BACKGROUND Rapid Eye Movement sleep deprivation (REMSD) of rats causes inflammation of
the liver and apoptotic cell death of neurons and hepatocytes. Studies also
suggest that REM sleep deprivation can cause muscle as well as cardiac
injury and neurodegenerative diseases.
OBJECTIVE AND METHODS The aim of this research was to determine whether REM sleep deprivation of rats would increase the levels of reactive oxygen species (ROS) in the hepatocytes and create oxidative stress in them. We selectively deprived the rats for REM sleep using the standard flower pot method.
RESULTS We observed that when rats were subjected to REM sleep deprivation, the levels of ROS in their hepatocytes increased ~184.33% compared to large platform control (LPC) group by day 9 of deprivation, but it returned towards normal level (~49.27%) after recovery sleep for 5 days. Nitric oxide synthase (iNOS) gene expression and protein levels as determined by real-time PCR and western blot analysis respectively were found to be elevated in hepatocytes of REM sleep deprived rats as compared to the LPC group. The level of nitric oxide (NO) in the hepatocytes of REMSD rats also increased by ~404.40% as compared to the LPC group but sleep recovery for 5 days normalized the effect (~135.35% compared to LPC group). We used a large platform control group as a reference group to compare with the REM sleep deprived group as the effect on the hepatocytes of both LPC group and cage control groups were not significantly different.
DISCUSSION We have analyzed the oxidative stress generated in the hepatocytes of rats due to REM sleep deprivation and further consequences of it. REMS deprivation not only increased the levels of ROS in the hepatocytes but also induced iNOS and NO in them. REM sleep deprived hepatocytes became more susceptible to oxidative stresses on further exposures. Furthermore, our study has great pathological and physiological.
Keywords: Reactive Oxygen Species; Nitric Oxide; Nitric Oxide Synthase; Catalase; Sleep, REM.
|Citation: Pandey A, Kar SK. Rapid Eye Movement sleep deprivation of rat generates ROS in the hepatocytes and makes them more susceptible to oxidative stress. 11(4):245. doi:10.5935/1984-0063.20180039|
|Received: May 20 2018; Accepted: October 29 2018|
Sleep is a natural physiological process which is needed for the survival of most of the living beings studied so far1. During sleep, repair of damaged tissue and organs takes place and therefore its deficiency increases the risk of heart and kidney diseases, high blood pressure, diabetes, and stroke2-5. In mammals, sleep is broadly categorized as two types, namely non-rapid eye movement sleep (NREM) and rapid eye movement sleep (REM). REM sleep is reported in all mammals and birds except few exceptions and seems necessary for the maintenance of certain essential body functions like memory consolidation6,7, brain maturation, spatial memory acquisition8, and maintenance of body physiology9. Prolonged loss of REM sleep can alter blood-brain barrier functions10 and can be fatal11,12. In terms of energy metabolism, there is no difference between wakefulness and when the animal is in REM sleep, which constitutes approximately 1/3rd of total sleep13. But extended sleep deprivation experiments in animals have shown that during sleep there is decreased cerebral glucose utilization14.
Most of the studies on REM sleep deprivation have analyzed the biochemical changes that take place in the brain and neurons11-13. Reports also suggest that REMSD can cause apoptosis of neuronal cells15,16 and muscle and cardiac injuries17,18, but what happens to other organs has remained unexplored? ROS are generated when metabolic processes take place in the tissues and the body has a natural system to maintain the balance using anti-oxidants present in the system or available from dietary sources.
The production of excessive ROS due to uncontrolled metabolic processes under stressful conditions can lead to a state, where maintenance and recovery to normal physiological levels become difficult. This can ultimately lead to cell death and tissue disintegration. Studies for testing the relationship between sleep and restoration of normal functioning of the body has been primarily done using total sleep deprivation. Some studies have indicated that ROS are generated due to sleep deprivation which is responsible for alteration of body physiology while others have shown no conclusive evidence of the generation of ROS due to total sleep deprivation.
One study, implicating the involvement of ROS, has reported that lipid peroxidation levels increased in the hippocampus of rodents due to sleep deprivation19, while other study showed that the antioxidant levels decreased in peripheral tissues of animals that were deprived of total sleep for 5 and 10 days and it was restored to normal levels only after rebound recovery sleep16. There are reports of increased superoxide dismutase (SOD) activity in rats which were subjected to total sleep loss (3-14 days) in comparison to control groups20. Some other studies suggest that sleep deprivation creates stress like conditions coupled with oxidative stress leading to decreased levels of glutathione in the whole brain21 and reduced SOD activity in the hippocampus and brainstem22.
Interestingly, there exists contradicting reports giving no conclusive evidence of oxidative damage either in the brain or peripheral tissues such as liver after either short-term (8hrs) or long-term (3-14 days) sleep deprivation. No increase in antioxidant enzymes like SOD, Catalase, Glutathione peroxidase or Malondialdehyde activity has been reported23. It was shown that prolonged wakefulness activates an adaptive stress pathway termed as the unfolded protein response, which momentarily protects against the detrimental consequences of ROS24-26 A recent report has shown a sharp increase in lipid peroxidation in the hepatocytes of sleep deprived rats25. Liver, being the metabolic hub, contributes significantly to the maintenance of body physiology26. Since hepatocytes are known to be involved in metabolism, detoxifying endo- and xenobiotics and synthesis of many proteins like albumin etc.27-29. Hence, we undertook this study to find out whether REMSD of rats would generate ROS in the hepatocytes of the liver and how the ROS generated in them would affect its physiological response.
Our previous study had shown that REM sleep deprivation induced acute phase response in the liver, increased the synthesis of pro-inflammatory cytokines like Interleukin (IL)1β, IL-6 and IL-12 and increased the levels of liver toxicity marker enzymes, alanine transaminase and aspartic transaminase circulating in the blood30IL-6 and IL-12 circulating in the blood of REM sleep deprived rats increased in proportion to the extent of sleep loss. But in contrast the levels of IFN-\u03b3 and a ~200. kDa protein, identified by N-terminal sequencing to be alpha-1-inhibitor-3(A1I3. In the present study, we have shown that REMSD of rats affects the level of ROS in the hepatocytes, increases the expression of NOS gene and the levels of the corresponding protein. Interestingly, we have also observed that the hepatocytes from REM sleep deprived rats become more susceptible to ROS upon further exposure. We also observed the increased production of NO by the hepatocytes after REM sleep deprivation.
MATERIALS AND METHODS
Male Wistar rats weighing between 220-260 gm were used in this study. Animals were housed in the institutional animal house facility with a 12 hr L:D cycle (lights on at 7.00 am). Food and water were provided ad libtium. All experiments were conducted as per the protocol approved by the University's Institutional Animal Ethics Committee.
Methods used for REM sleep deprivation and recovery
Animals were REM sleep deprived by the most widely used flower-pot method31,32. In this method, rats were kept individually on a small raised platform (6.5 cm diameter) surrounded by water in a bucket compared to LPC group where individual rats were kept on a bigger platform of 12.5 cm diameter under identical condition. Cage control (CC) rats were housed in cages under laboratory condition.
Although the animals kept on the small platform (6.5 cm diameter) could sit, crouch and have non-REM sleep, but due to the requirement of postural muscle atonia during REM sleep, they were unable to maintain their extended relaxed body posture on the small platform and tended to fall into the surrounding water. As a result, they wake up at the onset or prior to the appearance of REM sleep and thus are deprived of it. After stipulated days of deprivation, the recovery groups of rats were allowed to sleep uninterrupted in individual cages with a sufficient supply of food and water. The rats were sacrificed on different days (4th, 9th days) of REMSD and after 5 days of recovery and their hepatocytes were isolated for further analysis.
Hepatocytes were isolated from the liver of male Wistar rats from CC, LPC, and REM sleep deprived groups by a method used by Shen et al.33. Briefly, the abdomens of the rats were opened through a midline incision. The portal cannula was then placed on a suitable platform and liver was perfused with 0.02% EDTA (Sigma, cat No E9884) solution at 37º C, with a flow rate of 30 ml per minute for 15 minutes. Subsequently, collagenase II (3U/ml; Sigma, cat No C2674) in PBS solution (37°C) was recirculated through the liver at the same flow rate for 15 minutes. After perfusion, liver capsules were disrupted and digested, liver parenchyma was suspended in the ice-cold Hank's balanced salt solution (Sigma, cat no H6648). The resulting cell suspension was washed by centrifugation at 500rpm for 5 min 2-3 times and further centrifuged over 30% percoll (Sigma, P1644) at 100g for 5 min to obtain pure hepatocytes. The viability of the hepatocytes thus obtained as measured by trypan blue exclusion was ≥ 95%.
ROS measurement and optimization of dose for creating oxidative stress
Total ROS level in the hepatocytes obtained from CC, LPC and REMSD group of rats were estimated after labeling with 2',7'-dichlorodihydrofluorescein diacetate (DCF-DA, Sigma, Cat No D6883 ), which is a cell-permeable dye that becomes fluorescent after oxidation by different ROS such as hydroxyl radical, hydrogen peroxide, or peroxynitrite. Briefly, hepatocytes from different groups of rats were treated with 10µM-DCF-DA for 30 min followed by either measurement of ROS level or treatment with different doses 50-500µM of H2O2 (Sigma Aldrich, Cat No 04078) or 10-100mU/ml Xanthine (Sigma, Cat No X0626) /Xanthine oxidase (Sigma, Cat. no X1875) for induction of ROS.
Specific ROS inhibitors were also optimized for effective concentrations by treating the cells with 100-500U/ml Catalase (Sigma, cat No-C1345) and 10-50mU/ml Oxypurinol (Sigma, Cat No O6881). Whenever, ROS inhibitors have used the hepatocytes were treated with them first before DCF-DA treatment, following H2O2 or Xanthine/Xanthine oxidase treatments to induce oxidative stress condition similar to the protocol described for mouse osteoblasts and Human MG63 cells34the roles of ROS in RANKL expression and signaling mechanisms through which ROS regulates RANKL genes are not known. Here we report that increased intracellular ROS levels by H2O2 or xanthine/xanthine oxidase-generated superoxide anion stimulated RANKL mRNA and protein expression in human osteoblast-like MG63 cell line and primary mouse bone marrow stromal cells and calvarial osteoblasts. Further analysis revealed that ROS promoted phosphorylation of cAMP response element-binding protein (CREB. The cells were harvested at the indicated time points after incubation, washed three times with phosphate-buffered saline, and immediately analyzed by Flow cytometer using 488-nm excitation beam. The signals were obtained using a 530-nm bandpass filter (FL-1 channel) for DCF. Each determination is based on the mean fluorescence intensity of 5,000 cells.
Assay for hepatocellular NO production
Hepatocytes were seeded in24-well, flat-bottom plates (Falcon) at 105 cells/well and incubated at 37°C in humidified 5%CO2 conditions with and without 1µg/ml lipopolysaccharides (Sigma, Cat no L 2630). NO was measured indirectly by determination of the concentration of nitrite. Briefly, 100-µl aliquots were removed from hepatocytes cultures and incubated with an equal volume of Griess reagent (1%sulfanilamide,0.1%N-1-naphthyl ethylenediamine dihydrochloride in 2.5% phosphoric acid) from Sigma, Cat No G4410 for 10 min at 37°C. Absolute values of NO were determined using sodium nitrite as standard. Absorbance was read at 550 nm on a microtiter plate reader.
Isolation of RNA and TaqMan Real-time PCR
RNA was isolated from hepatocytes harvested from CC, LPC and REM sleep deprived group of rats and stored in RNA later (Sigma-Aldrich, Cat. R0901) using RNA purification kit (RNeasy Mini Kit, Qiagen, Germany). RNA concentrations and integrity was assessed by Nanodrop and Agilent 2100 Bioanalyzer (Agilent Technologies, Massy, France). Total RNA isolated from the hepatocytes were converted to cDNA using the reverse transcription PCR kit (Applied Biosystems, United States). GADPH served as a housekeeping gene while CC group served as calibrator Probes (Reporter dye FAM-labeled on 5' end and quencher VIC labeled on 3' end), PCR master mix, and PCR plate/tubes were obtained from Applied Biosystems, United States and it was used following the manufacturer's instructions. The catalogue numbers of gene probes were glyceraldehyde 3 phosphate dehydrogenase (GAPDH, Rn01749022_g1), iNOS (Rn02132634_s1) and master mix (Rn99999916-g1).
Western Blot Analysis
Western blotting was performed using a method described previously35HEK293, HeLa, and NIH-3T3 cells, we show that the anti-apoptotic protein Bcl-2 significantly inhibits transcriptional activation of various transcription factors, including NF kappa B, AP1, CRE, and NFAT. A Bcl-2 mutant lacking its BH4 domain (Delta BH4. Primary antibody (Santacruz Biotechnology, Inc, USA) used was at 1/1000 dilutions in 0.1% PBS-tween 20, whereas secondary antibody (Santacruz Biotechnology, Inc, USA) horseradish peroxidase-conjugated was used at 1/5000 dilutions in the same buffer. After following the published procedure the membranes were developed using enhanced chemiluminescence (ECL) reagent (Promega) and photographed using software (Photo and Imaging 2.0; Hewlett-Packard). The analysis of the images was performed with imaging software (Photoshop 8.0; Adobe).
We used LPC group of rats for comparative analysis as statistically CC and LPC control groups were found not to be significantly different (Tukey posthoc analysis followed by ANOVA). We used SPSS (Version 2.7.2) for one-way ANOVA and Tukey's HSD posthoc test for measuring out the effect across treatment groups. The p values ≥ 0.05 were considered significant.
Optimization of dose for induction of ROS using H2O2and Xanthine/Xanthine oxidase and it's suppression by Catalase or Oxypurinol treatment
We treated normal hepatocytes with different concentration of H2O2 or Xanthine/Xanthine oxidase in order to generate reactive oxygen species. Intracellular ROS level increased in a dose-dependent manner with treatments of different concentrations of H2O2 (50-500µM, Fig. 1a, ANOVA, F=354.91, df=4, p<0.001) or Xanthine/Xanthine oxidase (10-100 mU/ml, Fig. 1b, ANOVA, F=374.83, df=4, p<0.001). Based on the result obtained in this experiment we selected the dose of 200µM of H2O2 and 50 mU/ml of Xanthine/Xanthine oxidase to be optimal for the induction of oxidative stress in the hepatocytes. (Fig. 1a, 1b). Then we treated the normal hepatocytes with 200µM-H2O2 or 50 mU/ml- Xanthine/Xanthine oxidase for different time points ranging from 20-120 minutes to determine the optimal time required to induce maximum oxidative stress and observed that 60 minutes is quite sufficient to induce effective ROS production. (Fig. 1c, ANOVA, F=410.21, df=4, p<0.001 and Fig. 1d, ANOVA, F=864.77, df=4, p<0.001). Further, to establish the effective concentrations of ROS inhibitors like Catalase or Oxypurinol which were needed to scavenge the ROS that will be generated. We first, treated the normal hepatocytes with different doses of Catalase (100-500U/ml) or Oxypurinol (10-50µM) followed by treatment with 200µM-H2O2 or 50 mU/ml- Xanthine/Xanthine oxidase for 60 minutes. The doses of Catalase at 500U/ml, (Fig. 1e, 1f ANOVA, F=135.71, df=3, p<0.001) or Oxypurinol at 50µM, (Fig. 1f, ANOVA, F=128.36,df=3, p<0.001) were found to be quite effective in scavenging the ROS generated by treatments with H2O2 (200µM) or Xanthine/Xanthine oxidase (50 mU/ml) for 60 minutes, respectively. Therefore, these doses were used for further ROS stimulation and scavenging experiments. Our results showed that we were able to selectively induce the production of ROS after H2O2 (200µM) or Xanthine/Xanthine oxidase (50 mU/ml) treatment for 60 minutes and could quench them effectively by using specific ROS inhibitors like Catalase (500U/ml) or Oxypurinol (50µM).
ROS production in hepatocytes by REM sleep deprivation
ROS levels increased significantly with an increase in the number of days of REM sleep deprivation. We used LPC group of rats for comparative analysis as statistically there was no significant difference between CC and LPC control groups ruling out that experimental procedure of keeping rats on the large platform rather than in the cage itself had any effect (Fig. 2a, ANOVA, p<0.05). We observed ~123.04% change in production of ROS in the hepatocytes of the experimental group kept on small platform (6.5 cm diameter) on day 4 of deprivation which increased to~184.33 % on day 9 of deprivation compared to rats kept on LPC group. Sleep recovery of 5 days showed a reduction in the levels of ROS (~49.27% change) in the experimental group when compared to LPC control group indicating the redressal effects (Fig. 2a, ANOVA F=216.92, df=27, p<0.001). At the same time, when we exposed the hepatocytes collected from different groups of rats with ROS quenchers (500U/ml Catalase or 50µM Oxypurinol) which are known to reduce the oxidative stress, we got reduced levels of ROS in hepatocytes after days of deprivation (Fig. 2a, ~30.51% change in the presence of ROS quencher relative to ~123.04% change without it as compared to LPC) and after 9 days of deprivation (Fig. 2a, ~3.35% change in the presence of ROS quencher relative to ~184.33% change without it as compared to LPC). This confirmed that ROS were generated in the hepatocytes due to REMSD which further got quenched by inhibitors like Catalase or Oxypurinol.
Sleep loss increased the susceptibility of hepatocytes to further oxidative stress
REM sleep loss not only generated ROS in the hepatocytes of rats but also made them more susceptible to subsequent oxidative stress. We used LPC group of rats for comparative analysis as statistically the effect in CC and LPC control groups were found not to be significantly different. (Fig. 2b, 2c, ANOVA, p<0.05).We report here the increased production of ROS by hepatocytes after 4 day deprivation (~108.03% change) and 9 day deprivation (~252.22% change) compared to LPC group, when hepatocytes were exposed to 200µM-H2O2 for 60 minutes (Fig. 2b, ANOVA F=216.67, df=11, p<0.001 and Fig. 2c, ANOVA F=442.44, df=15, p<0.001). The levels of ROS became normal (~17.49% change compared to LPC) when the similar number of hepatocytes were treated with 500U/ml Catalase (Fig. 2b, 2c). Similarly, when hepatocytes from CC, LPC and REMSD groups after day 4 and 9 of deprivation and after 5Day recovery were treated with 50 mU/ml- Xanthine/Xanthine oxidase, different levels of ROS were induced in them. The levels of ROS observed after 4 days of REM sleep deprivation was~68.88% as compared to LPC group which increased to ~168.76% after 9 days of REM sleep deprivation (Fig. 2b, 2c). The treatment with 50 mU/ml- Xanthine/Xanthine oxidase + 50µM-Oxypurinol reduced the ROS levels on day 4 (~7.03% change) and day 9 (~4.12% change) compared to LPC group of rats (Fig. 2b, 2c). The ROS levels in hepatocytes collected from 5 Day recovery group were similar to that of CC and LPC groups (Fig. 2c). These experiments suggested that REM sleep deprivation made the hepatocytes more susceptible to further oxidative damages.
Expression of iNOS gene and protein circulation due to REM sleep deprivation
The iNOS gene expression got affected in hepatocytes of REMSD rats. We used LPC group of rats for comparative analysis as statistically CC and LPC control groups were found not be significantly different (Fig. 3a, 3b, 3c, ANOVA, p<0.05).We found increased expression of iNOS gene by ~7.96fold (log 10 value) by the 4th day of REM sleep deprivation, which increased further upto~14.04 fold (log 10 value) by the 9th day as compared to LPC control. After 5days of recovery sleep, the expression of the iNOS gene improved upto~6.93 fold (log 10 value) compared to LPC control (Fig. 3a, ANOVA F=357.99, df=6, p<0.001). Further, we measured iNOS protein levels in hepatocyte lysate using western blot. Our results show that iNOS protein level increased by~100.8% by 4th day which subsequently increased to ~166.1% by 9 days of REM sleep deprivation compared to that in the LPC group. (Fig. 3b, 3c, ANOVA F=393.94, df=4, p<0.001). The levels of iNOS protein returned to normal levels after 5days of recovery sleep (~46.8% change) compared to LPC controls (Fig. 3b, 3c).
Nitric oxide production in hepatocytes after REMSD
As production of iNOS protein increased in the hepatocytes due to REM sleep loss, we collected the hepatocytes from different groups of rats and measured NO levels in them. Our results show that NO levels were significantly increased in hepatocytes of the REMSD group rats on day 4 (~155.49% change) and day 9 (~404.40% change) than that of LPC group. Further, 5 days of sleep recovery showed redressal effects in terms of NO production but overall NO level was still quite high. (~135.35% change) than of LPC group (Fig. 4, ANOVA F=170.48, df=11, p<0.001). We used LPC group of rats for comparative analysis as statistically CC and LPC groups were found not to be significantly different (Fig. 4, ANOVA, p<0.05).
We subjected the rats to REM sleep deprivation using the flower-pot method and created the LPC and CC groups for comparison. We measured the total ROS level in the hepatocytes of all the groups by using DCF labeling technique. First, we collected the hepatocytes and standardized the method for induction of ROS in them by using H2O2 or Xanthine / Xanthine oxidase and later quenched them by using enzymes like Catalase or Oxypurinol. Lastly, we measured the expression levels of iNOS gene and the protein as well as NO level in hepatocytes after REM sleep deprivation. We used LPC group of rats as a reference group for statistical comparisons as CC and LPC groups had no significant differences in observed parameters. Our findings suggest that REM sleep deprivation of rats can increase ROS production in their hepatocytes which ultimately can induce apoptotic cell death36. Treatment of hepatocytes with H2O2 or Xanthine/Xanthine oxidase can selectively induce production of ROS inside the hepatocytes and it can be quenched by Catalase or Oxypurinol treatment. This gave us a tool to expose the hepatocytes from CC, LPC and REMSD group of rats and investigate how REM sleep deprivation affects their physiology and susceptibility to further oxidative stress.
The oxidative stress created by treatment of hepatocytes with H2O2 or Xanthine/Xanthine oxidase induced the ROS levels to a greater extent in the REMSD group of rats than in the CC and LPC group of rats indicating that REM sleep loss makes the hepatocytes more susceptible to further oxidative stress. Interestingly, when the rats were allowed to have 5 days recovery sleep after REM sleep deprivation their hepatocytes were found not to be as susceptible as before and responded to ROS stress like hepatocytes from CC and LPC groups.
The reason for this is not very clearly understood. Maybe the molecules which are responsible for stress response accumulated during REM sleep deprivation and increased the susceptibility further but they got alleviated to some extent when rats were allowed to have recovery sleep. Other studies have shown increased oxidative stress in brain cells of rats after REMSD20,37, which was also observed in other animal models38. Conditions like obstructive sleep apnea in humans and rodents were found to be positively correlated with increased ROS in the system39,40, and Parkinson disease associated with REM sleep loss was found to contribute to the colon oxidative stress and disruption of mitochondrial function41.
Sleep deprivation was further found to be associated with anxiety-like behavior in rats which can be reduced or prevented by administration of melatonin, which possibly reduces the oxidative stress and maintains the balance between GABAergic and glutamatergic transmission42. Administration of melatonin was found to reduce oxidative stress in rats by blocking down-regulation of γ-aminobutyric acid A-alpha-2 receptors.
These studies suggest that sleep deprivation is somehow directly or indirectly associated with the ROS levels in different tissues. In our previous study, we had also reported that REM sleep deprivation can create inflammation in the liver by increasing the levels of cytokines such as Interleukin-6 (IL-6), IL-12, IL-1β13 indicating that cytokines might be involved in the induction of iNOS genes. Results published by others earlier also suggest that cytokines like tumor necrosis factor alpha (TNF-α), IL-1β, and Interferon-gamma (INF-γ) synergistically activate iNOS gene expression in the liver35.
Lastly, our results show that hepatocytes form REMSD group of rats produced increased levels of NO which was reduced after sleep recovery. Previous studies had shown that NO exerts a protective effect by blocking TNF-α induced apoptosis and hepatotoxicity both in vivo and in vitro, by inhibiting the caspase-3-like protease activity43. Our unpublished results also show that REMSD can cause apoptosis in hepatocytes36. It suggests that might be excess of NO is being produced to counteract the oxidative stress generated due to sleep loss and it protects the hepatocytes from apoptotic death. Our present study is quite unique as it demonstrates that REM sleep deprivation can induce ROS in hepatocytes and make them more susceptible to further oxidative stresses. This shows how important REM sleep is for maintenance of body physiology and survival of animals.
Our results demonstrate that the REM sleep is very important for the well-being of animals and its loss can't only induce toxicity in the hepatocytes due to the production of ROS but also make them more susceptible to further oxidative stress and death due to apoptosis36. Apart from this, REMSD also can induce iNOS circulation in the system which would increase NO production, which may further affect the well-being of the hepatocytes.