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Introduction
Parkinson's disease (PD) is a motor function disorder
caused primarily by the loss of the neurotransmitter
dopamine (DA) in the central nervous system (CNS) as a result of
selective degeneration of nigral dopaminergic
neurons[1-3]. It is generally believed that both genetic and environmental
factors play roles in PD, but the exact mechanisms of
neuronal death have not been fully understood. Several
scientific reports demonstrate that apoptosis of the neurons in
the substantia nigra (SN) plays an important role in the
development of PD. A number of studies suggest that
oxidative stress contributes to dopaminergic neuron degeneration.
In previous studies, we found that the transcription factor
NF-κB plays a pro-apoptotic role in the excitotoxin-induced
apoptotic death of neurons in the SN par compact (SNpc),
possibly through upregulating p53 and
c-Myc[4-6]. Increased NF-κB immunoreactivity has been reported in the SNpc of
patients with PD, which suggests that NF-κB plays a role in
oxidative stress-induced dopaminergic neuron degeneration
in PD[7].
Although various treatments are successfully used to
alleviate the symptoms of PD, none of them prevents or halts
the neurodegenerative process of the disease. Clinical
studies have shown that ginkgo extracts, which have been widely
used as a dietary supplement in the United States, exhibit
several beneficial effects in a variety of CNS disorders,
including Alzheimer's disease (AD) and
PD[8-11].
The active components of Ginkgo biloba are
considered to be the ginkgo flavone glycosides, the polycyclic
lactone ginkgolides A, B, C, J, and
bilobalide[12]. EGb 761, a standard extract of
Ginkgo biloba (EGb) with potent
antioxidant properties, shows great benefits on the CNS, being
able to enhance peripheral and cerebral
circulation[13], and protecting neurons against a variety of insults.
Several studies have reported that EGb exerts beneficial
effects in AD and PD models. A recent study indicated that
that administration of EGb 761 can reduce β-amyloid
oligomers and restore cAMP respond element binding protein
(CREB) phosphorylation in the hippocampus of a transgenic
mouse model of AD[14]. EGb pretreatment is able to reverse
β-amyloid-peptide-induced isoprostane production in the rat
brain in vitro[15] and inhibit cerebral monoamine oxidase
(MAO) activity in vivo[16]. Wu and
Zhu[17] demonstrated that EGb could attenuate
1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced nigrostriatal
dopaminergic neurotoxicity in C57 mice. Another study suggested
that EGb offers dose-dependent protection against
6-hydroxydopamine (6-OHDA)-induced parkinsonism in rats.
The pretreatment of rats with EGb leads to a restoration of
compromised behavioral activity, levels of tyrosine
hydroxylase (TH), and neurotransmitter
DA[18]. Cao et
al[19] found that the combined use of
EGb with levodopa reduces the toxic effects of levodopa, and thus may be a therapeutic
strategy in the management of motor function in PD.
Bilobalide, a sesquiterpene lactone, which constitutes of
approximately 3% EGb 761, is quantitatively the major single
chemical constituent of EGb 761. The first pharmacological
action detected was a beneficial effect on cytotoxic brain
edema caused by triethyltin[20]. Bilobalide has now been
demonstrated to inhibit delayed ischemic neuronal death and
reduce infarct volume after focal cerebral ischemia and
ischemia-induced neuronal damage in
rodents[21-24]. Zhou et
al[25] demonstrated that bilobalide could protect against
β-amyloid toxicity and reactive oxygen species (ROS)-induced
apoptosis in PC12 cells. It has also been demonstrated that
bilobalide has potent inhibitory actions on the
N-methyl-D-aspartate-induced activation of phospholipase
A2 and the associated phospholipid breakdown in the
brain[26].
These studies suggest that bilobalide is a main active
component in the neuroprotective actions of
EGb, but it has not been reported whether bilobalide has a protective effect
on the neurodegeneration in PD or not. In the present study,
we explored the neuron protective effects of bilobalide in a
rat model of PD induced by 6-OHDA. We report that neuron
pathology and behavioral changes can be restored
effectively by pretreatment with bilobalide, suggesting that
bilobalide may be a candidate to alleviate the
Parkinson-related pathology.
Materials and methods
Animals Male Sprague_Dawley rats were obtained from
the Center for Experimental Animals, Soochow University
(Suzhou, China), and weighed 250_280 g at the start of the
experiment. The rats were housed under standardized
light/dark cycle (12 h) conditions with access to food and water
ad libitum. All procedures were performed in accordance
with the NIH Guidelines for the Care and Use of Laboratory
Animals.
Treatment with bilobalide Bilobalide was provided by
Zhong-liang CHEN (Department of Phytochemistry,
Shanghai Institute of Materia Medica, Chinese Academy of
Sciences, Shanghai, China), and the purity of this compound
was 98% (HPLC). Bilobalide was dissolved in DMSO and
then diluted with 0.9% sodium chloride. The final
concentration of DMSO was 1% (v/v). Bilobalide was administered
by intraperitoneal injection (ip) to rats once daily at doses of
5, 10, and 20
mg·kg_1·d_1 for 7 successive days, respectively.
PD model induced by 6-OHDA After 7 d of bilobalide
treatment, the rats were anesthetized with 400
mg/kg chloral hydrate (ip), and then placed on a stereotaxic instrument
(Stoelting, Wood Dale, IL, USA). A hole was drilled through
the skull to the top of the dura, and a 29 gauge stainless steel
needle was lowered to the left SNpc (coordinates: _5.2 mm
from bregma, 2.1 mm from midline, 7.8 mm from surface). A
solution of 6-OHDA with 0.1% ascorbic acid_saline (Sigma,
St Louis, MO, USA) was dissolved in 0.9% NaCl
(w/v) to a concentration of 4 µg/µL, and then infused unilaterally into
the left SNpc by a microinfusion pump delivering 2 µL (8 µg)
over a 5 min period. After infusion, the needle was kept in
place for 5 min to ensure diffusion. Sham-operated animals
received an equal volume of 0.9% NaCl delivered by the same
method.
Animal grouping 1 The rats were divided into 6 groups,
each consisting of 10 animals. Group 1 included
sham-operated rats pretreated with vehicle (S); group 2 included
sham-operated rats pretreated with 20 mg/kg
bilobalide (S+H_BB); group 3 included PD model rats pretreated with vehicle (M);
group 4 included PD model rats pretreated with 5
mg/kg bilobalide (M+L_BB); group 5 included PD model rats
pretreated with 10 mg/kg bilobalide (M+M_BB); and group 6
included PD model rats pretreated with 20 mg/kg bilobalide
(M+H_BB). The animals were used to evaluate the effect of
pretreatment with bilobalide on the behavioral changes and
neuron survival in 6-OHDA-lesioned rats.
Animal grouping 2 The rats were divided into 3 groups,
each consisting of 10 animals. Group 1 included
sham-operated rats pretreated with vehicle (control); group 2 included
PD model rats pretreated with vehicle (6-OHDA); and group
3 included PD model rats pretreated with 10 mg/kg bilobalide
(6-OHDA+M_BB). These animals were used to explore the
mechanism involved in the effects of bilobalide in
vivo.
Behavior studies On d 14 after a stereotaxic injection of
6-OHDA, the motor activity of the animals was tested for
locomotor activity in a computerized animal activity video
analyzer (Shanghai Jiliang Software Technology, Shanghai,
China). Each rat was placed in the chamber and its
locomotor activity was monitored by the activating camera and
viewed on the screen. The activities of the animals at 5 min
periods were recorded, and the data of the locomotion time
were collected by individuals who were trained in behavioral
observation.
On d 14 and 21 after infusion of 6-OHDA, the animals
were subject to rotational behavior
testing[27]. The rats were administered R-(2)-apomorphine hydrochloride (0.5 mg/kg
in 0.1% ascorbic acid_saline, subcutaneously) and placed in
a transparent cylindrical cage. Contralateral rotations (360°,
in short axis) over a 30 min interval from the initiation of
rotation were recorded.
Immunofluorescence of the brain sections
Twenty four days after the infusion of 6-OHDA, the rats were
anaesthetized and then perfused transcardially with 200 mL precooled
0.01 mol/L phosphate-buffered saline (PBS; pH 7.4), followed
by 200 mL perfusate containing 4% paraformaldehyde in 0.1
mol/L phosphate buffer (PB, pH 7.4). The brains were
postfixed overnight in the same paraformaldehyde fixative
and then transferred to 20% sucrose solution until they sank
to the bottom of the containers. The brains were snap frozen
and sectioned at 30 mm thickness with a cryostat (Leica
Microsystems GmbH, Wetzlar, Germany). Free-floating
sections were washed in 0.01 mol/L PBS 3 times for 10 min each
and incubated in PBS with 0.1% Triton X-100 for 1 h at room
temperature (RT).The brain sections were then blocked with
1% bovine serum albumin (BSA) for 1 h at RT and then
incubated with mouse monoclonal antibody TH (1:3000; Sigma,
USA), a primary antibody recognizing TH, in PBS
containing 0.1% Triton X-100 at 4 °C for 48 h. Sections were
subsequently rinsed in PBS and incubated for 1 h with the
secondary antibody (Cy3-conjugated donkey antimouse
immunoglobulin G [IgG]; Jackson ImmunoResearch, West Grove, PA,
USA). Sections were mounted on glass slides, coverslipped
with antifade mountant, and then observed with a
fluorescence microscope (Nikon, Tokyo, Japan).
Nissl's staining The brain sections were stained with
0.75% cresyl violet, dehydrated twice through graded alcohols
(70%, 95%, and 100%), cleared in xylenes 3 times for 5 min
each, coverslipped with resinous mountant, and then
observed with a light microscope.
Double immunofluorescence Approximately 6_24 h
after the 6-OHDA infusion, the rats were perfused, and the
brains were then frozen and sectioned according to the
protocol described earlier. The brain sections were rinsed with
PBS, incubated in PBS with 0.1% Triton X-100, blocked with
1% BSA in PBS, and then incubated with primary and
secondary antibodies sequentially. To examine if the activation
of NF-κB p65 occurs in nigral neurons, the brain sections
were incubated with a mouse monoclonal antibody against
TH (1:3000) and rabbit polyclonal antibody against
NF-κB p65 (1:500; Chemicon, Temecula, CA, USA), and then
incubated with the secondary antibodies
(fluorescein-isothiocyanate [FITC]-conjugated donkey antimouse IgG,
1:1000 and Cy3-conjugated donkey antirabbit IgG, 1:1000).
Sections were washed in PBS, mounted on glass slides,
coverslipped, and then examined with a laser confocal
system (Leica Microsystems GmbH, Wetzlar, Germany).
Terminal deoxynucleotidyl transferase-mediated dUTP
nick-end labeling The low molecular weight DNA fragments
as well as the single strand breaks ("nicks") in high
molecular weight DNA can be identified by labeling free 3'-OH
termini with modified nucleotides by an enzymatic reaction with
terminal deoxynucleotidyl transferase, which catalyzes
polymerization of nucleotides to free 3'-OH DNA ends in a
template-independent manner. This method labels these
hydroxyl groups with fluorescein-conjugated deoxynucleotides.
Fluorescein generates an intense signal that can be detected
by a fluorescence microscope.
Twenty four hours after the 6-OHDA infusion, the rat
brain sections were prepared according to the protocol
described earlier. The brain sections were incubated with a
mouse monoclonal antibody against TH, and reacted with a
secondary antibody (Cy3-conjugated donkey antimouse IgG).
Then the brain sections were mounted on polylysine-coated
glass slides. DNA damage was detected using a Fluorescein
FragEL DNA fragmentation detection kit (Calbiochem, San
Diego, CA, USA) according to the protocol of the manufacturer.
The sections were then washed in PBS, counterstained with
4',6'-diamidino-2-phenylindole dihydrochloride
(DAPI), and coverslipped. To determine the relationship between
NF-κB p65 and DNA damage, the brain sections were incubated
with rabbit polyclonal antibodies against NF-κB p65, and
subsequently reacted with secondary antibodies
(Cy3-conjugated donkey antirabbit IgG). DNA damage was then
detected as described earlier. Slides were observed with a laser
confocal system.
Cell counting Staining with a TH antibody can delineate
the SNpc in coronal sections. The sections were
immunofluorescent labeled for TH. Stained cells were counted within
the outlines, and total estimates were obtained. Labeled
profiles were counted only if the first recognizable profile of
the cell soma came into focus within the counting
frame[28]. Using every coronal section, the analysis was performed
starting with the first appearance of TH-positive neurons,
extending to the most caudal parts of the SNpc and
including both hemispheres. Sections were viewed under an
inverted fluorescence microscope. Each group included 6 rats,
and 6 brain sections from each rat were counted.
An estimation of the percentages of TH-positive
neurons having undergone recombination with NF-κB p65 was
obtained by montage images (FITC and Cy3) of the left SNpc
under a laser confocal system. The total number of
TH-positive neurons and total number of TH/NF-κB
double-positive neurons were counted for each section. The
number of double-labeled neurons divided by the total number
of TH-positive neurons was the percentage of recombination,
which showed the activation of NF-κB in each group. Each
group included 6 rats, and 6 brain sections from each rat
were counted.
Statistical analysis Data are presented as mean±SEM.
One-way ANOVA or the Student's unpaired two-tailed test
was used for the statistical analysis. Statistical significance
was set at P<0.05.
Results
Bilobalide restored 6-OHDA-induced impairment on
motor activity In the PD model group, the motor activity was
reduced significantly as compared to the sham-operated
group (S). The time spent on locomotion was significantly
decreased (76.9%). Different doses of bilobalide (L_BB,
M_BB, and H_BB) remarkably restored the locomotion time
(23.2%, 45.8%, and 58.0%) as compared to the PD model group (M)
respectively (P<0.05; Table 1). However, no significant
effects on motor activity were observed in the sham-operated
group treated with 20 mg/kg bilobalide as compared to the
sham-operated group (S).
Bilobalide inhibited apomorphine-induced circling
behavior The sham-operated animals failed to exhibit rotational
behavior upon apomorphine challenge. The PD model rats
exhibited contralateral rotations after the administration of
apomorphine (430±40 turns/30 min). Pretreatment with
different doses of bilobalide significantly reduced contralateral
rotations by a dose-dependent manner in the M+L_BB,
M+M_BB, and M+H_BB groups, as compared to the PD model rats
(P<0.05; Table 2). Similar inhibitory effects of
bilobalide on rotational behavior were observed at 2 and 3
weeks after 6-OHDA damage.
Bilobalide reduced 6-OHDA-induced loss of
dopaminergic neurons The loss of dopaminergic neurons in the SNpc
was examined with TH immunofluorescence and Nissl's
staining after 6-OHDA treatment. The infusion of 6-OHDA caused
a rapid and consistent loss of TH immunoreactivity in the
SNpc. By 24 d after 6-OHDA administration, a significant
loss of TH immunoreactivity was observed. Pretreatment
with bilobalide significantly reduced the 6-OHDA-induced
loss of TH-positive neurons in the SNpc (Figure 1).
We extended our observations with Nissl's staining 24 d
after 6-OHDA treatment. The results showed a remarkable
loss of Nissl's body in the 6-OHDA-lesioned SNpc, and
bilobalide substantially recovered the loss of nigral neurons
(Figure 2).
Bilobalide blocked 6-OHDA-induced activation of
NF-κB NF-κB is known as an important transcriptional factor,
playing a central role in the regulation of many immune and
inflammatory responses, as well as the control of cell
apoptosis. Recent evidence demonstrated the activation of
NF-κB in neuronal cells during neurodegenerative processes.
The activation of NF-κB was examined in
situ using immunofluorescence. The nuclear translocation of p65, a
family member of NF-κB, was detected with
immunofluorescence in dopaminergic neurons 24 h after 6-OHDA injection.
The results showed that 6-OHDA induced a higher
expression of NF-κB p65 in the nuclei of TH-positive neurons.
Pretreatment with bilobalide at a dose of 10 mg or higher
effectively blocked 6-OHDA-induced NF-κB p65 nuclear
translocation as revealed by immunofluorescence (Figure
3A). A quantitative analysis showed that the percentages of
NF-κB p65-positive dopaminergic neurons significantly
decreased from 37.5%±2.6% to 18.6%±1.7% by pretreatment
with bilobalide (P<0.05; Figure 3B).
Bilobalide reduced the numbers of terminal
deoxynucleotidyl transferase-mediated dUTP nick-end labeling-positive
nuclei in the SNpc There were few terminal deoxynucleotidyl
transferase-mediated dUTP nick-end labeling
(TUNEL)-positive nuclei in the contralateral SN of PD model animals or in
the ipsilateral SN of sham-operated animals. Increased
numbers of TUNEL-positive nuclei were observed 24 h after
6-OHDA infusion, which revealed apoptosis in the SNpc
(Figure 4A). A quantitative analysis showed that the
percentages of apoptosis in TH-positive neurons increased from
2.8%±0.4% to 55.7%±1.9% (P<0.05; Figure 4B). We detected
the co-expression of TUNEL and NF-κB p65 nuclear
translocation by double immunofluorescence. Elevated
NF-κB p65 nuclear translocation and TUNEL-positive nuclei were
observed 24 h after 6-OHDA damage (Figure 5). TUNEL and
p65 staining were colocalized in the nuclei of the SNpc
neurons. We found that pretreatment with bilobalide produced a
significant decrease of NF-κB p65 and TUNEL-positive nuclear
staining.
Discussion
A number of reports indicate that EGb has wide
pharmacological actions, such as
anti-anemia[29],
anti-edema[30],
anti-inflammation[31],
anti-hypoxia[32] and anti-oxidative
stress[33]. Wettstein et
al[34] reported that EGb should be considered
equally effective with second-generation cholinesterase
inhibitors in the treatment of mild to moderate Alzheimer's
dementia. Chandrasekaran et
al[22] reported that bilobalide exhibits protection against delayed ischemic neuronal death
similar to that observed with the administration of ginkgo
extracts. Consistent with this finding, Mdzinarishvili
et al[35] also demonstrated that bilobalide can prevent
ischemia-induced edema formation in vitro and
in vivo. Bilobalide could be an important active constituent of the extract. We
attempted to find out whether bilobalide is able to inhibit
neuronal necrosis and apoptosis induced by 6-OHDA in the
SNpc of rats. Our study demonstrates that bilobalide can
restore the locomotion, inhibit rotational behavior, and
reduce loss of dopaminergic neurons, suggesting that
bilobalide has neuroprotective effects and is able to improve
the pathological symptoms in PD model rats.
At present, the mechanisms by which bilobalide protects
neurons remain to be determined. In vitro and
in vivo studies indicate that bilobalide has multiple actions that may be
associated with neuroprotection, including preservation of
mitochondrial ATP synthesis[36, 37], inhibition of apoptotic
damage induced by staurosporine or by serum-free
media[38], suppression of hypoxia-induced membrane deterioration in
the brain[39], and regulation of mitochondrial gene
expression[23]. Zhou and
Zhu[25] demonstrated that bilobalide could
attenuate ROS-induced apoptosis in PC12 cell lines,
suggesting that bilobalide might be acting as a free-radical
scavenger.
NF-κB appeared to be elevated in the SN of the
post-mortem brains of PD cases[40,41]. The relationship of
NF-κB activation to the disease process, however, is unclear.
Several in vitro studies have reported the activation of
NF-κB in response to 6-OHDA
treatment[42]. The intrastriatal
administration of dopamine also produced oxidative damage to
striatal neurons and a robust activation of
NF-κB[43], but evaluations of the role of
NF-κB in in vitro PD models have not yielded consistent
results[44,45]. Some studies reported that
the neuroprotective effect offered by some pharmacological
agents is associated with the blockade of NF-κB activation,
suggesting that NF-κB plays a pro-apoptotic role in
PD[46_48]. In our previous study, 6-OHDA induced an increase in the
binding activity of NF-κB, which provided the first
biochemical evidence of the activation of NF-κB in animal models of
PD.
In this study, 6-OHDA-induced apoptosis of
dopaminergic neurons was accompanied by NF-κB activation. It was
implied that the activation of NF-κB p65 contributed to the
apoptosis of nigral neurons. A decrease in total cell
numbers and increase in apoptotic cells induced by 6-OHDA
were significantly attenuated by bilobalide pretreatment.
NF-κB activation and an increase in DNA fragmentation induced
by 6-OHDA were also significantly inhibited. Bilobalide may
block apoptosis of dopaminergic neurons through the
suppression of the expression of the NF-κB p65 protein and
decrease its nuclear translocation in the SNpc of rats.
In summary, the present study shows that apoptosis is
involved in 6-OHDA-induced dopaminergic neuronal death,
and that the activation of NF-κB plays an important role in
apoptosis. Pretreatment with bilobalide effectively
prevents the activation of NF-κB and produces a marked protective
effect on dopaminergic neurons against the toxicity induced
by 6-OHDA in the SNpc. Bilobalide may thereby provide a
therapeutic approach to rescue the dopaminergic neurons in
the process of PD.
Acknowledgment
Bilobalide was provided by Prof Zhong-liang CHEN from
the Department of Phytochemistry, Shanghai Institute of
Materia Medica, Chinese Academy of Sciences (Shanghai,
China).
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