Effect of endurance training on diurnal rhythms of superoxide dismutase activity, glutathione and lipid peroxidation in plasma of pinealectomized rats

Journal Pre-proof Effect of endurance training on diurnal rhythms of superoxide dismutase activity, glutathione and lipid peroxidation in plasma of pinealectomized rats Jana Tchekalarova, Tzveta Stoyanova, Zlatina Nenchovska, Natasha Ivanova, Dimitrinka Atanasova, Milena Atanasova, Katerina Georgieva

PII:

S0304-3940(19)30740-2

DOI:

https://doi.org/10.1016/j.neulet.2019.134637

Reference:

NSL 134637

To appear in:

Neuroscience Letters

Received Date:

31 July 2019

Revised Date:

4 November 2019

Accepted Date:

18 November 2019

Please cite this article as: Jana T, Tzveta S, Zlatina N, Natasha I, Dimitrinka A, Milena A, Katerina G, Effect of endurance training on diurnal rhythms of superoxide dismutase activity, glutathione and lipid peroxidation in plasma of pinealectomized rats, Neuroscience Letters (2019), doi: https://doi.org/10.1016/j.neulet.2019.134637

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Effect of endurance training on diurnal rhythms of superoxide dismutase activity, glutathione and lipid peroxidation in plasma of pinealectomized rats

Jana Tchekalarova1*, Tzveta Stoyanova1, Zlatina Nenchovska1, Natasha Ivanova1, Dimitrinka

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Atanasova1,2, Milena Atanasova3, Katerina Georgieva4

*Address for correspondence: Institute of Neurobiology, Acad. G. Bonchev Str., Bl. 23,

Bulgarian Academy of Sciences, Sofia 1113, Bulgaria; e-mail: [email protected]

Institute of Neurobiology, Bulgarian Academy of Sciences, Sofia 1113, Bulgaria

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Department of Biology, Medical University of Pleven, 1 Kliment Ohridski Str., Pleven 5800,

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Bulgaria

Department of Anatomy, Faculty of Medicine, Trakia University, Stara Zagora, Bulgaria

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Department of Physiology, Medical University-Plovdiv

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Graphical abstract

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Highlghts

 Exercise did not modify melatonin deficit in pinealectomy.

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 Exercise affected differently the SOD activity in the sham- and pin-treated group.  The SOD activity was higher at the beginning of the light period in the pin-ex group.

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 Exercise did not influence the GSH levels in the pinealectomized rats.  Exercise alleviated lipid peroxidation during the dark period in pinealectomized rats

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Abstract

Melatonin deficit is characterized by disturbed circadian rhythms of many physiological and biochemical parameters including markers of oxidative stress. Moderate endurance training

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exerts protection against oxidative stress. In the present study, we aimed to explore the impact of endurance treadmill training on disturbed rhythmic fluctuations of some markers of oxidative stress in pinealectomized rats. Animals were divided into four groups: sham-operated sedentary

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rats (sham-sed), a sham group with exercise (sham-ex), pinealectomized sedentary rats (pinsed) and pin rats with exercise (pin-ex). Animals were sacrificed by decapitation at 4-h intervals

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for biochemical analysis of plasma melatonin and markers of oxidative stress. The activity of superoxide dismutase (SOD) and the levels of glutathione (GSH) and lipid peroxidation

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demonstrated diurnal variations in the sham-sed group. The peak values of SOD were detected during the dark period that coincided with the peak plasma levels of melatonin in the sham-sed

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rats. The malondialdehyde (MDA) levels also showed a tendency to a progressive raise during the dark period. Pinealectomy was characterized by a remarkable melatonin deficit in plasma

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of sedentary rats, compromised fluctuations with decreased SOD activity and increased lipid

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peroxidation. While endurance training was unable to restore the melatonin deficit, it partly prevented the oxidative stress at selected time points in the pinealectomised rats. Our findings indicate the important role of endurance training against oxidative stress both in physiological conditions and melatonin deficit. Keywords: Melatonin deficit, Endurance training, Diurnal rhythms, SOD, GSH, MDA.

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Introduction

The diurnal fluctuations of melatonin release from the pineal gland, which is under control of the suprachiasmatic nucleus (SCN), are crucial for the oscillations in the body at the molecular, cellular and tissue level and for synchronizing physiological parameters with the light/dark cycle [1]. A reduced pineal secretion of melatonin can cause impaired synchronization of multiple physiological functions, including in sleep, blood pressure and homeostasis of

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hormones [2], [3], [4], [5]. Due to its regulatory control of the sleep-wake cycle, melatonin is broadly used in the therapy of impaired circadian rhythms associated with a jet-lag syndrome,

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prolonged light exposure, as well as age-related and seasonal changes.

Considered one of the strongest lipophilic free radical scavengers that neutralize free

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radicals and their harmful effect on cellular functions [6], [7], [8], the hormone melatonin, is with twice higher effectiveness compared to another powerful antioxidant vitamin E [9]. Its

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positive influence on the activity of antioxidant enzymes, including the superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) suggests that this hormone has a crucial impact

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on the regulation of oxidative stress in the body [10], [11]. Abnormal functional changes caused by the uncontrolled cellular oxidative stress due to free-radical processes can be observed both

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in the elderly as well as in earlier periods characterized by a melatonin deficiency [12], [13].

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In the light of the key role of the hormone as a synchronizer of the circadian rhythms of many biochemical and neurobiological parameters, it can be assumed that the release during the dark phase of the endogenous melatonin from the pineal gland is involved in the fluctuations of the markers of oxidative stress as well. Indeed, the products of lipid peroxidation and endogenous antioxidant signaling molecules are characterized by circadian dynamics in formation and activity, respectively, both in physiological and pathological states such as melatonin deficit in a variety of tissues [13]. The fluctuations in the production of lipid

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peroxides are closely related to the diurnal rhythm of activity of antioxidant enzymes such as SOD and catalase [14]. Thus, the peak levels of lipid peroxides, as well as the activity of antioxidant enzymes SOD and GSH-Px were detected at the beginning of the dark phase under physiological conditions in rats [13], [15]. However, under pathological conditions, the precise mechanism maintaining a synchronized circadian rhythm of melatonin release from the pineal gland and activity of the antioxidant system can be disrupted [16]. Lipid peroxidation and the activity of antioxidant enzymes SOD and GSH-Px were higher during the dark period and low

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during the light period but with significantly diminished amplitudes compared to controls in different tissues in rats with pinealectomy [13] suggesting that the endogenous melatonin is involved in the regulatory mechanism of antioxidant enzymes. Furthermore, nocturnal light can

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suppress both the release of melatonin and the antioxidant signaling system [12].

Exercise has a chronotropic activity and can influence the mammalian circadian clock

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[17]. So far, there is no experimental data about the effect of aerobic exercise on diurnal

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fluctuations of markers of oxidative stress in a model of melatonin deficiency. Experimental and clinical studies on the protective effect of this alternative approach against cellular oxidative stress provide a basis for a working hypothesis according to which the application of

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endurance training would have a positive effect on disturbed 24-hour fluctuations of markers of oxidative stress in melatonin deficiency. Therefore, we aimed to explore the influence of

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regular physical exercise on the presumably desynchronized rhythms of the markers of

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oxidative stress in a model of melatonin deficiency induced by pinealectomy.

2. Materials and methods 2.1. Animals Young adult male Wistar rats (250-300 g), obtained from the animal facility of the Institute of Neurobiology, Bulgarian Academy of Sciences were housed (n=3-4 per cage) in standard

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conditions at room temperature 20 oC, 50-60 % relative humidity and a constant light/dark cycle (12 h/12 h, lights on at 06:00 a.m.). Food (standard laboratory chow) and water were given ad libitum. The experimental procedures, approved by the local Ethics Committee of the Institute of Neurobiology, Bulgarian Academy of Sciences were performed following the European Communities Council Directive 2010/63/EU.

2.2. Experimental design

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The detailed experimental protocol is depicted in Scheme 1. One hundred and forty-four rats from the following four groups (n = 36 in each group with 6 subgroups n=6 for each time points of euthanasia – six time points) were used: sham-operated sedentary rats (sham-sed), sham

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group with exercise (sham-ex), sedentary rats with pinealectomy (pin-sed) and pinealectomized

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rats with exercise (pin-ex).

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2.3. Surgery procedure

Removal of the pineal gland was executed as previously reported [18] and according to the procedure described by Hoffmann and Reiter [19]. In brief, the pineal gland was removed

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through thin forceps in anesthetized with ketamine (40 mg/kg, i.p.) and xylazine (20 mg/kg, s.c.) rats. A similar procedure was applied over the sham groups but without the last action of

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the pineal gland removal. The removal of the pineal gland was confirmed for each rat by both

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morphological and biochemical analysis of plasma melatonin level post-mortem.

2.4. Exercise procedure The exercise procedure was initiated at least 15 days after surgery and was executed between ZT0 and ZT2 (at the very beginning of the light period). As running on a treadmill is a skill activity that the rats should develop before the experiment, all of them were trained 3 times a

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week on a treadmill (EXER-3R Treadmill, Columbus Instruments, Columbus, OH, USA) for 5 min daily for a week [20]. Such a workload induces no training adaptations, but familiarizes the animals with treadmill running and allows a selection of rats which run spontaneously [20]. Exercised rats were regularly trained on a treadmill with a velocity of 20 m/min, 0°slope (about 50 - 55% of VO2max), 5 days a week for 4 weeks. The exercise intensity was set according to the maximal lactate steady state, established for Wistar rats during treadmill running. The running duration was 20 min on day 1 and then gradually increased by 5 min every day. By the

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end of the first week, it reached 40 min a day and remained so until the end of the training period. The sedentary rats were handled every day and were placed on the treadmill for 5 min 3 days a week without running so that they experience similar stress stimuli to those of the

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training rats.

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2.5. Biochemical methods

The animals from the four groups were sacrificed 24 hours after the last exercise session under

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weak anesthesia with CO2 every 4th hour throughout the light/dark cycle, according to the zeitgeber time (ZT) as follows: at ZT0, ZT4, ZT8, ZT12, ZT16, and ZT20. In agreement with

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the nomenclature, zeitgeber time zero (ZT0) was accepted the moment that lights went on and zeitgeber time twelve (ZT12) when lights went off. Trunk blood, collected rapidly into plastic

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Vacutainer® Heparin Tubes, was centrifuged at 1500 rpm for 15 min at 4 oC. The plasma was stored at -70 oC until analysis. The euthanasia was conducted under dim red light at the time points during the dark phase to avoid the influence on the circadian rhythm of melatonin release in physiological conditions.

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2.5.1. Measurement of melatonin plasma levels Melatonin was measured by Elisa Plate Reader InfiniteF200Pro, TECAN, Austria and ELISA kit (Enzo, Switzerland) was used based on the instructions of the manufacturer. Each sample was measured in duplicate, and the average was calculated. The results were expressed as ng/ml.

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2.5.2. Measurement of markers of oxidative stress in plasma Cytosolic superoxide dismutase activity (SOD). SOD activity was determined with SOD assay kit (Cayman Chemical Company, USA). The absorbance was read at 440-460 nm. Each sample

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was measured in duplicate, and the average was calculated. The results were expressed as U/ml. Total glutathione (GSH). The amount of GSH was determined with the Glutathione assay kit

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(Cayman Chemical Company, USA). The absorbance was read at 405-414 nm. Each sample

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was measured in duplicate and the average was calculated. The results were expressed as µM. Lipid peroxidation. The extracted lipid peroxides were assayed with Thiobarbituric Acid

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Substances (TBARS) assay kit (Cayman Chemical Company, USA). The resulting TBARS were detected by reading the absorbance at 530-540 nm. Each sample was measured in

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duplicate and the average was calculated. The extent of lipid peroxidation was expressed as the

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amount of malondialdehyde (MDA) in µM.

Statistical analysis Experimental data were presented as mean±S.E.M. First, experimental data were checked for normality to estimate their appropriateness for parametric statistical tests. SigmaStat®11.0 software statistical package was used for the analysis of all experimental results. A three-way ANOVA with Surgery (sham/pinealectomy), Treatment (sedentary/exercise) and Time (ZT0,

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ZT4, ZT8, ZT12, ZT16, and ZT20) as independent factors with Bonferroni post hoc test was performed. Significance was set at p ≤ 0.05.

3. Results 3.1. Diurnal fluctuations of plasma melatonin levels Removal of the pineal gland was associated with impaired diurnal fluctuations of plasma melatonin levels (main effect of Surgery) at selected time points (main effect of Time) (Table

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1). Factor Treatment was without a significant effect on the melatonin level in plasma (P = 0.98) (Table 1). Post hoc test showed significantly different levels of plasma melatonin at selected time points in sedentary rats as follows: ZT8 vs. ZT12 (p = 0.009), ZT8 vs. ZT16 (p = 0.004),

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ZT8 vs. ZT20 (p = 0.005), respectively, within sham-sed group (Fig. 2). For exercised shamtreated rat, a significant increase of plasma melatonin level was detected at ZT 20 vs. ZT0(24)

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(p = 0.02) and ZT8 (p = 0.02), respectively. Pin-treated rats demonstrated a flattened pattern

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and a lack of diurnal rhythm of plasma melatonin levels (p > 0.05). Plasma melatonin levels in pin-sed group were significantly diminished at ZT12 (p = 0.03) and ZT16 (p = 0.009) when compared to the correspondent sham-sed group. The exercise did not influence plasma

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melatonin levels in pinealectomized rats.

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3.2. Diurnal fluctuations of plasma superoxide dismutase (SOD) activity

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Exercise affected differently the SOD activity in the sham- and pin-treated group (main effect of Treatment and Surgery, respectively) and this effect was independent on Time (Table 1). Post hoc test demonstrated a significant increase of SOD activity at ZT16 compared to ZT8 within sham-sed group (p = 0.02), while endurance training increased the enzyme activity in control at ZT8 (p = 0.05) (Fig. 3). Pinealectomy significantly diminished the activity of SOD during the dark period at ZT12 (p = 0.045) and ZT20 (p = 0.05), respectively, compared to the

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sham-sed group. Endurance training exerted a beneficial effect on the SOD activity in pinealectomized rats at the beginning of the light period (ZT0, p = 0.02) and ZT4 (p = 0.04), respectively.

3.3. Diurnal fluctuations in plasma glutathione (GSH) levels The plasma levels of GSH levels were affected by Time (main effect) (Table 1). Although

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Exercise per se did not affect GSH, a significant Treatment x Time interaction was detected (p = 0.05) (Table 1). Post hoc test demonstrated an increase of GSH levels at ZT12 in sham-ex group compared to sham-sed group (p = 0.007) (Fig. 4). Circadian fluctuations of GSH levels was showed both in the sham-sed group ZT0 vs. ZT8 (p = 0.02), ZT8 vs. ZT20 (p = 0.009) as

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well as the sham-ex group ZT0 vs. ZT12 (p = 0.002), ZT4 vs ZT12 (p = 0.003), ZT8 vs ZT12

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(p = 0.005), ZT12 vs ZT16 (p = 0.04), ZT12 vs ZT20 (p = 0.05), respectively. No changes in the GSH levels resulting from endurance training in pinealectomized rats were detected (p >

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0.05).

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3.4. Diurnal fluctuations in plasma malondialdehyde (MDA) levels There was a main effect of Surgery, Treatment and Time as well as Surgery x Treatment x Time

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interaction for plasma MDA levels (Table 1). Separated analysis by Surgery demonstrated that plasma lipid peroxidation was alleviated by exercise treatment at selected time points: ZT0 (p

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= 0.02) and ZT16 (p = 0.02), respectively, in the sham group (Fig. 5). Further, while pinealectomy was characterized by overall increased MDA levels mostly during the dark period at ZT12 (p = 0.001) and ZT16 (p = 0.004), respectively, pin-sed group compared to sham-sed group, exercise-induced flattened pattern and significantly decreased lipid peroxidation at ZT4 (p = 0.02), ZT12 (p = 0.004), and ZT16 (p = 0.004), respectively, was detected in pin-ex group compared to pin-sed group.

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4. Discussion In agreement with our recent study and literature data [4], [21], [21], in the present work, the plasma levels of melatonin were characterized by a diurnal rhythmic pattern showing increased levels after light off and low levels after light on in the sham-sed group. The endocrine function of melatonin is closely associated with the pineal gland where it is released in the blood while in other tissues, including the gastrointestinal tract it behaves as a paracrine hormone

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[22]. Removal of the pineal gland caused a drop in plasma melatonin levels during the dark phase and a flattened 24-h pattern [23], [24]. The long-term bright illumination also can induce melatonin deficiency both in experimental animals [4], [25], [26] and humans [27]. Endurance

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training was unable neither to modify the diurnal rhythm of melatonin release in the sham group nor the low hormonal level detected during the dark phase in pinealectomized rats. This result

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was expected because although this alternative treatment is well-known to exert its beneficial

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effects through a stimulatory impact on a variety of biochemical processes, exercise treatment cannot mimic melatonin activity on specific receptors, thereby triggering different intracellular processes. Moreover, Buxton et al. [28] reported in humans that repeated training during the

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resting (dark period) resulted in a shift of melatonin release from the pineal gland while exercise procedure applied during the active (light period) does not affect melatonin secretion.

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It is well-known from the literature that there is a close relationship between the

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circadian control of protein expression from the SCN and cell response to oxidative stress [29]. The antioxidant molecules are characterized by fluctuations in the activity or levels in plasma, brain, liver, kidney, heart, pancreas, gut [30]. In the present, the rhythmic activity of SOD in the sham-sed group study is in line with previously reported data [15] that coincided with a detected diurnal pattern of melatonin release. Nocturnal rats, characterized by a higher metabolism during the active dark period, are expected to have increased lipid peroxidation and

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a resulting increase of SOD activity engaged in the neutralization of the products of oxidative stress. The higher plasma levels of melatonin during the dark period might contribute to the enhanced activity of this antioxidant enzyme. This assumption is further confirmed with observed drop in the SOD activity over the whole dark period as a result of melatonin deficit in the pinealectomized group suggesting a pivotal role of endogenous hormone on this antioxidant anzyme. Endurance training triggers a chain of biochemical reactions resulting in increased

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oxidative stress, including a raise of superoxide anion radical (O2•–), due to the higher oxygen consumption [31]. The SOD enzyme appears the first antioxidant barrier against the production of reactive oxygen species (ROS). Removal of the pineal gland resulted in a flattened rhythm

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and significantly decreased the activity of this antioxidant enzyme during the dark period. This result agrees with literature data revealing that melatonin can enhance gene expression of

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antioxidant enzymes, including SOD both in physiological and pharmacological levels [10].

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Furthermore, diminished activity of SOD was detected in several brain structures of birds exposed to constant chronic light [30].

Exercise as an alternative beneficial treatment is reported to enhance SOD activity in

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humans [31]. However, exercise load, duration, and diet are crucial parameters that influence oxidative stress. Alternatively, the antioxidant system can be positively affected by aerobic

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training [32]. In the present study, although the exercise caused diurnal variations of SOD in

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physiological conditions, a tendency of increased enzyme activity, which was significantly higher compared to sedentary controls, was detected. Moreover, the endurance training enhancement of SOD activity during the light period in the pinealectomized group suggests that endogenous melatonin is not involved in the mechanism underlying its beneficial effect on the first defensive enzyme. The observed increased SOD activity as a result of endurance training might occur due to the protocol of training performed during the light period.

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One of the strongest natural scavengers of free radicals, melatonin is closely related to the glutathione cycle, including the reduced GSH and the antioxidant enzyme GPx [32]. Both melatonin and GPx are involved in the neutralization of the H2O2, which increased levels in the organism predispose to the production of more reactive and dangerous free radical .OH. Moreover, exogenously delivered melatonin contributes to increased activity of this important antioxidant enzyme and the levels of reduced GSH, respectively [10], [13]. Previous reports revealed that the circadian rhythmic pattern of GPx activity is closely related to that of the

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endogenous melatonin, which is substantial during the dark period [13]. Our data agree with literature data revealing that the variability in the reduced GSH is reciprocally associated with marker of lipid peroxidation [15]. In the present study, pinealectomy did not produce significant

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changes in the fluctuations of the reduced GSH in plasma, although previous reports in different tissues suggest a close link between melatonin and glutathione cycle [10], [34]. Aerobic

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exercise has been reported to alleviate oxidative stress through the increase of antioxidant

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enzyme activity at rest [35]. In our study, under control conditions, endurance training significantly raised the reduced GSH, which levels are closely related to the degradation of H2O2, at the onset of light off regimen. However, under pathological conditions, exercise did

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not influence the GSH levels in plasma suggesting a crucial role of endogenous melatonin in the beneficial effect of this alternative treatment.

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The fluctuations in the plasma MDA were negligible in physiological conditions

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(sham-sed group) in our experiments. However, under pathological state, diurnal fluctuations of lipid peroxidation appeared as a sequence of melatonin deficit. Rhythmicity in the lipid peroxidation in the brain was reported to exist both in control and pinealectomozed rats with a peak during the dark period [13], suggesting a lack of a close relationship between melatonin levels and MDA rhythms. Literature data considering circadian rhythms of lipid peroxidation are contradictive. Thus, fluctuations in the lipid peroxidation were detected in different tissues,

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including rat brain, liver, and heart [15], [36], [37]. However, peak values were reported during the light phase in the liver [37] but during the dark phase in the brain and heart [15], [36]. In agreement with our result showing a flattened pattern of MDA levels in the sham-veh group, a lack of diurnal variability in human serum MDA was detected previously [37]. Burmistrov et al. [37] showed that while the lipid peroxidation is low and without variability in controls from rat ovarian tissue, it is time-dependent and elevated under exposure to toxin toluene suggesting that the circadian oscillations of lipid peroxidation are associated with pathological conditions

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characterized by increased oxidative stress. In the present study, exercise treatment in physiological conditions exerted a significant time-dependent decrease in the MDA compared to the sham-sed group. Our results are in line

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with previous findings that chronic but not single training can alleviate lipid peroxidation in tissue brain homogenates [38]. The attenuation in the lipoperoxidation peaked at ZT16, which

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did not exactly coincide with the peak level of plasma melatonin, suggesting a lack of a direct

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link between the exercise treatment and the hormone. This assumption is confirmed by the effect of exercise on the MDA levels in pinealectomized rats, demonstrating a lack of circadian rhythm under pathological conditions. Thus, aerobic training exerted overall suppression of

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lipid peroxidation both during the light and the dark phases. In conclusion, our results revealed that while endurance training did not affect the low

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melatonin levels in rats with pinealectomy it alleviated oxidative stress in a time-dependent

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manner. Future experiments are needed to ascertain the precise mechanism underlying the beneficial activity of this alternative approach in melatonin deficiency, which possibly not involve the participation of the melatonin system.

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Acknowledgments This work was supported by the National Science Fund of Bulgaria (research grant # № DN

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03/10; DM 11/4).

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Text to figures

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Fig. 1 Experimental design.

) in plasma of sham-operated sedentary rats (sham-sed), sham rats exposed to long-term

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Fig. 2 Effect of pinealectomy and aerobic exercise on diurnal fluctuations of melatonin (ng.ml-

aerobic exercise (sham-ex), sedentary rats with pinealectomy (pin-sed) and pin-ex rats. Each group consisted of n=6-8 rats decapitated every 4 h. Results are expressed as means ± SEM. Three-way ANOVA followed by Bonferroni: at ZT12 *p = 0.03 and at ZT16 **p = 0.009, respectively, pin-sed compared to sham-sed group. The black box represents the dark phase of the light-dark cycle. ZT, zeitgeber time; ZT0 = ZT24.

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Fig. 3 Effect of pinealectomy and aerobic exercise on diurnal fluctuations of superoxide

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dismutase (SOD) activity (U.ml-1) in plasma of sham-sed, sham-ex, pin-sed and pin-ex rats.

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Results are expressed as means ± SEM. Three-way ANOVA followed by Bonferroni: at ZT8 *p = 0.05 sham-ex compared to sham-sed group; at ZT12 *p = 0.045 and ZT20 *p = 0.05,

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respectively, pin-sed compared to sham-sed group; at ZT0(ZT24) op = 0.02 and ZT4 op = 0.04,

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respectively, pin-ex compared to pin-sed group. Details as in Fig. 2.

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Fig. 4 Effect of pinealectomy and aerobic exercise on diurnal fluctuations of glutathione level (µM) in plasma of sham-sed, sham-ex, pin-sed and pin-ex rats. Results are expressed as means ± SEM. Three-way ANOVA followed by Bonferroni: at ZT12 **p = 0.007 sham-ex compared

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to sham-sed group. Details as in Fig. 2.

Fig. 5 Effect of pinealectomy and aerobic exercise on diurnal fluctuations of malondialdehyde

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(MDA) level (µM) in plasma of sham-sed, sham-ex, pin-sed and pin-ex rats. Results are expressed as means ± SEM. Three-way ANOVA followed by Bonferroni: at ZT0(24) *p = 0.02

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and ZT16 *p = 0.02, respectively, sham-ex vs. sham-sed group; at ZT12 ***p = 0.001 and ZT16 **p = 0.004, respectively, pin-sed compared to sham-sed group; at ZT4 oop = 0.02, ZT12 oop = 0.004, and ZT16 oop = 0.004, respectively, pin-ex compared to pin-sed group. Details as in Fig. 2.

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Table 1 Effect of Surgery, Exercise, and Time on plasma melatonin levels and components of oxidative stress (superoxide dismutase (SOD), glutathione (GSH), and malondialdehyde

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(MDA) level analyzed by a three-way ANOVA test.

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Table 1

Effect of Surgery, Exercise, and Time on plasma melatonin levels and components of oxidative stress (superoxide dismutase (SOD), glutathione (GSH), and malondialdehide (MDA) level analyzed by a

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three-way ANOVA test.

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Three-way ANOVA

Melatonin

Surgery [F1,121 = 9.831 P = 0.002]

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Treatment [F1,121 = 0.000766 P = 0.978]

SOD activity

Time [F5,121 = 2.440 P = 0.040] Surgery [F1,104 = 5.793 P = 0.018] Treatment [F1,104 = 4.013 P = 0.048] Time [F5,104 = 0.868 P = 0.506]

GSH

Surgery [F1,77 = 0.0171 P = 0.896] Treatment [F1,77 = 1.512 P = 0.224] Time [F5,77 = 3.172 P = 0.014]

26 Excerise x Time interaction [F5,77 = 2.379 P = 0.05]

Surgery [F1,112 =12.674 P < 0.001]

MDA

Treatment [F1,112 =13.026 P < 0.001] Time [F5,112 =11.796 P < 0.001]

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Surgery x Treatment x Time interaction [F5,112 = 11.914 P < 0.001]