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ORIGINAL ARTICLE
Year : 2022  |  Volume : 10  |  Issue : 1  |  Page : 9-14

Quercetin improves mood-related behaviors in mice subjected to paradoxical sleeplessness


Department of Pharmacology, Delta State University, Abraka, Nigeria

Date of Submission20-Oct-2021
Date of Decision02-Dec-2021
Date of Acceptance21-Mar-2022
Date of Web Publication01-Jul-2022

Correspondence Address:
Ms. Lily Oghenevovwero Otomewo
Delta State University, Abraka
Nigeria
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/njecp.njecp_41_21

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  Abstract 


Context: Food supplements are a widely consumed class of pharmaceuticals. Quercetin (QCT) is a bioflavonoid with reported benefits in various disease conditions. Aims: The present study sought to evaluate the potential protective role of QCT against anxiety-like and antisocial behavior in mice exposed to persistent wakefulness. Settings and Design: The sleep deprivation protocol used in this research was the multiple platforms over the water model. Subjects and Methods: Thirty male albino mice were randomly divided into five groups, each consisting of six mice: Group 1 was considered the naive group; Group 2 was considered the model control. Groups 3 and 4 received QCT (25 and 50 mg/kg; p. o.) and Group 5 received astaxanthin (50 mg/kg; p. o.) in addition to being sleep-deprived respectively. The mice in groups 2–5 received their respective treatment for 7 days but were subjected to a 72 h sleep deprivation from day 4. On day 8, behavioral activities were monitored, and then, animals were sacrificed 1 h after the drug administration. Brain samples were subsequently collected for the biochemical and histopathological analysis. Statistical Analysis Used: One-way analysis of variance. Results: The results indicate that persistent wakefulness-induced anxiety such as symptoms and depression-like behavior in mice. In addition, oxidative stress was significant in sleep-deprived group with an enhancement in activity of prooxidants. However, upon pre-treatment with QCT, such behaviors and suppression of antioxidant molecules were reversed. Conclusions: In conclusion, the present finding showed that QCT could attenuate the impairment of antioxidant enzymes, reduce anxiety, and depression-like behaviors caused by sleep deprivation in mice.

Keywords: Anxiety, quercetin, sleep deprivation, social behavior


How to cite this article:
Eduviere AT, Otomewo LO, Anyanwu OG, Igari FO, Okorigba OJ. Quercetin improves mood-related behaviors in mice subjected to paradoxical sleeplessness. Niger J Exp Clin Biosci 2022;10:9-14

How to cite this URL:
Eduviere AT, Otomewo LO, Anyanwu OG, Igari FO, Okorigba OJ. Quercetin improves mood-related behaviors in mice subjected to paradoxical sleeplessness. Niger J Exp Clin Biosci [serial online] 2022 [cited 2022 Aug 18];10:9-14. Available from: https://www.njecbonline.org/text.asp?2022/10/1/9/349561




  Introduction Top


Sleep is one of the most important identifying features of living things. Although reversible, sleep is considered a state of reduced perceptual engagement with the environment and proper sleep patterns is critical for overall health and wellness.[1] Circadian disturbances, usually caused by night shift work, exposure to light at night and other sleep-restricting lifestyle habits or medical conditions, increase the risk of metabolic, psychiatric, or neurodegenerative disorders.[2]

Quercetin (QCT) is a naturally occurring polyphenolic compound commonly used in dietary supplements.[3],[4],[5] Studies have posited that QCT possesses anti-oxidative, pro-oxidative, anti-obesity, anti-cancer, anti-inflammatory function, etc., and may also strengthen the activity of endogenous antioxidants.[6],[7],[8],[9] This study was therefore structured to evaluate the possible modulatory role of QCT on anxiety and depression symptoms in mice exposed to rapid eye movement (REM) sleep deprivation.

Sleep deprivation of the REM phase was carried out using a popular physical model of sleep deprivation known as the platform over water model. Since atonia (loss of muscle tone) is considered one of the core features of REM sleep, this model targets its disruption as the animal is likely to lose muscle tone as it proceeds into REM sleep and thus falls into the water below which consequently awakens it. Furthermore, the effect of this sleep deprivation on anxiety- and depression-like behavior was evaluated using the physical test apparatus known as the light/dark transition test, and the elevated plus maze test for the former, while the social interaction test (SIT) and forced swim test for the later.


  Subjects and Methods Top


Animals

Thirty adult Albino mice (male; 22–24 g) were obtained from the animal house of the College of Health Sciences, Delta State University, Abraka. Six animals (n = 6) were housed per plastic transparent cage laid with beddings. The total of five cages were then reserved in a room at ambient temperature and exposed to 12 h light/dark cycle. For the duration of the experiment, feed and water were made available to mice when necessary.

Treatment groups

The animals were then randomly allocated into five treatment groups and the different treatments were orally administered using an oral cannula:

Group 1 was considered the naive group, i.e., mice in this group were treated with 10 mL distilled water and were not sleep deprived.

Group 2 was considered the model control group, i.e., mice in this group were treated with 10 mL distilled water and were subjected to the sleep deprivation protocol afterwards.

Group 3 was considered the Q1 group, i.e., mice in this group were treated with 25 mg QCT and were subjected to the sleep deprivation protocol afterward.

Group 4 was considered the Q2 group, i.e., mice in this group were treated with 50 mg QCT and were subjected to the sleep deprivation protocol afterward.

Group 5 was considered the positive control group, i.e., mice in this group were treated with 50 mg astaxanthin and were subjected to the sleep deprivation protocol afterward. Astaxanthin was chosen as the positive control treatment because it is a known adaptogen with benefits that extend into the central nervous system.

The NIH guidelines were duly followed in the course of the experiment.

Experimental design

The REM phase of sleep was disrupted using the platform-over-water model where animals were suspended on a platform which was placed above water. Treatment lasted for 7 days and all groups except group 1 were subjected to 72 h of sleep deprivation protocol starting from day 4. On day 8, the various behavioral tests were carried out on mice from all groups. These tests include the:

Forced swim test and the SIT, which are recognized experimental tools for the evaluation of mood alterations (especially depressive-like) in animals by subjecting them to swimming as in the former or by testing their social behavior in the social interaction chamber as in the later.

Elevated plus maze test and the light/dark transition test, which are recognized pharmacological tools used to determine the anxiogenic or anxiolytic property of a substance by measuring the duration or frequency of entries into the closed or open arm of the elevated plus maze, and the light or dark compartment of the light/dark transition box, respectively. In this study, all behavioural tests were carried out for 5 min for each mouse.

Afterwards, a representative mouse from each group was selected for sacrifice and the brain tissue harvested. Brain supernatants and tissue slices were prepared for biochemical assays and histological analysis respectively. Note that the whole mouse brain was first harvested before the tissues of specific brain regions (caudate putamen and hippocampal CA1) were identified and homogenized following the standard operating procedures (1820, 2003). Thereafter, transverse sections of 5–6 μm thickness of the brain tissue were placed on slides and subjected to Hematoxylin and Eosin staining. Then, the tissues were examined under a light microscope at ×400 magnification and micrographs obtained using a digital camera. Furthermore, neuronal densities of the brain tissues were evaluated by simple estimation from micrographs. [Figure 1] below shows the experimental schedule in brief.
Figure 1: Schedule of experimental design

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Statistical analysis

Data were analyzed using the one-way analysis of variance. Student's Newman Keuls test was used to evaluate the differences. Data were presented as mean ± standard error of the mean (SEM), and P < 0.05 was considered statistically significant.


  Results Top


Behavioral response on an elevated plus maze in sleep-deprived mice treated orally with graded concentration of quercetin

As shown in [Table 1], sleep deprivation significantly (P < 0.05) increased anxiety-like behavior in mice shown by increased exploration of the closed arm of the elevated plus maze (EPM). QCT and astaxanthin treated groups spent longer times in the open arms and shorter times in the closed arms.
Table 1: Behavioural response on an elevated plus maze in sleep deprived mice treated orally with graded concentration of quercetin

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Behavioral response on a light/dark transition test in sleep-deprived mice treated orally with graded concentration of quercetin

The result revealed that anxiety was significantly (P < 0.05) elevated in sleep deprived mice by spending longer time in the dark compartment of the light/dark transition box. QCT group spent shorter times in the dark compartment and longer in the light compartments [Table 2].
Table 2: Behavioral response on a light/dark transition test in sleep-deprived mice treated orally with graded concentration of quercetin

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Malondialdehyde level in brain tissue samples harvested from sleep-deprived mice treated with quercetin

[Figure 2] displays the effect of QCT on brain malondialdehyde (MDA) levels in mice subjected to 72 h REM sleep deprivation. Brain tissue MDA levels were significantly (P < 0.05) elevated in sleep deprived mice. Both doses of QCT significantly (P < 0.05) reduced the levels of MDA in the brain tissues of mice exposed to sleep deprivation.
Figure 2: Effect of quercetin on the brain tissue malondialdehyde levels in sleep-deprived mice. Each result is expressed as mean ± standard error of the mean of grouped mice (n = 6). # indicates significant difference (P < 0.05) compared to the vehicle (not sleep deprived) group. * indicates significant difference (P < 0.05) compared to the vehicle + sleep deprivation group. (One-way analysis of variance followed by Student-Newman-Keuls post hoc test). VEH: Vehicle, AXT: Astaxanthin, QCT: Quercetin, SD: Sleep deprivation

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Glutathione level in brain tissue samples harvested from sleep-deprived mice treated with quercetin

[Figure 3] shows the effect of QCT on brain tissue Glutathione (GSH) concentration in mice subjected to REM sleep deprivation for 72 h. Brain tissue GSH concentration was significantly (P < 0.05) depleted in sleep-deprived mice. QCT treated groups revealed significantly (P < 0.05) replenished brain GSH concentration in mice brain tissues in a manner resembling dose dependence.
Figure 3: Effect of quercetin on brain tissue glutathione levels in sleep deprived mice. Each result is expressed as mean ± standard error of the mean of grouped mice (n = 6). # indicates significant difference (P < 0.05) compared to the vehicle (not sleep deprived) group. * indicates significant difference (P < 0.05) compared to the vehicle + sleep deprivation group. (One-way analysis of variance followed by Student-Newman-Keuls post hoc test). VEH: Vehicle, AXT: Astaxanthin, QCT: Quercetin, SD: Sleep deprivation

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Effect of quercetin on caudate putamen neurons of mice exposed to rapid eye movement sleep deprivation

[Figure 4] shows a photomicrograph of caudate putamen neurons of mice exposed to sleep deprivation following H and E staining. Slide NC shows normal neurons abundant in mice that were not sleep deprived. Slide N shows fewer viable neurons with the onset of necrosis. Slides Q25 and Q50 show the caudate putamen of mice pretreated with 25 and 50 mg/kg QCT respectively. It was observed that QCT administration caused a significant reduction in the rate at which caudate putamen neurons were undergoing necrosis due to sleep deprivation. Normal neurons are identified with a black arrow while neurons experiencing necrosis are identified with red arrows. Also, neuronal viability was also affected similarly as seen in [Figure 5].
Figure 4: Photomicrograph of the caudate putamen of mice after sleep deprivation. (NC) Vehicle only, (n) Vehicle + sleep deprivation, (Q25) QCT 25 mg/kg + sleep deprivation, (Q50) QCT 50 mg/kg + sleep deprivation, (AF1) AXT 50 mg/kg + sleep deprivation. Black arrow: Normal neuronal cells. Red arrow: Neuronal cells undergoing necrosis. Magnification: (×400). VEH: Vehicle, AXT: Astaxanthin, QCT: Quercetin, SD: Sleep deprivation

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Figure 5: Effect of quercetin on viable caudate putamen neurons in mice after exposure to sleep deprivation. Counts are based on the number of neuronal nuclei in three (3) rectangular boxes per slide, using the precalibrated Image J software. Each result is expressed as mean ± standard error of mean for grouped mice (n = 3). # indicates significant difference (P < 0.05) compared to the vehicle (not sleep deprived) group. * indicates significant difference (P < 0.05) compared to the vehicle + sleep deprivation group. (One-way analysis of variance followed by Student-Newman-Keuls post hoc test). VEH: Vehicle, AXT: Astaxanthin, QCT: Quercetin, SD: Sleep deprivation

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Effect of quercetin on hippocampal CA1 neurons of rapid eye movement sleep deprived mice

[Figure 6] shows a photomicrograph of hippocampal CA1 neurons of mice exposed to sleep deprivation following H and E staining. Slide NC shows normal neurons abundant in mice that were not sleep deprived. Slide N shows fewer viable neurons with the onset of necrosis. Slides Q25 and Q50 show the hippocampal CA1 of mice pretreated with 25 and 50 mg/kg QCT respectively. Normal neurons are identified with a black arrow while neurons experiencing necrosis are identified with red arrows. Also, neuronal viability was also affected similarly as seen in [Figure 7].
Figure 6: Photomicrograph of the hippocampal CA1 region of mice after sleep deprivation. (NC) Vehicle only, (n) Vehicle + sleep deprivation, (Q25) QCT 25 mg/kg + sleep deprivation, (Q50) QCT 50 mg/kg + sleep deprivation, (AF1) AXT 50 mg/kg + sleep deprivation. Black arrow: Normal neuronal cells. Red arrow: Neuronal cells undergoing necrosis. Magnification: (×400). VEH: Vehicle, AXT: Astaxanthin, QCT: Quercetin, SD: Sleep deprivation

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Figure 7: Effect of quercetin on viable hippocampal CA1 neurons in mice after exposure to sleep deprivation. Counts are based on the number of neuronal nuclei in three (3) rectangular boxes per slide, using the precalibrated Image J software. Each result is expressed as mean ± standard error of mean for grouped mice (n = 3). # indicates significant difference (P < 0.05) compared to the vehicle (not sleep deprived) group. * indicates significant difference (P < 0.05) compared to the vehicle + sleep deprivation group. (One-way analysis of variance followed by Student-Newman-Keuls post hoc test). VEH: Vehicle, AXT: Astaxanthin, QCT: Quercetin, SD: Sleep deprivation

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Behavioural response on a forced swim test in sleep deprived mice treated orally with graded concentration of quercetin

As shown in [Figure 8], sleep deprivation significantly (P < 0.05) increased the duration of immobility in the forced swimming test. Administration of QCT significantly (P < 0.05) attenuated this increase in immobility duration caused by sleep deprivation.
Figure 8: Behavioral response on a forced swim test in sleep deprived mice treated orally with graded concentration of quercetin. Each result is expressed as mean ± standard error of the mean of grouped mice (n = 6). # indicates significant difference (P < 0.05) compared to the vehicle (not sleep deprived) group. * indicates significant difference (P < 0.05) compared to the vehicle + sleep deprivation group. (One-way analysis of variance followed by Student-Newman-Keuls post hoc test). VEH: Vehicle, AXT: Astaxanthin, QCT: Quercetin, SD: Sleep deprivation

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Behavioural response on a social interaction test in sleep deprived mice treated orally with graded concentration of quercetin

The effect of QCT on depressive-like symptoms in mice subjected to REM sleep deprivation utilizing the SIT is shown in [Figure 9] below. Sleep deprivation significantly (P < 0.05) reduced the percentage of social preference of mice. Administration of both doses of QCT significantly (P < 0.05) enhanced the social behavior of mice.
Figure 9: Behavioral response on a social interaction test in sleep-deprived mice treated orally with graded concentration of quercetin. Each result is expressed as mean ± standard error of the mean of grouped mice (n = 6). # indicates significant difference (P < 0.05) compared to the vehicle (not sleep deprived) group. * indicates significant difference (P < 0.05) compared to the vehicle + sleep deprivation group. (One-way analysis of variance followed by Student-Newman-Keuls post hoc test). VEH: Vehicle, AXT: Astaxanthin, QCT: Quercetin, SD: Sleep deprivation

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  Discussion Top


The results from the present study show that mice subjected to 72-h sleep deprivation exhibited changes in behaviour and mood, together with a decrease in the level of GSH and increase in MDA level.

Anxiety-like behaviour was assessed using the elevated plus maze and the light/dark transition box. In both tests, sleep-deprived mice spent longer times in the closed arm and dark compartment respectively. This preference has been linked with anxiety in previous studies.[10],[11] Depressive-like symptoms were also assessed using the forced swimming and SIT. In both tests, sleep-deprived mice exhibited a significantly longer immobility time and antisocial behaviour respectively. These symptoms have been linked with mood disorders such as depression in previous studies.[12] The alterations in antioxidant and prooxidant levels in sleep deprived mice observed in this study have also been reported in previous studies.[11]

On the other hand, mice that were pretreated with QCT (25 and 50 mg/kg) exhibited anxiolytic property by significantly lowering degrees of anxiety-like behaviour and depressive-like symptoms. QCT must have done this through its ability to enhance antioxidant activity.[13] This agrees with existing literary evidence which have described QCT as an anxiolytic, an antidepressant and a potent antioxidant.[14],[15],[16],[17],[18]


  Conclusion Top


In conclusion, the present findings show that QCT could attenuate the impairment of antioxidant enzymes, reduce anxiety-and depression-like behaviours caused by 72 h sleep deprivation in mice.

Acknowledgement

Special thanks to the technical stafff of the Pharmacology laboratory.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
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    Figures

  [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6], [Figure 7], [Figure 8], [Figure 9]
 
 
    Tables

  [Table 1], [Table 2]



 

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