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Mechanism of Sini Freeze-Dried Powder to Improve Sleep Based on Gene
Expression in Drosophila Brain
Bian Hongsheng, Huang Lili, Jin Yang and Li Tingli
HeilongjiangUniversityofChineseMedicine,Harbin150040,China
Abstracts
Objective: By using gene chip technology, we observed the gene expression in Drosophilabrain through sleep deprivation caused by light stimulation and the intervention of Sini freeze-dried powder, in order to discuss the mechanism of Sini freeze-dried powder to improve sleep. Methods: We collect Wild-type (Canton S) female Drosophila melanogaster within 12 h of emergence without mating. After shallow anesthesia with CO2, flies are randomly divided into three groups (Control group, Sleep deprivation group and Administration group).We put the different groups of flies into glass tubes with basal medium, Continuous culture 3 days.In the beginning 4 days, we put administration group flies into the4%(Weight Ratio between Sini freeze-dried powder and basal medium) Sini freeze-dried powder medium. In the beginning 7 days, at 7:00, we put sleep
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deprivation group and administration group into the light control box. After pre-adapted 12 h, at 19:00, running light control box,starting sleep deprivation experiment. In the beginning 8 days, at 1:00, using the low-temperature freezing method, we obtain the three groups of flies' heads (N = 3 samples in parallel, each sample 30), for total RNA extraction and gene expression detection. Results: Theresults show that compared with the control group, the light stimulation sleep deprivation gene expression changesin Drosophila brain, show that the mechanism of light stimulation sleep deprivation related to gene expression of Drosophila brain; Compared with the sleep deprivation group, administration group gene expression changes in Drosophila brain, show that the mechanism of Sini freeze-dried powder to improve sleep related to Gene expression of Drosophila brain. Conclusion:The mechanism of Sini freeze-dried powder to improve sleep is related togene expression in Drosophila brain.
Keywords:Drosophila; Sini freeze-dried powder; Genechip
Introduction
Sini, a famous prescription, from the "Treatise on Febrile Diseases", was created by Zhang Zhongjing. With the development of research, Sini have expanded the scope of clinical applications and achieved remarkable results in the treatment of various diseases. Reports are not uncommon, especially in terms of improving sleep [1]. However, as with most traditional Chinese medicine researches, Most of Sini experiments are only at the level of the synergy of barbiturates, the phase of sleep, central neurotransmitter content, which has not been reported on the experimental study of molecular mechanism to improve sleep[2]. In recent years, Drosophila has been defined as a model organism by domestic and foreign scientists for sleep studies [3' 4]. In this experiment, wild-type Drosophila melanogaster is as a biological model. Using gene chip technology, we compare the parallel Control group, Sleep deprivation group and Administration group differences in brain gene expression in Drosophila, select gene closely related to improving sleep. We analyze the differentially expressed genes obtained, in order to reveal the molecular mechanism of Sini to improve sleep.
Materials and methods
The following methods of investigation were used the Wild-Type Canton S Flies (Drosophila melanogaster) provided by the College of Life Sciences, PekingUniversity.Sini freeze-dried powder: light yellow, mesh loose, the main components of Paeoniflorin (not less than 2.74%), Saikosaponins A (not less than 2.32%), Naringin (not less than 3.31%), Glycyrrhetinic acid (not Less than 2.70%). And main experimental apparatus ©Light Control Box (homemade)@PCR Instrument (MJ Company, Model: PTC-100)®Spectrophotometer (Nanodrop Company, Model: ND1000) . Main reagents©Cy3 NHS ester (GE healthcare company, batch number: PA13105)@aaUTP (Ambion Corporation, batch number: AM8436)@Gene Expression Hybridization Kit (Agilent Company, batch number: 5188 -5242) .etc. Experimental temperature: 24 ± 1°C; Humidity: 50 ~ 60%;Light: 12 h light - dark auto cycle (7:00 lights, turn off the lights 19:00).
Drosophila light stimulation sleep deprivation modelPut Drosophila selected into the light control boxes, light: 7:00-19:00, at 19:00 automatically turn off the light. From 20:00, at the top of every hour, light 10 minutes, other time as the dark period, this state continued until 7:00 the next day.In this way, during the day, flies have a normal activity. At night, Drosophila sleep is constantly interrupted and can not form a continuous sleep, so Drosophila sleep is deprived [5].
Sample SelectionSelect the wild-type Canton S female flies separated, after shallow anesthesia with CO2, randomly divided into three groups (Control group, Sleep deprivation group and Administration group). N = 90, continuous culture 3 days. In the first 4 days, we put administration group flies into the 4% (Weight Ratio between Sini freeze-dried powder and basal medium) Sini freeze-dried powder medium. In the first 7 days, at 7:00, we put sleep deprivation group and administration group into the light control box. After pre-adapted 12 h, at 19:00, running light control box, starting sleep deprivation experiment. In the first 8 days, at 1:00, using the low-
temperature freezing method, we obtain the three groups of flies' heads (N = 3 samples in parallel, each sample 30), stored in liquid nitrogen for later use. Drosophila brain microarray experimentReference to "Experiment Technical Manual Shanghai Biochip Co., Ltd"
Results and discussion
According to reports, more than 60% of human disease genes can be found in the Drosophila [6' 7].In recent years, Drosophila has been defined as a model organism by domestic and foreign scientists for sleep studies, because the fruit fly was shown to exhibit many important features of mammalian sleep[8].Similar to mammalian sleep, Drosophila also has a persistent sleep and elevated arousal threshold and is affected by central nervous system stimulants such as caffeine [9], by the way, fruit flies sleep relate to the circadian rhythms and regulation of internal environment.Circadian system can consolidate and strengthen most of the night sleep, homeostasis system work on Sleep after sleep deprivation.
Sleep deprivation is divided into total sleep deprivation, partial sleep deprivation and selective sleep deprivation.Sleep deprivation is sleep loss caused by various reasons, and cause a series of changes such as emotion, learning and memory, immune function [10]. The sleep of Human or Animal deprived experimentally is a feasible method ofphysiological significance and the necessity of sleep study. By the sleep deprivation experiments, we can study how the respond to sleep disorders, in order to infer the functional significance of the natural sleep state. By selecting or creating a suitable experimental animal model of sleep deprivation, it is significantly important for researching the effect of Sedatives and hypnotics andimprove sleep drugs.It is reported that light stimulation can interfere with Drosophila CRY which is a Deep Brain Circadian Photoreceptor, CRY mainly express in the central circadian pacemaker neurons in brain - the lateral neurons (LNs), light stimulation may directly or indirectly affect a complex series nerve conduction, making the show circadian oscillation of Clk expression, the role principle is also found in mammals [8].In summary, we copy Drosophila model of sleep deprivation in this study, usingthe method of light stimulus. The results show that (Reference to NCBI (http://www.ncbi.nlm.nih.gov/), Drosophila database (http://flybase.org/) The specific expression selected genes for analysis, description and classification.) only one eligible gene"Cecropin C", compared with those of control group, is differentially expressed in sleep deprivation group;CecC is a Drosophila protein-coding gene.Its molecular function is unknown.Experimental evidence suggests that in the biological process, it play the role of defense, which involves the reaction of defense gram-negative bacteria and Grampositive bacteria, and have an antibacterial effect in The Humoral Immune Response [11].Suttmann et al show that Cecropin has effective to anti-bacterial and anti-cancer cells, but has no effect on normal cells [12].In short, it is well known in the gene family, has anti-inflammatory effect, is an important immune response genes, is often examined in the Drosophila immune tests.
In this study, by light stimulation, result in fruit flies sleep disorders, make the Drosophila immune function. Show CecC up-regulated.But directly involve in the impact of flies sleep time, or indirectly affect the fruit fly by the Homeostasis system in Drosophila, we would also like to further study of this change.
Compared with those of control group, a total of 7 eligible genes are differentially expressed, among which the focus genes are Os-C and Obp19a in administration group. Os-C is a Drosophila protein-coding gene. It is reported that the combination with pheromone is the molecular function of the gene. In OS (-E,-F,-D,-C) gene family, four genes interact with Drosophila olfactory organs, Os-C, which is the fourth gene, is responsible for encoding 13-KDa protein, and the protein includes a putative nuclear import sequence and an accounting area [13]. Obp19a is a Drosophila protein-coding gene. Its molecular function: bind with odorant, help to capture and transport hydrophobic odorants. In the biological process, It responds to pheromones; involves in the sensory perception of chemical stimulus and olfactory behavior [14].
So far we have initially aware that the odorant binding proteins (odorant-binding protein, OBP) of the Insect can be divided into two categories: pheromone binding protein (Pheromone-binding protein, PBP) and general odorant binding protein (general odorant-binding protein, GOBP). PBP specifically exists in the hair sensilla of the lymph, which associate with the
pheromone, and GOBP exists in the cone receptors within the lymph, which is General odor-binding protein. Insects can feel volatile compounds as the information in air, for mating, feeding and looking for the reproductive sites [15-18]. The results show that after perceived by the peripheral receptor system, the pheromone signal is encoded, which is transmitted to the mesencephalic antennary lobe and olfactory lobe, through nerve fibers. The coded information finally decide on the courtship behavior of male insects. PBP plays an important role on the process of Insect sensitivity pheromone[19'20] 0
In summary, there are OS-C and Obp19a up-regulated genes in this experiment, maybe due to Sini freeze-dried powder continuely exist in Drosophila Peripheral feeling area, that Interfere with neural coding of chemical information. The normal behavioral response of female flies is not the information needed, thus showing behavioral response rate decreased and sleep time increased. Of course, the Drosophila immune system also is affected by the change, There are CecC, CecA1 and DptB up-regulated expression genes.And by analyze the administration group and sleep deprivation group, we believe that Os-C and Obp19a up-regulated expression genes in this experiment Odor stimulus has effect on the olfactory behavior, this way maybe affect the fruit flies sleep
In addition, we also screen out roX2, DptB, CecA1et al in the experiment. They are related with immune regulation. Therefore, we have reason to believe that fruit flies sleep behavior is associated with the olfactory behavior and immune system, but further study.
Through the study of Differential Gene Expression in Drosophila brain of control group, sleep deprivation group and administration group, we screen out a number of gene fragments related to improve the sleep of light stimulus females flies, prolong sleep time, and provide a theoretical basis for further research. Conclusions
The mechanism of Sini freeze-dried powder to improve sleep is related togene expression in Drosophila brain.
Corespondent author:
Liting Li, MD, Male, Heilongjiang Harbin, College of Pharmacy, Heilongjiang University of Chinese Medicine, Doctoral Supervisor. Main research interests: Natural drugs and compound biological activity. Email: [email protected]. Foundation item: National Natural Science Foundation of China (81073077) References
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The research of the treatment role of anemarrhena water decoction on the rat with chronic emotional stress by the empty bottles
Yunfeng DOU, Bing ZHANG, Tingli LI HeilongjiangUniversity of Chinese Medicine, Harbin,150040,China
Abstracts: Objective :To research the treatment role of Anemarrhena water decoction on the rats with chronic emotional stress induced by empety bottles.Methods:Making use of empty bottles stimulatecausethe chronic emotional stress model,Then observed behavioural changes(attacking,exploring,grooming) and weighted the rats by electronic animal scales befor the experiment,in the seventh day in the stimulus,in the fourteen day in the stimulus. .Used Enzyme-linked immunosorbent method to determine serum corticosterone in rats after stress. Results:The rats with chronic emotional stress manifested frequent attack,and modify behavior, Weight growth had striking decreased ,The content of cortical ketones in the blood serum had significant decreased, compared withtreatment group and model group :attack and modify behavior had significant increased, the weight growth had significant increased, the content of cortical ketones in the blood serum had significant decreased. Conclusion: The rhizoma anemarrhenae decoction for the improve lever function empty bottle stimulate induced chronic emotional stress for the treatment role.
Key words: rhizoma anemarrhenae, cortical ketones, empty bottle stimulate, Chronic emotional stress
Anemarrhenae is of cool nature, it tastes bitter and sweet,the rolo of clearing heat purging fire and nourishing yin for moistening dryness.Studies have shown that saponins from
