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ORIGINAL ARTICLE
Induction of oxidative stress and inhibition of superoxide
dismutase expression in rat cerebral cortex and cerebellum
by PTU-induced hypothyroidism and its reversal by curcumin
Srikanta Jena •Chinmay Anand •
Gagan Bihari Nityananda Chainy •
Jagneshwar Dandapat
Received: 19 July 2011 / Accepted: 4 November 2011 / Published online: 11 November 2011
ÓSpringer-Verlag 2011
Abstract The present study was carried out to elucidate
the effectiveness of curcumin in ameliorating the expres-
sion of superoxide dismutase (SOD) in cerebral cortex and
cerebellum of rat brain under 6-propyl-2-thiouracil (PTU)-
induced hypothyroidism. Induction of hypothyroidism in
adult rats by PTU resulted in augmentation of lipid per-
oxidation (LPx), an index of oxidative stress in cerebellum
but not in cerebral cortex. Curcumin-supplementation to
PTU-treated (hypothyroid) rats showed significant reduc-
tion in the level of LPx in both the regions of brain. The
decreased translated products (SOD1 and SOD2) and the
unchanged activity of SOD in cerebral cortex of PTU-
treated rats were increased on supplementation of curcumin
to the hypothyroid rats. Declined translated products of
SOD1 and SOD2 in cerebellum of PTU-treated rats were
alleviated on administration of curcumin to hypothyroid
rats. On the other hand, the decreased activity of SOD in
cerebellum of PTU-treated rats was further declined on
administration of curcumin to the hypothyroid rats. Results
of the present investigation indicate that curcumin differ-
entially modulates the expression of superoxide dismutase
in rat brain cortex and cerebellum under PTU-induced
hypothyroidism.
Keywords Cerebral cortex Cerebellum
Hypothyroidism Curcumin Oxidative stress
Antioxidant enzymes
Introduction
Although critical role of thyroid hormone (TH) in the
development and differentiation of brain is well established
[1,2], its role in adult brain is poorly understood. Recent
studies have established definite role of TH in adult brain
such as neurogenesis [3], expression of genes [4], phos-
phorylation of calmodulin dependent synaptosomal protein
[5] and anti-oxidant defences [6]. Several reports also
suggest that adult-onset of thyroid dysfunction is associated
with both neurological and behavioural abnormalities [7].
Moreover, increased generation of reactive oxygen species
(ROS), particularly in hypothyroidism, has been reported
both in humans [8] and rats [9], emphasising the importance
of THs for normal brain function. We have also reported
that TH has profound effect on antioxidant defences of
cerebellum of rat brain during development [2]. However,
information on regulation of antioxidant defences in adult
cerebral cortex and cerebellum is scanty. The cellular dys-
function due to hypothyroidism can be treated with T
3
or T
4
(levothyroxine); however, Mogulkoc et al. [10] reported
that thyroxine administration to hypothyroid rats resulted in
oxidative stress in several tissues. Therefore, administration
of exogenous antioxidant is a preferable therapeutic agent
against hypothyroid-induced oxidative stress. Curcumin, a
major phenolic compound derived from the rhizome of
Curcuma longa (turmeric) is known for its anti-inflamma-
tory, antioxidant, anti-cancerous and anti-amyloid proper-
ties [11–14]. The present communication aims to evaluate
the level of oxidative stress (OS) parameter, lipid
S. Jena (&)C. Anand J. Dandapat
Department of Biotechnology,
Utkal University, Bhubaneswar 751004,
Odisha, India
e-mail: jenasrikanta@yahoo.co.in
G. B. N. Chainy
Imgenex India Pvt. Ltd., E-5, Infocity,
Chandrasekharpur, Bhubaneswar 751024,
Odisha, India
123
Neurol Sci (2012) 33:869–873
DOI 10.1007/s10072-011-0853-4
peroxidation (LPx), and superoxide dismutase (SOD)
expression in different regions (cerebral cortex and cere-
bellum) of rat brain under 6-propyl-2-thiouracil (PTU)-
induced hypothyroidism. We also report the protective
effects of curcumin on the above-mentioned parameters.
Experimental design
Brain samples analyzed in the present study were of our
experimental model reported earlier [11]. In brief, experi-
ments were carried out on 150 ±10 day-old (weight about
350–400 g) adult male Wistar rats (Rattus norvegicus)
supplied by National Institute of Nutrition (Hyderabad,
India). The animals were maintained at 25 ±2°C under
standard conditions in the animal room. Animal care,
maintenance and experiments were performed under the
supervision of the Institutional Animal Ethics Committee
(IAEC). The rats were divided into three groups, each
group having five rats.
Group I: Control rats.
Group II: Rats were treated with PTU for 30 days.
Group III: Rats were treated with PTU along with cur-
cumin for 30 days.
Rats of group II were made hypothyroid by adminis-
tering 0.05% of PTU in their drinking water for 30 days
[15]. Group III rats were supplemented with 0.05% of PTU
in their drinking water along with oral administration of
curcumin (30 mg/kg body weight) one dose daily for
30 days [16]. Olive oil was used as the vehicle solution for
supplementation of curcumin. Group III animals were
orally administered with 1 ml of the desired concentration
of curcumin and the same amount of the vehicle solution
was also fed to control (group I) and hypothyroid (group II)
rats.
Materials and methods
Sample preparation
After the rats were killed by decapitation, cerebral cortex
and cerebellar region of the brain were dissected out and a
20% (w/v) homogenate was prepared in 50 mM phosphate
buffer, pH 7.4 with a Potter-Elvehjem type motor-driven
homogenizer. The crude homogenate was used for the
measurement of oxidative stress parameter, LPx. A part of
the crude homogenate was treated with Triton X-100 (final
concentration 0.1% v/v) and centrifuged at 10,000 9gfor
15 min at 4°C. The supernatant so obtained was used for
enzymatic (SOD) assay.
For western blotting analyses, a 10% homogenate of
cerebral cortex and cerebellar region of the brain were
prepared in 50 mM Tris–HCl buffer, pH 7.4, containing
150 mM NaCl, 104 mM PMSF, 100 lM E-64, 80 lM
aprotinin, 100 lM leupeptin, 1% triton X-100 and 0.1%
SDS to avoid protein degradation. The homogenates were
centrifuged at 1,000 9gfor 20 min at 4°C.
Determination of LPx level
Lipid peroxidation level was determined in crude homog-
enate samples. Lipid peroxidation was estimated by mon-
itoring the formation of thiobarbituric acid reactive
substances (TBARS) according to the method of Ohkawa
et al. [17]. Concentration of TBARS in samples was cal-
culated from its extinction co-efficient, 1.56 910
5
M
-1
cm
-1
and is expressed as nanomoles of TBARS
formed per milligram of protein.
Measurement of total SOD activity
Supernatant fraction (200 ll) was loaded to 1 ml syringe
column of Sephadex G-25 pre-equilibrated with phosphate
buffer (50 mM, pH 7.4). The sample loaded column was
centrifuged at 2,000 rpm for 2 min at 4°C in order to get
rid of low-molecular-weight interfering substances. The
activity of SOD was estimated in the eluted samples of the
column. The activity of SOD was determined according to
the method of Das et al. [18].
Western blotting analyses of SOD1 and SOD2
50 lg of total cellular proteins prepared for western blot-
ting analyses were resolved in 12% SDS-PAGE and
transferred to PVDF membrane (0.4 lm, PALL Life Sci-
ences) at 23 mA current for 1 h. The membrane was
blocked in 5% blocking solution for 1 h at room temper-
ature. The blot was then incubated with rabbit polyclonal
anti-G3PDH (1:1,000), anti-SOD1 (1:5,000) or anti-SOD2
(1:2,500) for 1 h at room temperature. The membrane was
washed three times (each 5 min) with washing solution and
subsequently incubated with HRP-conjugated anti-rabbit
goat IgG (1:7,500, Santa Cruz Biotechnology, Inc., USA)
for 1 h at room temperature. After washing, specific
immunoreactive proteins were detected with ECL kit
(Santa Cruz Biotechnology, Inc., USA) in X-ray film and
their expression level was measured by densitometry.
Statistical analyses
Quantitation of western blot bands was done using com-
puter assisted densitometry Image-Quant TL, image anal-
ysis software v2003. Relative densities were determined as
the ratio of AOEs band/G3PDH (internal control) band.
Data were presented as mean ±SD. The data were
870 Neurol Sci (2012) 33:869–873
123
analysed by one-way analysis of variance (ANOVA) fol-
lowed by Duncan’s new multiple range tests to find out the
level of significance among the mean values. The minimal
statistical significance was considered at pB0.05 levels.
Results
Level of LPx
The level of LPx did not change in cerebral cortex of PTU-
treated rats (group II), whereas a significantly increased
level of LPx was observed in cerebellum of group II rats
with respect to control rats (group I). Significantly
decreased level of LPx in both cerebral cortex and cere-
bellum was found after supplementation of curcumin to the
hypothyroid rats (group III) as compared to group II rats
(Fig. 1).
Activity of total SOD and western blotting of SOD1
and SOD2
An unchanged activity of SOD in cerebral cortex of
PTU-treated rats (group II) was observed as compared to
control rats (group I), whereas a significantly decreased
level of SOD activity in cerebellum of PTU-treated rats
(group II) was recorded with respect to control rats.
After administration of curcumin to hypothyroid rats
(group III), the activity of SOD in cerebral cortex was
significantly increased with respect to group II rats
(Fig. 2a), whereas the activity of SOD in cerebellum was
further decreased on supplementation of curcumin to
hypothyroid rats (group III) as compared to group II rats
(Fig. 2b).
The translated products of SOD1 and SOD2 were sig-
nificantly decreased in both cerebral cortex and cerebellum
of PTU-treated rats (group II) with respect to control rats
(group I). The expressions of SOD1 and SOD2 in the above
regions of rat brain were significantly increased on sup-
plementation of curcumin to the hypothyroid rats (group
III) as compared to group II rats (Fig. 3).
Discussion
Several studies suggest that deficiency of TH (hypothy-
roidism) modulates the antioxidant defence system [9–11].
However, Mogulkoc et al. [10] reported that thyroxine
administration to hypothyroid rats resulted in oxidative
stress in several tissues. Therefore, supplementation of
exogenous antioxidant may be a preferable therapeutic
agent against hypothyroid-induced oxidative stress.
Although antioxidant effects of curcumin have been
reported in several studies [9,11–13], information about
the regulation of expression of antioxidant genes in general
and SOD in particular in different regions of rat brain under
hypothyroid state by curcumin is lacking. In the present
study, effects of oral administration of PTU along with
curcumin on the level of LPx and SOD expression in
cerebral cortex and cerebellum have been investigated. It is
observed that the increased oxidative stress is region-spe-
cific under PTU-induced hypothyroidism, as evident from
the elevated level of LPx in cerebellar region only, but not
in cerebral cortex. This may be due to region-specific
expression of thyroid hormone receptor isoforms in hypo-
thyroid adult rats [19]. It has been reported that level of
LPx in hippocampus and amygdala regions of the rat brain
is elevated when hypothyroidism is induced by the treat-
ment of methimazole [20]. However, the level of LPx was
lowered both in cerebral cortex and cerebellum after sup-
plementation of curcumin to the PTU-treated rats. The
reason may be due to its antioxidative property against free
radical-induced oxidative stress. Curcumin has been
reported to be a potent antioxidant [9,12,13], chemopre-
ventive agent [13], which can also pass the blood–brain
barrier without damaging the brain cells [21]. Furthermore,
curcumin has been shown to act as a protective agent in
neurodegenerative disease which helps in reducing the
disrupted lipids [22] and inhibits O
2
2
and OH generation
by preventing oxidation of Fe
2?
to Fe
3?
through Fenton
reaction [16,22]. The elevated level of LPx in PTU-treated
rats was also decreased in crude homogenate of kidney
cortex on supplementation of curcumin [9], whereas, an
unaltered level of LPx was found in liver mitochondrial
Fig. 1 Effect of curcumin on
oxidative stress parameter, LPx
of cerebral cortex (a) and
cerebellum (b) of rat brain of
different experimental groups.
Groups Icontrol rats, II PTU-
treated rats, III PTU with
curcumin-treated rats. Data are
expressed as mean ±SD of five
animals. Data having different
superscripts differ significantly
(pB0.05) from each other
Neurol Sci (2012) 33:869–873 871
123
fraction in response to curcumin [11]. Results of these
studies depicted the differential effect of curcumin in
ameliorating the altered level of LPx in different tissues of
hypothyroid rats.
Reactive oxygen species have been associated with
many pathophysiological conditions such as cancer, Par-
kinson’s disease and Alzheimer’s disease [23]. Under
normal physiological conditions, a delicate balance exists
between the rate of ROS formation and the rate of their
neutralisation. Biological systems are equipped with cel-
lular enzymatic and non-enzymatic defence mechanism to
counteract such ROS. In enzymatic antioxidant-defence
system, superoxide dismutase is the first line of cellular
defence which converts the primary oxygen free radical,
O
2
2
(generated from the mitochondrial electron transport
chain) to H
2
O
2
and is further removed by catalase and
glutathione peroxidase [24]. In the present investigation,
decreased activity of SOD as a consequence of the declined
level of translated products (SOD1 and SOD2) in both the
regions (cerebral cortex and cerebellum) of brain suggests
that the accumulation of superoxide anion radicals might
be responsible for increased lipid peroxidation following
Fig. 2 Effect of curcumin on
the activity of SOD in cerebral
cortex (a) and cerebellum (b)of
rat brain of different
experimental groups. Groups
Icontrol rats, II PTU-treated
rats, III PTU with curcumin-
treated rats. Data are expressed
as mean ±SD of five animals.
Data having different
superscripts differ significantly
(pB0.05) from each other
SOD2
G3PDH
(C) SOD2
G3PDH
(D)
SOD1
G3PDH
(A)
G3PDH
SOD1
(B)
Fig. 3 Effect of curcumin on translate level of SOD1 (a,b) and
SOD2 (c,d) in cerebral cortex and cerebellum of rat brain of different
experimental groups. Groups Icontrol rats, II PTU-treated rats,
III PTU with curcumin-treated rats. Data are expressed as mean ±
SD of five animals. Data having different superscripts differ
significantly (pB0.05) from each other
872 Neurol Sci (2012) 33:869–873
123
PTU-treatment. The present findings are in good agreement
with our earlier observations where a reduction in expres-
sion of superoxide dismutase (SOD1 and SOD2) was
confirmed in cerebellum of developing rats in response to
hypothyroidism by western blotting and semiquantitative
RT-PCR [2]. Moreover, the possible reason for the
decreased activity of SOD may be due to a cellular adap-
tive response in hypothyroidism as ascorbic acid spared the
function of superoxide dismutase [9]. On supplementation
of curcumin to hypothyroid rats, the protein expressions
(SOD1 and SOD2) and the activity of SOD in both the
regions were ameliorated except in cerebellum where the
activity of SOD was decreased. Though findings of this
study could not explain the decreased activity of SOD in
cerebellum on supplementation of curcumin to hypothyroid
rats, similar pattern of SOD expression (SOD1 and SOD2)
was observed in kidney cortex, where an increased level of
translated product and decreased activity of the enzyme
were found under same experimental conditions [9]. Fur-
ther studies are needed to investigate the mechanism(s) of
action of curcumin in modulating the antioxidant-defence
system in rat brain under hypothyroidism.
Acknowledgments This work was supported by Department of
Biotechnology (DBT), Government of India. The first author is
extremely grateful to the DBT, Government of India for providing the
fellowship (Ref. No. DBT-JRF/05-06/123).
Conflict of interest The authors declare that there is no conflict of
interest.
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