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Journal of Molecular Neuroscience
ISSN 0895-8696
J Mol Neurosci
DOI 10.1007/s12031-012-9727-3
Curcumin has Neuroprotection Effect on
Homocysteine Rat Model of Parkinson
Zahra Mansouri, Masoumeh
Sabetkasaei, Fatemeh Moradi, Fatemeh
Masoudnia & Amin Ataie
1 23
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Curcumin has Neuroprotection Effect on Homocysteine
Rat Model of Parkinson
Zahra Mansouri &Masoumeh Sabetkasaei &
Fatemeh Moradi &Fatemeh Masoudnia &Amin Ataie
Received: 25 January 2012 /Accepted: 13 February 2012
#Springer Science+Business Media, LLC 2012
Abstract Parkinson’s disease (PD) is a progressive neuro-
logical disorder which is emanated by dopaminergic death
cell and depletion. Curcumin as a nontoxic matter has anti-
oxidant, anti-inflammatory, and antiproliferative activities,
and it involves antioxidant property same to vitamins C and
E. In this study, we investigated the neuroprotective properties
of the natural polyphenolic antioxidant compound, curcumin,
against homocysteine (Hcy) neurotoxicity. Curcumin
(50 mg/kg) was injected intraperitoneally (i.p.) once daily
for a period of 10 days beginning 5 days prior to Hcy
(2 μmol/μl) intracerebroventricular (i.c.v.) injection in rats.
The studies included immunohistological and locomotor
activity tests. These results suggest that homocysteine intra-
cerebroventricular administration (2 μmol/μl i.c.v.) may
induce changes in rat brain, and subsequently, polyphenol
treatment curcumin 50 mg/kg (i.p.) was capable in improving
locomotor function in insulted animal by protecting the ner-
vous system against homocysteine toxicity.
Keywords Curcumin .Homocysteine .
Intracerebroventricular .Rat .Locomotor activity .Stress
oxidative
Introduction
Parkinson’s disease (PD) is the second most common neu-
rodegenerative disorder after Alzheimer’s disease (AD)
(Hoehn 1987) with high prevalence in the world. Also, it
is progressive and posed patient in a debilitating condition
as well as includes extensive dopaminergic neuron degen-
eration in the substantia nigra pars compacta (Lotharius and
Brundin 2002) and the other subcortical nuclei with motor
and nonmotor symptoms. Motor symptoms are discriminated
by hypokinesia, rigidity, tremor, and postural imbalance
(Gomez-Tortosa et al. 1999), and nonmotor symptoms include
autonomic dysfunction, neuropsychiatric problems, and sen-
sory and sleep difficulties, which are common. Homocysteine
is considered a risk factor for multiple neurological disorders
including AD and PD (Gottfries et al. 1998; Mattson and Shea
2003; Reutens and Sachdev 2002).
Homocysteine (Hcy), a sulfur-containing amino acid
derived from the metabolism of methionine, is a risk factor
for vascular disease as well as brain atrophy. There is a tight
correlation between Hcy and cognitive impairment by epi-
demiological and longitudinal studies and probably is due to
cerebrovascular as well as neurotoxic mechanisms. Further-
more, it has been suggested that the involved pathological
mechanisms are apoptosis, neuronal death, oxidative stress,
overactivation of glutamate receptors, mitochondrial dys-
functions, and activation of caspases for all of neurodegen-
erative diseases (Mattson and Duan 1999; Mattson et al.
1999; Su et al. 1994). In spite of many research in this area,
the molecular mechanism of homocysteine-induced neuro-
toxicity has not been completely established at present.
Homocysteine can convey multiple neuropathological
effects, including cytosolic accumulation of calcium, induc-
tion of oxidative stress, apoptosis, and NMDA-mediated
excitotoxicity (Kim and Pae 1996;Krumanetal.2000;
Ho et al. 2002).
Z. Mansouri :F. Moradi :F. Masoudnia
Neuroscience Research Center,
Shahid Beheshti University of Medical Science,
Tehran, Iran
M. Sabetkasaei (*)
Department of Pharmacology, Neuroscience Research Center,
Shahid Beheshti University of Medical Science,
Tehran, Iran
e-mail: fkasaei@yahoo.com
A. Ataie
Department of Biology, Mazandaran University,
Babolsar, Iran
J Mol Neurosci
DOI 10.1007/s12031-012-9727-3
Author's personal copy
Apoptosis as an important physiological event causes cell
death with well-regulated activation and synthetic procedure
(Oppenheim 1991). Also, it can be severed by nonphysio-
logical agents, toxic substances, and drugs. The age of the
apoptotic cell has been diagnosed by morphological and
biological changes, such as cellular shrinkage, membrane
blebbing, chromatin condensation, and fragmentation of
chromatin DNA, with the small fragments moving into
membrane-bound vesicles. The apoptosis mechanism is
not fully understood; it is unequivocal that apoptosis is
resulted from the endogenous expression or posttranslation-
al activation of a set of proteins that are involved in an
intracellular signaling cascade. The increase in rate of apo-
ptosis has been reported in neurodegenerative disorders,
such as amyotrophic lateral sclerosis, PD, Alzheimer’s dis-
ease, and Huntington’s disease (Maler et al. 2003).
Polyphenols are natural substances that are extracted from
plants, fruits, and vegetables, including olive oil, red wine, and
tea (Ramassamy 2006). Recently, increasing interests have
been focused on identifying dietary compounds that can in-
hibit, retard, or reverse the multistage pathophysiological
events in PD pathology. The yellow pigment extracted from
the rhizome of Curcuma longa, curcumin, a polyphenolic
nonflavanoid compound, is the pharmacologically active sub-
stance of turmeric (Ganguli et al. 2000). Curcumin is nontoxic
and has antioxidant, anti-inflammatory, antiproliferative, and
antiapoptotic properties and activities (Aggarwal and Sung
2009). Curcumin is a putative therapeutic agent in the treat-
ment of neurodegenerative disorders such as PD (Jagatha et al.
2008;Kanthasamyetal.2005; Rajeswari and Sabesan 2008;
Zbarsky et al. 2005) and AD (Ma et al. 2009;Thomasetal.
2009). However, the mechanism underlying the neuroprotec-
tive effect of curcumin is far from clear.
Also, another possible supportive mechanism for this
substance is a scavenger of reactive oxygen species (ROS)
in insulted brain. Since the generation of ROS has been
correlated with the onset of PD, antioxidants may have a
therapeutic value, and we have supposed that in the curcu-
min therapy in this study. Therefore, the present study
investigated the neuroprotective effect of curcumin intra-
peritoneally (i.p.) against Hcy toxicity using behavioral
studies, as well as immunohistochemistry analysis.
Materials and Methods
Animals
Adult male Wister rats were purchased from Pasteur Institute
(Tehran, Iran) weighing between 250 and 300 g. The animals
were housed at 22°C in a controlled environment with a 12:12-
h light/dark cycle and were given access to standard laboratory
food and water ad libitum. All experiments were carried out in
accordance with the National Institutes of Health guidelines13
and were approved by the Research and Ethics Committee of
Shahid Beheshti Medical Science University (Tehran). We
used animals groups with eight animals per group.
Animals of the control group did not receive any injection.
In the vehicle group, vehicle of Hcy (phosphate-buffered
saline (PBS)) was injected intracerebroventricularly (i.c.v.),
and vehicle of curcumin (ethyl oleate) was injected (i.p.) for
10 days beginning 5 days prior to PBS injection. In the Hcy
group, Hcy (2 μmol/μl) was injected (intracerebroventricu-
lary), and the vehicle of curcumin (ethyl oleate) was injected
(i.p.) for 10 days beginning 5 days prior to Hcy injection. In
the Hcy-curcumin (Cur) group, curcumin was injected (i.p.)
for 10 days beginning 5 days prior to Hcy injection (i.c.v.). In
the curcumin group, curcumin (50 mg/kg) was injected during
10 days (i.p.), and the vehicle of homocysteine (PBS) was
injected (i.c.v.). Immunohistochemical and behavioral anal-
yses were performed 24 h after the last injection of
curcumin in the experimental groups.
Cannulation of Rat
For i.c.v. drug administration, rats were anesthetized using
ketamine (10 mg/kg) and placed in a stereotaxic apparatus.
Permanent 23 gauge stainless steel guide cannulae were
positioned in the lateral ventricle based on stereotaxic coor-
dinates taken from Paxinos and Watson atlas of rat brain
(Paxinos and Watson 2005) which were as follows: 1 mm
posterior to the bregma, 1.6 mm lateral to midline, and
3.6 mm ventral to the surface of the skull. The cannula
was fixed using dental cement, and two stainless steel
screws were placed into the skull. Rats were allowed 1 week
postsurgery to recover before performing the experiment.
Drugs were injected into the lateral ventricle 5 mm from the
surface of the cranium through a polyethylene tube (PE-20)
which was attached to a 10-μl Hamilton syringe.
Drugs
D-L-Homocysteine and curcumin were purchased from Sigma-
Aldrich, Germany. Ketamine and xylazine were obtained from
ALFASAN Co., Netherland. Hcy powder was dissolved in
hydrochloric acid (1 M) and diluted with PBS (Sigma-
Aldrich). The pH of the solution was regulated at 7.4 by the
addition of 0.1 N NaOH. Solutions of Hcy were prepared
freshly at a concentration of 2 μmol. The Hcy effective dose
(2 μmol/μl) was obtained from Lee et al. (2005).
The yellow powder of curcumin was dissolved in abso-
lute ethanol and stored as a stock solution (1%) at −20°C.
For injection, it was diluted with ethyl oleate (vehicle).
Curcumin dosage was selected on the basis of earlier reports
of its antioxidant effects (Sumanont et al. 2007; Shen et al.
J Mol Neurosci
Author's personal copy
2007; Ataie et al.). Curcumin (50 mg/kg) was injected
(intraperitoneally) once daily for a period of 10 days begin-
ning 5 days prior to Hcy (2 μmol/μl) (intracerebroventricu-
lar) injection in rats. We use four groups with eight animals
per group. Animals of the control group did not receive any
injection. In Hcy group, Hcy (2 μmol/μl) was injected intra-
cerebroventricularly. In Cur group, Cur (50 mg/kg) was
injected intraperitoneally. Behavioral and histochemical
analyses were performed 24 h after the last injection of
curcumin in the experimental groups.
Drug Administration
The injection needle was attached to a Hamilton syringe
through a polyethylene tube (PE-20). Homocysteine
(2 μmol/rat) or vehicle was injected into the lateral ventricle
in a volume of 1 μl/rat with a rate of 1 μl/min. Control group
did not receive anything. The injection needle was retained
in the guide cannula for an additional 1 min after injection to
facilitate diffusion of the drugs. The i.c.v. injections of the
drugs were made once daily.
Measurement of Locomotor Activities
The effects of Hcy alone or in combination with curcumin
on the rats’behavior were studied by an open-field appara-
tus. All experiments were carried out 24 h after the last
curcumin injections which began in 5 days following Hcy
(intracerebroventricular) injection in rats. The measurement
was started 3 min after the placement of animals into the
monitor in a quiet isolated place with a dim light. After the
rats were injected (i.c.v.) with homocysteine once a day, rats
were placed in the locomotor activity monitor (Ethovision-
XT; Noldus, Netherland). The changes in motor activity of
the animals were measured. Total distance and velocity were
determined. The locomotor activities were determined for
40 min postinjection (Lee et al. 2005).
Brain Histopathological Analysis
At the end of the behavioral experiments, the rats were deeply
anesthetized with a high dose of ketamine (150 mg/kg) and
perfused through the ascending aorta with 50–100 ml of 0.9%
saline followed by 100–200 ml of fixative solution containing
4% paraformaldehyde in 0.1 M phosphate buffer (PB, pH 7.4)
followed by 100 ml of 0.1 MPB containing 10% sucrose.
Following perfusion, the brains were removed from the skull;
the blocks of forebrain and brainstem were prepared, and after
final steps of preparation (30% sucrose for 2–3days),sections
were cut at a thickness of 50 μm on a freezing microtome
(Leica, Germany) and collected in PB (0.1 M). Every second,
a section was Nissl stained with 0.1% Bcl-2 (Sigma). The
tissue sections were deparaffinized in xylene. The slides were
stained with 0.1% cresyl violet according to the procedure in
Wilson and Gamble (2002) and viewed under a light micro-
scope (Labomed, USA) for the structure and morphology of
the cells. Microscopic images were obtained by a CCD
camera and DigiPro software. The cells also were counted in
different regions of substantia nigra (SN). The results are
represented as cell count per millimeter tissue.
Immunohistochemistry of Bax, Bcl-2, and TUNEL
Immunohistochemistry for Bax and Bcl-2 was carried out
on formalin-fixed, paraffin-embedded sections according to
the manufacturer’s instructions provided for each antibody.
Sections were deparaffinized and rehydrated. Antigen
retrieval was executed by microwaving in citrate buffer
(pH 6) for 1 and 2×5 min for Bax and Bcl-2, respectively.
The sections were quenched with 3% hydrogen peroxide
(H
2
O
2
) in absolute methanol and blocked with 10% normal
goat serum (NGS) + 1% bovine serum albumin in PBS for
Bax and Bcl-2 and with 5% NGS. Primary antibodies were
applied overnight at 4°C. These were either Bax rabbit
polyclonal antibody (abcam, 1/100) or Bcl-2 rabbit poly-
clonal antibody (abcam, 1/100. The sections were washed and
Control
Hcy
Hcy-Cur
Cur
0
2000
4000
6000
**
##
#
Total Distance(cm)
Control
Hcy
Hcy-Cur
Cur
0
2
4
6
8
#
#
**
#
Velocity(cm/s)
Fig. 1 Effect of curcumin (50 mg/kg) on locomotor activity (total
distance and velocity) after Hcy (2 μmol/μl) intracerebroventricular
injection in rats. Values are expressed as means ± S.E.M. (N08). One-
way ANOVA followed by Tukey’s Test was used for statistical analysis to
compare control group. **p<0.01 indicates significant decrease com-
pared to the control;
#
p<0.05,
##
p<0.01 different from Hcy groups
J Mol Neurosci
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then incubated with a ready-to-use antirabbit secondary anti-
body from Dako (EnVision Plus®), and color reaction was
developed using diaminobenzidine as the chromagen. The
slides were then counterstained with hematoxylin, dehydrated
using graded alcohols and xylene, and mounted with Per-
mount mounting medium (Entellan®, MERCK). Sections
used as negative controls were incubated with the primary
antibody diluents and PBS, instead of the primary antiserum.
Terminal deoxynucleotidyl transferase dUTP nick end
labeling (TUNEL) was performed on 5-μm thick paraffin-
embedded sections using the In Situ Cell Death Detection
Kit, POD (Roche Applied Science, Germany). Tissue sections
were deparaffinized in xylene, rehydrated, and immersed in
3% hydrogen peroxide to block the endogenous peroxidase
activity. After rinsing with PBS, sections were treated with
proteinase K solution at 37°C for 30 min to enhance the
staining, incubated for 60 min at 37°C with 50 μl of TUNEL
reaction mixture, and then incubated for 30 min at 37°C with
50 μl of converter-POD. Sections were rinsed in PBS, then
incubated for 10 min at 15–25°C with 50 μl of diaminobenzi-
dine substrate solution and rinsed again with PBS. Counter-
staining was achieved with 0.5% methyl green. For the
positive control, the sections were incubated in DNase1 solu-
tion at 15–25°C for 10 min, and for the negative control,
enzyme solution was omitted. Finally, the sections were dehy-
drated again, coverslipped as described above, and analyzed
under light microscopy. We perpetrated pictures of the histo-
pathological changes by light microscopy.
Neuronal Counting
For each animal, mesencephalic sections (interaural 2.9–
4.2 mm) were examined by a method as described by
Roghani and Behzadi (2001). Briefly, Nissl-stained neurons
of the substantia nigra pars compacta (SNC) were counted
manually (light microscopy, ×400) using a superimposed
grid to facilitate the procedure. At least two sections repre-
sentative of each of the four Paxinos–Watson planes (4.2,
3.7, 3.2, 2.97, interaural) were examined by scanning the
entire extent on each side. Counting was done blind to the
treatments received. The degree of immunostaining for Bax
and Bcl-2 was scored for five high-power field (×40 objec-
tive) per section, and the average score was calculated for
each section (Watson et al. 2001). Scoring was accom-
plished considering the presence of a section as the negative
control on each individual slide.
Statistics
The Bax, Bcl-2, the ratio of Bax to Bcl-2 immunostaining,
and Nissl-stained neurons of the SNC were determined at
each control, curcumin, homocysteine, and curcumin-
homocysteine groups by using the mean scales of Bax and
Bcl-2 in the same animal. All of the data are presented as
means ± standard error of the mean (S.E.M.) and analyzed
by ttests or one-way analysis of variance as appropriate
Control
Hcy
Hcy-cur
Cur
0
1
2
3
4
***
###
###
mean Bax staining semi-quantitative scale
a
b
Fig. 2 a Effect of curcumin
(50 mg/kg) on Bax protein
expression in the substantia
nigra in rat brain after
Hcy (2 μmol/μl)
intracerebroventricular
injection in rats. Values are
expressed as means ± S.E.M.
(N08). One-way ANOVA
followed by Tukey’s Test was
used for statistical analysis to
compare control group.
***p<0.001 indicates
significant increase compared
to the control,
###
p<0.001
different from Hcy groups.
bBax
J Mol Neurosci
Author's personal copy
using GraphPad Prism Software (version 5). p< 0.05 was
considered statistically significant.
Results
Locomotor Activity
I.c.v. treatment of homocysteine 2 μmol resulted into a
decrease in locomotor activity (total distance and velocity).
Pretreatment with curcumin (50 mg/kg) prevented this decrease
of locomotor activity. There was no significant change in
locomotor activity of animals of Hcy-Cur group compared to
control group (F(2, 18)06.251, p00.0087) for total distance
and (F(2, 19)07.892, p00.0032) for velocity (Fig. 1).
Bax/Bcl-2
To determine whether homocysteine leads to changes in
Bcl-2 family protein levels in the rat brain, we examined
the Bcl-2 and Bax protein immunostainings. As shown in
Fig. 2a, Bax protein was detected in control animals and in
homocysteine animals; Bax immunostaining significantly
Control
Hcy
Hcy-Cur
Cur
0
1
2
3
4
***
###
###
Bax/Bcl2 ratio
Fig. 4 Effect of curcumin (50 mg/kg) on Bax/Bcl-2 in the Substantia
nigra in rat brain after Hcy (2 μmol/μl) intracerebroventricular injec-
tion in rats. Values are expressed as means ± S.E.M. (N08). One-way
ANOVA followed by Tukey’s Test was used for statistical analysis to
compare control group. ***p< 0.001 indicates significant increase
compared to the control,
###
p<0.001 different from Hcy groups
Control
Hcy
Hcy-Cur
Cur
0
1
2
3
4
mean Bcl
2
staining semi-quantitative scale
***
###
##
**
a
b
c
Fig. 3 a Effect of curcumin
(50 mg/kg) on Bcl-2 protein
expression in the substantia
nigra in rat brain after Hcy
(2 μmol/μl) intracerebroven-
tricular injection in rats. Values
are expressed as means ± S.E.
M. (N08). One-way ANOVA
followed by Tukey’s Test was
used for statistical analysis to
compare control group. ***p<
0.001 indicates significant de-
crease compared to the control.
###
p<0.001 different from Hcy
groups. bBcl-2, ccresyl violet
J Mol Neurosci
Author's personal copy
increased in control groups (Fig. 2)(F(3, 96) 062.74,
p<0.001).
In contrast with Bax, Bcl-2 staining showed a vigor-
ous expression of this antiapoptotic protein in control
group, and this refers to the constitutive expression of
Bcl-2 protein in normal conditions, as shown in Fig. 3a.
Homocysteine exposure decreased Bcl-2 immunostain-
ing, and this decrement was statistically significant
(F(3, 127)083.60, p<0.001).
In Fig. 4, the Bax/Bcl-2 ratio was calculated for SN
tissue as explained above. The results showed that in
control group, this ratio is lower than homocysteine
groups which was significantly increased (F(3, 62)0
60.51, p<0.001).
Neuronal Counting
The results of neuronal counting showed that a significant
reduction was observed in homocysteine and also in Hcy-
Cur groups (F(3, 56)0170.5, p<0.001) in comparison with
the control group regarding the number of Nissl-stained
neurons on the left and right sides of SN. In addition, the
number of Nissl neurons on the left side of substantia nigra
was significantly higher in curcumin group in comparison
with the homocysteine-curcumin group.
Discussion
The aim of this study was to investigate the neuroprotective
effects of curcumin against homocysteine neurotoxicity in
substantia nigra in male rat. In this study, we evaluated the
possible neuroprotective mechanisms of curcumin to protect
substantia nigra cells against Hcy-induced oxidative stress.
We observed behavioral results which indicated that con-
secutive administration of homocysteine 2 μmol/μlfor
5 days significantly decreased locomotor activity (total dis-
tance and velocity) in comparison with the control groups.
This result is consistent with that of Lee et al. who reported
that they demonstrated a decrease in the level of locomotor
activity after acute homocysteine injection (Lee et al. 2005).
Curcumin treatment at dose 50 mg/kg/day could observe a
significant augmentation in locomotor function in Hcy-Cur
group in comparison with Hcy group (Fig. 1), although this
therapeutic matter had no histological and behavioral effects
(Figs. 1and 2). These results indicate that elevated homo-
cysteine could be associated with complications. Literature
data indicate that Hcy is toxic to neuronal cells (Lipton et al.
1997). Moreover, hyper Hcy has been implicated in neuro-
nal plasticity and neurodegenerative disorders in human
study (Mattson and Shea 2003). Concentration of Hcy in
the brain and cerebrospinal fluid is elevated in several
neurological diseases in human (Mattson and Shea 2003;
Eto et al. 2002; Regland et al. 1997; Yanai et al. 1983;
Quinn et al. 2004; Baydas et al. 2005)and experimental
animals (Algaidi et al. 2006; Streck et al. 2004). Numerous
studies have reported that homocysteine is elevated in levo-
dopa therapy for PD patients and suggested the substantial
role of homocysteine in causing various neurotoxic effects
(Muller et al. 1999; Muller et al. 2002; Muller et al. 2001;
Kuhn et al. 1998).
In the present study, we immunohistochemically investi-
gated the molecular response of the rat brain. The results
showed that the expression apoptosis regulatory proteins
Bax and Bcl-2 would be altered by homocysteine and
Control
Hcy
Hcy-Cur
Cur
0
50
100
150
200
250
***
***
###
*
$$
Neuronal count
a
b
Fig. 5 a Effect of curcumin
(50 mg/kg) on neuronal count
after Hcy (2 μmol/μl)
intracerebroventricular injection
in rats. Values are expressed as
means ± S.E.M. (N08). One-way
ANOVA followed by Tukey’s
Test was used for statistical anal-
ysis to compare control group.
***p<0.001, *p<0.05 indicate
significant decrease compared to
the control;
###
p<0.001 different
from Hcy groups;
$$
p<0.01 dif-
ferent from Hcy-Cur group. b
TUNEL. Representative photo-
micrographs of coronal staining
sections respectively from left to
right: the control group (Control),
the Hcy group (Hcy), the Hcy-
Cur group (Hcy-Cur), and the Cur
group (Cur). The arrow shows
the positive TUNEL spots. Scale
bar,40μm
J Mol Neurosci
Author's personal copy
curcumin (Figs. 2and 3). Hcy elevated the Bax/Bcl-2 ratio
in favor of apoptosis, and curcumin could (Fig. 4) decrease
the Bax/Bcl-2 ratio.
Apoptosis is a morphologically and biochemically well-
characterized form of programmed cell death to remove un-
necessary or damaged cells in various situations (Thompson
1995; Wyllie et al. 1980). Apoptosis leads to cell death and
differs from necrosis by distinct morphologic and biochemical
features (Orrenius et al. 2007; Zhang et al. 2001).
A key factor in determining cell death or survival following
apoptotic signals is the relative expression of Bax and Bcl-2
proteins. The interactions between these proapoptotic and
antiapoptotic proteins regulate the release of cytochrome c
and the propagation of apoptotic cascade (Deveraux et al.
2001; Korsmeyer et al. 1993;Oltvaietal.1993). The role of
these apoptotic proteins in adjusting the number of neural
precursors and postmitotic neurons during the development
of nervous system has been established (Krajewska et al.
2002; Mooney and Miller 2000; Pompeiano et al. 2000). In
the literature, there are two studies that investigated the in vivo
effect of Hcy on apoptosis (Baydas et al. 2005), and according
to the results, curcumin could inhibit Hcy-induced cell dam-
age when added together with Hcy instead of using it as a
pretreatment (Figs. 2,3,and4). Other studies, including our
own, showed that curcumin pretreatment prior to addition
of oxidative agent also showed potent antioxidant effects
(Ghoneim et al. 2002;Parketal.2008; Thiyagarajan and
Sharma 2004). However, we reached to a compromise
with previous reports in which their outcomes had been
proved that curcumin has a preventing role against cogni-
tive impairment and oxidative stress homocysteine-induced
in the rat brain (Ataie et al. 2010;Nurketal.2005). Also,
it has been illustrated that curcumin is able to protect
dopaminergic neurons against LPS-induced neurotoxicity
(Yang et al. 2008). Naturally occurring developmental cell
death largely exhibits morphological features of apoptosis
(Sun et al. 2002).
Result of the present study showed that Hcy was neuro-
toxic for rats. Histopathological results revealed that 5 days
after Hcy (i.c.v.) injection, Bax level was significantly
increased, and vice versa, Bcl-2 level was dramatically
decreased in the substantia nigra in comparison with the
vehicle and control groups (Figs. 2and 3). It has been
reported that hyperhomocysteinemia causes increases in
proapoptotic Bax levels and decreases in antiapoptotic
Bcl-2 levels in the rat brain (Baydas et al. 2005). We
proposed that the homocysteine effects are specified only
on the rat substantial nigra rather than in other regions of the
rat brain such as cortex and hippocampus.
Pretreatment of spiral ganglion neurons with curcumin
demonstrated that curcumin significantly reduced peroxyni-
trite (ONOO
–
)-induced damage in SGNs (Liu et al. 2011).
Therefore, regarding the role of increased Bax/Bcl-2 ratio in
initiating apoptotic cell death as a requirement for apoptosis,
the results of the present study emphasize that curcumin
exposure may decrease susceptibility to apoptosis of the
substantia nigra cells (Figs. 2a and 3a).
Also in this study, histopathological analysis showed that
Hcy reduced cell counting in the substantia nigra density in
comparison with the control groups (Fig. 5a). However, this
deficit was significantly improved by treatment with curcu-
min50mg/kg/day(Fig.5a). On the other hand, curcu-
min diminished apoptosis in TUNEL assay (Fig. 5b)
accompanied with an increase of Bcl-2 (Fig. 3b)level
and reversely for Bax protein (Fig. 2b). As illustrated in
Fig. 5b, animals treated with 2 μmol Hcy (i.c.v.)
resulted in the condensation of chromatin and in the
shrinkage of nuclei and apoptosis bodies compared with
the control group. However, pretreatment with curcumin at
50 mg/kg significantly decreased the nuclear condensation
and fragmentation. Curcumin (50 mg/kg) alone did not cause
apoptosis in substantia nigra.
Finally, we demonstrated that homocysteine has neuro-
toxic effects on dopaminergic neurons in substantial nigra,
and curcumin administration alleviates behavior symptoms
and apoptosis rate induced by homocysteine. No significant
change of the expression behavioral test, Bcl-2, and Bax
was observed in rats treated with curcumin alone. These
results indicate that pretreatment with curcumin has poten-
tial protective effects against oxidative stress in neuron cells,
which might make curcumin a suitable therapeutic agent for
the prevention and treatment of neurodegenerative diseases
associated with oxidative stress such as Parkinson.
Conclusion
The findings of our study reveal that curcumin at a dose of
50 mg/kg was able to significantly reverse behavioral and
biochemical changes caused by exposure of homocysteine
toward the control animal. Also, it caused an accretion in
cell count following homocysteine injection in the substan-
tial nigra. According to our result and consistent with the
reports, curcumin could be considered as a therapeutic agent
to prevent progressing of neurotoxic matter same to homo-
cysteine in feature.
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