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Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. References

Cellular defence against the formation of reactive oxygen species (ROS) involves a number of mechanisms in which antioxidant enzymes such as catalase (CAT) and superoxide dismutase (SOD) play an important role. The relation between sleep deprivation and oxidative stress has not yet been completely elucidated. Although some authors did not find evidence of this relationship, others found alterations in some oxidative stress markers in response to sleep deprivation. Thus, the objective of this study was to identify changes induced by sleep deprivation in the activity and gene expression of antioxidant enzymes in mice splenocytes, ideally corroborating a better understanding of the observed effects related to sleep deprivation, which could be triggered by oxidative imbalance. Splenocytes from mice sleep deprived for 72 h showed no significant difference in CAT and CuZnSOD gene expression compared with normal sleep mice. However, sleep-deprived mice did show higher MnSOD gene expression than the control group. Concerning enzymatic activity, CuZnSOD and MnSOD significantly increased after sleep deprivation, despite the expression in CuZnSOD remained unchanged. Moreover, CAT activity was significantly lower after sleep deprivation. The data suggest that the antioxidant system is triggered by sleep deprivation, which in turn could influence the splenocytes homoeostasis, thus interfering in physiological responses.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. References

Sleep deprivation (SD) is an increasing common occurrence in modern life and results in the predisposition of many diseases that can be induced by immune system deficiency, endocrine deregulation and oxidative stress (OS). Reactive oxygen species (ROS) generation is widely described in phagocytic cells, such as neutrophils and macrophages, as well as in cellular signalling pathways [1, 2]. Despite the activation of ROS respiratory burst to maintain the strength of the immune system, a number of cellular processes exist in which an imbalance occurs between the production and degradation of ROS, resulting in membrane lipid peroxidation (LPO), protein oxidation and nucleic acid injury, which culminates in cell death [3, 4].

It is widely known that OS enhances the development of many physiopathological conditions, such as neurodegenerative, vascular and chronic inflammatory diseases [4]. During oxidative phosphorylation in internal mitochondrial membranes, electrons from substrate oxidations are transferred to O2, process in which superoxide anions can be produced at either complex I and/or at the ubiquinone level; an excess of these species leads to OS [5]. An interesting consequence of the uncontrolled production of free radicals in the membrane of cell organelles (mitochondria, lysosomes and endoplasmic reticulum) is calcium (Ca2+) leakage, which occurs concomitantly with the antioxidant enzyme release to cytosol. These events lead to cytotoxicity and may cause cell death by apoptosis or necrosis [6, 7]. However, cells have a defence mechanism against the formation of ROS which includes antioxidant enzymes, for example, catalase (CAT) and superoxide dismutase (SOD).

Recent studies have shown that during the sleep–wake cycle, there is a differential regulation of genes encoding proteins involved in vesicular pool functioning, antioxidant defence and intracellular transport [8, 9]. These findings point to the importance of sleep in gene expression control. Thus, the objective of this study was to identify possible sleep deprivation (SD)-induced changes in activity and gene expression of antioxidant enzymes in splenocytes, which may help to explain the observed deficiency in the immune system observed after SD. Spleen cells were chosen because recently our group showed that SD results in the commitment of calcium homoeostasis in these cells [10], which, in turn, could be related to oxidative imbalance.

Materials and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. References

Animals

Male Swiss mice (n = 8 for each group), aged 4 months and weighing 30–35 g, from a colony maintained by the Department of Psychobiology – Universidade Federal de São Paulo (UNIFESP), were used in this study. Animals were maintained on a light:dark 12:12 cycle under controlled temperature conditions (20 ± 2 °C) with free access to food and water. Mice used in this study were maintained and treated in accordance with the guidelines established by the Ethical and Practical Principles of the Use of Laboratory Animals [11], and the study was approved by the Ethical Committee of the Universidade Federal de São Paulo (CEP UNIFESP n° 0183/08).

Sleep deprivation and experimental protocol

Animals were sleep deprived (SD group) using the classical platform technique adapted to mice. This method consists of placing the animals on a narrow platform (12 × 3 cm diameter each; 14 platforms per container) surrounded by water and placed inside a container (41 cm × 34 cm × 16.5 cm) for a period of 72 h, disrupting sleep when mice touch the water after muscle atonia [12, 13]. Mice from the control group (CT) remained in their cages for the same period and were allowed to sleep. After the SD period, mice were euthanized by cervical dislocation, their spleens were harvested and splenocytes were isolated [14].

Lipid peroxidation determination

Malondialdehyde (MDA) in spleen homogenate was determined by reaction with thiobarbituric acid. The amount of thiobarbituric acid reactive substances (mainly MDA) formed in each sample [15] was assessed by measuring the supernatant optical density at 535 nm using a Hitachi™ spectrophotometer (Hitachi Ltd., Tokyo, Japan). Results were expressed as nanomoles of MDA per gram of spleen dry tissue (nmol MDA/g).

Lipid peroxidative potential

We also analysed the lipid peroxidative potential (LPP) in another assay. Splenocytes were kept at 37 °C for 1 h to evaluate the production of MDA, which was quantified by reaction with thiobarbituric acid. Results were expressed as nanomoles of MDA per mg of protein using the molar extinction coefficient of 1.56 × 105/m/cm [16].

Sample preparation for enzymatic analysis

Spleens were macerated in Hanks buffer salt solution, and the homogenate was centrifuged in PO4K buffer. After, the supernatants were sonicated, centrifuged at 16000 g for 45 min at 4 °C and, CAT and SOD activity measurements were performed.

CAT and SOD activity determination

Spectrophotometric assays for CAT and SOD activities were performed according to Beutler [17] and Ewing and Janero [18], respectively. For mitochondrial SOD (MnSOD) activity measurement, the addition of 15 mm potassium cyanide (KCN) was necessary in the reaction medium to inhibit the cytosolic SOD (CuZnSOD) activity. Results were expressed as U (units)/mg Hb (haemoglobin).

Total protein measurement

To normalize the LPP levels and splenic antioxidant enzymes activity, the content of total proteins was measured in the respective homogenates using a Bio-Rad kit.

Gene expression

RNA was isolated using the TRIZOL® (Life Technologies, Carlsbad, CA, USA) method, following the manufacturer's protocol, and reverse transcribed using ImProm-IITM Reverse Transcriptase (Promega, Madison, WI, USA). Diluted cDNA was added to SYBR Green PCR Master Mix (Applied Biosystems, Warrington, UK) in addition to the respective primers for CAT, MnSOD and CuZnSOD (Table 1). The expression of the targeted genes was normalized using GAPDH as the endogenous control. Quantitative real-time PCR assays were performed using 96-well plates sealed with optical quality film (Applied Biosystems) on a DNA thermal cycler (Bio-Rad, Hercules, CA, USA) using the CFX96 Real-Time System. Gene expression was analysed by the 2−ΔΔCt method and is expressed in arbitrary units.

Table 1. Primers used for gene expression assays
GenesPrimers sequences
CAT

AGC GGA TTC CTG AGA GAG TG

GAG AAT CGA ACG GCA ATA GG

MnSOD

ACA CAT TAA CGC GCA GAT CA

AAT ATG TCC CCC ACC ATT GA

CuZnSOD

CCA GTG CAG GAC CTC ATT TT

CCT TTC CAG CAG TCA CAT TG

GAPDH

TGC ACC ACC AAC TGC TTA

GGA TGC AGG GAT GAT GTT

Statistical analysis

Comparisons were performed by Student's t-test, and all data are expressed as mean ± SEM. Calculations were performed using PrismTM version 4.03 for Windows (GraphPad Software Inc., La Jolla, CA, USA). The level of significance was set at P < 0.05.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. References

For verification of the OS in the spleen, we determined lipid peroxidation (LPO) levels between groups (CT and SD). As shown in Table 2, no difference was observed between SD and CT groups (n = 8, = 0.72). We also determined LPP by submitting spleens to oxidation, but differences between the groups were not observed (Table 2, n = 8, P = 0.44). Although no changes were observed that could indicate oxidative damage, we sought to verify antioxidant enzymes activity. We found lower CAT activity in the SD group than in CT group (Table 2, n = 8, P < 0.05). Moreover, the total SOD activity (mitochondrial and cytosolic) in spleen homogenates was higher in the SD than the CT group (Table 2, n = 8, P < 0.05).

Table 2. Determination of oxidative stress parameters by malondialdehyde (MDA) quantification and antioxidant enzymes activities in mice splenocytes
Oxidative stress parametersCT72 h SD
  1. LPO, Lipid peroxidation; LPP, Lipid peroxidative potential; CAT, catalase; SOD, superoxide dismutase; Mn, manganese; CuZn, copper zinc; CT, control group; SD, sleep deprived (n = 8/group Student t-test, *P < 0.05; **P < 0.01).

  2. Values are presented as mean ± SD.

MDA (LPO) (nmol/g dry tissue)8.87 ± 1.428.18 ± 4.61
MDA (LPP) (nmol/mg protein)20.26  ± 3.4819.59 ± 3.43
CAT (U/mg protein)5.75 ± 1.084.03 ± 0.88**
Total SOD (U/mg protein)14.25 ± 3.0318.8 ± 2.59**
MnSOD (U/mg protein)8.27 ± 1.9611.17 ± 2.15*
CuZnSOD (U/mg protein)5.53 ±0.927.62 ± 1.25**

Concerning SOD isoforms activity, we found a significant increase in MnSOD activity in the SD group (Table 2, n = 8, P < 0.05) compared with the CT group. We also observed a significant increase in CuZnSOD activity in the SD group (Table 2, n = 8, P < 0.01) compared with the CT group.

Moreover, we analysed gene expression of both antioxidant enzymes, CAT and SOD. We observed a significant increase in MnSOD gene expression after SD (Table 3, n = 8, P < 0.05); however, no differences were found in CuZnSOD and CAT expression normalized by GAPDH (Table 3, n = 8, P > 0.05). These results were confirmed using β-actin as housekeeping gene (data not shown).

Table 3. Gene expression of antioxidant enzymes in mice splenocytes
Gene expression (Arbitrary units)CT72 h SD
  1. CT, control; SD, sleep deprived.

  2. Values are presented as mean ± SD. The GADPH gene was used as reference (n = 8/group, Student t-test, *P < 0.05).

CAT1.06 ± 0.370.82 ± 0.22
MnSOD1.04 ± 0.351.67 ± 0.19*
CuZnSOD1.08 ± 0.450.815 ± 0.23

Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. References

A number of studies have shown that SD induces oxidative processes in several types of tissues [19-22], resulting in some cases, in cognitive impairment [23, 24] and behavioural changes [25]. Excessive endogenous formation of free radicals may be caused by the following different factors: increased activation of phagocytes; disruption of the normal processes of electron transfer in the mitochondrial respiratory chain, an increase in the concentration of transition metal ions and decreased levels of antioxidant defences [3, 26, 27]. Additionally, Gopalakrishnan et al. [27] found that, after deprivation of total (8 h) and paradoxical (3–14 days) sleep, rats did not show differences in their generation of LPO in brain and liver tissues and skeletal muscle. These data confirm our findings in determining LPO in spleen tissue, as well as stimulating the generation of tissue with the LPP (Table 2).

Kim et al. [28] showed changes in levels of corticosterone and pro-inflammatory cytokines as well as increased levels of OS markers and neurodegenerative processes in the hippocampus of rats after sleep restriction and other forms of SD. D'Almeida et al. [21] demonstrated that, after 96 h of SD using a single-platform method, rats had reduced glutathione levels in the brain. We also observed a significant reduction in CAT activity in the experimental model of SD by 72 h, and measurements of the total SOD activity performed showed a significant increase after SD (Table 2). These results suggest that SD may cause an augmented production of ROS which, in turn, results in an imbalance of antioxidant defence. According to Everson et al. [20], a reduction of 23% and 36% in liver CAT activity was observed in rats that were sleep deprived for 5 and 10 days, respectively, by the disc-over-water method. Atli et al. [29] demonstrated that the imbalance of metallic ions and low cytosolic pH decreases CAT activity. As shown in our previous study [10], splenocytes from SD mice have ruptured lysosomes, which could lead to a lower cytosolic pH and a consequent reduction in CAT activity.

Mitochondria are considered key organelles in the generation of inline image, which in turn activates the phosphorylation chain to ATP production by the subsequent capture of Ca2+ [30]. MnSOD is localized inside mitochondria, and when it is overexpressed, it can affect the function of this organelle. Rodriguez and Ochoa [31] observed a 15–20% reduction in ATP levels in fibrosarcoma cells when MnSOD was overexpressed. The significant increase in MnSOD activity (Table 2) and the increased mRNA expression of this enzyme (Table 3) after SD suggest a compensatory response to control the higher generation of mitochondrial superoxide anion. Goldsteins et al. [32] showed that an increased activity of CuZnSOD in the mitochondrial intermembrane space from blood lymphocytes of mice resulted in a higher production of peroxides. The experiment also indicated that there was a subsequent release of cytochrome c that contributed to the activation of the apoptotic pathways. Considering that we found increased levels of CuZnSOD activity in splenocytes of mice after 72 h of SD when compared with the CT group (Table 2), it is possible that SD could also trigger cell death by related mechanisms. In fact, we recently showed a mitochondrial calcium gradient disruption after SD that could be another factor contributing to cell death [10].

Some studies have shown that the reaction of cytochrome c with hydroperoxides results in the formation of oxoferryl cytochrome c radical, a highly reactive species that can oxidize proteins, DNA, lipids and endogenous antioxidants [33-37]. Because we did not find differences in gene expression of CAT and CuZnSOD (Table 3), it is possible that the formation of hydroperoxides radicals by mitochondrial dysfunction may have primarily triggered post-transcriptional mechanisms that have fewer effects in gene expression. Accordingly, Naidoo et al. [9] observed that SD causes ER stress, which in turn could alter formation and/or maturation of enzymes, as CAT and CuZnSOD, without changing gene expression.

Sarsour et al. [38] demonstrated that cell cultures exposed to concentrations of 21% O2 showed increased mitochondrial ROS generation with subsequent increases in MnSOD activity, as compared to the cultures exposed at 4% O2. Andreazza et al. [39] showed the reduced activity of complex I-III in the hypothalamus of SD mice, suggesting that sleep plays an important role in energy metabolism. In our work, higher MnSOD activity and expression that were observed may be the consequence of excessive ROS generation. Considering that nowadays the redox-sensitive transcription factor NF-E2-related factor 2 (Nrf2) is known to play an important role in activating antioxidant enzymes [40], another possible explanation for the increase in MnSOD activity and expression could be the activation of Nrf2 induced by SD.

Mitochondrial Ca2+ overload as a result of cellular excitotoxicity has been associated with the generation of inline image, inducing the release of pro-apoptotic mitochondrial proteins [5]. This process occurs through the fragmentation of DNA, lipids and proteins, as well as through the loss of membrane integrity and ionic imbalance, which culminates in cell death by apoptosis or necrosis [41, 42]. Although several studies show that sleep is essential for the maintenance of health and that its deprivation implicates in many metabolic, endocrine and immune disorders, further studies of cellular and molecular mechanisms are necessary to understanding the impacts of SD inside the cell. Our work showed that SD produces an imbalance in the redox status in spleen cells confirmed by the increase in SOD activity and expression, and reduction in CAT activity. These changes could be related to dysfunction in mitochondrial metabolism and vulnerability in cell signalling pathways, which may explain some of the cytotoxic effects of SD.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. References
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