| Objective:Determine the jujubosidesA mechanism to improve fruit flies sleep functionPart one part is based on drosophila model of jujubosidesA improve fruit flies. Study of effects of sleep1.JujubosidesA glycosides to the age of 7-day-old wild type strain of CS drosophila melanogaster sleep time and sleep-wake rhythm.Select 7-day-old wild type strain of CS drosophila melanogaster, were randomly divided into blank group, zizyphus jujube RenZao nucleoside group A (JA),diazepam group,use of DAMS flies activity monitoring system for monitoring method,to sleep time,sleep time extension rate and sleep-wake rhythm here to observe,explore zizyphus jujube RenZao glycosides to the age of 7,A wild type strain of CS drosophila melanogaster sleep and sleep-wake rhythm.2.Investigat the dose effect relationship of jujubosidesA on the 24 hours sleep of 7-day-old wild type CS strain drosophila.Select 7-day-old wild-type CS strain drosophila for the study,and divided into 0.25,0.5,1,2,and 4 mg/ml dose groups randomly. Choose sleep time,sleep duration rate and sleep wake rhythm as observation index to investigat the dose effect relationship of jujubosidesA on the 24 hours sleep of 7-day-old wild type CS strain drosophila by DAMS Drosophila activity monitoring system.3.Investigat the time effect relationship of jujubosidesA on the 24 hours sleep of 7-day-old wild type CS strain drosophila.Select wild-type CS strain drosophila for the study,dose with the optimum dosage lmg/ml according to the dose effect relationship and dose for 1、2、3、4、5、6 days respectively.Choose sleep time, sleep duration rate and sleep wake rhythm as observation index to investigate the time effect relationship of jujubosidesA on the 24 hours sleep of 7-day-old wild type CS strain drosophila by DAMS Drosophila activity monitoring system.Compared with the blank group,3 group d sleep time prolong the highest rate.Part two,effect of jujubosidesA on mechanical sleep deprived Drosophila sleep duration and sleep-wake rhythm.Select 7-day-old wild-type CS strain drosophila for the study,and divided the Drosophila into blank group,model group(SNAS group)and administration group(JA group)randomly.Copy the sleep deprivation model with SNAS mechanical stimulation sleep deprivation device and DAMS drosophila activity monitoring system.Choose sleep time, sleep duration rate and sleep wake rhythm as observation index to investigate the sleep duration and sleep-wake rhythm of jujubosidesA on the 24 hours’sleep of 7-day-old wild type CS strain drosophila.Part three:Investigate the mechanism of jujubosidesA in improving sleep function of Drosophila.1.Effect of jujubosidesA on the contents of 5-HT,DA and GABA in the brain of Drosophila.Select 7-day-old wild-type CS strain drosophila for the study, and divided the Drosophila into blank group,model group(SNAS group)and administration group(JA group)randomly.Choose 5-HT, DA and GABA in the brain of Drosophila as the observation index,study the effect of jujubosidesA on the content change of the brain neurotransmitter in Drosophila by Enzyme linked immunosorbent assay(ELISA).2.Effects of jujubosidesA on metitepine which is a 5-HT receptor antagonist in Drosophila sleep and sleep wake rhythmSelect 7-day-old wild-type CS strain drosophila for the study,and divided the Drosophila into blank group, MEP group and JA+MEP group randomly. Choose sleep time, sleep duration rate and sleep-wake rhythm as observation index to explore the effects of metitepine on sleep and sleep-wake rhythm of Drosophila and the effect of drug intervention by DAMS drosophila activity monitoring system.3.Study on the relationship between Gsa-cAMP-pka signaling pathway and the physiological structure of sleep wake in DrosophilaElect 7-day-old wild-type CS strain drosophila for the study, and divided the Drosophila into blank group, model group(SNAS group)and administration group(JA group)randomly.Real-time PCR was used to detect the relative expression of GSa, AC and PKA three genes in cDNA samples. Explore the relationship between Gsa-cAMP-pka signaling pathway and the physiological structure of sleep wake in Drosophila. Part four:Effects of jujubosidesA on sleep duration and sleep-wake rhythm of Gsa gene deleted DrosophilaSelect 7-day-old wild-type CS strain drosophila and Gsa gene deleted Drosophila, and divided the Drosophila into wild-type group, Gsa gene deleted group and JA group randomly.Choose sleep time and picture of sleep-wake rhythm as observation index to explore the sleep duration and sleep-wake rhythm in Gsa gene deleted Drosophila and study the effects of jujubosidesA on sleep duration and sleep-wake rhythm of them.Results:Part one:Study on the effect of jujubosidesA on the 24 hours sleep of 7-day-old wild type CS strain drosophila. Investigat the dose effect relationship of jujubosidesA on the 24 hours’sleep of 7-day-old wild type CS strain drosophila.Experimental results show that:Compared with the blank group. When the concentration of jujubosidesA is 0.25mg/mL-2mg/mL,AST and NAST are both extended, and there are extremely significant differences(p<0.01). When the concentration reached lmg/mL, AST, DAST and NAST are extended, and have extremely significant difference(p<0.01).Compared with the blank group, When the concentration of jujubosidesA was lmg/ml, the sleep time extension rate was highest of drosophila.The sleep-wake rhythm picture shows that:In the blank group, Drosophila sleep present the fragmentation during the day and there are less sleep fragmentation but more continuous sleep in the night. Jujubosides A affects sleep time by reduction of the continuous activity time and sleep fragmentation of drosophila during the day and in the night,jujubosides A affects sleep fragmentation less. When the concentration of jujubosides A was lmg/mL,the effect was most obvious.Investigat the time-effect relationship of jujubosidesA on the 24 hours’sleep of 7-day-old wild type CS strain drosophila.Experimental results show that:Compared with the blank group, the AST,NAST of drosophila are extended(p<0.05) in the 2d group and the AST, NAST and DAST are extended with a very significant difference(p<0.01) in the 3d group. The AST, DAST and NAST are shorten in the 4d group.Sleep-wake rhythm figure display that Drosophila’circadian sleep time are increased,but it was mainly increased in the daytime. In the 3d group, DAST and NAST are increased, the sleep fragmentation is reduced and sleep-wake rhythm in drosophila is not affected. In the 4d group and 5d group, the sleep fragmentation of Drosophila is increased during the day significantly and reduced in the night. In the 6d group, the sleep fragmentation of Drosophila is increased during the day and at night significantly.Part two, effect of jujubosidesA on mechanical sleep deprived Drosophila’sleep duration and sleep-wake rhythm.Experimental results show that:Compared with the blank group, AST and NAST are reduced with significant differences(P<0.01) in the SNAS group. Compared with the SNAS group, AST and NAST are increased with significant differences(P<0.01) in the JA group.Sleep-wake rhythm figure display that:Compared with the blank group, NAST was reduced significantly in the SNAS group, concentrative sleep rebound was emerged after 7 o’clock and the rhythm of sleep has changed, in the JA group, NAST sleep fragmentation was increased concentrative sleep rebound was still emerged after 7 o’clock and DAST has extended. Investigate the mechanism of jujubosidesA in improving sleep function of Drosophila.1.Effect of jujubosidesA on the contents of 5-HT,DA and GABA in the brain of Drosophila.Experimental results show that:1. Compared with the blank group, the content of 5-HT decreased(p< 0.05) in the SNAS group. Compared with the SNAS group, the content of 5-HT increased in the JA group obviously.2. Compared with the blank group, the content of DA decreased significantly(p< 0.01) in the SNAS group. Compared with the SNAS group, the content of DA has no significant difference in the JA group.3. Compared with the blank group, the content of GABA decreased significantly(p< 0.01) in the SNAS group. Compared with the SNAS group, the content of GABA has no significant difference in the JA group.2.Effects of jujubosidesA on metitepine which is a 5-HT receptor antagonist in Drosophila sleep and sleep wake rhythmExperimental results show that:Compared with the blank group, AST and NAST were reduced (P<0.01) with significant difference in the SNAS group and the MEP group has no significant difference. In the SNAS group, concentrative sleep rebound was emerged after 7 o’clock and the rhythm of sleep has changed. In the MET group, sleep-wake rhythm is not obvious. Compared with the MET group, AST has increased (P<0.05) with significant difference in the JA+MEP group.3.Study on the relationship between Gsa-cAMP-pka signaling pathway and the physiological structure of sleep wake in DrosophilaExperimental results show that:Compared with the blank group, the relative content of Gsa, AC3 and PKA gene has increased with significant difference in the SNAS group. Compared with the SNAS group, the relative content of Gsa, AC3 and PKA gene has decreased with significant difference in the JA group.Effects of jujubosidesA on sleep duration and sleep-wake rhythm of Gsa gene deleted DrosophilaExperimental results show that:Compared with the wild type, AST, DAST, and NAST have no significant difference in the Gsa gene deleted group. Compared with the Gsa gene deleted group, AST and DAST were increased (P<0.01) with significant difference in the JA group. Sleep-wake rhythm figure display that:Compared with the wild type, in the Gsa gene deleted group,sleep fragmentation was increased. At about 19:00 o’clock, there is no centralized activity and the rhythm is not obvious. Compared with the Gsa gene deleted group, the sleep fragmentation was decreased and the sleep time becomes longer in the JA group. At about 19:00 o’clock, concentrated activities emerged and the rhythm has changed.Conclusion:Jujuboside A can effectively improve the sleep time of sleep deprived Drosophila and reduce the sleep fragmentation. The mechanism of action is related to many factors as for 5-HT content, a neurotransmitter in the brain of Drosophila, and the signaling pathway with 5-HT mediated. Also, it may be associated with Gsagene expression. |
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