentration 5 lM) was added to the
cell suspension. The suspension was shaken at 37°C for
1 h, and then centrifuged twice at 1,000 rpm for 5 min.
The cells were re-suspended in HEPES buffer solution,
containing NaCl 132, KCl 3, glucose 10, HEPES 10 and
CaCl
2
2 mM, pH 7.4 and finally, the fluorescence inten-
sity was measured by Varian Cary Eclipse spectrofluo-
rometer with excitation and emission wavelengths of 340
and 500 nm, respectively.
Statistical Analysis
All data were average of triplicate analysis. The data were
recorded as means ±S.E.M. Analysis of variances was
performed by ANOVA procedures. P\0.05 was consid-
ered as significant difference (*or
#
P\0.05,**or
##
P\0.01 and *** or
###
P\0.001).
Results
S. sahendica Attenuated H
2
O
2
-Induced Apoptosis
in PC12 Cells
AO/EB staining discriminates live cells from dead ones on
the basis of membrane integrity. AO is a cell-permeable
nucleic acid selective cationic dye which is taken up by
both viable and nonviable cells and emits green fluores-
cence if intercalated into double stranded nucleic acid. EB
intercalates and stains DNA, providing a red–orange fluo-
rescence. Although it does not stain healthy cells, it can be
used to identify cells that are in the early or final stages of
apoptosis which have much more permeable membranes.
The result obtained from AO/EB double staining is repre-
sented in Fig. 1a. In this method, the extent of apoptosis
was determined based on fluorescence emission and mor-
phological aspect of chromatin condensation in stained
nuclei. Apoptotic cells had orange to red nuclei with con-
densed or fragmented chromatin, whereas viable cells had
uniform bright green nuclei with organized structure. In
this method, necrotic cells showed uniformly red nuclei.
Analysis of the stained cells indicated that pretreatment of
cells with S. sahendica has significantly and dose-depen-
dently decreased the extent of cell apoptosis compared to
the cells exposed to H
2
O
2
(Fig. 1b).
2218 Neurochem Res (2011) 36:2216–2226
123
S. sahendica Decreased Bax/Bcl-2 Ratio in PC12 Cells
Bcl-2 family proteins are important regulators of various
apoptotic pathways. Bcl-2 and Bcl-XL are inhibitors of
cytochrome crelease from the mitochondria into the
cytosol. However, Bax acts as a pro-apoptosis factor by
destroying mitochondrial integrity [27,28]. Here by H
2
O
2
treatment, the protein level of Bcl-2 was downregulated
while the level of Bax was upregulated. As shown in
Fig. 2, the ratio of Bax/Bcl-2 increased by 6.58 fold in the
presence of H
2
O
2
, compared to control. In PC12 cells
pretreated with 10, 25, 50 and 100 lg/ml of S. sahendica
extract, decrease of Bax/Bcl-2 ratio were 1.44, 1.98, 2.85
and 4.13 time, respectively, compared to the H
2
O
2
-treated
cells.
S. sahendica Inhibited Caspase-3 Activation and PARP
Cleavage in Differentiated PC12 Cells
Another important data, confirming the protective effect of
S. sahendica was obtained from Western blot analysis of
caspase-3. Apoptosis is induced by the cleavage of a subset
of cellular proteins by proteases of the caspase family.
Caspase-3 is a key effector caspase, which is responsible for
cleaving a large number of protein substrates including
PARP-1[29]. PARP-1 plays an important role in the cellular
Fig. 1 Analysis of apoptosis by
using AO/EB double staining.
aThe cells were exposed to
different concentrations of
S. sahendica for 24 h followed
by exposure to 150 lMofH
2
O
2
for 24 h. bThe number of
stained cells was counted in 10
randomly selected fields.
Viability was calculated as the
percentage of living cells in
treated cultures compared to
those in control cultures. Each
value represents the mean
±S.E.M (n =3). *significantly
different from untreated cells.
#
significantly different from
H
2
O
2
-treated cells
Bax
Bcl2
β-actin
20 kDa
45kDa
28 kDa
A
B
Fig. 2 Bax:Bcl-2 ratio in PC12 cells of experimental groups.
aDiverse response of Bax and Bcl-2 to S. sahendica (10, 25, 50
and 100 lg/ml) in PC12 cells pretreated for 24 h and then exposed to
H
2
O
2
(150 lM) for 24 h. Twenty lg proteins were separated on SDS–
PAGE, Western blotted, probed with anti-Bax and -Bcl-2 antibodies
and reprobed with anti-b-actin antibody. (One representative Western
blot was shown; n =3). bThe densities of Bax and Bcl-2 were
measured and the Bax:Bcl-2 ratio was calculated. The mean of three
independent experiments is shown. *significantly different from
control cells.
#
significantly different from H
2
O
2
-treated cells
Neurochem Res (2011) 36:2216–2226 2219
123
defense against oxidative stress [30]. To verify whether
S. sahendica extract interferes apoptosis via suppressing
caspase-3 activation, we measured the level of cleaved
caspase-3 by Western blot analysis in the presence of this
extract. As shown in Fig. 3a and b, H
2
O
2
induced the
appearance of cleaved active caspase-3, arguing for
involvement of caspase-3 in H
2
O
2
-induced cell death in
PC12 neurons. In cells pretreated with 10, 25, 50 and
100 lg/ml of S. sahendica extract, the level of caspase-3
was decreased by 2.5, 0.3.36, 4.48 and 7.49 time, respec-
tively, compared to H
2
O
2
-treated cells, demonstrating the
ability of this plant to suppress H
2
O
2
-induced apoptosis in
differentiated PC12 cells. In order to confirm the suppres-
sion of caspase-3 activation by different concentrations of
S.sahendica, we examined the level of PARP cleavage by
Western blot analysis. As shown in Fig. 3c and d, in PC12
cells treated with H
2
O
2
, accumulation of the 89- and
24-kDa PARP cleaved fragments are present, hence, con-
firming the activation of caspase-3, that responsible for
PARP cleavage. While in PC12 cells pretreated with dif-
ferent concentration of S. sahendica bands of cleaved PARP
(89 and 24 kDa) were weaker compared to H
2
O
2
-treated
cells.
S. sahendica Restored H
2
O
2
-Induced MMP Collapse
in PC12 Cells
We assessed the effects of S. sahendica extract on MMP,
which is an important mitochondrial parameter controlling
key cellular processes. The decrease of the MMP induced
by H
2
O
2
was evaluated by determining the cellular reten-
tion of Rh123, which is known to be concentrated in the
mitochondria and quenched at a high MMP level. As MMP
decreases, Rh123 is released, causing dequenching and an
increase in Rh123 fluorescence [31]. As shown in Fig. 4,
addition of S. sahendica extract (10, 25, 50 and 100 lg/ml)
caused a dose-dependent increase of MMP after 24 h
preincubation.
Procaspase-3 35 kDa
17 kDa
Caspase-3
45 kDa
β-actin
PARP
PARP
PARP
β-actin 45 kDa
89 kDa
24 kDa
119 kDa
A
B
C
D
Fig. 3 The Cleavage of procaspase-3 and PARP in PC12 cells
pretreated with S.sahendica.aProcaspase-3 and cPARP response to
S. sahendica (10, 25, 50 and 100 lg/ml) in PC12 cells pretreated for
24 h and then exposed to H
2
O
2
(150 lM) for 24 h. Twenty lg
proteins were separated on SDS–PAGE, Western blotted, probed with
anti-caspase-3 and -PARP antibodies and reprobed with anti-b-actin
and/or—Lamin-B2 antibodies, respectively. (One representative
Western blot was shown; n =3). bThe densities of Procaspase-3,
caspase-3 and dPARP bands were measured and the ratios to their
related loading controls were calculated. The mean of three indepen-
dent experiments is shown. *significantly different from control cells.
#
significantly different from H
2
O
2
-treated cells
2220 Neurochem Res (2011) 36:2216–2226
123
S. sahendica Inhibited H
2
O
2
-Induced Intracellular ROS
Generation in Neuron-Like PC12 Cells
ROS are produced as a by-product of cellular metabolic
pathways and function as a critical second messenger in a
variety of intracellular signaling pathways. Thus, a defect
or deficiency in the anti-oxidant defense system and/or the
excessive intracellular generation of ROS leads to cell
oxidative stress. Oxidative stress activates some factors and
leads to apoptosis. To determine whether S. sahendica may
attenuate the ROS generation, intracellular ROS level was
measured. Treatment of PC12 cells with H
2
O
2
(150 lM)
increased intracellular ROS generation significantly, while
in PC12 cells pretreated with S. sahendica extract intra-
cellular ROS generation was inhibited effectively (Fig. 5).
S. sahendica Inhibited H
2
O
2
-Induced Cytochrome
cRelease in PC12 Cells
Release of cytochrome cfrom mitochondria is a key ini-
tiative step in the mitochondria apoptotic pathway. Chan-
ges in MMP lead to release of cytochrome cinto cytosol
[32]. To further confirm the role of the mitochondrial
apoptotic pathway in mediating H
2
O
2
-induced apoptosis,
we examined the distribution of cytochrome cwithin PC12
cells undergoing H
2
O
2
-induced apoptosis while treated
with S.sahendica. As shown in Fig. 6, a dose-dependent
accumulation of cytochrome cin the mitochondria
observed in PC12 cells pretreated with S. sahendica extract
while in cells treated only with H
2
O
2
, this accumulation of
cytochrome cwas observed in cytosol.
S. sahendica Inhibited H
2
O
2
-Induced Intracellular
Calcium Elevation in PC12 Cells
Accumulation of intracellular Ca
2?
induced by excitotoxic
agent leads to mitochondrial membrane depolarization,
increased production of oxygen free radicals and caspase
or calpain dependent cell death. To establish whether
S. sahendica attenuated accumulation of intracellular Ca
2?
induced by H
2
O
2
, we examined the Ca
2?
levels. As shown in
Fig. 7,S. sahendica in a dose-dependent manner attenuated
elevated intracellular Ca
2?
levels.
S. sahendica Inhibited H
2
O
2
-Induced Calpain-2
Expression in PC12 Cells
Calpain, calcium-dependent cysteine protease, is ubiqui-
tously expressed in neurons, both in cytosolic and in syn-
aptic terminals [33]. It plays a pivotal role in the
excitotoxic signal transduction cascade leading to DNA
Fig. 4 Effect of Salvia species on MMP level. The PC12 cells were
incubated 24 h with or without S. sahendica (10, 25, 50 and 100 lg/ml)
and then exposed to H
2
O
2
(150 lM) for 6 h. MMP level were measured
with Rhodamine 123. The mean of three independent experiments is
shown. *Significantly different from control cells.
#
Significantly
different H
2
O
2
-treated cells
Fig. 5 Inhibitory effect of
S. sahendica on H
2
O
2
-induced
intracellular ROS. The PC12
cells were incubated 24 h with
or without S. sahendica (10, 25,
50 and 100 lg/ml) and then
exposed to H
2
O
2
(150 lM) for
6 h. Intracellular levels of ROS
were measured with DCFH-DA.
The mean of three independent
experiments is shown
Neurochem Res (2011) 36:2216–2226 2221
123
fragmentation [34]. To verify whether S. sahendica extract
inhibits apoptosis via calpain-2 suppressing, we measured
the level of calpain-2 by western blot analysis in the
presence of this extract. As shown in Fig. 8,H
2
O
2
induced
the expression of calpain-2, arguing for involvement of
calpain-2 in H
2
O
2
-induced cell death in PC12 neurons.
In cells pretreated with 10, 25, 50 and 100 lg/ml of
S. sahendica extract, the level of calpain-2 was decreased
by 1.38, 1.56, 1.67 and 1.89 time, respectively, compared
to H
2
O
2
-treated cells, demonstrating the ability of this plant
to suppress calpain-2 expression in differentiated PC12
cells. Thus, it appears that S. sahendica could suppress
expression of calpain activated by H
2
O
2
.
S. sahendica Decreased H
2
O
2
-Induced Caspase-12
Expression in Differentiated PC12 Cells
Caspase-12 is an activator caspase that is residual in ER.
Activation of caspase-12 eventually leads to induction of
caspase-3 and initiation of apoptotic cascade [36–38]. To
determine whether S. sahendica extract suppressed cas-
pase-12 expression and following apoptotic cascade, we
measured the level of caspase-12 by western blot analysis.
As shown in Fig. 9,H
2
O
2
induces the cleaved of caspase-
12. It is reason for the involvement of caspase-12 in H
2
O
2
-
induced cell death in PC12 cells. In cells pretreated with
10, 25, 50 and 100 lg/ml of S. sahendica extract, the level
of caspase-12 was decreased by 1.15, 1.26, 1.54 and 1.75
time, respectively, compared to H
2
O
2
-treated cells.
Cytochrome C (M) 14 KDa
14 KDa
Cytochrome C (C)
45 KDa
β-actin
A
B
Fig. 6 Western blot analysis to measure the effects of S. sahendica
on cytochrome cin PC12 cells. aCytochrome cresponse to
S. sahendica (10, 25, 50 and 100 lg/ml) on mitochondrial and
cytosolic fractions (shown as M and C, respectively) from PC12 cells
pretreated for 24 h and then exposed to H
2
O
2
(150 lM) for 24 h.
Twenty lg proteins were separated on SDS–PAGE, Western blotted,
probed with anti-cytochrome cantibody and reprobed with anti-b-actin
antibody. (One representative Western blot was shown; n =3). bThe
density of cytochrome cbands in mitochondrial fractions and ccytosolic
fractions were measured and the ratio was calculated. The mean of three
independent experiments is shown. *significantly different from control
cells.
#
significantly different from H
2
O
2
-treated cells
Fig. 7 Effect of S. sahendica on H
2
O
2
-induced intracellular cal-
cium level. The PC12 cells were incubated 24 h with or without
S. sahendica (10, 25, 50 and 100 lg/ml) and then exposed to H
2
O
2
(150 lM) for 6 h. Intracellular levels of calcium were measured with
Fura-2/AM. The mean of three independent experiments is shown
Calpain-2 80 kDa
Β-actin 45 kDa
A
B
Fig. 8 Western blot analysis to measure the effects of S. sahendica
on the expression of cleaved calpain-2 in PC12 cells. acalpain-2
response to S. sahendica (10, 25, 50 and 100 lg/ml) on PC12 cells
pretreated for 24 h and then exposed to H
2
O
2
(150 lM) for 24 h.
Twenty lg proteins were separated on SDS–PAGE, Western blotted,
probed with anti-calpain-2 antibody and reprobed with anti-b-actin
antibody. (One representative Western blot was shown; n =3). bThe
density of calpain-2 band was measured and the ratio was calculated.
The mean of three independent experiments is shown. *significantly
different from control cells.
#
significantly different from H
2
O
2
-
treated cells
2222 Neurochem Res (2011) 36:2216–2226
123
Discussion
ROS are produced in all mammalian cells, partly as a result
of normal cellular metabolism, and partly due to activation
of membrane-bound enzyme systems in response to exog-
enous stimuli. A disturbance in the intracellular balance
between ROS production and the activities of the anti-oxi-
dant defense systems leads to accumulation of ROS in cells,
referred to as oxidative stress [39]. Several studies have
reported the association of neural oxidative stress with
neurodegenerative disorders such as AD [1,2,4]. The main
sources of ROS in AD brain are mitochondrial dysfunction,
interneuronal Abaccumulation and redox-active metals
[40]. The goal of this study is to investigate the triggered
mechanisms by Salvia sahendica against oxidative stress
induced by H
2
O
2
. Accordingly, cells exposed to H
2
O
2
were
used for an in vitro model of oxidative stress [41].
Several lines of evidence reveal that Salvia species have
therapeutic properties including antioxidant activities
[15–19]. In a previous study from our group, we reported
the antioxidant and antiglycating effects of S. sahendica on
oxidative stress induced by H
2
O
2
. The HPLC analysis of
S. sahendica revealed that the major compound of this
extract is Rosmarinic acid (67.12 mg/g) [16]. Rosmarinic
acid is a major natural phenolic compound of the genus
Salvia and has many biological activities such as inhibition
of HIV-1, antitumor, antihepatitis and hepatoprotection as
well as anticoagulation and anti-inflammation. In addition,
rosmarinic acid can inhibit the activity of xanthine oxidase
and is used to scavenge the surplus free radicals in the
body. Some experiments have reported a strong capacity of
rosmarinic acid in scavenging free radicals [42]. Attenua-
tion of ROS in the present study in PC12 cells pretreated
with S. sahendica confirms that.
ROS are involved in the regulation of different signal
transduction pathways including mitochondrial pathways
of apoptosis. It has been well documented that mitochon-
drial membrane is one of the first targets of ROS that leads
to initiation of apoptosis cascades [39]. ROS change
mitochondrial membrane permeability, which is known as
mitochondrial permeability transition (MPT). MPT is
controlled by nonspecific pores in the mitochondrial inner
membrane called mitochondrial permeability transition
pores (MPTPs) [43]. Opening of MPTPs leads to disruption
of MMP that is considered a ‘‘universal feature of cell
death’’ and is often considered the ‘‘point of no return’’ in
the cell death cascade of events [44]. It triggers the release
of apoptogenic proteins into the cytosol, including cyto-
chrome c[45,46]. In the cytosol, cytochrome cinteracts
with its adaptor molecule, Apaf-1, resulting in recruitment
of pro-caspase-9 in the presence of dATP or ATP [47].
Caspase-9, in turn, cleaves and activates pro-caspase-3.
This effector caspase is responsible for the cleavage of
various proteins such as PARP that eventually, leads to
biochemical and morphological process of apoptosis [29].
Several studies consistently indicated involvement of Bcl2
family proteins in mediating and/or inhibiting cytochrome
crelease [48,49]. In the present study, our finding indi-
cated that H
2
O
2
can cause increase in ROS level in PC12
cells, a very user-friendly cell line that is used for studying
neuronal signaling pathways and other neurobiochemical
events [50]. As described above, elevated ROS levels
induced by H
2
O
2
eventually lead to triggering the intrinsic
pathway of apoptosis [39]. So we expected the expression
of apoptotic factors. To demonstrate the mechanisms or
factors were induced and/or suppressed by neuroprotective
effect of S. sahendica, we measured the expression levels
of Bax, Bcl-2, cytochrome c, Caspase-3 and PARP in this
pathway. Bax/Bcl-2 ratio is a parameter for description of
apoptotic state. Increase of this ratio in PC12 cells treated
with H
2
O
2
indicated that cells have undergone apoptosis,
while in samples pretreated with S. sahendica, Bax/Bcl-2
ratio decreased approximately to about control samples in a
dose-dependent manner. Thus, it appears that the ability of
S. sahendica to attenuated ROS formation inhibits
the pores formation in mitochondrial. Otherwise, this
Cleaved Caspase-12 48 kDa
β-actin 45 kDa
A
B
Fig. 9 Western blot analysis to measure the effects of S. sahendica
on the expression of cleaved caspase-12 in PC12 cells. aCaspase-12
response to S. sahendica (10, 25, 50 and 100 lg/ml) on PC12 cells
pretreated for 24 h and then exposed to H
2
O
2
(150 lM) for 24 h.
Twenty lg proteins were separated on SDS–PAGE, Western blotted,
probed with anti-caspase-12 antibody and reprobed with anti-b-actin
antibody. (One representative Western blot was shown; n =3). bThe
density of caspase-12 band was measured and the ratio was
calculated. The mean of three independent experiments is shown.
*significantly different from control cells.
#
significantly different
from H
2
O
2
-treated cells
Neurochem Res (2011) 36:2216–2226 2223
123
inhibitory effect of S. sahendica is intensified by decrease
of Bax levels. Therefore, we expected that cytosolic level
of cytochrome cin samples pretreated with S. sahendica
decreased compared to samples treated with H
2
O
2
,a
hypothesis that was confirmed by our data. Decrease in
cleavage of caspase-3 and PARP in PC12 cells pretreated
with S. sahendica demonstrated that apoptosis pathway
was suppressed by this plant. So it seems that S. sahendica
increases ROS degradation by anti-oxidant defense system
induction [16] and can inhibit pore formation and stabilizes
the mitochondrial membrane, which suppresses cyto-
chrome crelease to the cytosol.
Moreover, several studies indicated that elevated levels
of ROS increase calcium release from internal stores. Two
cytoplasmic organelles involved in Ca
2?
homeostasis: ER
and mitochondria. Change in mitochondrial membrane
permeability induced by ROS leads to leakage of Ca
2?
from mitochondrial matrix to the cytosol and elevates
intracellular levels of Ca
2?
[51]. A number of cellular
stress conditions including depletion of intracellular Ca
2?
stores and oxidative stress lead to accumulation of unfol-
ded or misfolded proteins in ER lumen, which is referred
to as ER stress [10,11]. ER stress couples with the
apoptosis pathway via caspase-12 which is predominantly
localized at the ER. Caspase-12 is specifically activated by
disturbance of ER homeostasis such as ER stress and
reduction of intracellular calcium ion store but not by
other apoptotic stimuli [35–38]. Caspase-12 is activated by
direct proteolytic cleavage by a cytosolic calcium-acti-
vated endopeptidase namely calpain. Inactive calpain
resides in cytosol. Upon activation of calpain by elevated
intracellular Ca
2?
, calpain translocates to the ER mem-
brane and cleaves procaspase-12. Following its cleavage at
the ER, caspase-12 directly processes downstream casp-
ases in the cytosol [52–54]. In this study, we clarified that
the intracellular Ca
2?
, calpain and caspase-12 expression
levels in PC12 cells were affected by H
2
O
2
. Our
results clearly showed that in PC12 cells pretreated with
S. sahendica, intracellular levels of Ca
2?
decrease in a
dose-dependent manner compared to cells treated with
H
2
O
2
. Thus, it appears that S. sahendica at first step
suppresses H
2
O
2
-induced Ca
2?
release from internal
storage by attenuation of ROS levels in cells. Moreover,
our data reveal that calpain levels in PC12 cells pretreated
with S. sahendica decrease compared to sample treated
with H
2
O
2
, significantly. As calpain cleaved caspase-12,
we expected the decrease of cleaved caspase-12. Based on
our results, caspase-12 in PC12 cells pretreated with
S. sahendica is less cleaved than cells treated only
with H
2
O
2
. Taken together, these results suggest that
S. sahendica inhibits the ER stress induced by ROS in
PC12 cells treated by H
2
O
2
. From the other side, during
apoptosis, activated Bax translocates from the cytoplasm
to both mitochondria and the ER to interact with Inositol-
3-phosphate receptors (IP
3
R
5
). Bax counteracts Bcl2
resulting in further increase of Ca
2?
release and acceler-
ated apoptosis. By localizing to both ER and mitochon-
dria, Bcl2 may prevent apoptotic cross-talk between these
two organelles by lowering the amount of free Ca
2?
in
the ER, inhibiting ER Ca
2?
release from IP
3
R
5
or by
increasing the tolerance of mitochondria to high Ca
2?
loads [55]. It is reported that either the overexpression of
Bcl
2
or the loss of Bax leads to reduced resting ER Ca
2?
concentrations and a secondary decrease in Ca
2?
uptake in
mitochondria [56,57].
In conclusion, according to our investigations there are
three possible mechanisms by which S. sahendica exerts its
neuroprotective effect against oxidative stress induced by
H
2
O
2
in PC12 cells: The first one is exerted by induction of
anti-oxidant enzyme that we reported previously. Accord-
ing to our data, H
2
O
2
elevates ROS production in PC12
cells while pretreatment of cells with S. sahendica extract
decreased ROS in PC12 cells by activation of catalase and
superoxide dismutase enzymes as we reported previously
[16]. Activation of anti-oxidant enzymes is the first step of
the cell defense system. The second mechanism includes
mitochondrial pathway of apoptosis. In this defense
mechanism, it seems that S. sahendica stabilizes MMP and
suppresses the cytochrome crelease from mitochondrial
matrix to the cytosol. Release of cytochrome cis the initial
process of intrinsic pathway of apoptosis. So, activation of
downstream factors in this pathway will be suppressed.
Third mechanism by which S. sahendica protects cells
involves ER. In the presence of ROS, intracellular levels of
calcium elevates that is one of the conditions that leads to
ER stress. S. sahendica attenuates levels of intracellular
calcium in two ways: (1) stability of mitochondrial mem-
brane by S. sahendica inhibits the release of Ca
2?
to the
cytosol because mitochondria play a role in calcium buf-
fering in cells. (2) Suppression of Ca
2?
leakage induced by
ROS from ER to cytosol. Attenuation of intracellular levels
of Ca
2?
by S. sahendica prevents triggering of apoptosis
pathway mediated by calpain and consequently caspase-12,
an ER membrane specific protease. So, S. sahendica by
attenuation of ROS levels blocks apoptosis cascade that
may be triggered by ROS.
Regarding the extent of traditional use, broad distribu-
tion and oral consumption of this plant in Iran, it seems that
it is a suitable candidate for treatment of patient with
neurodegenerative disorders. Further studies in animal
models of AD are in progress in our laboratory.
Acknowledgments The authors thank Solaleh Khoramian Tusi for
her excellent technical assistance. This work was supported by Shahid
Beheshti University of Medical Sciences Research Funds.
Conflict of interest The authors have no conflict of interest.
2224 Neurochem Res (2011) 36:2216–2226
123
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