Abstract: The present study has been designed to investigate the possible modulating role of vitamin D3 on chronic exercise-induced apoptosis in skeletal (gastrocnemius) muscle of rats. Rats were run for 8 weeks on a treadmill at a speed of 36 m min-1 to induce apoptosis in their skeletal muscle. Vitamin D3 (0.03 μg/100 g and 0.06 μg/100 g, s.c.) treatment, thrice weekly, was started three days prior to subjecting the animals to chronic exercise and was continued for 8 weeks of the experimental protocol. Oxidative stress was assessed by measuring the levels of TBARS, super oxide anion generation and reduced glutathione in gastrocnemius muscle. Further, serum level of corticosterone (glucocorticoid), a potent indicator of stress, was measured. Moreover, the apoptotic cell death was assessed using DNA gel electrophoresis. Chronic treadmill exercise produced profound DNA smearing. The levels of TBARS, super oxide anion generation and serum corticosterone were noted to be increased and the level of GSH was noted to be decreased in rats subjected to chronic treadmill exercise. However, treatment with vitamin D3 markedly reduced DNA smearing. Moreover, the chronic exercise-induced increases in TBARS level, superoxide anion generation and corticosterone levels were significantly attenuated by treatment with vitamin D3. In addition, vitamin D3 markedly increased the exercise-induced decrease in GSH level. It suggests that antioxidant potential of vitamin D3 and its novel efficacy in modulating exercise-induced corticosterone levels in rats may contribute to its anti-apoptotic effect.
INTRODUCTION
The term apoptosis is derived from a Greek word meaning falling off leaves from tree and is described as programmed cell death (PCD) (Geske and Gerschenson, 2001). It is a genetically controlled, biochemical and morphological response of cells to commit suicide (Steller, 1995) evoked through internal (Taniike et al., 2008) as well as external pathological or physiological signals (Hetts, 1998). It plays an integral role in a variety of biological events, including morphogenesis, tissue homeostasis and removal of unwanted or harmful cells (Schultz and Harrington, 2003). The morphological features of apoptosis include changes in plasma membrane asymmetry and attachment, condensation of cytoplasm and nucleus and inter-nucleosomal cleavage of DNA (Ameisen, 2002).
Exercise has been reported to induce apoptosis in the skeletal muscle of mice and rats (Sandri et al., 1995). Strenuous exercise stimulates a variety of signals, such as, increase in reactive oxygen species (ROS), glucocorticoids (Yuan et al., 2003), which in turn have been suggested to be the possible reason for increased apoptosis after acute intense exercise.
Vitamin D3, formed in the skin during the exposure of 7-dehydrocholesterol to sunlight, undergoes a hydroxylation on C-25 in the liver and a subsequent hydroxylation in the kidney to form 1,25-dihydroxyvitamin D3 [1,25(OH)2D3], which is the biologically active form of vitamin D (DeLuca, 1974). Until the 1980s, the function of vitamin D was considered largely in the area of calcium, phosphorus and bone metabolism. Continued pursuit has resulted in the understanding that vitamin D3 is a multi-potential vitamin having influence on numerous target cells. Its pro-differentiating, anti-proliferating and immuno-modulatory roles have instigated extensive work in the field of cancer, hypertension, diabetes (Holick, 2004), psoriasis (Morimoto, 1986), arthritis (Cantorna et al., 1998) and autoimmune disorders (Cantorna et al., 2004).
Few reports propose vitamin D3 as a pro-apoptotic agent (Park et al., 2000; Narvaez and Welsh, 2001), while others describe a protective role of vitamin D3 against apoptosis (Manggau et al., 2001; Riachy et al., 2002). The effect of 1,25(OH)2D3 is undoubtedly dose dependent, apparent even in nano-molar concentration and time dependent, maximal after a 24 h pre-incubation (Ravid et al., 2002). Physiological concentration of 1,25(OH)2D3 do not induce apoptosis in primary melanocytes despite a cell growth inhibitory effect (Sauer et al., 2003). Proposed mechanisms of the anti-apoptotic effect of 1,25(OH)2D3 involve reduction in the release of mitochondrial cytochrome-c (DeHaes et al., 2003) and formation of Sphingolipid breakdown product sphingosine-1-phosphate (S1P) (Manggau et al., 2001), resulting in a changed Bcl-2/Bax ratio (Sauer et al., 2005) and by the up-regulation of Bcl-2 expression (Wang et al., 1999). Pretreatment with vitamin D3 has been reported to inhibit efficiently but not completely UVB-induced activation of poly (ADP-ribose) polymerase (PARP) (DeHaes et al., 2003). PARP, a DNA repairing enzyme enhances the survival of cells (Burkle, 2001). 1,25(OH)2D3 has been reported to inhibit caspase-3 activation and protects the cells from caspase-independent cell death-induced by hyper-osmotic and oxidative stress (Diker-Cohen et al., 2003). 1,25(OH)2D3 regulates the expression of death receptors and protects cancer cells from apoptosis induced by death ligands (Zhang et al., 2005). Pretreatment of hippocampal cells with vitamin D3 (close to physiological level) in vitro has been reported to inhibit cell death (Obradovic et al., 2006).
It is reported that the anti-oxidant properties of vitamin D3 in vivo (Sardar et al., 1996). Its efficacy as an effective anti-neoplastic agent and its anti-oxidant potential in murine lymphoma was also subsequently reported by us (Mukhopadhyay et al., 2000). Vitamin D at physiological concentration has been found to protect cell proteins and membranes against oxidative damage (Ravid and Koren, 2003) and there is evidence that it may stabilize chromosomal structure and enhance repair of double strand breaks (Chatterjee, 2001). Active form of vitamin D3 has been recognized as an anti-oxidative agent through their induction of metallothionein, a potent free radical-scavenging protein (Hanada et al., 1995).
The present study therefore aims to investigate the possible role of Vitamin D3 in chronic treadmill exercise-induced apoptosis in skeletal muscle (gastrocnemius) of rats. This is also an effort to elucidate whether vitamin D3’s antioxidant potential can possibly contribute to its anti-apoptotic efficacy. Vitamin D3 has been reported to decrease glucocorticoid levels in vitro (Obradovic et al., 2006). We have attempted to understand if a cross-talk exists between exercise-induced glucocorticoid levels and vitamin D3 in vivo and can this contribute to its possible effect on apoptosis.
MATERIALS AND METHODS
The experimental protocols used in the present study were approved by institutional animal ethical committee. Male Wister albino rats weighing about 200-225 g were maintained on the standard rat food (Kissan Feed Ltd. Chandigarh, India) and water ad libitum. They were raised in departmental animal house and regulated for temperature (25±0.5°C), humidity (50-60%) and were exposed to 12 h cycle of light and dark.
Experimental Protocol
In the present study, 5 groups were employed and each group comprised
of 8 animals.
Group 1 (Normal Control, n = 8)
The rats were not allowed to treadmill exercise but were handled daily
to the noise of the treadmill.
Group 2 (Chronic Exercise Control, n = 8)
The rats were subjected to chronic treadmill exercise for 8 weeks.
Group 3 (Chronic Exercise + Vehicle Control, n = 8)
The rats were treated with propylene glycol (10%, used as vehicle, s.c.),
thrice weekly, starting three days prior to subjecting the animals to
chronic treadmill exercise and this was continued throughout the duration
of the exercise.
Group 4 (Chronic Exercise + Vitamin D3, 0.03 μg/100
g, n = 8)
The rats were treated with vitamin D3 (0.03 μg/100 g,
dissolved in 10% propylene glycol, s.c.), thrice weekly, starting three
days prior to subjecting the animals to chronic treadmill exercise and
was continued throughout the duration of exercise.
Group 5 (Chronic Exercise + Vitamin D3, 0.06 μg/100
g, n = 8)
The rats were treated with vitamin D3 (0.06 μg/100 g,
dissolved in 10% propylene glycol, s.c.), thrice weekly, starting three
days prior to subjecting the animals to chronic treadmill exercise and
was continued throughout the duration of exercise.
Animals of Groups 2, 3, 4 and 5 ran on a level motorized rodent treadmill (Patiala Sports Industries, Patiala, India) 5 days weekly for 8 weeks. During the 1st 4 weeks, the speed of the treadmill and duration of the running sessions were gradually increased from a speed of 10 m min-1 for 10 min, to a running speed of 36 m min-1 for 60 min by the end of the 4th week. For the next 4 weeks, a 5 min warm up session at a speed of 20 m min-1 was followed by the 60 min running session at a speed of 36 m min-1. Animals of the normal control group (Group 1) were handled daily and subjected to the noise of the running treadmill by placing their cages next to the treadmill when their other partners ran on the treadmill. This procedure was designed to minimize any possible confounding effect of external factors (e.g., handling, treadmill noise) on the dependent variables. At the end of 8th week, exercised animals as well as normal control animals were sacrificed and gastrocnemius tissues were quickly removed. Blood was collected prior to their sacrifice by retro orbital bleeding for serum preparation.
Gastrocnemius Muscle DNA Extraction and Gel Electrophoresis
The concentration of DNA was determined spectrophotometrically (DU 640
Spectrophotometer, Beckman Coulter Inc. CA, USA) at 260 nm. Protein contamination
of DNA was assessed by determining the ratio of absorbance at 260 and
280 nm, which should be more than 1.75. To detect the inter-nucleosomal
cleavage, 10-12 μg of extracted DNA was added to equal volume of
loading dye and it was loaded in the well. Electrophoresis was carried
out using 1.8% agarose gel in 1x TBE buffer for 1.15 h at 400 mA, 50 V
and 3 W in submarine electrophoresis unit (Pharmacia Biotech, Freibury,
Germany). Ethidium bromide (0.5 μg mL-1) was added to
the gel for DNA detection.
Estimation of Tissue Thiobarbituric Acid Reactive Substances (TBARS)
Level
The whole gastrocnemius tissue TBARS level was measured by the method
of Ohkawa et al. (1979). Tissue was homogenized in Tris buffer
(pH-8.3) and the homogenate was mixed with sodium dodecyl sulphate (SDS),
acetic acid (pH-3.5) and thiobarbituric acid (TBA). The test tubes were
incubated at 95°C for 60 min and then cooled. n-Butanol: pyridine
(15:1 v/v) mixture was added to the test tubes and centrifuged at the
4,000 g for 10 min. The absorbance of developed colour in organic layer
was measured spectrophotometrically at 532 nm. The absorbance was extrapolated
from standard curve generated using tetra-methoxy propane as standard.
Results were expressed as μmole mg-1 of protein and protein
content was estimated by the method of Lowry et al. (1951).
Estimation of Tissue Superoxide Anion Genaration Level
The whole gastrocnemius tissue super oxide anion level was measured by
the method of Wang et al. (1998). Weighed amount of tissue was
placed for 1.5 h in buffer at 37°C containing 100 mM L-1
of nitrobluetetrazolium (NBT). NBT reduction was stopped by addition of
0.5 N HCl. The tissue was minced and homogenized in a mixture of 0.1 N
NaOH and 0.1% SDS in water containing 40 mg L-1 diethylene
triamine penta acetic acid. The mixture was centrifuged at 20,000 g for
20 min and the resultant pellet was re-suspended in 1.5 mL of pyridine
and kept at 80°C for 1.5 h to extract formazan, an adduct formed after
reaction of NBT with super oxide anions. The mixture was centrifuged at
10,000 g for 10 min and the absorbance of the formazan was determined
specrtophotometrically at 540 nm. The amount of reduced NBT was calculated
using the formula: Amount of reduced NBT = A x V / T x Wt x ε
x l, where A is the absorbance, V is the volume, T is the time for which
the tissue was incubated with NBT, Wt is the blotted wet weight of the
tissue, ε is the extinction coefficient (0.72 L/mM/mm) and l
is the length of the light path. Results were expressed as Reduced NBT
picomole/minute per mg.
Estimation of Reduced Glutathione (GSH) Level
The whole gastrocnemius tissue GSH level was measured by the method of
Beutler et al. (1963). Tissue homogenate was taken and the proteins
were precipitated with 10% w/v chilled trichloroacetic acid. Samples were
kept in ice bath and were centrifuged after 30 min at 1000 g for 10 min
at 4°C. GSH levels were measured in the supernatant, which was mixed
with 0.3 M disodium hydrogen phosphate solution. Freshly prepared DTNB
(5,5’-dithiobis-2-nitrobenzoic acid) solution (40 mg/100 mL in 1% w/v
sodium citrate) was added just before measuring the absorbance spectrophotometrically
at 412 nm. Different concentrations of GSH standard was also used to prepare
a standard curve simultaneously. Results were expressed as micro mole/mg
of protein.
Estimation of Serum Corticosterone Level
The estimation of corticosterone in serum was carried out by the method
of Silber et al. (1958). Serum was treated with freshly prepared
chloroform: methanol mixture (2:1, v/v), followed by chloroform. The samples
were vortexed for 30 sec and centrifuged at 2000 rpm for 10 min. The chloroform
layer was carefully removed with the help of a syringe and was transferred
to a fresh tube. The chloroform extract was then treated with 0.1 N NaOH
by vortexing rapidly and NaOH layer was immediately removed. The samples
were then treated with 30 N H2SO4 by vortexing vigorously.
After phase separation, chloroform layer on the top was removed using
the syringe and discarded. The tubes containing H2SO4
layer were kept in dark for 30-60 min and thereafter, fluorescence measurements
were carried out in a spectrofluoremeter (ELCO ®SL-174) with excitation
and emission wavelengths set at 472 and 532.2 nm, respectively. Blank
was prepared without adding sample. Different concentration of corticosterone
standard was used to prepare standard curve. Results were expressed as
micro gram/deciliter.
Statistical Analysis
All data was expressed as mean±SEM. Statistical significance was
calculated using one-way ANOVA. Tukey’s multiple range test was employed
as post hoc test for multiple comparison. Statistical significance was
indicated if p<0.05.
Drugs and Chemicals
Vitamin D3 was obtained as a gift sample from Ind-Swift Ltd.
Parwanoo (HP), India. DNA, proteinase K, RNAase, Agarose gel, BSA, glutathione,
5,5’-dithiobis-2-nitrobenzoic acid were purchased from SRL Pvt. Ltd.,
Mumbai, India. Corticosterone was purchased from Sigma, St. Louis, MO,
USA. All other chemicals were obtained from SD. Fine Chem Ltd., Mumbai,
India and were of analytical grade.
RESULTS
Effect of Vitamin D3 on Gastrocnemius Muscle DNA Smearing
There was apparently no DNA smearing in the gastrocnemius muscle of the
Normal Control Group (Group 1), as is evident from Fig. 1. Chronic treadmill
exercise produced significant gastrocnemius muscle DNA smearing in Chronic
Exercise Control Group (Group 2), as compared to Normal Control Group
(Fig. 2). The administration of vehicle did not alter the chronic treadmill
exercise-induced DNA smearing in Vehicle Control Group (Group 3), as compared
to Chronic Exercise Control Group (Group 2). However, the administration
of Vitamin D3 reduced chronic exercise-induced DNA smearing
significantly. Interestingly, this attenuating effect of the vitamin was
more pronounced in the group receiving 0.06 μg/100 g dose (Group
5), than in the one receiving the lower dose of 0.03 μg/100 g, (Group
4), thus exhibiting dose dependency of the vitamin, as is evident from
Fig. 2.
| Fig. 1: | Electrophoretic pattern of DNA extracted from gastrocnemius
muscle of Normal Control Group rats. L1, L2, L3 and L4 represent lanes.
No DNA smearing was observed in Group 1. Wells were loaded with 12
μg of DNA |
| Fig. 2: | Electrophoretic pattern of DNA extracted from gastrocnemius
muscle of rats. L1, L2, L3, L4 and L5 represent DNA extracted from
gastrocnemius muscle of Group 1, 2, 3, 4 and 5 rats respectively.
Chronic exercise produce DNA smearing in Chronic Exercise Control
Group as compare to Normal Control Group and vehicle has no modulating
effect on DNA smearing. However, Vitamin D3 treatment reduces
DNA smearing significantly in a dose dependant manner and higher dose
exhibiting more efficacies. Wells were loaded with 12 μg of DNA |
| Table 1: | Effect of vitamin D3 on TBARS level, superoxide anion
(Reduced NBT) generation, GSH level and corticosterone level |
Results are expressed as mean±SEM (n = 8). ap<0.05 vs normal control group (Group 1); bp<0.05 vs chronic exercise control group (Group 2); cp<0.05 vs chronic exercise + vitamin D3 (0.03 μg/100 g) treated group (Group 4)
Effect of Vitamin D3 on Gastrocnemius Muscle TBARS Level
Chronic treadmill running causes significant increase in gastrocnemius
muscle TBARS level in Chronic Exercise Control Group (Group 2), as compared
to Normal Control Group (Group 1), as can be seen in Table 1. TBARS level
in the Vehicle Control Group (Group 3), exhibited no difference from that
of Groups 2 animals. However, treatment of Vitamin D3 reduces
chronic exercise-induced increase in TBARS level significantly in a dose
dependent manner (Table 1).
Effect of Vitamin D3 on Gastrocnemius Muscle Super Oxide
Anion Generation Level
Table 1 shows the gastrocnemius muscle superoxide anion generation in
the different experimental groups. As is evident from the Table 1, chronic
treadmill running causes significant increase in super oxide anion production
(Group 2), compared to that of the Normal Control Group (Group 1). Administration
of the vehicle had no effect on this exercise-induced increase in super
oxide anion production. However, administration of Vitamin D3
reduces chronic exercise-induced increase in super oxide anion production
significantly, the higher dose being more effective than the lower.
Effect of Vitamin D3 on Gastrocnemius Muscle Reduced Glutathione
(GSH) Level
Chronic treadmill running causes significant decrease in gastrocnemius
muscle GSH level in Chronic Exercise Control Group (Group 2), in comparison
to the Normal Control Group (Group 1), as can be observed in Table 1.
Administration of the vehicle did not alter chronic treadmill running-induced
decrease in GSH level in Group 3 as compared to Group 2 animals. However,
administrations of Vitamin D3 increase the chronic exercise-induced
decrease in GSH level significantly, again in a dose dependent manner
(Table 1).
Effect of Vitamin D3 on Serum Corticosterone Level
Chronic treadmill running causes significant increase in serum corticosterone
level, as was seen in Group 2 animals (Table 1). Administration of the
vehicle did not modulate chronic treadmill running-induced increase in
corticosterone level in the Vehicle Control Group (Group 3), as compared
to the Chronic Exercise Control Group (Group 2). However, both groups
of vitamin D3 treated animals demonstrated marked significant
reduction in chronic exercise induce-increase corticosterone production.
Here also the higher dose of the vitamin was more effective in attenuating
the elevated serum corticosterone level, as is evident from Table 1.
DISCUSSION
The active gene directed process of cell death, apoptosis, is characterized by fragmentation of chromosomal DNA (Steller, 1995). A characteristic pattern of this is DNA ladder formation (Wyllie, 1980) and can be recognized by electrophoresis on an agarose gel. Some reports have investigated the influence of acute intense exercise on apoptosis in skeletal muscles of both young normal and dystrophin-deficient (mdx) mice and have measured the apoptotic changes by DNA gel electrophoresis (Sandri et al., 1995). Chronic treadmill running at a speed of 36 m min-1, starting from 10 m min-1, for 8 week, has been used to induce apoptosis in the skeletal (gastrocnemius) muscle of rats in this study. The DNA smearing noted in the Exercise Control Group (Group 2) may thus reflect apoptosis-induced-necrotic cell death, as observed in other similar studies (Gold et al., 1994).
Two different doses of vitamin D3 were employed in the present study, keeping in view the abundance of reports on both pro (Park et al., 2000; Narvaez and Welsh, 2001) and anti-apoptotic role of the vitamin D3 (Manggau et al., 2001; Riachy et al., 2002). However, since most studies report calcium-mediated toxicity of high doses of vitamin D in rats (Kitagawa et al., 1992), 0.03 μg/ 100 g and 0.06 μg/100 g, thrice weekly doses were chosen for the present study. Studies with such doses have also been reported to have beneficial effect on apoptosis (Manggau et al., 2001; Obradovic et al., 2006). This is of utmost importance because the hypercalcaemic action of vitamin D3 is considered a major limitation in its therapeutic use (Studzinski et al., 1993). The antiapoptotic effect of Vitamin D by upregulating antiapoptotic protein Bcl-2 has been demonstrated (Duque et al., 2004). The complete checking of DNA smear formation in Group 5 and the near-normal pattern in Group 4 animals in this study definitely speak of the potential of vitamin D3 as an anti-apoptotic agent.
Strenuous exercise has been reported to stimulate a variety of signals, such as, increase in ROS and glucocorticoids secretion, which in turn can potentially induce apoptosis (Bejma and Ji, 1999). Therefore, estimation of parameters such as DNA gel electrophoresis and TBARS, ROS, GSH and Corticosterone (Glucocorticiods) in this study stand justified.
Free radicals are toxic agents created as by-products of metabolic activity. Under aerobic conditions, the participation of oxygen in redox reactions in unavoidable and ROS such as super oxide anion, hydroxyl radical, hydrogen peroxide, lipid peroxides and nitric oxide are produced (Sjodin et al., 1990). It has been documented that exercise can increase the generation of ROS in biological tissues like skeletal muscles and myocardium and also increase metabolic rate and oxygen consumption (Reid et al., 1992; Bejma et al., 2000). Increased production of ROS during intense exercise has thus been suggested to be the possible reason for increased apoptosis after exercise (Phaneuf and Leeuwenburgh, 2001). Rats exercising on treadmill have been reported to show an increase in ROS above resting levels (Bejma and Ji, 1999). One of the predominant processes resulting from oxidative stress includes oxidative lipid peroxidation, measured as TBARS (Carlson and Sawada, 1995). Study results corroborate with the above mentioned studies and show that chronic treadmill exercise leads to a considerable increase in TBARS concentration, an index of lipid peroxidation, in the gastrocnemius muscle of rats.
In this study, vitamin D3 administration significantly attenuated the exercise-induced lipid peroxidation (TBARS) and this effect was significantly more pronounced in the higher dose of Vitamin D3 treated animals (Group 5). It has been suggested that ROS influences apoptosis mainly through the modulation of the mitochondrial mediated pathway. It has also been hypothesized that a high oxidative stress level destabilizes the mitochondrial membrane homeostasis and therefore induces the formation of mitochondrial membrane permeability pores and release of pro-apoptotic factors such as cytochrome-c (Pollack and Leeuwenburgh, 2001). Vitamin D3, a highly lipophilic compound, may accumulate in cell membranes, stabilize the membranes, decrease membrane fluidity and thus decrease lipid peroxidation (Mukhopadhyay et al., 2000). Thus, one of the possible underlying mechanisms of the anti-apoptotic effect of the vitamin could be its inhibitory effect on lipid peroxidation.
The generation of super oxide anion (ROS) in the gastrocnemius muscle can be successfully quantified by measuring the amount of NBT reduction (Wang et al., 1998). This indicator has been used in the present study as NBT reduction is very sensitive to even low levels of super oxide anion as a result of the accumulation of blue formazan in the tissue over time. This study reveals that chronic treadmill exercise drastically increases the production of super oxide anion in the gastrocnemius muscle and vitamin D3 treatment reduces this exercise-induced increase in super oxide anion level to near-normal values, the higher dose exhibiting a greater efficacy.
The thiol system and the antioxidant enzymes are a major cell defense against acute oxygen toxicity. GSH is known to maintain the intra-cellular redox balance in mammalian cells (Uhlig and Wendel, 1992) and produce an ameliorative effect on cellular injury (Meister, 1988). GSH can react with reactive oxygen metabolites (ROMs) in a number of ways. It reduces H2O2, one of the major ROS, to form water, itself getting oxidized to form glutathione disulphide, GSSG (Ross et al., 1985).
The observed significant decrease in GSH level in the chronic exercise control group thus, is in support of the above mentioned reports. Also, GSH has been reported to prevent DNA damage caused by certain free radicals (Sarkar et al., 1994). In addition, exercise-induced decreased GSH levels, can trigger mitochondria to release caspase-activating proteins, such as cytochrome-c and AIF (Apoptosis Inducing Factor) into the cytosol, marking early apoptotic event (Green and Reed, 1998). Thus, decrease in GSH, along with heightened pro-oxidant status, may contribute to the DNA damage observed in exercise-induced apoptosis in gastrocnomius muscle of rat.
Treatment of vitamin D3 causes significant increase in reduced glutathione (GSH) levels, the higher dose again showing greater ability in elevating GSH levels. Dietary vitamin D3 has been reported to possess more antioxidant efficacy than vitamin E. It effectively modulates GSH and enzymes of the cellular antioxidant defense system, i.e. superoxide dismutase, glutathione peroxidase, glutathione reductase and glucose 6-phosphate dehydrogenase (Sardar et al., 1996). It has also been shown that high levels of oxidative stress, induced by dietary selenium and vitamin E deficiencies, increase caspase-like activity and DNA fragmentation (as measured by DNA gel electrophoresis) in chick skeletal myocytes (Nunes et al., 2003). Thus, decreased lipid peroxidation and super oxide anion, in association with elevated GSH levels, undoubtedly speaks of the controlled pro-oxidant status and improved antioxidant defense brought about by vitamin D3 treatment in this study.
Increased secretion of glucocorticoids during intense exercise could also be suggested as one of the positive factors for increased apoptosis after acute intense exercise (Phaneuf and Leeuwenburgh, 2001). Corticosterone (Glucocorticoids) overload in rats has been reported to result in the impairment of mitochondrial oxidative energy metabolism in brain (Katyare et al., 2003). The present study recorded significantly higher level of corticosterone following chronic treadmill exercise, which is well in agreement with the above mentioned studies.
Vitamin D3 has been reported to inhibit in vitro dexamethasone (specific agonist of glucocorticoid receptor) induced apoptosis in hippocampal cells (Obradovic et al., 2006). In the present study, vitamin D3 effectively decreased the level of exercise-induced serum corticosterone. Therefore, the protective effect of vitamin D3 in exercise-induced apoptosis of gastrocnemius muscle may additionally be due to the vitamin’s inhibitory effect on glucocorticoids.
ACKNOWLEDGMENTS
The authors are thankful to Dr. Monica Gulati, Prof. and Head, Department of Pharmaceutical Sciences, Lovely Professional University, Phagwara, Punjab for her support. We extend our gratitude to Prof. Manjeet Singh, Director Academics and Shri. Parveen Garg, Chairman, I.S.F. College of Pharmacy, Moga, Punjab for their inspiration and constant support.