Download EFFECT OF RESVERATROL AND ENVIRONMENTAL

EFFECT OF RESVERATROL AND ENVIRONMENTAL ENRICHMENT ON BIOMARKERS OF
OXIDATIVE STRESS IN YOUNG HEALTHY MICE
*Mustapha Shehu Muhammad1, Rabiu Abdussalam Magaji2, Aliyu Mohammed2, Ahmed-Sherif Isa2. and
Mohammed Garba Magaji3.
1
Department of Human Physiology, College of Medical Sciences, Gombe State University,
Gombe, Nigeria.
2
Department of Human Physiology, Faculty of Medicine, Ahmadu Bello University, Zaria,
Nigeria.
3
Department of Pharmacology and Therapeutics, Faculty of Pharmaceutical Sciences,
Ahmadu Bello University, Zaria, Nigeria.
*Corresponding author’s emails: [email protected]; [email protected].
+2347032527295
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ABSTRACT
Resveratrol (RESV) and Environmental Enrichment (EE) have been separately reported to protect organisms
against various diseases. This study investigated the potential benefit of the combination of RESV and EE on
biomarkers of oxidative stress in young healthy mice. Fifty mice of both sexes were randomly divided into five
groups of 10 animals each: group I served as control, group II were maintained on alternate day feeding, group
III received RESV 50 mg/kg, suspended in caboxymethylcellulose orally per kg/day. Group IV received CMC
and kept in an Enriched Environment, group V received RESV + EE. The treatment lasted for 28 days. The
animals were sacrificed 24 hours after the last treatment and brain samples were collected for biochemical
evaluation. The results obtained showed a significant decrease (P < 0.05) in malondialdehyde concentration in
EE group and RESV treated group kept EE when compared to the control. A significant decrease was also
observed in glutathione peroxidase activity in all the treatment groups when compared to the control. A
significant decrease in GPx activities in RESV, EE and RESV + EE treated groups in male and female mice
when compared to the control groups respectively. However, a significant increase in GPx activities was
observed in EE group in male mice and EODF, RESV groups in female mice when compared to RESV + EE
groups respectively. In conclusion, the result of our study indicates that EE possesses antioxidant properties by
decreasing MDA concentration and attenuating lipid peroxidation in the brain of young albino mice.
Key words: Oxidative stress; Environmental enrichment; Resveratrol; Lipid peroxidation; Antioxidant
enzymes
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Introduction
Oxidative stress is an imbalance between the reactive oxygen species generated and the antioxidant defense
system to detoxify the reactive intermediates (Balaban et al. 2005). Reactive oxygen species (ROS) are
chemically reactive molecules that consist of oxygen ions and peroxides which include hydrogen peroxide,
singlet oxygen, nitric oxide, peroxynitrite, and superoxide free radicals which are toxic to the cell and may lead
to cell death (Rege et al. 2013). The antioxidant enzymes known to protect the cells and tissues against oxidative
injury caused by reactive oxygen species, such as superoxide dismutase (SOD), catalase, peroxidases, and nonenzymatic free radical scavengers (ascorbic acid, 𝛼-tocopherol, and GSH) convert the reactive oxygen species to
water and oxygen, which are stable molecules (Uttara et al. 2009; Rege et al. 2013).
The number of age-related chronic diseases such as cancer, obesity, diabetes and neurodegenerative disorders
has been on the increase progressively over the years and oxidative stress has been reported to induce changes in
the signaling pathways that may induce cellular responses such as inflammation, cell proliferation, cell survival
and death (Kregel and Zhang 2007). Oxidative stress has also been observed to play a significant role in
mediating the pathophysiology of numerous ill health conditions such as obesity and diabetes mellitus thereby
forming a link between obesity, cerebrocortical reactive oxygen species, impaired brain functions and the risk
for developing neurodegenerative disorders such as Alzheimer’s disease (Balaban et al. 2005; Luchsinger and
Mayeux 2007; Naderali et al. 2009).
Resveratrol (3, 5’, 4-trihydroxystilbene), a natural polyphenolic compound found mainly in the skin of grapes,
mulberries, peanuts and red wine has previously possesses numerous health benefits such as increase in lifespan
in yeast; (Saccharomyces cerevisiae), nematode worm (Caenorhabditis elegans), fruitfly (Drosophila
melanogaster), and short-lived fish (Nothobrachius furzeri) through a direct Sirtuin 1 dependent mechanism or
indirectly through activation of MAPK (Howitz et al. 2003; Bauer et al. 2004; Wood et al. 2004; Viswanathan et
al. 2005; Valenzano et al. 2006; Hawley et al. 2010). However, the effects and mechanisms of action of
resveratrol in life extension is currently a serious issue of debate. Studies have shown that mice fed with high fat
diet supplemented with high level of resveratrol consistently have improved health parameters and metabolic
alterations similar to what is observed with CR, including glucose homeostasis, endurance, survival and
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improvement in motor and cognitive function (Lagouge et al. 2006; Pearson et al. 2008; Muhammad et al.
2014), thus, resveratrol is said to be caloric restriction mimetic. However, the life-span-prolonging effect of
resveratrol has been seriously debated (Miller et al. 2011), but the mechanisms responsible for the antioxidant
effect of resveratrol against oxidative stress need further investigation.
In recent times, resveratrol has emerge as a valuable biomolecule in ameliorating the delirious effect of
oxidative stress and lipid peroxidation in numerous disease models by activating nuclear factor erythroid-related
factor 2/antioxidant responsive element (Nrf2-ARE) pathway, a major cellular antioxidant enzyme system that
transcribe the synthesis/ up-regulation of GSH, heme-oxygenase 1 (HO1) and superoxide-dismutases (SODs)
(Chen et al. 2005; Ungvari et al. 2009; Ren et al. 2011; Li et al. 2015; Soeur et al. 2015). It also enhances
immune function and up-regulate signalling molecules that are linked to multiple pathways, such as elevating
cAMP levels, peroxisome proliferator activated receptor-gamma coactivator 1a (PGC-1α) activity and NF-κB
(Baur et al., 2006; Park et al., 2012; Lai et al., 2016). Resveratrol has recently been found to up-regulate AMPA
receptor protein expression mediated by AMP-activated protein kinase (AMPK) and subsequent downstream
phosphoinositide 3-kinase (PI3K)/Akt signaling (Wang et al. 2015). However, Buendia et al. (2016) reported
that a phase III clinical trial has been performed with resveratrol to study its effect in mild moderate AD
(NCT00678431) with other clinical trials actively recruiting participants at the moment (NCT01504854,
NCT01716637, NCT01219244).
Environmental enrichment (EE) is defined as a sustained and progressive increase in cognitive and sensorimotor
stimuli with aggregated voluntary physical activity and complex social interactions (Anastasia et al. 2009). It
has been shown to possess many benefits such as neuroprotection both in health and disease conditions
especially in ageing and animal models of neurodegenerative disorders (Faherty et al. 2005; Nithiantharajah and
Hannan 2006; Laviola et al. 2008). Although the mechanism of EE neuroprotection has not been settled, the
synthesis and release of trophic factors (TFs) has been suggested to play a crucial role in mediating the
neuroprotective effects of EE (Nithiantharajah and Hannan 2006). It has been observed to alter the expression of
TFs and their receptors in several brain areas and induces astrogliogenesis (Spires et al. 2004; Steiner et al.
2006). Environmental enrichment has also been found to cause increased brain-derived neurotrophic factor
(BDNF) expression in the striatum and glia cell-line derived neurotrophic factor (GDNF) mRNA in the
substantia nigra (SN) in animals (Bezard et al. 2003; Faherty et al. 2005).
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Numerous studies have been carried out on both resveratrol and EE with much attention been focused on their
beneficial role on aging and age related disorders to the neglect of their possible beneficial role especial in
young healthy animals (Muhammad et al. 2014). The physiological effects of resveratrol on antioxidant system
have been extensively studied in animal models of various disease conditions such as obesity, diabetes and
neurodegenerative disorders. The role of resveratrol in modulating the antioxidant system either by direct
scavenging property or activation of pathways that up-regulate cells’ natural antioxidant defences has not been
well settled especially in young healthy animals. Many health benefits related to polyphenols arise from their
various antioxidant activities and most phenolic compounds such as resveratrol act as a primary antioxidant,
chelator and superoxide anion scavenger (Zhang et al. 1999; Noguchi et al. 2000; Hosoda et al. 2006). Although
resveratrol was reported to behave as a poor scavenger of ROS in vitro, it functions as a potent antioxidant in
vivo (Baur and Sinclair 2006). The influence of gender in modulating physiological functions has been well
established. Studies have demonstrated the role of resveratrol and EE in oxidative stress in various diseased
models. Moreira et al. (2013) reported the effect of resveratrol in rat brain mitochondria biogenesis and
oxidative stress with gender playing a minimal role. Elucidating the role of EE and resveratrol in oxidative
stress in apparently healthy male and female mice could give an idea whether gender could influence the role of
EE and resveratrol in modulating brain oxidative stress in apparently healthy animals. Hence this study
evaluates the effects of RESV and EE on antioxidant enzymes and lipid peroxidation in young healthy male and
female mice.
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Materials and methods
Chemicals/Reagents
Resveratrol (60g) of analytical grade was purchased from Candlewood Stars Incorporated, Danbury, USA
(Batch No: MR120718). Carboxymethylcellulose CMC (10 g) (Product no: 27929, BDH Chemicals LTD, Poole
England) was obtained from Department of Pharmacology and Therapeutics, Ahmadu Bello University, Zaria.
Trans-resveratrol, due to its low solubility in water, was suspended in 10 g/L CMC (Juan et al. 2002).
Animal care and management
Fifty (50) Swiss albino mice of both sexes, 4-5 weeks of age and weighing 15-22 g were used for this study. The
animals were obtained from the Institute of Veterinary Research, Vom, Jos, Plateau State-Nigeria. The animals
were housed in standard polypropylene cages in groups of five in a temperature and humidity controlled
environment subject to a 12 h light/dark cycle and fed with standard laboratory animal feed and water ad
libitum. Dietary delivery groups were given controlled access to food, water was available ad libitum. The mice
were allowed to acclimatize to the environment of the behavioural laboratory for the period of one week before
commencement of the experiment. All experimental protocols were in accordance with the Ahmadu Bello
University Research policy; and ethic and regulations governing the care and use of experimental animals (NIH
Publication no. 85-23, revised 1996). The experiments were conducted in a quiet laboratory between hours of
900 h to 1600 h.
Animal housing and management
The enriched cage (66 cm long × 46 cm wide × 38 cm high) as described by Harburger et al. (2007) was used in
the study. The cage contained tubes, ramps, stairs, and different ‘toys’ (hard plastic balls, cubes, cones, and
sticks). The toys were changed twice a week to continuously encourage exploration of the environment. The
complexity (number of objects) of the housing facility was increased progressively: every 2 days, two to four
objects were added to the environment. Ten days after housing animals in the EE, the complexity of the cage is
expected to be maximal but the positions of the objects were changed continuously after every 2 days (Anastasia
et al. 2009). Five (5) mice were housed together to allow social interactions. The impoverished condition
consisted of normal cages (45cm long x 30cm wide x 18cm high) made without objects or running wheels,
housing five (5) animals per cage.
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Animals were kept in EE housing for four weeks while receiving the appropriate treatment, as described by
Steiner et al. (2006). The control animals were administered with 10 ml/kg of 1% carboxymethylcellulose
(CMC) and kept under good housing condition.
Animal groupings
The animals were divided into five (5) groups each comprising of ten (10) animals of both sexes per group, i. e
five (5) males and five (5) females mice kept in separate cages to prevent mating. Group I animals received 10
ml/kg caboxymethylcellulose (control group) 50mg/kg per body weight, group II animals were maintained on
every other day feeding (EODF) with standard laboratory animals diet, group III animals received resveratrol
50mg/kg orally as described in a previous study by Blanchet et al. (2008), group IV animals received CMC and
were kept in an enriched housing (EE), group V animals received resveratrol 50mg/kg orally and were kept in
EE housing. All treatment will last for 4 weeks.
Biochemical assessments
The animals were sacrificed by decapitation and brain samples were collected using surgical incision and rinsed
with ice-cold isotonic saline. The brain samples were then grinded in a cold glass mortar and homogenized with
ice-cold 100 mM phosphate buffer (pH 7.4; 1 g of tissue/9 ml). The homogenates (10% w/v) were then
centrifuged at 10,000 × g for 15 min and the supernatants so formed were used for the following biochemical
analysis in the Department of Chemical Pathology, Ahmadu Bello University Teaching Hospital, Zaria, Nigeria.
Assessment of lipid peroxidation
The level of thiobarbituric-acid reactive substance, Malondialdehyde (MDA), as an index of lipid peroxidation
was evaluated in brain samples using Mouse Malondialdehyde (MDA) ELISA Kit (WKEA MED SUPPLIES
CORP, U.S.A) according to the manufacturer’s instructions. The principle is based on the reaction of MDA with
thiobarbituric acid (TBA); forming an MDA-TBA2 adduct that absorbs strongly at 532 nm (Janero, 1990).
Assessment of anti-oxidants enzymes
Superoxide dismutase activity:
The activity of Superoxide Dismutase enzyme (SOD) in the mice brain sample was determined using Mouse
SOD ELISA assay kit (Product NWK-SOD02, Specificity: Cu/Zn, Mn and Fe Superoxide Dismutase,
Sensitivity: 5 U/mL). The SOD assay was carried out according to the manufacturer’s instructions. The assay kit
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is based on the principle of superoxide inhibition of autooxidation of hematoxylin as described by Martin et al.
(1987).
Catalase activity:
Catalase (CAT) activity was assessed using Mouse CAT activity assay kit (NWLSS). CAT activity was assessed
using NWLSS CAT activity assay kit (Product NWK-CAT01, Specificity: Catalase, Sensitivity: 6.0 U
Catalase/mL). Catalase enzyme activity was measured based on the principle of catalase consumption of H 2O2
substrate at 240 nm (Beers and Sizer 1952).
Glutathione Peroxidase Activity:
GPx activity was assessed using NWLSSTM cGPx (GPx1) ELISA assay kit (Product NWK-GPX02, Specificity:
Glutathione peroxidase, Sensitivity: 12.5 pg/ml). The NWLSS™ cGPx assay is based on a sandwich EnzymeLinked Immunosorbent assay (ELISA), where sample GPx concentration is determined by comparing the 450
nm absorbance of sample wells to the absorbance of known standards (Takebe, 2002).
Statistical analysis
Data obtained were expressed as Mean ± SEM. Statistical analysis was carried out using SPSS version 22 and
all analysis was done using one way analysis of variance followed by Tukey’s post-hoc multiple comparison test
to examine the effect of treatments on MDA, SOD, CAT and GPx on combined sex groups and two way
analysis of variance followed by Bonferroni test for multiple comparison to examine the effect of treatments and
sex on MDA, SOD, CAT and GPx. Values of P < 0.05 were considered significant.
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Results
Malondialdehyde (MDA) assay
The concentration of MDA in the various treatment groups and the control was presented in figure 1. A
significant decrease was observed (F (5, 50) = 3.7, P = 0.007) in the EE group (1.47 ± 0.08 IU/L) and RESV +
EE group (1.48 ± 0.05 IU/L) when compared to the control group (1.78 ± 0.06 IU/L).
Assessment of superoxide dismutase activity
The activity of Superoxide dismutase enzyme (SOD) in the treatment and the control groups was presented in
figure 2. There was no significant statistical difference observed in SOD activities in the various treatment
groups when compared to the control group.
Assessment of catalase activity
The activity of catalase (CAT) in the treatment and the control group was presented in figure 3. There was no
significant statistical difference observed in the CAT activities in the various treatment groups when compared
to the control group.
Assessment of glutathione peroxidise activity
The activity of Glutathione Peroxidase enzyme (GPx) in the treatment and the control group was presented in
figure 4. A significant decrease in the activities of GPx was observed [F (5, 50) = 24.5, P = 0.001] in EODF
(49.90 ± 1.57 IU/L), RESV (48.20 ± 1.29 IU/L), EE (50.29 ± 1.54 IU/L), and RESV + EE (41.73 ± 1.07 IU/L)
treatment groups when compared to the control group (58.00 ± 1.82 IU/L). However, a significant increase in
GPx activities was also observed in EODF (49.90 ± 1.57 IU/L), RESV (48.20 ± 1.29 IU/L) and EE (50.29 ±
1.54 IU/L) treatment groups when compared to RESV + EE group (41.73 ± 1.07 IU/L).
Sex dependent effect of resveratrol and EE on lipid peroxidation and antioxidant enzymes
Two way ANOVA revealed a sex-dependent effect [F (5, 44) = 9.9, P = 0.0001], F test for groups and what
about the different comparison bases on sex the one reported is based on groups effect of resveratrol and EE on
lipid peroxidation and antioxidant enzymes (Table 1). Further analysis using Bonferroni post hoc comparison
showed a significant decrease in GPx activity in RESV and RESV + EE treated groups in male mice when
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compared to the control. A significant increase in GPx activity was also observed in male mice in EE treated
group when compared to RESV + EE.
In the female treated mice, a significant decrease in GPx activity was also observed in RESV, EE and RESV +
EE treated groups when compared to the control. However, a significant increase in GPx activity was observed
in female mice in EODF and RESV treated groups when compared to RESV + EE group. Overall, there was no
significant statistical difference on the interaction between sex and group in the concentration and activities of
MDA, SOD, CAT and GPx in all the treatment groups respectively.
10
2.0
Malondialdehyde (IU/L)
1.8
1.6
*
*
EE only
Resveratrol
+ EE
1.4
1.2
1.0
0.8
0.6
0.4
0.2
0.0
Control
EODF
Resveratrol
only
Groups
Figure 1: Effect of resveratrol and EE on malondialdehyde concentration in mice
Results are presented as Mean ± SEM; CMC: carboxymethylcellulose; EODF: every other day feeding; EE:
environmental enrichment; n=10; * Significance (P < 0.05) when compared to the control
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Superoxide Dismutase (IU/L)
2.5
2.0
1.5
1.0
0.5
0.0
Control
EODF
Resveratrol
only
EE only
Resveratrol +
EE
Groups
Figure 2: Effect of resveratrol and EE on superoxide dismutase in mice
Results presented as Mean ± SEM; CMC: carboxymethylcellulose; EODF: every other day feeding; EE:
environmental enrichment; n=10 * Significance (P < 0.05)
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70.0
Catalase (IU/L)
60.0
50.0
40.0
30.0
20.0
10.0
0.0
Control
EODF
Resveratrol
only
EE only
Resveratrol
+ EE
Groups
Figure 3: Effect of resveratrol and EE on catalase activity in mice
Results presented as Mean ± SEM; CMC: carboxymethylcellulose; EODF: every other day feeding; EE:
environmental enrichment; n=10; * Significance (P < 0.05)
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70.0
Glutathione Peroxidase (IU/L)
60.0
*#
*#
50.0
*#
*
40.0
30.0
20.0
10.0
0.0
Control
EODF
Resveratrol
only
EE only
Resveratrol +
EE
Groups
Figure 4: Effect of resveratrol and EE on glutathione peroxidase activity in mice
Results presented as Mean ± SEM; CMC: carboxymethylcellulose; EODF: every other day feeding; EE:
environmental enrichment; n=10; * Significance (P < 0.05) when compared to control; # Significance (P < 0.05)
when compared to RESV + EE
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Table 1: Sex dependent effect of resveratrol and EE on lipid peroxidation and antioxidant enzymes
Groups
SOD
Male
CAT
GPx
MDA
Female
Male
Female
Male
Female
Male
Female
Control
1.92 ± 0.09
1.97 ± 0.18
67.00 ±1.30
60.33 ± 2.84
56.80 ± 1.62
60.00 ± 4.36
1.76 ± 0.07
1.80 ± 0.15
EODF
1.66 ± 0.05
1.76 ± 0.07
65.00 ± 1.00
60.80 ± 2.46
49.20 ± 2.06
50.60 ± 2.58#
1.64 ± 0.09
1.70 ± 0.07
Resveratrol only
1.84 ± 0.12
1.60 ± 0.11
61.20 ± 1.59
60.40 ± 2.48
48.20 ± 1.31*
48.20 ± 2.39*#
1.52 ± 0.09
1.60 ± 0.10
EE only
1.72 ± 0.07
1.63 ± 0.19
59.60 ± 1.12
63.67 ± 0.88
51.60 ± 1.60#
45.67 ± 2.19*
1.44 ± 0.10
1.43 ± 0.14
Resveratrol + EE
1.76 ± 0.08
1.80 ± 0.13
58.00 ± 2.86
63.80 ± 1.28
43.40 ± 1.36*
39.80 ± 1.66*
1.52 ± 0.09
1.50 ± 0.05
Results presented as Mean ± SEM; CMC: carboxymethylcellulose; EODF: every other day feeding; EE: environmental enrichment; n=10;
* Significance (P < 0.05) when compared to the control; # Significance (P < 0.05) when compared to RESV + EE group
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Discussion
The present study evaluated the effect of EE, resveratrol as CR mimetic and the combined effect of both on lipid
peroxidation and antioxidant enzymes in young healthy mice. The results obtained for
malondialdehyde
concentration demonstrated a significant decrease in MDA concentration in EE group when compared to the
control. This corroborated the findings of Cechetti et al. (2012) and Marmol et al. (2015) who reported a
significant decrease in free radical content and basal TBARS in the hippocampus cerebral cortex of rats. A
significant decrease in malondialdehyde concentration was also observed in resveratrol treated group kept in
enriched environment (RESV + EE) when compared to the control. These findings suggest that EE attenuates
lipid peroxidation which is the major end point of oxidative stress.
The results obtained for the antioxidant enzymes indicated significant decrease in Glutathione Peroxidase (GPx)
activity in all the treatment groups when compared to the control group. This finding was contrary to the results
obtained by Robb et al. (2008) on the effect of administration of dietary resveratrol, who observed increase in
expression and activities of MnSOD, GSH-Px and CAT in mouse brain. While Robb et al. observed upregulation of MnSOD by administering resveratrol via a high fat diet which could possibly tilt the balance in
favour of oxidant status through liberation of reactive species; resveratrol was suspended in CMC and
administered orally to the animals in our study. Robb attributed the increase in MnSOD to citrate cynthase
activity; a citric acid cycle enzyme whose activity is commonly measured as a marker of mitochondrial number.
Interestingly, in the same study conducted by Robb et al., resveratrol failed to up-regulate the levels and
activities of MnSOD, CAT, and GPx through a standard laboratory diet, which agreed with our findings. Other
factors that may account for the differences observed between our studies include; the dosage used and the
species of animal employed in the study. While Robb et al., administered 200 mg/kg to BL6 mice, we used a
dose of 50mg/kg/body weight administered orally to Swiss albino mice.
Nuclear factor E-related factor 2 (Nrf2), a transcription factor that plays key role in maintaining redox
homeostasis via its interaction with Kelch-like ECH-associated protein 1 (Keap1). Under oxidative stress, Nrf2
is released from the Nrf2/Keap1 complex and translocates to the nucleus; where it induced the expression of a
battery of genes encoding diverse cytoprotective proteins, including antioxidant enzymes (Taguchi et al. 2011).
Resveratrol scavenges ROS through modulation of glutathione biosynthesis via Nrf2 antioxidant response
element signalling (Rahman 2008). This could also be a reason why resveratrol failed to up-regulate the brain
16
natural antioxidant enzymes via modulation of Nrf2. Resveratrol has also been reported to behave as a poor
scavenger of ROS in vitro. It however functions as a potent antioxidant in vivo. The in vivo antioxidant property
of resveratrol probably arises from its ability to increase nitric oxide (NO) synthesis, which in turn functions as
an in vivo antioxidant, scavenging superoxide radicals due to the presence of unpaired electron (Das and Maulik
2006). Resveratrol was reported to induce NO synthesis and lower oxidative stress in the ischemic reperfused
heart, brain, and kidney (Hatori et al. 2002). The affinity of NO for O 2– was observed to be far greater than the
affinity of superoxide dismutase (SOD) for O2–, hence, NO may compete with SOD for O2–, thereby removing
O2– and sparing SOD and other endogenous or primary antioxidant enzymes for other scavenging duties (Das
and Maulik 2006). This may explain why resveratrol failed to up-regulate the activities of the indigenous
antioxidant enzymes as observed in this study.
The effect of EE on GPx activity also conflicted the findings of Cechetti et al. (2012) who reported upregulation of antioxidant enzymes by EE. Although, we worked on naive animals, Cechetti et al. studied the
effect of EE on cognitive functions and oxidative stress induced by cerebral hypoperfusion in rats which may
shift the oxidative status to pro-oxidants leading to up-regulation of antioxidants enzymes by EE to covert the
reactive oxygen species, thereby reducing peroxidative injury. However, a significant increase in GPx activities
was observed in EODF, RESV, and EE treated groups respectively when compared to RESV + EE treated
group. This demonstrated that individual treatment with either RESV or EE tends to up-regulate the activities of
GPx more than the combine treatment with RESV + EE. A possible explanation for could be that, although EE
has been established to be neuroprotective by ameliorating the deleterious effect of oxidative stress especially in
neurodegenerative disorders, it has been shown to mask the estrogen-like beneficial role of RESV in cognitive
function in mice (Muhammad et al. 2014). This could be a possible reason why the combined treatment of
RESV and EE failed to up-regulate GPx activities and other antioxidant enzymes beyond the individual
treatments with RESV and EE respectively. The decrease in GPx activities in EODF group when compared to
the control and its increase when compared to RESV + EE treatment group confirmed its beneficial role in
antioxidant status of the body system.
The sex-dependent effects of resveratrol and EE on antioxidant enzymes have demonstrated a significant
decrease in GPx activities in RESV and RESV + EE treated groups in male mice and RESV, EE and RESV +
17
EE treated groups in female mice when compared to their control groups respectively. As stated earlier while
discussing the combined gender treatments, the decrease in GPx activities in both male and female mice in the
treatment groups may be due to ability of RESV and EE to induce the synthesis of substances such NO which
scavenge reactive oxygen species and spare cells natural antioxidant enzymes for other scavenging functions.
However, a significant increase in GPx activities was observed in EE treated group in male mice and EODF,
RESV treated groups in female mice when compared to the RESV + EE treated groups respectively. The high
GPx levels observed in the EE treated group in male mice could be due mild stress elicited by EE in the male
mice which suggest that the female mice are physiologically more protected from oxidative stress than males,
since they do not need to synthesize large amounts of GPx to counter oxidative disturbance (Mortol et al. 2015).
Freitas et al. (2005) reported an increase in catalase activity in association with glutathione, providing
neuroprotection against the increased lipid peroxidation in the hippocampus of male rats. The increased in GPx
activities in the resveratrol female treated mice may also be due to the oestrogen-like effect of resveratrol on
antioxidants enzymes. This demonstrates that both EE and resveratrol have a sex dependent effect on GPx
activity as observed in our study. Taken together, there was no significant difference in the interaction between
sex and group in the concentration and activities of MDA, SOD, GPx and CAT respectively.
An important point of consideration in this study is that contrary to other studies where resveratrol and EE were
found to up-regulate antioxidant enzymes activities were carried out in disease conditions or animal models of
certain disorders such as obesity, hyperlipidemia, ethanol induced oxidative damage and cerebral hypoperfusion
(Robb et al. 2008; Das et al. 2010; Timmers et al. 2011; Cechetti et al. 2012) we used naïve animals. As we
reported previously in our previous ostudy (Muhammad et al. 2014), there is little evidence that both RESV and
EE enhances normal physiological functions in healthy individuals in the absence of pathological or age-related
decline in normal body functions which indicates that resveratrol and EE confers some protection on the brain
and other body systems from the effects of aging, oxidative, physical and chemical damage (Story et al. 2012;
Muhammad et al. 2014); this could possibly be an explanation for the absence of an observed effect of
resveratrol and EE on the up-regulation of antioxidant enzymes activities compared to the control in our study.
Conclusion
In conclusion, the result of our study indicates that EE possesses antioxidant properties by attenuating lipid
peroxidation which is the major end point of oxidative stress by decreasing MDA concentration in the brain of
young healthy Swiss albino mice over a period of four weeks. We also found that both RESV and EE decrease
18
GPx activities in the combined gender group and across the sex groups. Finally, we have also demonstrated the
beneficial role of RESV and EE by increasing GPx activities independently than their combined treatment in
male and female mice respectively.
.
Acknowledgments
The authors wish to thank Malam Muhammad Mu’azu in the Department of Pharmacology and Therapeutics,
Ahmadu Bello University, Zaria, Nigeria, for his assistance in training and handling of the animals, Mr. Olu
Aiyegbuisi of the Department of Chemical Pathology, Ahmadu Bello University Teaching Hospital Zaria,
Nigeria for biochemical investigations. They also appreciate Gombe State University, Nigeria, for providing
financial assistance for the study.
Compliance with ethical standards
All experimental protocols were in accordance with the Ahmadu Bello University Research policy; and ethic
and regulations governing the care and use of experimental animals (NIH publication no. 85-23, revised 1996).
Conflict of Interests
The authors declare that there is no conflict of interests regarding the publication of this paper.
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