Method for screening sleep promoting medicine by utilizing sleep awakening frequency of zebra fish

Abstract

The invention belongs to the technical field of medicine screening, discloses a method for screening a sleep promoting medicine by using zebra fish sleep awakening frequency, and particularly discloses a method for screening a sleep improving medicine, which comprises the following steps: by taking zebra fish as a model organism, and taking zebra fish sleep and / or awakening frequency as an evaluation index, screening a sleep promoting medicine by using zebra fish as a model organism; and screening medicines capable of promoting sleep. The method for screening the sleep-improving medicine by utilizing the sleep and / or awakening frequency of the zebra fish is particularly suitable for rapidly screening the sleep-improving medicine. The used zebrafish is an international universal spinal model animal, and has the advantages of clear background, easiness in obtaining, stable and controllable quality, high experimental result credibility, good repeatability and convenience in large-scale popularization and application.

Description

Technical Field

[0001] The invention belongs to the technical field of drug screening, and specifically relates to a method for screening sleep-promoting drugs by utilizing the sleep-wake frequency of zebrafish. Background Art

[0002] Sleep disorders have become a huge hidden danger threatening human health. At present, drug treatments for sleep disorders mostly use sedatives and hypnotic drugs, but they are often accompanied by many side effects such as memory loss and dependence. Therefore, finding sleep-regulating drugs with few side effects and good efficacy is still an urgent problem that needs to be solved in current clinical practice.

[0003] Since in vitro cell models cannot truly simulate the complex sleep behavior of the body, the models currently used for sleep drug screening research are mainly rats and mice. However, the habit of mice to stay awake during the day and come out at night has certain limitations in simulating the natural sleep rhythm of humans, and there are problems such as long cycles and high costs in large-scale drug screening. Therefore, it is particularly necessary to develop a high-throughput, low-cost screening system. Zebrafish is listed as the third largest model vertebrate after mice and rats by the US NIH, and has the advantages of high egg production, short experimental cycle, and low cost. It is a very good model for high-throughput drug screening; previous studies have confirmed that zebrafish and humans have basically the same sleep performance. Zebrafish have very regular circadian rhythm phenomena like humans, especially in the slow wave sleep and propagation wave sleep (similar to rapid eye movement sleep) stages (Nature, 2019). The two are basically the same. Zebrafish is a good alternative model for sleep-related drug screening.

[0004] At present, screening technology based on the cell level cannot simulate the real sleep state, and the screening method based on the whole mammalian animal model has problems such as long cycle and high cost. Therefore, it is necessary to find a drug screening animal model that can simulate the real sleep state and has a short drug screening cycle and low cost. Summary of the invention

[0005] The present invention aims to overcome the problems of long cycle, high cost and low throughput in the existing screening process of drugs affecting sleep behavior, and provide a method for screening drugs for improving sleep by using the sleep or wakefulness frequency of zebrafish, so as to achieve high-throughput, low-cost and rapid screening of drugs.

[0006] The first aspect of the present invention aims to provide a method for screening drugs for improving sleep.

[0007] The purpose of the second aspect of the present invention is to provide application of the method of the first aspect of the present invention in the evaluation of the sleep-improving efficacy of a drug or food.

[0008] In order to achieve the above object, the technical solution adopted by the present invention is:

[0009] The first aspect of the present invention provides a method for screening drugs for improving sleep, comprising the following steps: using zebrafish as a model organism and the frequency of zebrafish sleep and / or wakefulness as an evaluation index to screen drugs that can promote sleep.

[0010] As an internationally used vertebrate model animal, zebrafish is easy to implement high-throughput, low-cost drug screening. It is an important supplement to the current sleep-related drug research and development system, and is more in line with the international experimental animal welfare ethics "3R principle" (reduction, substitution and optimization).

[0011] In some embodiments of the present invention, zebrafish are divided into a blank control group, a positive drug control group, and a test substance group. The zebrafish are treated according to the groups, and the circadian sleep behavior of each group of zebrafish is recorded using a zebrafish behavior trajectory tracking device; the circadian sleep behavior of each group of zebrafish is converted into the frequency of sleeping or waking in unit minutes using R language statistical analysis, and the sleep and wakefulness frequencies of each group of zebrafish are analyzed for significance of differences.

[0012] In some embodiments of the present invention, the zebrafish behavior trajectory tracking device includes a zebrafish behavior analysis system.

[0013] In some embodiments of the present invention, the effect of the drug is determined as follows: under the premise that the experiment is effective, for sleep frequency, when the average awakening frequency of the test substance group is greater than that of the blank control group and there is a significant difference therebetween, then the test substance is determined to have an effect on improving sleep, otherwise it has no effect on improving sleep; for awakening frequency, when the average awakening frequency of the test substance group is less than that of the blank control group and there is a significant difference therebetween, then the test substance is determined to have an effect on improving sleep, otherwise it has no effect on improving sleep.

[0014] In some embodiments of the present invention, the effectiveness of the experiment is determined as follows: for sleep frequency, the average awakening frequency of the positive drug control group must be greater than that of the blank control group and there is a significant difference compared with the blank control group, and the result of this experiment is determined to be valid; for awakening frequency, the average awakening frequency of the positive drug control group must be less than that of the blank control group and there is a significant difference compared with the blank control group, and the result of this experiment is determined to be valid.

[0015] In some embodiments of the present invention, the blank control group is zebrafish treated with embryo culture medium.

[0016] In some embodiments of the present invention, the embryo culture medium includes NaCl, KCl, CaCl2·2H2O, MgSO4·7H2O and NaHCO3.

[0017] In some embodiments of the present invention, the treatment process of each treatment group is: placing zebrafish in a well plate containing embryo culture medium, positive drug solution or test substance solution, and standing for 20 to 48 hours without feeding or changing the solution.

[0018] In some embodiments of the present invention, in the method, the light intensity during the treatment of each treatment group is 200-300 lux, the temperature is 27-30° C., and the light / dark cycle is 14 / 10 h.

[0019] In some embodiments of the present invention, the zebrafish is 5-6 day old zebrafish larvae, which have normal activity and good adaptability.

[0020] Zebrafish have basic biological structures and physiological functions that are highly similar to mammals, and the homology rate with human disease-related genes is as high as 82%. Especially in terms of sleep rhythm, compared with the nocturnal habits of mice, the sleep rhythm of zebrafish is closer to that of humans. In sleep evaluation research, compared with the 30-45 days of mouse experiments, the evaluation time of zebrafish can be shortened to 24 hours. At the same time, due to the low breeding cost, short development cycle, and high number of single spawning times of zebrafish, multiple test products can be evaluated at the same time, which is more economical than mice.

[0021] In some embodiments of the present invention, a co-solvent control group may be provided in the method, wherein the co-solvent includes a reagent that helps the positive drug or test substance solution.

[0022] The second aspect of the present invention provides the use of the method of the first aspect of the present invention in the evaluation of the sleep-improving efficacy of a drug or food.

[0023] The beneficial effects of the present invention are:

[0024] The method of screening sleep-improving drugs using the sleep or wakefulness frequency of zebrafish is particularly suitable for rapid screening of sleep-improving drugs. The zebrafish used is an internationally common vertebrate model animal with a clear background, easy to obtain, stable and controllable quality, high credibility of experimental results, good repeatability, and convenient for large-scale promotion and application.

[0025] The method of the present invention can use a 96-well plate to simultaneously perform large-scale monitoring of the sleep behavior of 96 5- to 6-day-old zebrafish, and the screening and evaluation can be completed within 24 to 48 hours. Compared with the traditional mammalian model, the screening efficiency of the method of the present invention is greatly improved, and the screening cost is greatly reduced, which is an important supplement to the current drug screening system. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 This is a line graph of the sleeping and waking frequency of zebrafish in Example 1.

[0027] Figure 2 This is a line graph of the sleeping and waking frequency of zebrafish in Example 2.

[0028] Figure 3This is a line graph of the zebrafish sleep and wakefulness frequency in Example 3. DETAILED DESCRIPTION

[0029] The present invention is further described in detail below through specific examples.

[0030] It should be understood that these embodiments are only used to illustrate the present invention and are not used to limit the scope of the present invention.

[0031] In order to make the purpose, technical scheme and advantages of the embodiments of the present invention clearer, the technical scheme in the embodiments of the present invention will be described clearly and completely below. If the specific conditions are not specified in the embodiments, they are carried out according to conventional conditions or conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be purchased commercially.

[0032] The features and performance of the present invention are further described in detail below in conjunction with the embodiments.

[0033] Example 1

[0034] A method for screening sleep-improving drugs using zebrafish sleep or wakefulness behavior, comprising the following steps:

[0035] (1) 5- to 6-day-old zebrafish larvae were used as experimental animals.

[0036] (2) The zebrafish were divided into a blank control group, a solvent control group (as needed), a positive drug control group, and a test substance group. The zebrafish behavior trajectory tracking equipment was used to record the sleep behavior of each group of zebrafish. The recording time was 24 to 48 hours (10 hours in the dark: 14 hours in the light).

[0037] (3) After the experiment, the circadian sleep behavior of each group of zebrafish was converted into the frequency of sleep or wakefulness per minute using R language statistical analysis, and the difference in sleep or wakefulness activity frequency between the positive drug control group and the test substance group and the blank control group (or cosolvent control group) was analyzed.

[0038] (4) Determination of the validity of the results: For sleep frequency, the average awakening frequency of the positive drug control group must be greater than that of the blank control group (or solvent control group) and there must be a significant difference (p<0.05) compared with the blank control group (or solvent control group), proving that the results of this experiment are valid; for awakening frequency, the average awakening frequency of the positive drug control group must be less than that of the blank control group (or solvent control group) and there must be a significant difference (p<0.05) compared with the blank control group (or solvent control group), proving that the results of this experiment are valid.

[0039] (5) Determination of sleep improvement effect: For sleep frequency, the average awakening frequency of the test substance group must be greater than that of the blank control group (or solvent control group) and there is a significant difference (p<0.05) compared with it, and the test substance is considered to have an effect on improving sleep; for awakening frequency, the average awakening frequency of the test substance group must be less than that of the blank control group (or solvent control group) and there is a significant difference (p<0.05) compared with it, and the test substance is considered to have an effect on improving sleep.

[0040] Application Example 1

[0041] The method of Example 1 is used to screen whether the drug to be tested has the effect of improving sleep, as follows:

[0042] 1. Experimental organisms

[0043] The zebrafish broodstock used in the experiment were raised in a circulating water culture system at 28±1°C and a light / dark cycle of 14 / 10h, with a stocking density of approximately 20 fish / 10L of water.

[0044] Male and female fish were paired at a ratio of 2:1, and spawning was performed using the light-induced method. The harvested fertilized eggs were cultured with embryo culture medium (formula see Table 1) and placed in a constant temperature incubator with a light intensity of 200 lux and a temperature of 28.5±0.5°C. The light / dark cycle of the incubator was 14 / 10h. 5-6 day old (5-6 dpf) fry were selected as experimental fish.

[0045] Table 1 Zebrafish embryo culture medium

[0046]

[0047] 2. Sample preparation

[0048] The test water was embryo culture medium. The solution used in the blank control group and the solution used in the positive drug group (melatonin) were prepared according to Table 2.

[0049] Table 2 Solution preparation

[0050] Group Blank control group Positive drug group 1 Positive drug group 2 Positive drug group 3 concentration Embryo culture medium 10μM 20μM 40μM

[0051] 3. Animal distribution

[0052] The test container is a transparent flat-bottom 96-well plate. According to the experimental grouping, zebrafish are distributed to each well of the 96-well plate, with one experimental fish placed in each well and 200 mL of test solution in each well. Each group has 16 replicate wells. The test time is 24 hours, and no feeding or water change is performed during the test. During the test, the light, temperature and light environmental factors are the same as the incubation conditions.

[0053] 4. Behavioral monitoring

[0054] The zebrafish behavior analysis system (Viewpoint ZebraLab, Viewpoint behaviortechnology) was used to monitor the activity of zebrafish larvae in 96-well plates for 24 hours. The infrared monitoring background threshold was set to 35, the high activity threshold was set to 25, the inactivity threshold was set to 4, the distance unit was millimeter (mm), and the movement frequency of larvae per minute (frequency / min) was recorded.

[0055] 5. Statistics

[0056] The frequency of sleep or wakefulness in each group was counted using R language.

[0057] 6. Determination of sleep improvement effect

[0058] The results of sleep or wake frequency in each group are as follows Figure 1 As shown in Table 3, according to the sleep or wakefulness behavior data, the three concentration groups of the positive drug (melatonin) can significantly promote sleep (p<0.05), that is, the positive drug melatonin has a good effect in promoting sleep.

[0059] Table 3 Sleep or wakefulness frequency in each group

[0060]

[0061] Application Example 2

[0062] 1. Experimental organisms

[0063] Same as Example 1.

[0064] 2. Sample preparation

[0065] Melatonin was used as a positive drug (40 μM), caffeine, a neurostimulant drug that can cause insomnia, was used as a test substance, and the test water was used as embryo culture medium. According to Table 4, a blank control group, a positive drug group (melatonin), and a test substance group (caffeine) were prepared.

[0066] Table 4 Solution preparation

[0067] Group Blank control group Positive drug group Caffeine group concentration Embryo culture medium 40μM 0.2g / L

[0068] 3. Animal distribution

[0069] The test container is a transparent flat-bottom 96-well plate. According to the experimental grouping, zebrafish are distributed to each well of the 96-well plate, with one experimental fish placed in each well and 200 mL of test solution in each well. Each group has 16 replicate wells. The test time is 24 hours, and no feeding or water change is performed during the test. During the test, the light, temperature and light environmental factors are the same as the incubation conditions.

[0070] 4. Behavioral monitoring

[0071] The zebrafish behavior analysis system (Viewpoint ZebraLab, Viewpoint behaviortechnology) was used to monitor the activity of zebrafish larvae in 96-well plates for 24 hours. The infrared monitoring background threshold was set to 35, the high activity threshold was set to 25, the inactivity threshold was set to 4, the distance unit was millimeter (mm), and the movement frequency of larvae per minute (frequency / min) was recorded.

[0072] 5. Statistics

[0073] The frequency of sleep or wakefulness in each group was counted using R language.

[0074] 6. Determination of sleep improvement effect

[0075] The results of sleep or wake frequency in each group are as follows Figure 2 As shown in Table 5, according to the sleep or wakefulness behavior data, the negative test substance concentration group (caffeine) could not significantly promote sleep (p<0.05), and caffeine had no effect on improving sleep (p>0.05).

[0076] Table 5 Sleep or wakefulness frequency in each group

[0077]

[0078] Application Example 3

[0079] 1. Experimental organisms

[0080] Same as Example 1.

[0081] 2. Sample preparation

[0082] Melatonin was used as a positive drug (40 μM), alprazolam, a drug for treating insomnia, was used as a test substance, and the test water was used as embryo culture medium. According to Table 6, a blank control group, a positive drug group (melatonin), and a test substance group were prepared.

[0083] Table 6 Solution preparation

[0084] Group Blank control group Positive drug group Alprazolam group concentration Embryo culture medium 40μM 0.1g / L

[0085] 3. Animal distribution

[0086] The test container is a transparent flat-bottom 96-well plate. According to the experimental grouping, zebrafish are distributed to each well of the 96-well plate, with one experimental fish placed in each well and 200 mL of test solution in each well. Each group has 16 replicate wells. The test time is 24 hours, and no feeding or water change is performed during the test. During the test, the light, temperature and light environmental factors are the same as the incubation conditions.

[0087] 4. Behavioral monitoring

[0088] The zebrafish behavior analysis system (Viewpoint ZebraLab, Viewpoint behaviortechnology) was used to monitor the activity of zebrafish larvae in 96-well plates for 24 hours. The infrared monitoring background threshold was set to 35, the high activity threshold was set to 25, the inactivity threshold was set to 4, the distance unit was millimeter (mm), and the movement frequency of larvae per minute (frequency / min) was recorded.

[0089] 5. Statistics

[0090] The frequency of sleep or wakefulness in each group was counted using R language.

[0091] 6. Determination of sleep improvement effect

[0092] The results of sleep or wake frequency in each group are as follows Figure 3 As shown in Table 7, according to the sleep or wakefulness behavior data, the test substance concentration group can significantly promote sleep (p<0.05), and the test substance has the effect of improving sleep.

[0093] Table 7 Sleep or wakefulness frequency in each group

[0094]

[0095]

[0096] The embodiments of the present invention are described in detail above in conjunction with the accompanying drawings, but the present invention is not limited to the above embodiments. Various changes can be made within the knowledge of ordinary technicians in the relevant technical field without departing from the purpose of the present invention. In addition, the embodiments of the present invention and the features in the embodiments can be combined with each other without conflict.

Claims

1. A method for screening a drug for improving sleep, comprising the following steps: Zebrafish was used as a model organism, and the frequency of zebrafish sleep and / or wakefulness was used as an evaluation indicator to screen drugs that can promote sleep.

2. The method according to claim 1, characterized in that The method specifically includes: dividing zebrafish into a blank control group, a positive drug control group, and a test substance group, treating the zebrafish according to the groups, and using a zebrafish behavior trajectory tracking device to record the circadian sleep behavior of each group of zebrafish; using R language statistical analysis to convert the circadian sleep behavior of each group of zebrafish into the frequency of sleeping or waking in a unit minute, and performing a significant difference analysis on the frequency of sleeping and waking of each group of zebrafish.

3. The method according to claim 2, characterized in that The effect of the drug is determined as follows: Under the premise that the experiment is effective, for sleep frequency, when the average awakening frequency of the test group is greater than that of the blank control group and there is a significant difference compared with it, then the test drug is judged to have the effect of improving sleep, otherwise it has no effect on improving sleep; for awakening frequency, when the average awakening frequency of the test group is less than that of the blank control group and there is a significant difference compared with it, then the test drug is judged to have the effect of improving sleep, otherwise it has no effect on improving sleep.

4. The method according to claim 3, characterized in that The validity of the experiment is determined as follows: for sleep frequency, the average awakening frequency of the positive drug control group must be greater than that of the blank control group and there is a significant difference compared with the blank control group, and the results of this experiment are determined to be valid; for awakening frequency, the average awakening frequency of the positive drug control group must be less than that of the blank control group and there is a significant difference compared with the blank control group, and the results of this experiment are determined to be valid.

5. The method according to any one of claims 1 to 4, characterized in that: The blank control group was treated with embryo culture medium to treat zebrafish.

6. The method according to claim 5, characterized in that The treatment process of each treatment group is as follows: the zebrafish are placed in a well plate containing embryo culture medium, positive drug solution or test substance solution, and left to stand for 20 to 48 hours without feeding or changing the solution.

7. The method according to claim 6, characterized in that In the method, the light intensity during the treatment of each treatment group is 200-300 lux, the temperature is 27-30° C., and the light / dark cycle is 14 / 10 h.

8. The method according to claim 5, characterized in that The zebrafish are 5-6 day old zebrafish larvae.

9. The method according to any one of claims 1 to 4, characterized in that: A co-solvent control group may also be provided in the method, wherein the co-solvent includes a reagent that helps dissolve the positive drug or the test substance.

10. Use of the method according to any one of claims 1 to 9 in evaluating the sleep-improving efficacy of a drug or food.

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