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24Effect of Schisandrin on rhythm of sleep:wake of wild-type CS drosophila
Lili Huang, Hongsheng Bian, Shuang Yu, Tingli Li* Pharmaceutical College, Heilongjiang university of Chinese Medicine, Harbin, Heilongjiang, China
Abstracts Objectives: The aim of this study is to explore the effect of Schisandrinon the sleep-wake activity in fruit flies. Methods: Canton Sflies aged 6 days were collected by CO2anesthesia and located separately in 32tubes, each with a filter paper. The filter paper was saturated with different concentration Schisandrin or placebo.The total sleep, the average duration ofsleep bouts, and the number of sleep bouts during nighttime and daytime were calculated basedon the sleep definition as a period of 5 or moreminutes of behavioralimmobility.Comprehensive microarray analysis of gene expression in flies head were performed using GeneChip DrosophilaGenome arrays as described in the Affymetrix GeneChip ExpressionAnalysis Technical Manual.Results:Compared to control, Schisandrin (0.635 mg/ml and 1.27 mg/ml) had significantly reduced the sleep time of daytime of female and male drosophila, and no effect onnighttime sleep time. Schisandrin (2.54 mg/ml, 5.08 mg/ml and 1.27 mg/ml) had significantly not only decreased the sleep time of daytime of female and male drosophila, but also the sleep time of nighttime in comparison with control. Schisandrindecreased sleep duration during daytime due to a decrease in the number of sleep episodes, and the reduction in sleep time during nighttime is associated with andecrease of duration of sleep episodes.Moreover, Schisandrinalso significantly reduced the time spent sleeping of lightdeprivation on male flies (p<0.01), and had no effect on the females compared to sleep-deprived flies. Approximately 170 genes (fold change cut-off >2) are differentially expressed in flies administered by feeding with Schisandrin compared to control, including 84 down-regulated and 86 up-regulated. Expression changes associated with catabolism, redox reactions, circadian rhythms, transmembrane transport and immune response. Conclusion: Schisandrin significantly reduced the sleep duration in female and male fruit flies. Schisandrin make changes accordingly in drosophila gene expression. These results suggested that Schisandrin have an effect at the molecular level of sleep regulation.
Keywords: Drosophila, Schisandrin, Sleep: wake
Introduction
Schisandra consists of the dried ripe fruit of Schisandra Chinensis (Turcz.) Baill.Schisandrin is one of the lignan components of Schisandra, which is clinically prescribed to improve work efficiency and sleep. In previous research, we had found that decoction of Schisandra prolonged the time spending sleep of free-moving rat[1]. Furthermore, using the method of serum pharmacochemistry of Chinese Medicine, we found that Schisandrin was the mainly constituents absorbed into blood of rats after oral administration decoction of Schinsandra[2].
The complexity of sleep in mammals has led to a movement toward examining simpler model systems, such as nematodes, files and fish, that exhibit sleep and harbor technical advantages not evident in more conventional rodent or primate models. A sleep-like state has been described in drosophila melanogaste, and a five minute period of inactivity has been found to be a reliable indicator of sleep[3' 4]. Many features of drosophila sleep are independent of changes in spontaneous movement and include elevated arousal threshold, homeostatic regulation, electrophysiologic correlates, and conserved responses to sleep-wake requlator drugs[3' 5]. In addition, genetic screens have identified shared genes and pathways of sleep control with their mamalian relations[6' 7]. Drosophila as a model is used for studies of sleep, and it is an ideal model system for understanding the molecular mechanisms of sleep. The power of drosophila genetics can be used to facilitate the molecule mechanisms of sleep regulatory.
The aim of this study is to explore the effect of Schisandrinon the sleep-wake activity in fruit flies, then compare gene expression using microarrays in drosophila administered by feeding with and without Schisandrin at the same time.
Materials and Methods
Source and culture of flies
Wild-type drosophila melanogaster of the Canton S strain were cultured at 25°C,50-60% humidity, in a 12 h light/dark (LD) cycle, on a standard foodcontaining yeast,corn flour, sugar and agar. Propionic acid wasadded to prevent fungal growth. Flies were obtained fromCollege of Life Sciences, Peking University.
Procedure
Flies aged 6 days were collected by CO2anesthesia and individually placed into 65-mm glass tubes plugged on one end by filter paper and the other end by a porous cap, allowing air passage. The flies were starved for 4 hours before collection. The filter paper was saturated with solution that consist of Tween 80 0.4 ml, Schisandrin 0, 6.4, 12.7, 25.4, 50.8, 101.6 mg, respectively, volume to 10 ml with 5% sucrose solution. The saturated filter paper without Schisandrin was defined as Control. Each glass tube is placed into the Monitoring System devices (DAMS) from Trikinetics (Waltham, MA) that contains a series of 32 infrared emitter-detector pair, one for each tube. Generally, flies subjected to certain conditionswere given a day for adaptation, and data from the second day of the recording were used for analysis, that is 7-day-old flies.
Sleep deprivation was conducted by light stimulation. The process is as follows: 6-day-old flies were placed into glass as mention aboved. The flies were allowed to recover and adapt for 1 day. Light stimulation start the dark phase of the light:dark cycle of the second day at 7:00 pm. The all dark phase was insteaded of rthym of 10 min light/50 min dark alternately, continuing until 7:00 am the thire day. The light intensity is 650 Lux. During baseline, sleep deprivation and recovery, flies remained in the DAMS monitor. Since locomotor activity during sleepdeprivationwas continuously recorded, the extent of sleep loss could be calculated for eachindividual fly.
Female fly aged 6 days were anesthesiaed and placed in the tube with Schisandrin (2.54mg/ml) and No-Schisandrin filter paper. Flies head were collected at 7 am, after the flies were cultured in medium with and without Schisandrinfor 36 h.Sixty flies for each experimental group(Schisandrin and Control) were collected and divided into two pools (n=30flies/pool). Total RNA was extracted from fly heads of each pool. The two pools were used to hybridize twoindependent sets of Affymetrix Drosophila Genome Arrays. RNA extraction was performed using Trizol according to the manufacturer'sinstructions. Final RNA concentrations were determinedspectrophotometrically. Microarrays labeling, hybridization, andexpression analysis were performed according to the AffymetrixGeneChip Expression Analysis Technical Manual. The following criteria were used to select over-represented GO categories: (1) a representation increased at least twofold in theexperimental set compared with the reference; (2) overrepresentedwith a p <0.05 after false discovery rate correction.
Behavioral Analysis
For sleep analysis, 7-day-old flies were monitored in light/darkconditions at 25°C. An awake fly moves and back and forth in the tube, periodically breaking the infrared beam. Each crossing was counted by the Trikineticssystem as a movement. Locomotor activity of individual flies was monitored and collected continuously in 5 min bins using the Drosophila Activity Monitoring System devices (DAMS) from Trikinetics (Waltham, MA). The total sleep, the average duration ofsleep bouts, and the number of sleep bouts during nighttime and daytime were calculated basedon the sleep definition as a period of 5 or moreminutes of behavioralimmobility.
Data Analysis
Results are shown as the mean±S.E.M. Comparisons were carried out using ANOVA for continuous variables with normal distributions. When significant results were obtained, the analysis was followed by Tukey HSD test for multiple comparisons. The significance level was established at p<0.05. All statistical analyses were performed via SPSS 17.0 for Windows.
Results and Discussion
As shown in Table 1 and Table 2, compared to control, Schisandrin (0.635 mg/ml and 1.27 mg/ml) had significantly reduced the sleep time of daytime of female and male drosophila, and no effect onnighttime sleep time. Schisandrin (2.54 mg/ml, 5.08 mg/ml and 1.27 mg/ml) had significantly not only decreased the sleep time of daytime of female and male drosophila, but also the sleep time of nighttime in comparison with control. In female drosophila, the decreased sleep time was greater especially in the daytime than in the nighttime (45% vs. 20%, 37% vs. 26%, 32% vs. 16%).The sleep were calculated basedon the sleep definition as a period of 5 or moreminutes of behavioral inactivity (no beam breaks). During daytime, the decrease in sleep amount in flies induced by Schisandrin was mainly due to a decrease in the number of sleep episodes ratherthan in their duration, on the contrary, Schisandrin (2.54 mg/ml) significantly prolonged the duration of sleep episodes. During nighttime, the decrease in sleep amount in flies induced by Schisandrin was mainly due to a decrease in the duration of sleep episodes ratherthan in their number, on the contrary, the number of sleep episodes in flies administered by different concentration Schisandrin increased, but only Schisandrin (5.08 mg/ml) has significantly differences. Thus, Schisandrin affects the sleep amount under controled the circadian regulation.
Table 3 and Table 4 shows the effect of Schisandrin and light stimulation on sleep time among flies in the separate female and male. As seen in the table, light stimulation in the dark phase had significant deprived the sleep time of nighttime of flies in female and male. The post hoc of Tukey test showed that the three groups had significant differences in sleep duration of nighttime of male flies (Light stimulaion+Schisandrin (341.56±22.34) < Light stimulation (428.91±23.76) < Control (551.56±14.72)). The rankings according to sleep time of nighttime of female flies were Control (611.46±16.51)> Light stimulation (469.58±12.74) > Light stimulaion+Schisandrin (457.50±15.19), but the difference between Light stimulaion+Schisandrin and Light stimulaion for sleep duration did not reach significance. As shown in Table 3 and Table 4, no significant differences were obtained for sleep duration of female flies during sleep recovery between the Light stimulaion+Schisandrin and Light stimulation groups, but there were significant differences among male flies. In agreement with previous studies[8], the present study showed that after sleep deprivation by light stimulation, flies showed a large compensatory increase in sleep the next day. In addition, differences observed in male and female sleep behavior in flies administrated by Schisandrin during sleep deprivation and recovery. Previous studies found that the differences inmale and female sleep patterns may be related to sex-specificdifferences in metabolic needs [9].
Using whole-genome arrays, approximately 170 genes (fold change cut-off >2) are differentially expressed in flies administered by feeding with Schisandrin compared to control, including 84 down-regulated and 86 up-regulated. Expression changes associated with catabolism, redox reactions, circadian rhythms, transmembrane transport and immune response.
Table 1 The effect of Schisandrin on the time spending in sleep of female drosophila (* ±S.E.M.)_
Group Concentratio Sleep Duration (min)
(mg/ml) All day 24h Daytime 12h Nighttime 12h
Schisandrin 0.635 793.54±20.79 206.25±18.69** 587.29±13.76
1.27 699.35±40.00* 156.09±22.88** 543.26±28.71
2.54 629.19±35.63** 155.32±16.50** 473.87±24.63**
5.08 612.42±33.63** 177.58±81.26** 434.84±26.56**
10.16 683.23±32.67** 190.48±15.72** 492.74±25.49*
Control - 873.13±24.24 281.56±18.48 591.56±12.67
Comparisons between Schisandrin and Control, *p<0.05, **p<0.01
Table 2 The effect of Schisandrin on the time spending in sleep of male drosophila (x ±S.E.M.)_
Group Concentratio Sleep Duration (min)
(mg/ml) All day 24h Daytime 12h Nighttime 12h
Schisandrin 0.635 810.83±33.77** 303.96±18.34** 506.88±19.44
1.27 824.38±33.22** 297.92±18.60** 526.46±21.29
2.54 700.00±40.80** 254.17±20.48** 442.50±24.52**
5.08 710.78±50.05** 290.94±24.13** 419.84±29.56**
10.16 713.13±30.00** 322.19±17.71* 390.94±22.32**
Control - 978.91±23.05 399.53±15.40 576.25±12.81
Comparisons between Schisandrin and Control, *p<0.05, **p<0.01
Table 3 The effect of Schisandrin on the time spending in sleep of female drosophila by light
stimulation (x ±S.E.M.)
Group All day 24h 7-day-old Daytime 12h Nighttime 12h 8-day-old 7:00am- 9:00am
Light 634.38±25.45**# 176.88±17.29**## 457.50±15.19** 31.46±5.67*
stimulaion+Schisandrin #
Light stimulation 762.50±21.00** 292.92±18.08 469.58±12.74** 29.17±6.13*
Control 902.71±27.17 291.25±17.04 611.46±16.51 14.17±3.72
Compared to Control, *p<0.05, **p<0.01. Compared to Light stimulation, ## p<0.01.
Table 4 The effect of Schisandrin on the time spending in sleep of male drosophila by light
stimulation (x ±S.E.M.)
Group All day 24h 7-day-old Daytime 12h Nighttime 12h 8-day-old 7:00am- 9:00am
Light 566.25±40.15**# 224.68±20.51**## 341.56±22.34**# 42.03±5.96#
stimulaion+Schisandrin # #
Light stimulation 764.53±37.36** 335.62±22.26 428.91±23.76** 62.76±6.03*
Control 919.84±223.40 368.28±16.49 551.56±14.72 41.56±5.10#
Compared to Control, *p<0.05, **p<0.01. Compared to Light stimulation, # p<0.05, ## p<0.01.
Conclusions
In summary, Schisandrin significantly reduced the sleep duration in female and male fruit flies. Schisandrin make changes accordingly in drosophila gene expression. These results suggested that Schisandrin have an effect at the molecular level of sleep regulation. References
[1]. Huang LL LT, Guo LQ, Sun CY. The effects of Schisandra Fruit on the sleep of freely moving rats. Pharmacology and Clinics of Chinese Materia Medica, 2007, 23(5) : 126-127
[2]. Huang LL L, Liu J. Decoction of Fructus Schisandrae's constituents absorbed into blood of rats after oral administration. Zhong Hua Zhong Yi Yao Za Zhi, 2009, 24(7) : 846-848
[3]. Shaw PJ, Cirelli C, Greenspan RJ, Tononi G. Correlates of sleep and waking in Drosophila melanogaster. Science, 2000, 287(5459) : 1834-1837
[4]. Hendricks JC, Finn SM, Panckeri KA, et al. Rest in Drosophila is a sleep-like state. Neuron, 2000,25(1) : 129-138
[5]. Nitz DA, van Swinderen B, Tononi G, Greenspan RJ. Electrophysiological correlates of rest and activity in Drosophila melanogaster. Curr Biol, 2002, 12(22) : 1934-1940
[6]. Doronkin S, Reiter LT. Drosophila orthologues to human disease genes: an update on progress. Prog Nucleic Acid Res Mol Biol, 2008, 82 : 1-32
[7]. Bushey D, Cirelli C. From genetics to structure to function: exploring sleep in Drosophila. Int Rev Neurobiol, 2011, 99 : 213-244
[8]. Huber R, Hill SL, Holladay C, Biesiadecki M, Tononi G, Cirelli C. Sleep homeostasis in Drosophila melanogaster. Sleep, 2004, 27(4) : 628-639
[9]. Harbison ST, Sehgal A. Quantitative genetic analysis of sleep in Drosophila melanogaster. Genetics, 2008, 178(4) : 2341-2360
Sleep-awakening Physiological Structure in Different Day-old Wild-typeCS
Drosophila
Li Tingli, Zhang Ji
(College of Pharmacy of Heilongjiang University of Chinese Medicine, Harbin, China)
Abstracts. Study Objectives: Fruit flies genetic advantage to promote sleep molecular mechanism research, and evaluate its sleep parameters are often just total sleep time, in order to better explore different phenotype fruit flies and wild type fruit flies sleep regulation process difference, we adopt large sample to determine 7 age male and female flies sleep - awakening physiological structure, more male and female flies sleep-awakening physiological structure of the difference. . Reveal different day age drosophila sleep - awakening physiological structure changes.
Design: Choose wild type Canton S fruit flies for experimental object, using fruit flies activity monitoring system (DAMS), respectively record total sleep time, sleep times, each time sleep duration, and drew the 24 hours of sleep structure and activity rhythm diagram to clarify drosophila sleep - awakening physiological structure.
Results: 1.7 days of age female fruit fly all day long on average sleep time is 606 minutes, and sleep times is 17 times, each time sleep duration is 47 minutes; 7 age male fruit fly all day long on average sleep time is 9 minutes, sleep times is 23 times, each time sleep duration is four minutes. 2. Female flies with age increase round-the-clock total sleep time gradually raised, gradually increase the number of sleep, every time sleep duration gradually reduced; Male fruit flies with age increase round-the-clock total sleep time no obvious change, sleep times increase gradually, every time sleep duration reduced gradually. 3. Along with the age increasing drosophila activity rhythm sex is not obvious, night and day, alternating activity peak disappear.
Conclusion: 7 age drosophila sleep - awakening physiological structure stability, rhythmic good, different day age wild type CS drosophila sleep-awakening physiological structure changes, along with the increase of day age drosophila sleep-awakening rhythm strength is reduced, sleep consolidate sex is abate, sleep fragmentation increased obviously.
Keywords: Drosophila, sleep - awakening physiological structure, Biological rhythm, DAMS
Introduction
In 2000, two independent lab published data show the fruit fly class sleep state characteristics. Although different approaches, but have found it surprising similarity: wild type fruit flies in the light and dark alternate and continuous dark two conditions male and female are for the performance of the night long time of fixed[1][2], opens the fruit fly as sleep medicine research model system of the new era. And evaluate its sleep parameters are often just total sleep time, has certain limitation, because one day of the sleep may be assigned to different time periods. If sleep
