Markers of oxidative stress lipid peroxidation and glutathione and activities and levels of antioxidant enzymes SOD, cata-lase, glutathione peroxidase, glutathione reductase and transfer
Trang 1O R I G I N A L A R T I C L E
Organ and tissue-dependent effect of resveratrol and exercise
on antioxidant defenses of old mice
Bui Thanh Tung1•Elisabet Rodriguez-Bies2• Hai Nguyen Thanh1•
Huong Le-Thi-Thu1•Pla´cido Navas2• Virginia Motilva Sanchez3•
Guillermo Lo´pez-Lluch2
Received: 28 October 2014 / Accepted: 20 April 2015
Ó Springer International Publishing Switzerland 2015
Abstract
Background Oxidative stress has been considered one of
the causes of aging For this reason, treatments based on
antioxidants or those capable of increasing endogenous
antioxidant activity have been taken into consideration to
delay aging or age-related disease progression
Aim In this paper, we determine if resveratrol and
exer-cise have similar effect on the antioxidant capacity of
different organs in old mice
Methods Resveratrol (6 months) and/or exercise
(1.5 months) was administered to old mice Markers of
oxidative stress (lipid peroxidation and glutathione) and
activities and levels of antioxidant enzymes (SOD,
cata-lase, glutathione peroxidase, glutathione reductase and
transferase and thioredoxin reductases, NADH cytochrome
B5-reductase and NAD(P)H-quinone acceptor
oxidore-ductase) were determined by spectrophotometry and
Western blotting in different organs: liver, kidney, skeletal
muscle, heart and brain
Results Both interventions improved antioxidant activity
in the major organs of the mice This induction was
ac-companied by a decrease in the level of lipid peroxidation
in the liver, heart and muscle of mice Both resveratrol and
exercise modulated several antioxidant activities and pro-tein levels However, the effect of resveratrol, exercise or their combination was organ dependent, indicating that different organs respond in different ways to the same stimulus
Conclusions Our data suggest that physical activity and resveratrol may be of great importance for the prevention
of age-related diseases, but that their organ-dependent ef-fect must be taken into consideration to design a better intervention
Keywords Antiaging Antioxidant Resveratrol Exercise Old mice
Introduction Imbalance in the activity of antioxidant enzymes and the production of free radicals by metabolic activities, mainly associated with mitochondria, have been associated with the aging process by the free radical theory of aging [1,2] The main radicals in cells are derived from reactive oxygen species (ROS) and have been considered active factors in aging and aging research because of their potential in-volvement in many degenerative diseases These ROS are highly reactive and damage many biological macro-molecules such as DNA, RNA, protein and lipids [3] For this, antioxidant enzymes constitute an important defense system to clear up the harmful ROS in vivo and to prevent oxidative damage of macromolecules
Resveratrol (trans-3,40,5-trihydroxystilbene) (RSV) is a naturally occurring phytoalexin found in red wine, berries and peanuts RSV has shown many positive effects on biological systems ranging from cancer chemoprevention [4], prevention of inflammation [5] and antioxidant
& Bui Thanh Tung
tungasia82@yahoo.es
1 School of Medicine and Pharmacy, Vietnam National
University, Hanoi, Floor 5 Building Y1, 144 Xuan Thuy,
Cau Giay, Hanoi, Vietnam
2 Centro Andaluz de Biologı´a del Desarrollo, Universidad
Pablo de Olavide-CSIC, CIBERER, Instituto de Salud Carlos
III, Carretera de Utrera Km 1, 41013 Seville, Spain
3 Departmento de Farmacologı´a, Facultad de Farmacia,
Universidad de Sevilla, 41012 Seville, Spain
DOI 10.1007/s40520-015-0366-8
Trang 2capacity [6] Although its effect has been studied for more
than a decade, the molecular mechanisms of RSV remain
elusive [7] Although a direct activation seems to be
un-likely, the modulation of sirtuins by regulation of NAD?/
NADH ratio and the activation of the AMPK-dependent
pathway seem to be the main mechanisms involved in the
RSV effect on cells and organisms affecting longevity,
metabolism regulation, cancer, inflammation, etc [7, 8]
During the last years, the activation of the DNA
damage-dependent pathway [9, 10] by activating ATM and the
regulation of the different pathways affected by this kinase
probably indicate a common mechanism of action for all
the effects of RSV on the cell and organisms Furthermore,
several investigations have demonstrated the role and
protective effect of RSV against certain forms of oxidant
damage, through a hydrogen-electron donation from its
hydroxyl groups [11] or by increasing the expression of
antioxidant enzymes [12] Therefore, RSV may be an
im-portant dietary factor to improve health and prolong the
average lifespan in animals [13,14]
Physical activity is associated with better health mainly
by its effect on muscle strength and the cardiovascular
system On the other hand, physical activity can also
positively affect physiological endogenous antioxidant
defenses in old subjects It reduces the production of
oxi-dants and oxidative damage, improves antioxidant defense
system and increases the resistance of organs and tissues
against the deleterious action of free radicals [15]
Fur-thermore, the physical activity level correlates closely with
antioxidant enzymatic activities, especially related to the
glutathione-dependent system in the liver and brain [16]
Furthermore, recently we have described the positive
re-lationship of coenzyme Q10-dependent prevention of LDL
oxidation and physical activity in elderly people [17,18]
The aim of the present study was to evaluate the
pos-sible antiaging properties of RSV and/or physical activity
in old mice by modulating endogenous antioxidant
ac-tivities and enzyme levels in different organs Thus, the
content of glutathione, sulfhydryl group and lipid damage
through malondialdehyde (MDA) and the activity and
levels of superoxide dismutase (SOD), catalase (CAT),
glutathione peroxidase (GPx), glutathione reductase (GR),
glutathione-S-transferase (GST), NAD(P)H-quinone
ac-ceptor oxidoreductase (NQO1), NADH cytochrome b5
re-ductase (CytB5Rase) and thioredoxin reductase (TrxR) in
different mice organs were determined Furthermore, the
antioxidant ratio (R) indicated as the activity of SOD
re-lated to the sum of the activities of CAT and GPx, which
has been related to cell senescence [19,20] was also
de-termined The different responses of organs to the same
stimulus and their relationship with the induction of
en-dogenous antioxidant systems are discussed
Materials and methods Animals and feeding regimen Male mice (C57BL/6 J) at the age of 18 months were used for these experiments The experiments were of duration
6 months until their killing Thus, at the end of the study, the mice were 24 months of age (old mice) A total of 16 mice were used, divided into four groups: Control no trained (Control-NT), Control trained (Control-T), Resveratrol no trained (RSV-NT) and Resveratrol trained (RSV-T) The animals were fed with a basal diet (Teklad Global Diet chow 2014S, Harlan) and kept in a thermo-statically controlled cage holder at 22°C with a 12 h lighting cycle All animals were maintained according to a protocol approved by the Ethical Committee of the University Pablo de Olavide and following the interna-tional rules for animal research
Training consisted of running at a speed of 20 m/min,
20 min/day, 5 days/week, for all the time The group Control was fed a liquid containing ethanol in water (180 lL ethanol/100 mL H2O) and the group RSV was fed
a liquid containing resveratrol [180 lL of a dilution of
55 mg/mL trans-resveratrol in ethanol in100 mL H2O, reaching a concentration of 100 mg/L (0.01 % RSV)] in opaque bottles to avoid light-dependent decomposition Drinking water was changed twice a week for both groups Taken into consideration an average drinking of 4–5 mL/day and the weight of the animals, the calculated dose of RSV was around 500 lg/animal/day (16.67 mg/ kg/day) Animals were killed by cervical dislocation and dissected The brain, kidney heart, muscle and liver were frozen in liquid nitrogen and stored at -80°C until the analysis
All animals were maintained according to a protocol approved by the Ethical Committee of the University Pablo
de Olavide of resolution 03/09 and following the interna-tional rules for animal research
Body weight Mice’s body weight was measured every 2 weeks to check for a possible influence of physical performance and RSV Treatment of sample
Frozen tissue from the brain, kidney heart, muscle and liver was homogenized in nine volumes of ice-cold tissue lysis buffer containing 150 mM sodium chloride, 1.0 % NP-40,
50 mM Tris, pH 8.0, and 1 mM PMSF (phenylmethylsul-fonyl fluoride) with protease inhibitors (Sigma) Ho-mogenates were centrifuged at 10009g for 10 min at 4°C
Trang 3Single-use aliquots of the homogenates were stored at
-80°C before measurements The protein concentration
was determined by the Bradford’s method
Measurement of antioxidant activities and oxidative
damage
The SOD activity was spectrophotometrically measured
using the method developed by Marklund and Marklund
[21] Briefly, SOD was detected on the basis of its ability to
inhibit superoxide-mediated oxidation of pyrogallol One
unit was determined as the amount of enzyme that inhibited
oxidation of pyrogallol by 50 %
CAT activity was measured by following the rate of
disappearance of H2O2at 240 nm [22] One unit of CAT
activity is defined as the amount of enzyme catalyzing the
degradation of 1 lmol H2O2per min and specific activity
corresponding to transformation of H2O2(in nmol) per min
per mg protein
The whole amount of glutathione, reduced (GSH) plus
oxidized (GSSG) forms, was determined by the method
suggested by Anderson [23] The amount of glutathione
was expressed as nmol per mg total protein
The GPx activity was determined in a coupled assay
with glutathione reductase by measuring the rate of
NADPH oxidation at 340 nm using H2O2as the substrate
[24] GR activity was determined by following the
oxida-tion of NADPH at 340 nm as described by Carlberg and
Mannervik [25] GT activity was determined by Habig’s
methods [26] based on the conjugation of
1-chloro-2,4-dinitrobenzene (CDNB) with reduced glutathione
Enzy-matic activity was calculated by using the extinction
co-efficient of 9.6 mM-1 cm-1 for CDNB and expressed as
nmol/min/mg protein
Total CytB5Rase activity was assayed by measuring the
rate of potassium ferricyanide reduction
spectrophotomet-rically, according to the method of Strittmatter and Velick
[27] The enzyme activity was calculated using the
ex-tinction coefficient of 6.22 mM-1 cm-1 for NADH and
expressed as nmol/min/mg protein
NQO1 activity was determined spectrophotometrically
by monitoring the reduction of the standard electron
ac-ceptor, 2,6-dichlorophenol-indophenol (DCPIP) at 600 nm
as described by Benson et al [28] in the absence or
pres-ence of dicoumarol The dicoumarol-inhibitable part of
DCPIP’s reduction was calculated as NQO1 activity using
the extinction coefficient of 21.0 mM-1 cm-1 and
ex-pressed as nmol DCPIP reduced/min/mg protein
TrxR activity was determined by the method of Hillet
et al [29] and based on the reduction of 5,50-dithiobis
(2-nitrobenzoic acid) (DTNB) determined by the increase
in absorbance at 412 nm A unit of activity was defined as 1.0 nmol 5-thio-2-nitrobenzoic acid (TNB) formed/min/mg protein
Protein thiol (SH) groups were estimated by Ellman’s method [30] Briefly, 0.5 ml of sample homogenate was added to a cuvette containing 0.5 ml phosphate buffer (0.1 M, pH 7.4); 0.2 ml of 3 mM 5,5-dithiobis (2-ni-trobenzoic acid) was then added to start the reaction After 10 min, absorbance was measured at 412 nm The amount of SH groups was calculated according to the formula: mol SH/ml = [(Dsample/14,150)/dilution fac-tor]/ml
Lipid peroxidation assay was performed by determining the reaction of malondialehyde with two molecules of 1-methyl-2-phenylindole at 45°C as described by Ge´rard-Monniern et al [31] Peroxidized lipids are expressed as nmol MDA equivalents/mg protein
All the biochemical analyses and enzyme activities were determined in triplicate per sample Activity or determi-nation per sample was considered as the mean of these three determinations Data are the result of the mean of the samples from four animals per treatment (n = 4)
Immunoblotting analysis Homogenates of samples were separated by 10 % (v/v) SDS-PAGE and then transferred to nitrocellulose mem-branes and subjected to immunoblot analyses using the primary antibodies anti-Cu, Zn-SOD (SOD1), anti-CAT, (Santa Cruz Biotechnology), anti-CytB5Rase (rabbit poly-clonal antibody kindly provided by Dr J M Villalba, Universidad de Co´rdoba, Spain), anti-GPx1, anti-TrxR1, anti-TrxR2 (Acris Antibodies, Germany) or anti-NQO1 (Abcam, Cambridge, UK) and secondary antibodies horseradish peroxidase-conjugated goat rabbit or anti-sheep antibodies (Calbiochem, Germany) Protein expres-sion levels were corrected for whole protein loading de-termined by staining membrane with Ponceau S Protein expressions were visualized by the ChemiDocTM XRS? System and compiled with Image LabTM 4.0.1 Software (Bio-Rad Laboratories)
Statistical analysis The results were analyzed by two-way ANOVA using SigmaPlot 10.0 program (Systat Software Inc.) All values were expressed as mean ± SE The critical significance level a was established at 0.05 and, then, statistical sig-nificance was defined as P \ 0.05
Trang 4RSV and exercise decreased oxidative damage in old
mice
To evaluate the levels of some oxidative damage markers,
we determined the level of glutathione and MDA in
dif-ferent tissues The results are shown in Table1
The highest levels of glutathione were found in the liver,
whereas other tissues showed similar lower levels around
6 nmol/mg proteins In this case, exercise increased the
level in the heart, muscle and liver without any effect on
the brain and the kidney On the other hand, RSV increased
the level in the brain, muscle and liver without any effect
on the kidney and the heart Combined RSV with exercise
only had an effect on the liver and heart
The lLevels of MDA, indicating lipid peroxidation, were
higher in the brain and lower in the heart, kidney, and liver
Exercise decreased the level MDA in the liver, whereas
RSV decreased MDA only in muscles A combination of
both produced a clear decrease in the heart, muscle and liver
Antioxidant activities are improved by RSV and exercise
in old mice
We determined the activity of several antioxidant enzymes
in the different tissue (Tables2,3and4)
Kidney and liver showed the highest CAT activity,
whereas heart showed the lowest levels (Table2)
Inter-estingly, in those organs showing the highest activity,
kidney and liver, it was further increased by exercise RSV
only induced activity in kidney, whereas the combination
of both increased the activity in both organs A trend
to-ward increase in muscle was found, but without reaching
statistical significance
In the case of SOD, the response was different Liver showed the highest activity, but exercise or RSV decreased
it whereas their combination increased it Heart activity was also induced by both as well as its combination Ex-ercise combined with RSV slightly increased its activity in brain A trend toward an increase was also found in muscle, but without significance Other important enzymatic ac-tivity involved in eliminating H2O2, GPx, was higher in kidney, muscle and liver and lower in heart and brain (Table3) Interestingly, GPx activity increased in all or-gans after RSV treatment or training This increase was significant in the liver, heart and kidney in the case of training, whereas it was significant in the brain, heart and liver in the case of RSV The combination of both in-creased significantly the activity only in the kidney and liver
Interestingly, GR and GST activities did not respond to exercise or RSV as GPx GR was not affected, whereas GST only increased in the heart and liver with exercise, RSV and their combination
Other interesting antioxidant activities linked to an-tioxidant protection in cell membranes were also affected
by exercise or RSV depending on the organ (Table4) CytB5Rase was induced by exercise in kidney and by the combination of exercise and RSV in kidney and liver No effect was found with RSV alone On the other hand, NQO1 activity was strongly induced in liver by both ex-ercise and RSV, whereas in the heart exex-ercise induced while RSV decreased it
Thioredoxin reductase activity was only determined in the kidney and liver Exercise, RSV and its combination increased significantly TrxR activity in kidney However, the contrary effect was found in the liver which showed a trend toward a decrease with exercise or RSV that was significant when both were combined
Table 1 Oxidative stress
markers in old mice Control-NT Control-T RSV-NT RSV-T
Glutathione Brain 5.67 ± 0.14 5.60 ± 0.46 6.97 ± 0.50* 6.16 ± 0.54 Heart 6.19 ± 0.22 8.44 ± 0.91* 7.49 ± 0.65 7.69 ± 0.39* Kidney 5.85 ± 0.69 6.45 ± 0.43 6.34 ± 0.69 6.52 ± 0.55 Muscle 6.52 ± 0.44 10.27 ± 0.52* 8.70 ± 0.65* 7.75 ± 0.72 Liver 21.4 ± 5.8 37.9 ± 0.1* 35.3 ± 0.3* 38.5 ± 2.3* MDA
Brain 4.66 ± 0.24 4.68 ± 0.53 4.82 ± 0.38 4.71 ± 0.29 Heart 0.47 ± 0.06 0.31 ± 0.11 0.34 ± 0.05 0.29 ± 0.06* Kidney 0.51 ± 0.12 0.55 ± 0.03 0.50 ± 0.07 0.52 ± 0.03 Muscle 1.07 ± 0.05 1.14 ± 0.02 0.69 ± 0.06* 0.85 ± 0.05* Liver 0.45 ± 0.11 0.32 ± 0.06* 0.38 ± 0.07 0.32 ± 0.07* Values are the mean ± SE * Significant difference vs Control-NT levels, P \ 0.05 Glutathione (sum of oxidized and reduced form) and MDA levels are indicated as nmol/mg protein
Trang 5Antioxidant protein levels are differentially affected
by RSV and/or exercise in old mice
Our experience shows that in many cases, increase of
en-zymatic activity is not accompanied by higher protein
levels and vice versa [12,32,33] For this reason, we also
determined changes in the protein levels of antioxidant
enzymes as indicated in the tables (Fig.1)
CAT levels were only induced by exercise and/or RSV
in muscle, whereas other organs did not respond to these
stimuli However, SOD1 did show modifications at the
protein level in all the organs studied, with kidney and
muscle being the most affected Interestingly, in brain and liver, exercise induced SOD1 expression, but this effect was avoided when combined with RSV In the case of GPx1, only the liver showed response to all the stimuli, whereas the brain only showed higher levels when both exercise and RSV were combined In the case of GR, the response varied from lower levels in the brain to higher levels in the heart after exercise and RSV combination
In the case of the NAD(P)H-depending enzymes, CytB5Rase and NQO1, induction of activity found in kid-ney was accompanied by higher levels of the protein in this organ, whereas in the heart this increase was found when
Table 2 Antioxidant CAT and
SOD activities in old mice Control-NT Control-T RSV-NT RSV-T
CAT Brain 2.07 ± 0.09 2.04 ± 0.05 2.11 ± 0.02 2.21 ± 0.07 Heart 3.37 ± 0.43 3.14 ± 0.39 3.35 ± 0.29 2.88 ± 0.34 Kidney 195 ± 22 236 ± 21* 250 ± 13* 276 ± 18* Muscle 44.4 ± 5.4 57.9 ± 5.2 56.3 ± 5.1 58.1 ± 2.6 Liver 77 ± 3 110 ± 13* 85 ± 9 104 ± 7* SOD
Brain 2.34 ± 0.12 2.39 ± 0.08 2.55 ± 0.1 2.77 ± 0.09* Heart 7.51 ± 0.35 10 ± 0.35* 9.25 ± 0.34* 8.62 ± 0.21* Kidney 2.21 ± 0.25 2.35 ± 0.21 2.41 ± 0.14 2.50 ± 0.08 Muscle 19.4 ± 4.8 26.7 ± 4.2 27.7 ± 1.8 32.5 ± 1.3* Liver 1542 ± 386 1298 ± 260 1470 ± 246 1761 ± 370 Values are the mean ± SE * Significant difference vs Control-NT levels, P \ 0.05 Activities are indi-cated as nmol/min/mg protein
Table 3 Antioxidant GPx, GR
and GST activities in old mice Control-NT Control-T RSV-NT RSV-T
GPx Brain 17.4 ± 0.5 19.7 ± 0.6 21.5 ± 1.5* 19.7 ± 0.6 Heart 13.6 ± 2.4 16.1 ± 2.1* 16.1 ± 1.4* 14.7 ± 0.7 Kidney 30.3 ± 1.5 39.8 ± 3.8* 36.5 ± 1.4 39.5 ± 1.8* Muscle 28.7 ± 0.5 30.5 ± 0.8 30.1 ± 1.3 28.5 ± 1.1 Liver 35.1 ± 12.8 79.2 ± 4.3* 69.9 ± 4.3* 85.7 ± 13.1* GR
Brain 2.30 ± 0.18 2.09 ± 0.08 2.19 ± 0.09 1.99 ± 0.14 Heart 4.84 ± 1.09 5.39 ± 0.61 5.30 ± 0.28 4.09 ± 0.55 Kidney 24.1 ± 0.9 22.5 ± 0.3 22.5 ± 1.2 26.6 ± 1.4 Muscle 8.36 ± 1.12 5.84 ± 1.10 9.42 ± 1.15 8.15 ± 0.89 Liver 28.2 ± 1.3 28.9 ± 1.6 27.0 ± 2.1 24.2 ± 0.5 GST
Brain 90.7 ± 2.4 92.4 ± 1.4 97.4 ± 2.7 99.9 ± 4.7 Heart 18.4 ± 0.6 12.4 ± 0.6* 13.6 ± 0.5* 12.8 ± 0.8* Kidney 35.1 ± 7.2 28.4 ± 1.0 31.5 ± 1.8 32.9 ± 1.9 Muscle 50.4 ± 7.6 54.2 ± 1.6 51.9 ± 3.7 54.5 ± 2.0 Liver 751 ± 212 1094 ± 106* 1130 ± 118* 897 ± 119 Values are the mean ± SE * Significant difference vs Control-NT levels, P \ 0.05 Activities are indi-cated as nmol/min/mg protein
Trang 6RSV and exercise were combined and was accompanied by
lower levels of protein However, in the case of NQO1, the
decrease in the activity found in heart when RSV and
ex-ercise were combined, was accompanied by lower levels of
protein However, the contrary was found in liver, higher
activity accompanied by lower amount of protein
TRxR proteins showed a complex response to RSV and/
or exercise Remarkably, in kidney, RSV or exercise seems
to decrease the levels of TRxR1, but when combined these
levels increased In liver, this protein responded with
higher presence induced by exercise but not by RSV,
although the activity was inhibited by both interventions
Discussion
It is widely considered that the deleterious and irreversible
changes produced by free radicals throughout the life of the
organism are one of the main factors involved in aging [1]
Thus, free oxygen radicals have been proposed as
impor-tant causative agents of aging For this reason, in the theory
of aging coined by Harman [34] in 1956, it is postulated
that aging is produced by oxidative reactions caused by a
higher production of free radicals or a lower capacity to
eliminate them or to repair their oxidative effect Abundant
evidences show that a variety of ROS and other free radical
are truly involved in the occurrence of molecular damage,
which can lead to structural and functional disorders, dis-eases and death
RSV has been shown to have potent antiaging and health-promoting activities by modulating antioxidant ac-tivities in cells among other effects [12] The same anti-aging effect by modulating endogenous antioxidant capacity has been also associated with caloric restriction that is considered to be mimicked by RSV [32,35] On the other hand, physical activity ameliorates age-related pairments by reducing the oxidative damage and also im-proving antioxidant defense systems Many antioxidants and antioxidant activities are involved in the protection against oxidative damage These endogenous enzymatic antioxidant defenses include CAT, SOD, CytB5Rase, NQO1, glutathione, GPx, GR, GST and TrxR
Lipid peroxidation is one of the main events induced by oxidative stress and is particularly active in biomembranes like mitochondria Polyunsaturated fatty acids (PUFAs) are one family of the most important components of cell membranes in living systems Free radicals attack PUFAs leading to the formation of highly reactive electrophilic aldehydes, including MDA, 4-hydroxy-2-nonenal (HNE), and the most abundant products Nohl’s study has reported accumulation of lipid peroxidation products during aging [36] Furthermore, we have found that the antiaging effect
of CR is more effective when the source of fat is rich in monounsaturated and saturated fatty acids than when rich
in PUFAs [37–39] In accordance with these studies, we have found that MDA levels increase with aging in mice liver In this study, we also found that RSV and/or exercise can protect these membranes in the muscle, heart and liver
in old mice indicating an induction of the endogenous antioxidant systems in these organs by mainly affecting SOD and GPx activities and levels of glutathione (Table1)
In this study, we show that RSV and/or exercise can affect endogenous antioxidant activities in different ways depending on the organ Taking into consideration the different roles and locations of each enzyme, this effect can reflect the adaptive mechanisms of these organs against a mild oxidative stress induced by exercise or regulated by RSV Our results agree with previous work by Wong et al [40] which showed that long-term RSV intake attenuates oxidative damage in tissues specially affected during aging such as liver, heart or kidney Moreover, the previous study
of Thirunavukkarasu and coworkers [41] showed that ex-ercise increases glutathione-dependent activities Similarly,
in the present study, administration of RSV and exercise improved the activity of GPx in old mice
However, the complex relationship between antioxidant activities can induce wrong conclusions from activity or protein levels We show that in many cases, activity is not accompanied by similar changes at the protein levels
Table 4 Antioxidant CytB5Rase, NQO1 and TrxR activities in old
mice
Control-NT Control-T RSV-NT RSV-T
CytB5Rase
Brain 245 ± 2 254 ± 5 258 ± 6 228 ± 1
Heart 772 ± 91 730 ± 72 715 ± 63 890 ± 69*
Kidney 148 ± 15 165 ± 13* 150 ± 8 197 ± 21*
Muscle 4.1 ± 0.5 5.3 ± 0.2 4.4 ± 1.0 5.7 ± 0.8
Liver 328 ± 33 351 ± 28 336 ± 26 381 ± 42*
NQO1
Brain 5.3 ± 0.1 5.7 ± 0.3 7.4 ± 0.4* 6.8 ± 0.7*
Heart 16.3 ± 3.5 19.0 ± 2.9* 12.3 ± 2.7* 13.5 ± 2.5*
Kidney 1.1 ± 0.2 2.4 ± 0.2* 1.6 ± 0.4 3.1 ± 0.9*
Muscle 0.5 ± 0.0 0.5 ± 0.0 0.7 ± 0.1 0.6 ± 0.1
Liver 5.4 ± 0.9 11.7 ± 0.5* 7.7 ± 1.5* 9.8 ± 1.7*
TrxR
Brain Not determined
Heart Not determined
Muscle Not determined
Kidney 1.6 ± 0.4 2.3 ± 0.3* 3.0 ± 0.5* 3.0 ± 0.3*
Liver 5.2 ± 0.7 4.6 ± 0.3 4.4 ± 0.5 2.8 ± 1.0*
Values are the mean ± SE * Significant difference vs Control-NT
levels, P \ 0.05 Activities are expressed as nmol/min/mg protein
Trang 7Furthermore, the relationship between antioxidant
ac-tivities must also be taken into consideration In fact, it was
proposed that the imbalance in the ratio of SOD to CAT
and GPx results in the accumulation of H2O2that through
the Fenton reaction results in the formation of hydroxyl
radicals which are highly reactive and damage
macromolecules such as DNA, protein and lipids For this reason, the balance in the activity of these enzymes has been directly related to cell senescence [19,42] Interest-ingly, a previous work demonstrated that the R ratio [R = SOD/(CAT ? GPx) in activities] increases in liver along with age [12] The results shown in this manuscript
Fig 1 Protein expression of
antioxidant enzymes in different
organs in old mice Indicated
organs were processed as
indicated in ‘‘ Materials and
CAT, SOD1, GPX1, GR,
CytB5Rase, NQO1, TRxR1 and
TRxR2 proteins determined by
Western blotting Quantification
was performed considering
Ponceau Red staining as loading
control Results refer to the
levels found in each organ of the
control group *Significant
differences vs control levels in
each organ P \ 0.05
Trang 8demonstrate that both exercise and RSV decrease this ratio,
indicating a higher protective effect in the liver However,
R was not affected in the other organs
In conclusion, our results indicate that both RSV and
exercise improve in different manner the activities of
en-dogenous antioxidant enzymes such as CAT, SOD1, GPx,
GR, GST, NQO1 in old mice, and in some cases preventing
the decrease of these activities associated with aging
Consequently, RSV supplementation and a higher physical
activity should be strongly encouraged in older people, not
only to improve physical function, avoid sarcopenia and
maintain higher independence, but also to attenuate
ox-idative damage caused by aging However, we cannot
ex-trapolate the effects of these interventions in one or few
organs to the whole organism A deeper study of the
regulation of antioxidant enzymatic activities and
expres-sion in relationship with aging is needed
Acknowledgments We thank Almudena Velazquez Dorado and
Ana Sanchez Cuesta for their technical support The group was
fi-nanced by the Andalusian Government as the BIO177 Group through
FEDER funds (European Commission) The research was financed by
the Spanish Government Grant DEP2012-39985 (Spanish Ministry of
Economy and Competitiveness) Tung Bui Thanh received a
fellow-ship from the AECID program (Spanish Ministry of Foreing Affair).
ERB, PN and GLL are also members of the Centro de Investigacio´n
Biome´dica en Red de Enfermedades Raras (CIBERER), Instituto
Carlos III.
Conflict of interest On behalf of all authors, the corresponding
author states that there is no conflict of interest.
Human and Animal Rights All animals were maintained
accord-ing to a protocol approved by the Ethical Committee of the University
Pablo de Olavide of resolution 03/09 and following the international
rules for animal research This article does not contain any studies
with humans performed by any of the authors.
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