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Introduction
The brain is the most vulnerable organ to ischemic insult
because of its high metabolic rate, relative low oxygen stores,
and insufficient reserves of high-energy carbohydrates,
which usually result in severe functional loss in the central
nervous system. The re-establishment of blood flow is
essential to repair ischemic brain tissue damage. However,
ischemia-reperfusion injury is the most imperative problem
during the course of repairing of ischemic brain tissue damage.
Neuronal apoptosis has been implicated in the
pathophysiology of brain ischemia and reperfusion
injury[1_3]. Accord-ingly, protection from the abnormally increased neuronal
apoptosis will be beneficial to the therapy.
The polyphenolic compound resveratrol
(trans-3,4',5-trihydroxystilbene, Res) is a naturally occurring
phytochemical which has been found in more than 70 plant species,
including human food products like grapes, peanuts, berries,
and some herbs. The physiological function of this
polyphenol is thought to serve as a phytoalexin, protecting plants
against environmental stress or pathogenic attack, and as a
strong antioxidant to reduce the oxidation of lipoproteins in
animals[4_6]. Notably, it can also protect the blood vessels
from atherosclerosis and inhibit the platelet aggregation and
cyclo-oxygenase[4,7,8]. Recent studies have demonstrated
the ability of Res to exert protective effects against brain
injury induced by ischemia_reperfusion in
gerbils[9], and epidemiological studies have also shown that the
consumption of Res-enriched red wine is significantly correlated with
a reduction in the incidence of age-related macular
degenera-tion, Alzheimer's disease, and
stroke[10_12]. Furthermore, our study concluded that Res had significant protective effects
on injury induced by oxygen-glucose
deprivation/reperfu-sion (OGD/RP) insult in primary cultured neurons of
neonatal rats[13].
In this study, we examined the influence of Res on the
neuronal apoptosis for further investigation of its effects on
the injury induced by OGD/RP in primary cultured neurons
of neonatal rats, and investigated the possible protective
mechanisms.
Materials and methods
Animals and reagents Sprague-Dawley rats (200_250 g)
purchased from the Animal Center of the Institute of Field
Surgery of the Third Military Medical University in China
(Chongqing, China; Specific-pathogen free Grade II,
Certificate No scxk 20020003) were kept in a regulated environment
(23±1 oC, 50%±2% humidity) with 12 h light/dark cycle (light
on 8:00_20:00). Newborn Sprague-Dawley rats (less than 72
h) were incubated in the animal room. Res (purity
³98% via HPLC) was provided by Hunan Huaguang Biological
Products (Huaihua, China). The concentration of solvent (DMSO)
in the final culture media was less than 0.05%
(v/v), which was safe to the culture in the control group. Nimodipine
(Nim, No 95060120, molecular weight: 418.4) was obtained
from Zhengzhou Chemical Pharmaceutical Factory
(Zheng-zhou, China). Dulbecco's modified Eagle's medium
(DMEM)/F12, and fetal bovine serum (FBS) were purchased from
Hyclone (Logan City, Utah, USA). Hydroxyethyl piperazine
ethanesulfonic acid (HEPES), Fura-2/AM, ethyleneglycol
bis(2-aminoethyl ether)tetraacetic acid (EGTA), and Triton
X-100 were from Sigma (St Louis, MO, USA). The Annexin
V/ fluorescein isothiocyanate (FITC) kit was purchased from
Jingmei Biotechnology (Shenzhen, China).
Primary cortical neuron culture[14]
The cortices taken from 1_3 d old newborn Sprague-Dawley rats were collected
into cold D-Hanks' solution and dissected free of meninges
and blood vessels. The cerebral tissues were minced and
incubated in 0.125% trypsin at 37 oC for 30 min. Then the
DMEM/F12 medium with fetal bovine serum was added to
terminate digestion. The whole cell suspension was filtered
with a nylon mesh (200 mesh, hole width: 95 µm), the filtrate
was centrifuged at 3 000×g for 10 min, then the sediment was
resuspended with DMEM/F12 containing 20% FBS, 100
kU/L benzylpenicillin, and 100 mg/L streptomycin. The cells were
adjusted to approximately
1×109/L and planted into 96-well
plates or dishes, which were previously coated with 10 mg/L
poly-L-lysine for 24 h, at 37 oC in an atmosphere of 5%
CO2 and 95% O2. Arabinosylcytosine (5 µg/mL) was added on
the third day after incubation to prevent the growth of
non-neuronal cells. After 24 h, the culture was changed with the
normal medium and refreshed every 2_3
d[15,16]. Under these conditions, the cultures typically contained more than 95%
neurons as assessed by staining with an antibody directed
against neuron-specific enolase.
OGD/RP and treatment OGD were performed on mature
cultures at d 10 using the previously described
methods[17,18]. Briefly, the cells exposed to glucose-free Earl's solution
[116.4 mmol/L NaCl, 5.4 mmol/L KCl, 1.8 mmol/L
CaCl2, 0.8 mmol/L MgSO4, 2.6 mmol/L
NaH2PO4, 26.2 mmol/L
NaHCO3, and 20.1 mmol/L HEPES (pH 7.4)] containing different
concentrations of Res (0.1, 1.0, and 10.0 µmol/L, respectively) or
solvent were cultured at 37 oC in an incubator bubbled in an
atmosphere of 5% CO2 and 95% N2
(OGD) for 2 h. Then the culture medium was changed into normal Earl's solution (with
the addition of 5.6 mmol/L glucose into the glucose-free
Earl's solution) bubbled in an atmosphere of 5%
CO2 and 95% O2 (RP) for another 12 h.
Apoptosis assay[19] Neuronal apoptosis was assayed by
flow cytometry with the Annexin V/FITC kit. In brief,
1×106 single cells per sample were collected after OGD for 2 h and
the following RP for 12 h and washed twice with the buffer;
Annexin V/FITC was then added. After incubation for 10
min at room temperature in the dark, the cells were washed
and resuspended, propidium iodide was then added to a
final concentration of 1 mg/L. The percentage of apoptosis
was detected and 1×104 cells were counted for each sample
by flow cytometry.
Morphological evaluation by electron microscope
Morphological observation was conducted in 4 groups: control
group, OGD/RP group, OGD/RP+Nim 1.0 µmol/L group and
OGD/RP+1.0 µmol/L Res group. The cortical neurons of the
newborn rats were incubated in a 6-well plate, in which glass
slides were placed in advance. After being treated with OGD
for 2 h and RP for 12 h according to the method previously
described, the glass slides full of growing neurons were
harvested from the 6-well plate, rinsed gently 3 times with cold
phosphate-buffered saline (PBS), fixed for 1 h at 4
oC with
2.5% glutaral, then rinsed twice with 0.1
mol/L PBS, each time lasting more than 30 min. Then they were fixed for 1 h with
1% osmic acid, rinsed twice with 0.1 mol/L
PBS, dehydrated with different concentrations (30%, 50%, 70%, 80%, 90%,
and 100%) alcohol, replaced with isoamyl acetate, dried with
CO2 at critical point, and sherardized in a vacuum. Finally,
the morphological changes of the neurons were observed
and photographed by transmission electron microscope
(JEOL, Tokyo, Japan).
Measurement of intracellular free calcium
concentration ([Ca2+]i)
The cultured cells were incubated with 0.125%
trypsin to obtain dispersed single cell suspension. The cell
suspension was then incubated with Fura-2/AM at a
concentration of 10 µmol/L in the dark for 50 min at 37
oC. After rinsing twice with Hank's solution containing bovine sera
albumin, the cells were resuspended in Hank's solution and
used to measure the [Ca2+]i with RF-5000 spectrometry. The
fluorescent dye was measured with an emission wavelength
of 500 nm and the maximum absorption at 340 nm when the
calcium bound. The concentration of the
[Ca2+]i was represented as
Kd×(F_Fmin
)/(Fmax_F)[20], where
Kd is 224 nmol/L (the
Kd value of Fura-2/AM),
F is the measured fluorescence intensity, while
Fmax and Fmin refers to the maximum and
minimum values at 340 nm after adding Triton X-100 (0.98 g/L)
and EGTA (5 mmol/L), respectively.
Real-time RT-PCR analysis The transcription of
caspases-3 and -12 were detected by real-time RT-PCR. The
total RNA of primary cultured neurons was isolated using
Trizol reagent (Invitrogen, Carlsbad, CA, USA) and purified
with the RNeasy mini kit (Qiagen, Palo Alto, CA, USA). RNA
was spectrophotometrically quantified by measuring the
optical density of samples at 260/280 nm, redissolved in
30_50 µL diethyl pyrocarbonate water (RNA concentration of
each samples was 50 ng/µL), and stored at -80
oC.
Primer preparations were devised according to the
sequence searched on GenBank. The nucleotide sequence of
the primers used in this experiment were as follows: (1)
caspase-3 (GenBank Access No NM_012922.2): sense
5'-CAG AGC TGG ACT GCG GTA TTG A-3', antisense 5'-AGC
ATG GCG CAA AGT GAC TG-3'; (2) caspase-12 (GenBank
Access No NM_130422.1): sense 5'-CTG GCC CTC ATC ATC
TGC AA-3', antisense 5'-TGG ACG GCC AGC AAA CTT-3';
and (3) β-actin (GenBank Access No V01217.1): sense
5'-TGA CAG GAT GCA GAA GGA GA-3', antisense 5'-TAG AGC
CAC CAA TCC ACA CA-3'. The predicted length of the
PCR product was 300 bp.
Total RNA was reverse transcribed with MuL V reverse
transcriptase and oligo (dT) primers. The SYBR green DNA
PCR kit (Applied Biosystems, Foster City, CA, USA) was
used for the real-time PCR analysis. The relative differences
in expression among the groups were expressed using cycle
time (Ct) values as follows: the
Ct values of the interested genes were first normalized with
β-actin of the same sample, and then the relative difference between the control and each
treatment group was calculated and expressed as a relative
increase, setting the control at 100%.
Statistical analysis All the data were expressed as
mean±SD. The data were analyzed statistically using the
SPSS 11.5 for Windows statistical program (SPSS, Chicago,
IL, USA) by one-way ANOVA or Student's t-test.
P<0.05 was considered statistically significant.
Results
Effects of Res on apoptosis Neuronal apoptosis was
assayed after OGD/RP injury. There was a very low
level (11.1%) of neuronal apoptosis under normal conditions. After
OGD/RP insult, the percentage of apoptosis significantly increased
by 49.1% (P<0.01). Similar to Nim treatment, the addition of
0.1, 1.0, and 10.0 µmol/L Res during OGD/RP markedly
reduced the percentage of cell apoptosis to 41.7%, 40.8%,
and 37.4%, respectively. The anti-apoptotic effect of 10.0
µmol/L Res was more significant than that of 1.0 µmol/L Nim
and 0.1 or 1.0 µmol/L Res (P<0.05). The vehicle had no effect
on cell apoptosis induced by OGD/RP (Figure 1A_B).
Transmission electron microscope observation
Electron microscope evidence showed that the nucleus of
normal cortical neurons were big, round, or oval. The
euchromatin distributed homogeneously, the structures of
intracytoplasmic mitochondria and rough endoplasmic reticulum
were clear, and the ribosomes were abounded (Figure 2A).
On the contrary, the nucleus of cortical neurons subjected
to OGD/RP were irregular, a chromatin mass formed, the
perinuclear space obviously became thicker, mitochondria
swelled and vacuolized, cristae arrangement became
disordered, mitochondria membranes were ruptured, and the
endocytoplasmic reticula were expanded. Some neurons were
even obviously broken to pieces and the membrane dissolved
(Figure 2B). However, pretreatment with 1.0 µmol/L Nim
during the course of OGD/RP insult could obviously relieve the
neuronal damage compared with those of OGD/RP alone.
Most of the nuclear chromatins distributed homogeneously,
the ultramicrostructure was similar to that of normal neurons
with swollen mitochondria mitigated, lamellar cristae in
mitochondria became clear, and the ribosomes became more
abundant (Figure 2C). Pretreatment with 1.0 µmol/L Res during
the course of OGD/RP insult relieved neuronal
morphological damage compared with those of OGD/RP alone, and the
ultramicrostructure was similar to that of the OGD/RP+1.0
µmol/L Nim group (Figure 2D).
Effects of resveratrol on
[Ca2+]i At the end of OGD/RP
insult, the significant elevation of
[Ca2+]i, which was nearly
3.5-fold of that under normal conditions, was observed in
the insulted neurons. Nim, a Ca2+ blocker used to relax the
cerebral vasculature, could to some extent depress the
elevation of [Ca2+]i in OGD/RP-insulted cells. Similarly, Res
also obviously inhibited the elevation of
[Ca2+]i induced by OGD/RP in a concentration-dependent manner. Consistent
with in the findings of the apoptosis experiment, the
inhibitory effect of 10.0 µmol/L Res on the elevation of
[Ca2+]i was more significant than that of 1.0
µmol/L Nim and 0.1 or 1.0 µmol/L Res (Table 1).
Real-time RT-PCR analysis of selected gene expression
The real-time RT-PCR analysis showed that OGD/RP insult
significantly caused an increase in the transcription of
caspases-3 and -12 in cultured neurons, which were
approximately 6-fold of that under normal conditions. The vehicle
did not affect the selected gene transcriptions. Similar to
that of the elevation of
[Ca2+]i, Nim could also blunt the
overexpression of the selected genes, and Res treatment also
obviously depressed the overexpression of caspases-3 and
-12 mRNA induced by OGD/RP in a
concentration-dependent manner. The depressing effect of 10.0
µmol/L Res was also more remarkable than that of 1.0
µmol/L Nim (Table 2).
Discussion
It has been reported that the neuroprotective effect of an
antioxidant in ischemic brain injury is involved in neuronal
apoptosis[1], and during early reperfusion, the apoptotic
mechanisms are engaged in vulnerable neurons, such as the
cortex and hippocampus[20]. Combined with our previous
findings that Res decreases lactate dehydrogenase leakage
and improves cell survival in the OGD/RP model of cultured
neonatal rat cortical neurons[13], we further studied the
protective effects of Res, including its anti-apoptotic effect on
the same model, which imitated ischemia-reperfusion
performance in the brain. We found that OGD/RP insult caused a
remarkable increase in the neuronal apoptotic percentage,
which was depressed by the addition of Res in a
concentration-dependent manner and elicited an obvious abnormal
neuronal morphological change, which was ameliorated by
the addition of Res. All the facts indicated that the
anti-apoptotic activity of Res contributed to the beneficial effect
on neuronal injury in this model.
Abnormalities of [Ca2+]i homeostasis, especially the
[Ca2+]i overload, have been linked to neuronal apoptosis induced
by ischemia-reperfusion[21]. The increasing
[Ca2+]i leads to the activation of many pivotal cellular processes that can
alter the cellular functions and even lead to cell injury and
death, including neuronal
apoptosis[22,23]. Our results showed that OGD/RP insult caused an elevated
[Ca2+]i in the subjected neurons, and pretreatment with Res inhibited cell
apoptosis concentration-dependently, which was consistent
with its anti-apoptotic effect in the same model. The results
suggest that the effect of Res on neuronal apoptosis may
be, at least partly, involved in its depressing activity in the
abnormal elevated [Ca2+]i of neurons. However, the
mechanism of the inhibitory effects of Res on
[Ca2+]i remains to be elucidated. It has been reported that Res inhibits
Ca2+ influx in isolated rat ventricle myocytes and thrombin-stimulated
human platelets by inhibiting L-type
Ca2+ channels or store-operated
Ca2+ channels[24,25], respectively, indicating the
possibility that Res may have a direct inhibitory effect on
[Ca2+]i in neurons by acting
Ca2+ channels. On the other hand, Res is well known as an antioxidant depressing the
production of reactive oxygen
species[26]. It has been established that the
Ca2+ overload can be elicited by oxidative
stress in neurons subjected to ischemia_reperfusion
injury[27]. The facts suggest another possibility: the depressing effect
of Res on the [Ca2+]i of neurons may indirectly originate from
its antioxidant activity in our model. We deduce that the
inhibitory effects of Res on Ca2+ overload originated from
both the direct acting on the Ca2+ channel and its antioxidant
activity.
It was interesting that the effects of Res (1.0
µmol/L) on apoptosis and the elevated
[Ca2+]i of neurons were equal to
that of Nim, a high-lipid solubility
Ca2+ blocker used to relax the cerebral
vasculature[28], at the same concentration; more
significant effects could be elicited by increasing the
concentration of Res. Furthermore, a higher concentration of
Res can be reached in the brain tissue after
administration[9]. This result seems to indicate the clinical potential of Res for
brain ischemic diseases.
The progress of apoptosis is regulated in an orderly way
by a series of signal cascades under certain circumstances.
An activation of caspases, the aspartate-specific cysteine
proteases and members of the interleukin-1 β-converting
enzyme family, is a critical step in neuronal
apoptosis[29]. Caspase-3 is one of several caspases that integrate various
cell death signals and initiate the cleavage of key cellular
substrates, such as gelsolin, fodrin, actin, focal adhesion
kinase, and poly adenosine diphosphate
ribose[30]. In the present study, the transcription of caspase-3 in neurons was
increased by OGD/RP insult, consistent with previous
reports that showed significant increases in the transcriptional
activity of caspase-3 gene during neuronal
apoptosis in vitro[31] and after traumatic brain injury
in vivo[32] or transient cerebral
ischemia[33] or brain ischemia_reperfusion
injury[34]. Caspase-12 plays a key role in many nervous system diseases,
including brain ischemia_reperfusion
injury[34]. The activated caspase-12 then activates procaspase-9, and the
activated caspase-9 in turn activates its downstream substrates,
including procaspase-3, to elicit cell
apoptosis[35]. Notably, our study also showed that OGD/RP insult could cause an
increase in the expression of caspase-12 mRNA to the same
extent of the overexpression in caspase-3 mRNA (about
6-fold), and pretreatment with Res could remarkably depress
the overexpression of caspases-3 and -12 mRNA in a
concentration-dependent manner, which were parallel with its
effects on the elevated
[Ca2+]i and the elevated apoptosis of
neurons induced by OGD/RP performance. The results
suggest that the protective effects of Res on neuronal apoptosis
induced by OGD/RP may be also related to its downregulation
of caspases-3 and -12 mRNA.
In conclusion, the results of the present study
demonstrated that Res could attenuate rat cortical neuronal
apopto-sis induced by OGD/RP. The mechanisms are, at least partly,
due to the inhibition of the calcium overload and the
over-expression of caspases-3 and -12 mRNA.
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