Objective: To investigate the effects of 72 hours continuous sleep deprivation (SD) on circadian clock gene expression and oxidative stress in the rat liver. Methods: Twenty healthy male Sprague-Dawley rats were divided into 2 groups (n = 10 each) using a random number table: normal control group (group C), sleep deprivation group (group SD). Group SD was treated with a modified multiple platform water environment method. After 72 hours sleep deprived, the levels of AST (Aspartate transaminase ) and ALT (Alanine aminotransferase) in serum were determined. The contents of malondialdehyde (MDA), the activities of superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) in the liver tissue of the rats were examined in both two groups. The expression levels of CLOCK, BMAL1 and CRY1 protein in liver tissue were examined by Western blotting. Results: Compared with group C, the content of MDA, and the levels of AST and ALT in serum were significantly increased (P < 0.05); SOD activity was significantly decreased (P < 0.05); GSH-Px activity was significantly decreased (P < 0.01); and the expression of CLOCK, BMAL1 and CRY1 protein was downregulated in group SD. Conclusion: 72 hours continuous sleep deprivation can downregulate the expression of circadian clock gene and promote oxidative stress in rats.
With the development of modern society and the industry competition, lack of sleep, insomnia and other sleep disorders are increasingly becoming a major public health problem. Sleep disorders not only increase the incidence of accidents in work, but also cause great harm to human health. Sleep deprivation affects many physiological functions, including organ hormone secretion, cognitive ability, memory and immune function. A considerable body of clinical evidence also suggests that sleep deprivation has a close relationship with the expression of circadian clock genes and oxidative stress [
Circadian rhythm is an important physiological process to maintain the homeostasis of organism. The circadian rhythm consists of a set of clock genes, such as CLOCK, BMAL, PER and CRY, which is regulated by the transcription-translational feedback loop composing of the biological clock gene and their encode protein. The CLOCK/BMAL complex activates the expression of PER and CRY. The activated PER and CRY in turn inhibit the activity of the CLOCK/ BMAL complex. In addition to this, recent biochemical and physiological studies have confirmed that sleep deprivation as a strong stressor for the body, increase the level of oxidative stress of the brain, resulting in cognitive dysfunction and memory loss [
Over the past decades, a mounting body of evidence has accumulated that sleep deprivation is associated with the development of many diseases (including cancer, neurological diseases and cardiovascular disease), but little is known regarding whether oxidative stress and circadian oscillations are important consequences of sleep deprivation in digestive system, especially in liver. As we all know, the liver is the main energy metabolism organ, and the liver can maintain energy homeostasis by regulating the expression of circadian clock gene to control the physiological activities [
SPF (specific pathogen free) grade male Sprague-Dawley rats were provided by Hubei Provincial Center for Disease Control and Prevention (Wuhan, China). MDA level, SOD and GSH-Px activities were measured by using commercial kits from Jiancheng Bio-engineering Institute (Nanjing, China). The RIPA buffer and bicinchoninic acid (BCA) assay kit were purchased from Beyotime Biotechnology (Shanghai, China.). The anti-CLOCK, anti-BMAL1, anti-CRY1 and anti- GAPDH antibody were purchased from Gene Tex (Texas, America).
All animal procedures were approved by the ethics committee of Wuhan University and were carried out in accordance with the NIH (National Institutes of Health) Guidelines for the Care and Use of Laboratory Animals. Twenty healthy male Sprague-Dawley rats, weighing 200 - 250 g, aged 10 -12 weeks, were divided into 2 groups (n = 10 each) using a random number table: normal control group (group C), sleep deprivation group (group SD). All rats were fed in a climate-controlled room (22˚C - 24˚C) on a 12/12-h light-dark schedule (lights on at 7 AM) with a free access to food and water. All rats were allowed to acclimate for one week in the aquarium without sleep deprivation before starting the experiments.
Rats in group SD were sleep deprived for a 72-h period using the columns-in-water model (modified multiple platform water environment method). Sleep consists of two main stages non-rapid eye movement (REM) and REM sleep. This method is reported to interfere with both non-REM and REM sleep, but mainly affect REM sleep. The cylindrical aquarium (diameter: 70 cm, height: 40 cm) contained 10 columns (platform diameter: 7 cm, height: 8 cm, with platform 1 cm above the water level), spaced 7 cm apart (edge to edge) [
After 3 days, all rats were sacrificed, the liver tissues and body were weighed, and serum was collected for determination of liver function.
Using the rate method to determine the levels of AST and ALT, the instrument was Modular P800 automatic biochemical analyzer (Roche), and its reagents, standards and quality control products were supported by Roche company too.
All rats were sacrificed after three days of sleep deprivation. The liver samples were taken and washed with ice salt water. The liver tissue was weighed, 4˚C homogenated, and then 3000 r/min 15 min centrifuged. 10% wet weight per volume liver tissue was prepared to determine the oxidative stress. The contents of MDA, the activities of SOD and GSH-Px in the supernatant were performed by assay kits according to the manufacturer’s protocol.
The expression levels of CLOCK, BMAL1 and CRY1 were detected by Western blotting. The total protein was extracted by using RIPA lysis buffer (50 mM Tris, pH 7.4, 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate) and protease inhibitors were added immediately before use. The protein concentration of extracted samples was measured using the BCA protein assay kit. After adding 4 times volume loading buffer, the simples were boiled for 10 minutes to denature. Sample protein containing 20 μg total protein was subjected to SDS-PAGE using 12% resolving gel, followed by transfer to nitrocellulose membranes. After blocking with 5% skimmed milk for 1 hour, the nitrocellulose was then incubated with anti-CLOCK, anti-BMAL1, anti-CRY1 and anti-GAPDH antibody (dilution 1:1000) overnight at 4˚C, and then incubated with the secondary antibodies (dilution 1:5000). The bands were detected by the Odyssey image analyzer. The gray values of target proteins were used to reflect the level of expressions by software Image J.
The data were reported as mean ± standard deviation, analyzed using SPSS version 19.0 software, and compared by the unpaired Student t test. Statistical significance was defined as P < 0.05.
There was no significant difference between the groups before the experiment on the weights of body and liver (P > 0.05). At the end of the experiment, the weights of body and liver of the rats in the sleep deprivation group were decreased than group C, and the differences were statistically significant (P < 0.05) (
At the end of the experiment, the levels of AST and ALT in serum of the rats in the sleep deprivation group were increased than group C, and the differences were statistically significant (P < 0.01) (
Group | Body weight (g) | Liver | |||
---|---|---|---|---|---|
before | after | weight (g) | coefficient | ||
Group C | 242.7 ± 13.2 | 248.4 ± 14.3 | 10.01 ± 2.02 | 3.48 ± 0.22 | |
Group SD | 238.9 ± 11.5 | 235.1 ± 10.6* | 8.43 ± 1.76* | 3.25 ± 0.18 | |
Compared with group C, *P < 0.05.
Group | AST (U/L) | ALT (U/L) | AST/ALT |
---|---|---|---|
Group C | 166.4 ± 38.3 | 35.7 ± 6.21 | 4.50 ± 1.13 |
Group SD | 244.2 ± 70.1** | 64.2 ± 14. 9** | 3.98 ± 0.92 |
Compared with group C, **P < 0.01.
Compared with group C, the content of MDA was significantly increased (P < 0.05), the activity of SOD was significantly decreased (P < 0.05), the activity of GSH-Px was significantly decreased (P < 0.01) in group SD (
Compared with group C, the expression of CLOCK, BMAL1 and CRY1 protein were downregulated in group SD (
Circadian rhythms refer to physiological processes that occur with a repeating period of approximately 24 hours, which are ubiquitously present in prokaryotes, fungi, algae, plants and mammals [
metabolism, as glucose, bile acids, lipids, drug metabolism and enzyme expression and they are all subject to timed circadian control. Thus, liver local biological clock gene can maintain the liver homeostasis by regulating the absorption and metabolism of nutrients [
Ferrell JM et al. found that even short-term destruction of the daily sleep pattern dramatically altered the balance of bile acids metabolism, and lipid homeostasis to contribute to dyslipidemia [
Studies have shown that sleep deprivation is a strong oxidative stress response. Chang HM et al. found that sleep deprivation predisposed liver to oxidative stress and phospholipid damage [
Besides producing increasingly, the scavenging ability reduction is another reason for ROS accumulation. SOD is among the most important members of the antioxidant defense system because of its special physiological activity. GSH-Px plays an important role in the antioxidant system because of its unique sulfhydryl group to accelerate the removal of free radicals in the body. In the liver tissue, GSH-Px also can accelerate the removal of free radicals to reduce liver injury. Everson et al. found that GSH-Px activity in the liver was significantly lower than that in the control group after 5 days continuous SD [
These changes were associated with widespread suppression of core clock gene expression and RNA rhythms of lipid metabolic, indicating the presence of sleep-clock-metabolism axis in liver, which mediated pathophysiology of sleep deprivation. We observed 72 hours continuous sleep deprivation in rats, and found that the expression levels of CLOCK, BMAL1 and CRY1 gene in liver tissue were significantly down-regulated by western blotting. It was confirmed that sleep deprivation could significantly affect the expression of biological clock gene in liver tissue, which resulted in the disorder of rhythmic biochemical reaction and the internal environment, leading liver injury.
Oxidative stress and biological clock genes are closely related. Recent studies have shown that several pathways for protecting cells from oxidative stress may be regulated by circadian rhythms, including genes involved in glutathione biosynthesis [
Li, T., Cao, R.N., Xia, R. and Xia, Z.Y. (2017) Effects of 72 Hours Sleep Deprivation on Liver Circadian Clock Gene Expression and Oxidative Stress in Rats. Yangtze Medicine, 1, 194-201. https://doi.org/10.4236/ym.2017.14020