Establishment method and application of bee sleep rhythm injury model
By combining light and dark cycles with caffeine induction, a bee sleep rhythm disorder model was established, which solves the problems of complex operation and high cost in existing technologies and provides a stable sleep rhythm disorder model for the research and treatment of sleep rhythm disorder-related diseases.
Patent Information
- Application Number
- CN202511245211.6
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-09-02
- Publication Date
- 2025-11-28
AI Technical Summary
Existing animal models of sleep rhythm disorder are complex to operate, costly, and produce unstable results. They are difficult to effectively simulate the physiological and psychological effects of sleep rhythm disturbances and also cause significant physiological and psychological stress to animals.
A method combining light-dark alternation with caffeine induction was used to establish sleep rhythm damage models in bees with and without bacteria by culturing mature bees under light-dark alternation and feeding them caffeine-sucrose solutions. A mixed solution was prepared using intestinal bacterial fluid and sterile pollen, and the caffeine concentration and light-dark cycle were adjusted to control the degree of sleep damage.
It simplifies the model building process, reduces costs, and provides a stable model of sleep rhythm disruption that can significantly affect the sleep duration, frequency, and activity level of bees. It is suitable for screening methods or drugs for the prevention or treatment of sleep rhythm disruption-related diseases and can also be used to study the specific mechanisms of sleep rhythm disruption.
Smart Images
Figure CN121014587A_ABST
Abstract
Description
Technical Field
[0001] This application relates to the field of biotechnology. Specifically, this application relates to a method for establishing and applying a model of bee sleep rhythm disruption induced by alternating light and dark conditions combined with caffeine. Background Technology
[0002] Disruptions to sleep rhythms have significant negative impacts on both physical and mental health. Physiologically, they can lead to impaired immune function, endocrine disorders, and metabolic disturbances, thereby increasing the risk of various diseases. Psychologically, long-term sleep rhythm disturbances are closely associated with mood disorders such as anxiety and depression, often manifesting as mood swings, poor concentration, and memory loss. In recent years, in-depth research has revealed the potential link between sleep rhythm disturbances and various neurodegenerative diseases, such as Alzheimer's disease and Huntington's disease, providing important evidence for a deeper understanding of their pathogenesis.
[0003] In scientific research on sleep rhythms, it is often necessary to construct sleep rhythm impairment models in experimental animals to further study the mechanisms of sleep rhythm impairment and treatment and recovery methods. Currently, there are various methods for constructing animal models of sleep rhythm impairment, but they all have several limitations. For example, the chronic social frustration model can effectively simulate the impact of long-term social stress on sleep rhythms in humans, but it causes significant psychological stress to animals and may lead to changes in other non-target behaviors; the empty bottle stimulation model is relatively simple to operate and can induce emotional stress responses, but individual differences in stress responses may affect the consistency of experimental results; the electric shock model can quickly induce stress responses, but it causes significant physiological trauma to animals and may interfere with other physiological indicators; drug-induced models can study the mechanisms of action of specific drugs on sleep rhythms, but there are issues of time-sensitivity and dependence; gene editing models can delve into the regulatory mechanisms of specific genes on sleep rhythms and have high scientific value, but they are technically demanding and costly, and may also be accompanied by non-target gene effects.
[0004] Therefore, exploring methods that can be easily operated and cost-effectively constructed to build stable sleep rhythm disorder models is of vital importance and significance for the study of sleep rhythm disorder mechanisms and treatment. Summary of the Invention
[0005] To overcome the shortcomings of existing technologies and reduce the cost of model building, this application aims to provide a stable and efficient method for establishing a bee sleep rhythm disorder model, which can be used to screen new methods or drugs for the prevention or treatment of sleep rhythm disorder-related diseases, and can also be applied to explore the specific mechanisms of sleep rhythm disorder-related diseases.
[0006] The specific technical solution of this application is as follows:
[0007] 1. A method for establishing a bee sleep rhythm disorder model, characterized by comprising the following steps:
[0008] Animal selection: Mature bees were selected as subjects for establishing a bee sleep rhythm disorder model;
[0009] Preparation of caffeine sucrose solution: Caffeine is dissolved in a sterile sucrose solution to prepare a caffeine sucrose solution.
[0010] Model establishment: The mature bees were cultured under alternating light and dark conditions while being fed the caffeine-sucrose solution to establish a bee sleep rhythm disorder model;
[0011] The bee sleep rhythm damage model is a bacterial bee sleep rhythm damage model. In the animal selection step, intestinal bacterial solution is prepared using the intestinal contents of normal bees. Then, bees that have been cultured in the dark until they emerge are fed a mixed solution containing the intestinal bacterial solution. Subsequently, they are cultured again to obtain mature bees.
[0012] 2. A method for establishing a bee sleep rhythm disorder model, characterized by comprising the following steps:
[0013] Animal selection: Mature bees were selected as subjects for establishing a bee sleep rhythm disorder model;
[0014] Preparation of caffeine sucrose solution: Caffeine is dissolved in a sterile sucrose solution to prepare a caffeine sucrose solution.
[0015] Model establishment: The mature bees were cultured under alternating light and dark conditions while being fed the caffeine-sucrose solution to establish a bee sleep rhythm disorder model;
[0016] The bee sleep rhythm damage model is a sterile bee sleep rhythm damage model. In the animal selection step, bees that have been cultured in the dark until they emerge are fed a mixed solution containing sterile pollen, sterile sucrose solution and sterile buffer solution, and then recultured to obtain mature bees.
[0017] 3. The method according to claim 1, wherein the mixed solution further comprises sterile pollen, sterile sucrose solution and sterile buffer solution.
[0018] 4. The method according to item 1, characterized in that intestinal bacteria are prepared using the intestinal contents of more than 20 normal bees.
[0019] 5. The method according to items 1 and 2, characterized in that the time period of the light-dark alternation is light:dark = 8-16h:16-8h:
[0020] Preferably, the time period for the light-dark alternation is light:dark = 12h:12h.
[0021] 6. The method according to items 1 and 2, characterized in that the illuminance during the alternating light and dark light cycle is 500-12000 lux (Lux):
[0022] Preferably, the illumination intensity during the alternating light and dark light cycle is 2500-6000 lux.
[0023] 7. According to the methods described in items 1 and 2, the concentration of caffeine in the caffeine sucrose solution is 20 mg / L to 2000 mg / L.
[0024] Preferably, the concentration of caffeine in the caffeine sucrose solution is 200 mg / L to 1000 mg / L.
[0025] 8. The method according to items 1 and 2, characterized in that the re-culturing time is 1 to 13 days:
[0026] Preferably, the reculturing time is 3 to 8 days.
[0027] 9. The method according to items 1 and 2, characterized in that, during the reculturing process, a sterile sucrose solution is fed.
[0028] 10. The method according to any one of claims 1 to 9, characterized in that the concentration of the sterile sucrose solution is 40 w / v% to 60 w / v%.
[0029] Preferably, the concentration of the sterile sucrose solution is 50% (w / v).
[0030] 11. The method according to any one of items 1 to 10, characterized in that the sterilization of the sterile sucrose solution by filtration is carried out using a filter membrane with an aperture of 0.22 μm.
[0031] 12. The method according to items 1 to 10, characterized in that the sterilization conditions of the sterile pollen are: sterilization with an electron beam of intensity of 20 to 40 kGy for 3 to 5 hours.
[0032] 13. The method according to items 1 to 10, characterized in that the sterile buffer solution is a buffer solution for maintaining the activity of bee intestinal bacteria:
[0033] Preferably, the sterile buffer solution is sterile PBS buffer.
[0034] 14. The method according to any one of items 1 to 10, characterized in that the mature bee is a 2-15 day old bee:
[0035] Preferably, the mature bee is a 3- to 9-day-old bee.
[0036] 15. The method according to any one of items 1 to 10, characterized in that the mature bee is a bee of the Apidae family.
[0037] 16. The method according to item 15, wherein the mature bee is a honeybee or a bumblebee.
[0038] 17. The method for establishing a fungal bee sleep rhythm disorder model according to any one of items 1 to 16 is used in screening methods or pharmaceutical compositions for the prevention or treatment of sleep rhythm disorder-related diseases.
[0039] 18. The application according to item 17, wherein the pharmaceutical composition comprises a chemical agent and / or a biological agent.
[0040] 19. The application of the method for establishing the fungal bee sleep rhythm disorder model according to any one of items 1 to 16 in the study of the disease mechanism related to sleep rhythm disorder. The effects of the invention
[0041] The method for establishing a bee sleep rhythm disorder model in this application uses mature bees as the model subjects. It can establish both germ-infected and germ-free bee sleep rhythm disorder models, effectively inducing sleep rhythm disorder in bees, reducing total sleep duration, sleep frequency, sleep latency, and significantly increasing activity. The degree of sleep disorder in the model can be adjusted by changing the caffeine concentration. Compared with other common animal models in the prior art, this method is simple to operate, low in cost, has a short modeling cycle, produces significant sleep rhythm disorder symptoms with adjustable disease severity, is suitable for large-scale sleep rhythm disorder model experiments, and can be used to screen new methods or drugs for the prevention or treatment of sleep rhythm disorder-related diseases. It can also be applied to explore the specific mechanisms of sleep rhythm disorder-related diseases. Attached Figure Description
[0042] Figure 1 This study investigated the effect of light intensity during the photoperiod on the activity status of a model of disrupted sleep rhythms in bees with fungi.
[0043] Figure 2 This study investigated the effect of light intensity during the photoperiod on the activity level of a model of disrupted sleep rhythm in bees with fungal infection.
[0044] Figure 3 The effect of light intensity during the photoperiod on 24-hour sleep duration in a model of disrupted sleep rhythm in bees with fungi.
[0045] Figure 4 The effect of light intensity during the photoperiod on the number of sleeps in a 24-hour sleep rhythm disorder model of bees with fungal infection.
[0046] Figure 5The effect of light intensity during the photoperiod on the sleep latency of a model of disrupted sleep rhythm in bees with fungi.
[0047] Figure 6 The effect of different concentrations of caffeine on the survival rate of a bee model of impaired sleep rhythm in bees with fungal infection.
[0048] Figure 7 The effects of different concentrations of caffeine on the activity state of a model of impaired sleep rhythm in bees with fungi.
[0049] Figure 8 The effect of different concentrations of caffeine on activity levels in a model of impaired sleep rhythm in bees with fungal infection.
[0050] Figure 9 The effects of different concentrations of caffeine on 24-hour sleep duration in a model of disrupted sleep rhythm in bees with fungi.
[0051] Figure 10 The effect of different concentrations of caffeine on the number of sleeps in a 24-hour sleep rhythm disorder model of bees with fungal infection.
[0052] Figure 11 The effect of different concentrations of caffeine on sleep latency in a model of impaired sleep rhythm in bees with fungi.
[0053] Figure 12 The effect of 200 mg / L caffeine concentration on the activity status of a sterile bee sleep rhythm disorder model.
[0054] Figure 13 The effect of 200 mg / L caffeine concentration on activity levels in a sterile bee sleep rhythm disorder model.
[0055] Figure 14 The effect of 200 mg / L caffeine concentration on 24-hour sleep duration in a sterile bee sleep rhythm disorder model.
[0056] Figure 15 The effect of 200 mg / L caffeine concentration on the number of sleeps in a sterile bee sleep rhythm disorder model over 24 hours.
[0057] Figure 16 The effect of 200 mg / L caffeine concentration on sleep latency in a sterile bee sleep rhythm disorder model.
[0058] Figure 17 The effect of 200 mg / L caffeine concentration on the activity status of a bumblebee sleep rhythm disorder model.
[0059] Figure 18 The effect of 200 mg / L caffeine concentration on activity levels in a bumblebee sleep rhythm disorder model.
[0060] Figure 19The effect of 200 mg / L caffeine on the 24-hour sleep duration of a bumblebee sleep rhythm disorder model was determined.
[0061] Figure 20 The effect of 200 mg / L caffeine concentration on the number of sleep episodes in a 24-hour sleep rhythm disorder model of bumblebees.
[0062] Figure 21 The effect of 200 mg / L caffeine concentration on sleep latency in a bumblebee sleep rhythm disorder model.
[0063] Figure 22 Comparative study on the effects of caffeine and modafinil on the establishment of a model of sleep rhythm disorder in bees with bacterial infection.
[0064] Figure 23 Comparative study on the effects of caffeine and modafinil on the establishment of a model of sleep rhythm disorder in bees with bacterial infections.
[0065] Figure 24 Comparative study on the effects of caffeine and modafinil on the establishment of a model of sleep rhythm disorder in bees, comparing sleep duration.
[0066] Figure 25 Comparative study on the effects of caffeine and modafinil on the establishment of a model of sleep rhythm disorder in bees with bacterial infections, including sleep frequency.
[0067] Figure 26 Comparative study on the effects of caffeine and modafinil on the establishment of a model of sleep rhythm disorder in bees and sleep latency. Detailed Implementation
[0068] The following description provides exemplary embodiments of this application, including various details to aid understanding, and should be considered merely exemplary. Therefore, those skilled in the art will recognize that various changes and modifications can be made to the embodiments described herein without departing from the scope and spirit of this application. Similarly, for clarity and brevity, descriptions of well-known functions and structures are omitted in the following description.
[0069] On the one hand, this application provides a method for establishing a bee sleep rhythm disorder model, which includes the following steps:
[0070] Animal selection: Mature bees were selected as subjects for establishing a bee sleep rhythm disorder model;
[0071] Preparation of caffeine sucrose solution: Caffeine is dissolved in a sterile sucrose solution to prepare a caffeine sucrose solution;
[0072] Model establishment: The mature bees were cultured under alternating light and dark conditions while being fed the caffeine-sucrose solution to establish a bee sleep rhythm disorder model.
[0073] In one specific embodiment, the mature bee is a 2- to 15-day-old bee, for example, a 2-day-old bee, a 3-day-old bee, a 4-day-old bee, a 5-day-old bee, a 6-day-old bee, a 7-day-old bee, a 8-day-old bee, a 9-day-old bee, a 10-day-old bee, an 11-day-old bee, a 12-day-old bee, a 13-day-old bee, a 14-day-old bee, a 15-day-old bee, etc., preferably a 3- to 9-day-old bee.
[0074] In this application, "bee" refers to the general term for insects in the order Hymenoptera other than ants. The species of bee in this application are not limited. For example, it may belong to the families Apidae, Sparganidae, Ichthyidae, Trichogrammae, Vespidae, and Sand Bees, and further may belong to genera such as Apiidae, Bumblebee, Wheat Bee, Stingless Bee, Mason Bee, and Leafcutter Bee under the family Apiidae.
[0075] In one specific embodiment, the mature bee is a bee of the Apidae family, more preferably a honeybee or a bumblebee.
[0076] The genus *Apis* of the family Apidae is collectively known as honeybees. Their defining characteristic is their ability to collect nectar and pollen and process it into honey. The most common are the Chinese honeybee (a type of Eastern honeybee) and the Italian honeybee, Carniolan honeybee, and European black honeybee (both Western honeybees). Other examples include the dwarf honeybee, black dwarf honeybee, giant honeybee, black giant honeybee, Sabah honeybee, Suvelasian honeybee, and Green Slave honeybee. This application does not limit the types of honeybees used, but prefers those that are easily purchased and obtained.
[0077] Bumblebees, encompassing over 500 species of social insects in the genus *Bumblebee* of the family Apidae, are characterized by their robust bodies densely covered with long, black, yellow, or multicolored hairs. They are widely distributed throughout the world except for Antarctica and Oceania. They nest and live in colonies with structures identical to honeybees, feeding on pollen and nectar from flowering plants. Major species include the Yellow Bumblebee, the Short-headed Bumblebee, and the Picea Bumblebee. This application does not limit the species of bumblebees used, but prefers those that are easily purchased and obtained.
[0078] In one specific embodiment, the concentration of caffeine in the caffeine sucrose solution is 20 mg / L to 2000 mg / L, for example, it can be 20 mg / L, 30 mg / L, 40 mg / L, 50 mg / L, 60 mg / L, 70 mg / L, 80 mg / L, 90 mg / L, 100 mg / L, 110 mg / L, 120 mg / L, 130 mg / L, 140 mg / L, 150 mg / L, 2... 00mg / L, 250mg / L, 300mg / L, 350mg / L, 400mg / L, 450mg / L, 500mg / L, 550mg / L, 600mg / L, 650m g / L, 700mg / L, 750mg / L, 800mg / L, 850mg / L, 900mg / L, 950mg / L, 1000mg / L, 1050mg / L, 1100mg The concentrations of caffeine, such as 1150 mg / L, 1200 mg / L, 1250 mg / L, 1300 mg / L, 1350 mg / L, 1400 mg / L, 1450 mg / L, 1500 mg / L, 1550 mg / L, 1600 mg / L, 1600 mg / L, 1700 mg / L, 1700 mg / L, 1800 mg / L, 1800 mg / L, 1900 mg / L, 1900 mg / L, 1950 mg / L, and 2000 mg / L, can be varied to adjust the severity of the disease in the model. However, high concentrations of caffeine can increase bee mortality, shortening the operational experimental period for the bee sleep rhythm disorder model. To ensure a relatively obvious sleep rhythm disorder symptom while avoiding insufficient bee survival, a caffeine concentration of 200 mg / L is preferred for establishing the bee sleep rhythm disorder model.
[0079] In one specific implementation, the light-dark cycle every 24 hours is light:dark = 8-16h:16-8h, for example, light:dark = 8h:16h, light:dark = 8.5h:15.5h, light:dark = 9h:15h, light:dark = 9.5h:14.5h, light:dark = 10h:14h, light:dark = 10.5h:13.5h, light:dark = 11h:13h, light:dark = 11.5h:12.5h, light:dark = 12h:12h, light:dark = 12.5h. Stable sleep rhythm disruption models can be established under different light-dark alternation cycles, such as 11.5h, 13h:11h, 13.5h:10.5h, 14h:10h, 14.5h:9.5h, 15h:9h, 15.5h:8.5h, and 16h:8h. For ease of operation, a light-dark alternation cycle of 12h:12h is preferred for establishing the bee sleep rhythm disruption model.
[0080] In one specific embodiment, the light intensity of the alternating light and dark light cycle is 500–12000 Lux, for example, it can be 500 Lux, 600 Lux, 700 Lux, 800 Lux, 900 Lux, 1000 Lux, 1500 Lux, 2000 Lux, 2500 Lux, 3000 Lux, 3500 Lux, 4000 Lux, 4500 Lux, 5000 Lux, 5500 Lux, 6000 Lux. Stable sleep rhythm impairment models can be established under different light intensities, including Lux, 6500 Lux, 7000 Lux, 7500 Lux, 8000 Lux, 8500 Lux, 9000 Lux, 9500 Lux, 10000 Lux, 10500 Lux, 11000 Lux, 11500 Lux, and 12000 Lux. A light intensity of 2500 Lux is preferred for establishing the beehive sleep rhythm impairment model.
[0081] In one specific embodiment, the bee sleep rhythm damage model is a bacterial bee sleep rhythm damage model. In the animal selection step, intestinal bacterial solution is prepared using the intestinal contents of normal bees, and then the emerging bees are fed a mixed solution containing the intestinal bacterial solution to obtain mature bees, which are bacterial bees.
[0082] In one specific implementation, a normal bee refers to a bee with a normal and well-developed gut microbiota. The species of the normal bee can be the same as or different from the bee used to establish the bee sleep rhythm disorder model. Preferably, the normal bee is the same species as the bee used to establish the bee sleep rhythm disorder model.
[0083] In one specific embodiment, the intestinal bacterial culture is collected as follows: Glycerol-PBS buffer is added to a sterile centrifuge tube one. 20-30 normal bees are dissected, and their intact intestines are placed in the centrifuge tube one. The intestines are thoroughly homogenized, and the mixture of intestinal contents and glycerol-PBS buffer is aspirated from the sterile centrifuge tube (discarding the intestinal tissue). This mixture is then collected in a centrifuge tube two to obtain the intestinal bacterial culture. Since there may be differences in the intestinal flora among individual bees, using 20-30 normal bees minimizes the impact of these individual differences.
[0084] After collecting the intestinal bacterial culture, the culture was aliquoted into several sterile centrifuge tubes and stored at -80°C for later use. This aliquoting method allows for the use of only a small tube at a time, avoiding repeated freeze-thaw cycles.
[0085] In one specific implementation, the process of cultivating bees that have emerged from the hive is as follows: select honeycomb combs, take out bee pupae that are dark in color and have not grown hair, place the bee pupae in an incubator under dark conditions, at a temperature of 30-40°C and a humidity of 60-80%, and incubate them overnight with sterile sucrose solution. Observe the emergence status of the bee pupae on the second day (if they have not emerged, incubate them for another night) to cultivate bees that have emerged from the hive.
[0086] In one embodiment, the mixed solution further comprises sterile pollen, sterile sucrose solution, and sterile buffer solution.
[0087] In one specific embodiment, the mixed solution consists of the intestinal bacterial solution, sterile pollen, sterile sucrose solution, and sterile buffer solution.
[0088] In one specific embodiment, after feeding the newly emerged bees the mixed solution, they are cultured for 1 to 13 days, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13 days, preferably 3 to 8 days. More preferably, a sterile sucrose solution is fed during the re-culture process to obtain mature bees, which are germ-infected bees. This culture stage is an induction and adaptation period, which can better improve the survival ability of the bees and increase the operability of subsequent induction.
[0089] In one specific embodiment, the bee sleep rhythm damage model is a sterile bee sleep rhythm damage model. In the animal selection step, newly emerged bees are fed a mixed solution containing sterile pollen, sterile sucrose solution and sterile buffer solution to obtain mature bees, which are sterile bees.
[0090] The second mixed solution of this application does not contain intestinal bacterial fluid. In one specific embodiment, the second mixed solution consists of sterile pollen, sterile sucrose solution, and sterile buffer solution.
[0091] In one specific embodiment, after feeding the newly emerged bees the second mixed solution, they are cultured for 1 to 13 days, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13 days, preferably 3 to 8 days. More preferably, they are cultured with a sterile sucrose solution during the re-culture process to obtain mature, sterile bees. This culture stage is an induction and adaptation period, which can better improve the bees' survival ability and increase the operability of subsequent induction.
[0092] In one specific embodiment, after obtaining the mature bees, sterile bee verification is performed. The specific process is as follows: 2 to 5 bees are taken out as verification bees, and the intestinal contents of each verification bee are taken out and the total number of bacteria is measured to verify that the mature bees are sterile bees.
[0093] In one specific embodiment, the process for sterile bee verification is as follows: 2-5 bees are selected as verification bees, dissected, and their intestines are placed in 100 μL of 50% glycerol (prepared with PBS). The intestines are then ground with a pestle, and 10 μL of the mixture is diluted with 10% PBS. 3 and 10 4 Take 100 μL of the sample, spread it on a plate, and incubate for 24 hours (35℃, 5% CO2). Then count the bacteria. If the final total bacterial count in the bee's intestinal contents is less than 10... 5 These can be considered sterile bees. The colonization level of gut bacteria may vary among different bee individuals, and the plating count method itself may have some degree of randomness; therefore, 10⁻⁶ dilutions were used here. 3 and 10 4 The multiple is to ensure that reliable data can be obtained from the final coating results.
[0094] In one specific embodiment, the sterile buffer is a buffer for maintaining the activity of bee gut bacteria, and is more preferably a sterile PBS buffer.
[0095] The sterile pollen of this application can ensure that bees consume more food, achieve better colonization results, and provide nutrients such as fats and amino acids for bee growth. Sucrose is the main carbon source in the bee's diet.
[0096] In one specific embodiment, the sterilization conditions for sterile pollen are as follows: sterilization with an electron beam of intensity of 20-40 kGy for 3-5 hours, wherein the intensity of the electron beam can be, for example, 20 kGy, 22 kGy, 24 kGy, 26 kGy, 28 kGy, 30 kGy, 32 kGy, 34 kGy, 36 kGy, 38 kGy, 40 kGy, etc., and the sterilization time can be, for example, 3 hours, 3.2 hours, 3.4 hours, 3.6 hours, 3.8 hours, 4 hours, 4.2 hours, 4.4 hours, 4.6 hours, 4.8 hours, 5 hours, etc.
[0097] In one specific embodiment, the preparation process of sterile pollen is as follows: the pollen is dispensed into self-sealing bags, sealed tightly, and sterilized by electron beam with an intensity of 20-40 kGy for 3-5 hours to obtain sterile pollen.
[0098] In one specific embodiment, the concentration of the sterile sucrose solution is 40% (w / v) to 60% (w / v), for example, it can be 40% (w / v), 42% (w / v), 44% (w / v), 46% (w / v), 48% (w / v), 50% (w / v), 52% (w / v), 54% (w / v), 56% (w / v), 58% (w / v), 60% (w / v), etc., preferably 50% (w / v).
[0099] In one specific embodiment, sucrose is added to ultrapure water to obtain a sucrose solution, which is then filtered through a 0.22 μm filter membrane to remove bacteria, resulting in the sterile sucrose solution.
[0100] On the other hand, this application also provides the application of the germ-bearing bee sleep rhythm disorder model established by the aforementioned method and / or the sterile bee sleep rhythm disorder model established by the aforementioned method in screening methods or pharmaceutical compositions for the prevention or treatment of sleep rhythm disorder-related diseases; preferably, the pharmaceutical composition includes chemical agents and / or biological agents.
[0101] Furthermore, this application also provides an application of the germ-bearing bee sleep rhythm disruption model established by the aforementioned method and / or the sterile bee sleep rhythm disruption model established by the aforementioned method in studying the disease mechanisms related to sleep rhythm disruption. It should be understood that the research is for non-therapeutic purposes.
[0102] This application provides methods for establishing models of sleep rhythm disruption in bees under both sterile and non-sterile conditions, which can efficiently induce sleep rhythm disruption in bees, resulting in reduced sleep duration, fewer sleep episodes, prolonged sleep latency, increased activity levels, and increased activity volume.
[0103] Furthermore, this application can regulate the severity of the disease in the model by altering the caffeine concentration. As the caffeine concentration increases, specific disease manifestations become more pronounced, including reduced sleep duration, fewer sleep episodes, prolonged sleep latency, increased activity levels, and increased activity levels. However, high caffeine concentrations can increase bee mortality, shortening the operational testing period for the sleep rhythm disorder model. To ensure a relatively clear sleep rhythm disorder symptom model while avoiding insufficient bee survival, it is recommended to use a caffeine concentration of 200 mg / L for establishing the bee sleep rhythm disorder model. Example
[0104] The method for establishing a bee sleep rhythm disorder model of this application will be described in detail below with reference to the accompanying drawings in the embodiments of this application. The described embodiments are preferred embodiments and not all embodiments of this application. Based on the embodiments of this application, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this application.
[0105] Example 1: The effect of light intensity during alternating light and dark periods on the establishment of a bee sleep rhythm disorder model
[0106] 1. Pre-experimental preparation
[0107] Weigh 500g of sucrose and dilute to 1L with ultrapure water to obtain a 50% (w / v) sucrose solution. Filter the solution through a 0.22μm filter membrane for sterilization to obtain the required 50% (w / v) sterile sucrose solution for the experiment. Aliquot a portion of this solution into 2ml sterile centrifuge tubes for later use. Aliquot the purchased pollen (rapeseed pollen from Qinghai Province) into resealable bags, ensuring strict sealing. Sterilize the bags with an electron beam at 30kGy for 4 hours to obtain sterile pollen.
[0108] Dissolve caffeine (sigema, catalog number 27620) in a pre-prepared 50% (w / v) sterile sucrose solution to prepare a 200 mg / L caffeine sucrose solution. All caffeine sucrose solutions must be sterilized by filtration through a 0.22 μm filter membrane and dispensed into 2 ml sterile centrifuge tubes for later use.
[0109] Prepare several sterile 1.5mL centrifuge tubes and add 200μL of glycerol-PBS buffer [50% (v / v) glycerol: 1×PBS buffer = 1:1]. Take 20-30 normal worker bees from a hive, dissect the bees to obtain intact intestines, and place them into separate centrifuge tubes. Homogenize the intestines on ice using an electric grinder at 6000rpm for 30s to ensure thorough homogenization. Take a sterile 50mL centrifuge tube and pipette the contents of all the 1.5mL centrifuge tubes into a separate 50mL centrifuge tube. Mix the contents thoroughly and aliquot the resulting intestinal bacterial solution into several new sterile 1.5mL centrifuge tubes. Store the mixture at -80℃ for later use. Use only the smaller tubes needed to avoid repeated freeze-thaw cycles.
[0110] 2. Selection and cultivation of experimental bees
[0111] Select suitable honeycomb frames. Using a sterilized awl or tweezers, gently pry open the pupa caps at random points on the honeycomb frame to observe the maturity of the pupae. Continue prying open the caps around pupae that are darker in color and have not yet grown hair. When prying open the pupa caps, be careful to clean the surrounding area as much as possible to avoid bringing honeycomb debris with the pupae. Hold the sterilized tweezers firmly together and insert them from above the pupae, then open them to the sides. Gently pinch the thorax of the pupae to remove them, avoiding physical damage as much as possible. Note that pupae that have already developed wings should not be used.
[0112] Place the selected bee pupae into plastic square boxes that have been disinfected with 84 disinfectant and 75% alcohol. Insert 2mL centrifuge tubes (with holes punched in the tube walls) containing sterile sucrose solution into the side or top of the box. Each square box can hold 150-200 bee pupae. Place the square boxes containing bee pupae in an incubator at 35℃ and 60-80% humidity overnight.
[0113] On the second day, observe the emergence of bee pupae (if they have not emerged, culture them for another night). Gently remove the emerged bees and divide them into disposable transparent culture cups. Take a new sterile 2mL centrifuge tube and add about 0.1mL of sterile pollen using a spatula sterilized with an alcohol lamp. Add 990μL of sterile PBS to the centrifuge tube containing 0.1mL of sterile pollen, and use a pipette to aspirate and vortex to completely dissolve the pollen. Then add 10μL of normal worker bee intestinal fluid and vortex to mix. Finally, bring the volume to 2ml with 50% sterile sucrose solution and shake the centrifuge tube thoroughly. Feed each newly emerged bee a tube of the above sucrose-pollen mixture containing normal bee intestinal bacteria. Continue to incubate in a constant temperature incubator for two days, then switch to 50% (w / v) sterile sucrose solution and continue culturing for 3 days. Afterward, induce sleep rhythm damage. Note that during this stage, the bees must be cultured in a dark environment without any light source.
[0114] 3. Animal grouping and experimental design
[0115] The aforementioned bees were selected as experimental bees to investigate the effect of photoperiod light intensity on the establishment of a bee sleep rhythm disruption model. They were divided into six groups: a control group, a 0 Lux light intensity + 200 mg / L caffeine group, a 500 Lux light intensity + 200 mg / L caffeine group, a 2500 Lux light intensity + 200 mg / L caffeine group, a 6000 Lux light intensity + 200 mg / L caffeine group, and a 12000 Lux light intensity + 200 mg / L caffeine group. Each group consisted of 30 bees, further divided into six subgroups of 5 bees each. Each biological replicate group was aliquoted into 90mm diameter disposable plastic culture dishes (with pre-drilled ventilation and feeding tube holes on the sides). The dishes were inverted for incubation. For the control group, each dish contained sterile pollen and a 2ml tube of pre-prepared sterile sucrose solution; a feeding tube was punched in the centrifuge tube before insertion. For all other groups, each dish contained sterile pollen and a 2ml tube of pre-prepared 50% (w / v) sucrose solution containing 200mg / L caffeine; a feeding tube was punched in the centrifuge tube before insertion. Each culture dish was clearly labeled with its group and order information.
[0116] 4. Experimental observation and analysis
[0117] The marked culture dishes were placed in pre-prepared artificial light source biological incubators with corresponding light intensities according to their groups. They were laid flat, with humidity set at 60% and temperature at 35 degrees Celsius. The photoperiod was set to light:dark = 12:12h (7:00-19:00:19:00-7:00). The light intensity during the photoperiod for the control group was 2500 Lux, for the group with 500 Lux light intensity + 200 mg / L caffeine it was 500 Lux, for the group with 2500 Lux light intensity + 200 mg / L caffeine it was 2500 Lux, for the group with 6000 Lux light intensity + 200 mg / L caffeine it was 6000 Lux, for the group with 10000 Lux light intensity + 200 mg / L caffeine it was 12000 Lux, and for the group with 0 Lux light intensity + 200 mg / L caffeine it was incubated in the dark for 24 hours.
[0118] Under the above conditions, the bees were cultured for 96 hours (4 days). Then all incubators were set to 24-hour dark culture. In the dark culture environment, the bees were cultured for 96 hours (4 days). During the last 24 hours of the 4-day dark culture, the complete 24-hour images of bee sleep and activity were collected by the night vision camera in the incubator.
[0119] After completing the above image acquisition, the images were analyzed using the Ethovision XT17 animal behavior analysis system to statistically analyze the bees' sleep duration, number of sleeps, sleep latency, and activity levels every 30 minutes.
[0120] 5. Experimental Results
[0121] The results are as follows Figures 1-5 As shown, compared with the control, the 500 Lux light intensity + 200 mg / L caffeine group, the 2500 Lux light intensity + 200 mg / L caffeine group, the 6000 Lux light intensity + 200 mg / L caffeine group, and the 12000 Lux light intensity + 200 mg / L caffeine group all showed significantly reduced sleep duration and number of sleeps, significantly prolonged sleep latency, and increased activity levels during both light and dark periods, with a significant increase in overall activity. The 0 Lux light intensity + 200 mg / L caffeine group showed no significant changes in sleep duration, number of sleeps, sleep latency, or activity levels, but a slight increase in activity. Comparisons between different light intensity groups showed no significant differences in sleep duration, number of sleeps, sleep latency, or activity levels among the groups, but the 0 Lux light intensity + 200 mg / L caffeine group showed significantly lower activity levels than the other light intensity groups.
[0122] The above results indicate that the caffeine-induced light-dark alternation culture provided in this application is a necessary condition for establishing a stable bee sleep rhythm disorder model. The light intensity range of the light-dark alternation photoperiod, from 500 Lux to 12000 Lux, can all be used to establish the bee sleep rhythm disorder model. Scientific research shows that a photoperiod light intensity of 2000-3000 Lux is generally the optimal light condition for bee growth and development. To ensure the stability of other growth and development conditions besides sleep rhythm disorder in the established model, a photoperiod light intensity of 2500 Lux is preferred for establishing the bee sleep rhythm disorder model. In the figure, * indicates p < 0.05; ** indicates p < 0.01; *** indicates p < 0.001; **** indicates p < 0.0001.
[0123] Example 2: Effects of different concentrations of caffeine on a model of disrupted sleep rhythms in bees with fungi.
[0124] 1. Pre-experimental preparation
[0125] Same as in Example 1, except that the sterile sucrose solution is dispensed into 2 ml and 5 ml centrifuge tubes respectively; the 50% (w / v) sucrose solution containing caffeine needs to be prepared with caffeine concentrations of 5 mg / L, 20 mg / L, 50 mg / L, 200 mg / L, 500 mg / L, 1000 mg / L, 1500 mg / L, 1800 mg / L and 2000 mg / L, and after preparation, it is dispensed into 2 ml and 5 ml centrifuge tubes respectively.
[0126] 2. Selection and cultivation of experimental bees
[0127] The selection and cultivation of experimental bees were the same as in Example 1.
[0128] 3. Animal grouping and experimental design
[0129] The selected bees were divided into 10 large groups: control group, caffeine concentration 5 mg / L group, caffeine concentration 20 mg / L group, caffeine concentration 50 mg / L group, caffeine concentration 200 mg / L group, caffeine concentration 500 mg / L group, caffeine concentration 1000 mg / L group, caffeine concentration 1500 mg / L group, caffeine concentration 1800 mg / L group, and caffeine concentration 2000 mg / L group, with 130 bees in each large group. They were divided into two batches: the first batch of 100 bees and the second batch of 30 bees. The first batch was divided into 5 cages with 20 bees per cage, and each cage was a biological replicate. The second batch was divided into 6 small groups with 5 bees per small group, and each small group was a biological replicate. The small groups were divided into 90 mm diameter disposable plastic culture dishes (the culture dishes had ventilation holes and feed tube holes pre-made on the side) and inverted for culture. In the control group, sterile pollen was sprinkled into each petri dish and bee cage, and a pre-prepared sterile sucrose solution was inserted. Before insertion, feeding holes were made in the centrifuge tubes; one 2ml tube of sterile sucrose solution was inserted into the petri dish, and two 5ml tubes of sterile sucrose solution were inserted into the bee cages. In the other groups, sterile pollen was sprinkled into each petri dish, and a pre-prepared 50% (w / v) sucrose solution containing caffeine was inserted according to different concentrations. Before insertion, feeding holes were made in the centrifuge tubes; one 2ml tube of sterile sucrose solution containing the corresponding concentration of caffeine was inserted into the petri dish, and two 5ml tubes of sterile sucrose solution containing the corresponding concentration of caffeine were inserted into the bee cages. The group and order information were clearly marked on each petri dish and bee cage.
[0130] 4. Experimental observation and analysis
[0131] Place the marked petri dishes and bee cages in a biological incubator with a pre-prepared artificial light source of 2500 Lux. The petri dishes should be laid flat, while the bee cages can be stacked. The humidity should be set to 60%, the temperature to 35 degrees Celsius, and the photoperiod should be set to light:dark = 12:12h (7:00-19:00:19:00-7:00).
[0132] Under the above conditions, the bees were cultured for 96 hours (4 days). All incubators were then set to 24-hour dark culture. During the final 24 hours of the 4-day dark culture, complete images of bee sleep and activity were captured on the culture dishes using a night vision camera inside the incubator. After the 4-day dark culture, the survival rate of bees in cage-cultured bees was statistically analyzed by group. Following the image acquisition, the images were analyzed using the Ethovision XT17 animal behavior analysis system to statistically analyze bee sleep duration, sleep frequency, sleep latency, and activity levels every 30 minutes.
[0133] 5. Experimental Results
[0134] The results are as follows Figure 6As shown, compared with the control, there was no significant difference in bee survival rate in the caffeine concentration groups of 5 mg / L, 20 mg / L, 50 mg / L, 200 mg / L, 500 mg / L and 1000 mg / L; the bee survival rate in the 1500 mg / L, 1800 mg / L and 2000 mg / L groups was significantly lower than that in the control group. Figure 7-11 The results showed that, compared with the control, bees in the caffeine concentration groups of 20 mg / L, 50 mg / L, 200 mg / L, 500 mg / L, 1000 mg / L, 1500 mg / L, 1800 mg / L, and 2000 mg / L exhibited significantly reduced sleep duration and sleep frequency, significantly prolonged sleep latency, and increased activity levels during both light and dark periods, with a significant increase in overall activity. Furthermore, the significance of these differences increased with increasing caffeine concentration. Compared with the control, the 5 mg / L caffeine concentration group showed no significant changes in sleep duration, sleep frequency, sleep latency, or activity level, and no change in activity level.
[0135] The above results indicate that this application can regulate the disease severity of the sleep rhythm disorder model by changing the caffeine concentration. As the caffeine concentration increases, the specific disease manifestations become more pronounced, including reduced sleep duration, reduced sleep frequency, prolonged sleep latency, increased activity levels, and increased activity. However, high caffeine concentrations increase bee mortality, shortening the operational experimental period of the sleep rhythm disorder model. To avoid insufficient bee survival while ensuring relatively obvious sleep rhythm disorder symptoms in the model, a caffeine concentration of 200 mg / L is preferred for establishing the bee sleep rhythm disorder model. In the figure, * indicates p < 0.05; ** indicates p < 0.01; *** indicates p < 0.001; **** indicates p < 0.0001.
[0136] Example 3: Construction of a sterile bee sleep rhythm disorder model
[0137] 1. Pre-experimental preparation
[0138] The preliminary preparations for the experiment were the same as in Example 1.
[0139] 2. Selection and cultivation of experimental bees
[0140] The selection of experimental bees was the same as in Example 1.
[0141] On the second day, observe the emergence of the bee pupae (if they have not emerged, culture them for another night). Gently remove the emerged bees and divide them into disposable transparent culture cups. Culture sterile bees according to the following steps: In a clean bench, take a new sterile 2mL centrifuge tube and add about 0.1mL of sterile pollen using a spatula that has been sterilized with an alcohol lamp. Add 1000μL of sterile PBS to the centrifuge tube containing 0.1mL of sterile pollen, and use a pipette to blow and vortex to completely dissolve the pollen. Then, bring the volume to 2ml with 50% (w / v) sterile sucrose solution, shake well, and feed each newly emerged bee with the above sterile sucrose pollen mixture. Place in a sterile constant temperature incubator for two days, then switch to conventional 50% (w / v) sterile sucrose solution and continue culture for 3 days. After that, induce sleep rhythm damage. Note that the bees at this stage must be cultured in a dark environment without light.
[0142] 3. Animal grouping and experimental design
[0143] The selected bees were divided into two main groups: a control group and a sterile bee model group, with 30 bees in each main group. These were further divided into six subgroups of five bees each, forming a biological replicate. Each subgroup was individually aliquoted into 90mm diameter disposable sterile plastic culture dishes (with pre-drilled ventilation and feeding tube holes on the sides) and incubated upside down. In the control group, each dish was sprinkled with sterile pollen and a 2ml sterile sucrose solution was inserted. A feeding tube hole was made in the centrifuge tube before insertion. In the sterile bee model group, each dish was sprinkled with sterile pollen and a 2ml sterile sucrose solution containing 200mg / L caffeine was inserted. A feeding tube hole was made in the centrifuge tube before insertion. Each culture dish was clearly labeled with its group and order information.
[0144] 4. Experimental observation and analysis
[0145] Place the marked culture dishes in a sterile artificial light source biological incubator with a light intensity of 2500 Lux. The culture dishes are laid flat, the humidity is set to 60%, the temperature is 35 degrees Celsius, and the photoperiod is set to light:dark = 12:12h (7:00-19:00:19:00-7:00).
[0146] Under the above conditions, the bees were cultured for 96 hours (4 days). Then, all incubators were set to 24-hour dark culture. In the dark culture environment, the bees were cultured for 96 hours (4 days). During the last 24 hours of the last 4 days of dark culture, the bees' sleep and activity images were collected from the culture dishes using a night vision camera inside the incubator. After the above image collection was completed, the images were analyzed using the EthovisionXT17 animal behavior analysis system. The sleep duration, number of sleeps, sleep latency, and activity status and activity level of the bees every 30 minutes were statistically analyzed.
[0147] 5. Experimental Results
[0148] The results are as follows Figure 12-16 The results showed that, compared with the control group, the sleep duration and number of sleeps in the sterile honeybee model group were significantly reduced, the sleep latency was significantly prolonged, the activity state was more active in both the light and dark periods, and the overall activity level was significantly increased.
[0149] The above results indicate that this application demonstrates that a sleep rhythm disorder disease state can be induced in sterile bees through alternating light and dark culture at 2500 Lux and caffeine at a concentration of 200 mg / L. Specific manifestations include reduced sleep duration, reduced sleep frequency, prolonged sleep latency, increased activity levels, and increased activity levels. Therefore, this demonstrates that a sterile bee sleep rhythm disorder model can be established through alternating light and dark culture at 2500 Lux and caffeine at a concentration of 200 mg / L. In the figure, * indicates p < 0.05; ** indicates p < 0.01; *** indicates p < 0.001; **** indicates p < 0.0001.
[0150] Example 4: Construction of a sleep rhythm disruption model in bumblebees with fungi
[0151] 1. Pre-experimental preparation
[0152] The sterile sucrose solution, sterile pollen, and caffeine-related solution were obtained in the same manner as in Example 1.
[0153] Prepare several sterile 1.5mL centrifuge tubes and add 200μL of glycerol-PBS buffer (50% glycerol: 1×PBS buffer = 1:1). Take 20-30 normal bumblebees from their nests, dissect them, and obtain intact intestines. Place each intestine into a separate centrifuge tube and homogenize on ice using an electric grinder at 6000rpm for 30s to ensure thorough homogenization of the intestinal tissue. Take a sterile 50mL centrifuge tube and pipette the intestinal contents from all the 1.5mL tubes (discarding the intestinal tissue). Collect the contents into a 50mL centrifuge tube. Aliquot the mixed intestinal bacterial solution into several new sterile 1.5mL centrifuge tubes and freeze at -80℃ for later use. Use only the smaller tubes when needed, avoiding repeated freeze-thaw cycles.
[0154] 2. Selection and cultivation of experimental bumblebees
[0155] Select a suitable honeycomb and observe the maturity of the larvae inside based on their color. Using sterilized tweezers, gently pry open the pupa cap on the darker-colored combs, ensuring the surrounding area is thoroughly cleaned to remove any honeycomb debris. Hold the sterilized tweezers firmly together and insert them from above the pupa, then open them to the sides. Gently pinch the pupa's thorax to remove it, avoiding any physical damage. Note that winged pupae should not be used.
[0156] Place the selected bee pupae into small plastic cups that have been sterilized with 84 disinfectant and alcohol, with only one pupae in each cup. Insert a 2mL centrifuge tube (with holes punched in the tube wall) containing sterile sucrose solution into the side or top of the cup. Place the box containing the bee pupae in an incubator at 25-30℃ and 50% humidity overnight. Observe the emergence of the pupae for several days (bee pupae generally emerge in about three days).
[0157] After the bee pupae emerge, take a new sterile 2mL centrifuge tube and add approximately 0.1mL of sterile pollen using a spatula sterilized with an alcohol lamp. Add 990μL of sterile PBS to the centrifuge tube containing the 0.1mL of sterile pollen, and use a pipette to aspirate and vortex to completely dissolve the pollen. Then add 10μL of normal bumblebee intestinal flora solution and vortex to mix. Finally, bring the volume to 2ml with 50% (w / v) sterile sucrose solution, shake well, and feed each newly emerged bumblebee with the above sucrose-pollen mixture containing normal bee intestinal flora solution. Continue to incubate in a constant temperature incubator for two days, then switch to the conventional 50% (w / v) sterile sucrose solution and continue incubation for 3 days. Afterward, induce sleep rhythm damage. It is important to note that the bees at this stage must be incubated in a dark environment without any light source.
[0158] 3. Animal grouping and experimental design
[0159] The bumblebees selected were divided into two main groups: a control group and a bumblebee model group. Each main group contained 18 bumblebees, further subdivided into 6 smaller groups of 3 bumblebees each, forming a biological replicate. Each group was aliquoted into 90mm diameter disposable sterile plastic culture dishes (with pre-drilled ventilation and feeding tube holes on the sides) and incubated upside down. In the control group, each dish was sprinkled with sterile pollen and a 2ml sterile sucrose solution was inserted. A feeding tube hole was punched in the centrifuge tube before insertion. In the bumblebee model group, each dish was sprinkled with sterile pollen and a 2ml sterile sucrose solution containing 200mg / L caffeine was inserted. A feeding tube hole was punched in the centrifuge tube before insertion. Each culture dish was labeled with its group and order information.
[0160] 4. Experimental observation and analysis
[0161] Place the marked culture dishes in a biological incubator with a pre-prepared artificial light source of 2500 Lux. The culture dishes are laid flat, the humidity is set to 60%, the temperature is 29 degrees Celsius, and the photoperiod is set to light:dark = 12:12h (7:00-19:00:19:00-7:00).
[0162] Under the above conditions, bumblebees were cultured for 96 hours (4 days). Then, all incubators were set to 24-hour dark culture. In the dark culture environment, the bumblebees were cultured for 96 hours (4 days). During the last 24 hours of the dark culture, the bumblebees' sleep and activity images were collected from the culture dishes using a night vision camera inside the incubator. After the above image collection was completed, the images were analyzed using the EthovisionXT17 animal behavior analysis system. The bumblebees' sleep duration, number of sleeps, sleep latency, and activity status and activity level every 30 minutes were statistically analyzed.
[0163] 5. Experimental Results
[0164] The results are as follows Figure 17-21 The results showed that, compared with the control group, the bumblebee model group with fungi had significantly reduced sleep duration and number of sleeps, significantly prolonged sleep latency, more active status during the photoperiod, and significantly increased overall activity.
[0165] The above results indicate that this application demonstrates that a sleep rhythm disorder disease state in bumblebees can be induced by alternating light and dark culture at 2500 Lux and caffeine at a concentration of 200 mg / L. Specific manifestations include reduced sleep duration, reduced sleep frequency, prolonged sleep latency, increased activity, and increased activity levels. Therefore, this demonstrates that a sleep rhythm disorder model in bumblebees can be established through alternating light and dark culture at 2500 Lux and caffeine at a concentration of 200 mg / L. In the figure, * indicates p < 0.05; ** indicates p < 0.01; *** indicates p < 0.001; **** indicates p < 0.0001.
[0166] Example 5: Comparison of the effects of caffeine and modafinil on the establishment of a model of sleep rhythm disorder in bees with bacteria
[0167] 1. Pre-experimental preparation
[0168] The preparation of sterile pollen, sterile sugar water, caffeine solution, and intestinal bacteria solution is the same as in Example 1.
[0169] Preparation of 1 g / L modafinil sterile saccharide solution: Weigh 50 mg modafinil (Sigma, M6940) into a 1.5 ml centrifuge tube, add 1 ml of methanol to dissolve it into a 50 mg / ml modafinil stock solution, take 400 μl of the modafinil stock solution into a 50 ml centrifuge tube, add sterile saccharide solution to make up to 20 ml, mix well, and dispense into 2 ml centrifuge tubes for later use.
[0170] Preparation of sterile sugar solution for methanol control group: Take 400ul of methanol into a 50ml centrifuge tube, add sterile sugar solution to make up to 20ml, mix well, and dispense into 2ml centrifuge tubes for later use.
[0171] 2. Selection and cultivation of experimental bees
[0172] The selection and cultivation of experimental bees were the same as in Example 1.
[0173] 3. Animal grouping and experimental design
[0174] The selected bees were divided into four main groups: control group, methanol control group, modafinil-induced group, and caffeine-induced group, with 30 bees in each main group. These were further divided into six subgroups of five bees each, forming a biological replicate. The subgroups were then placed into disposable plastic culture dishes with a diameter of 90 mm (the sides of the culture dishes had been pre-made with ventilation holes and feeding tube holes) and inverted for culture. In the control group, sterile pollen was sprinkled into each petri dish, and a 2ml tube of pre-prepared sterile sucrose solution was inserted. A feeding well was punched in the centrifuge tube before insertion. In the methanol control group, sterile pollen was sprinkled into each petri dish, and a 2ml tube of pre-prepared sterile sucrose solution containing 1g / L modafinil was inserted. A feeding well was punched in the centrifuge tube before insertion. In the modafinil group, sterile pollen was sprinkled into each petri dish, and a 2ml tube of pre-prepared sterile sucrose solution containing 200mg / L modafinil was inserted. A feeding well was punched in the centrifuge tube before insertion. In the caffeine group, sterile pollen was sprinkled into each petri dish, and a 2ml tube of pre-prepared sterile sucrose solution containing 200mg / L modafinil was inserted. Each petri dish and bee cage was clearly labeled with the group and order information.
[0175] 4. Experimental observation and analysis
[0176] Place the marked culture dishes in a biological incubator with a pre-prepared artificial light source of 2500 Lux. The culture dishes should be laid flat, the humidity should be set to 60%, the temperature to 35 degrees Celsius, and the photoperiod should be set to light:dark = 12:12h (7:00-19:00:19:00-7:00).
[0177] Under the above conditions, the bees were cultured for 96 hours (4 days), and then the incubator was set to 24-hour dark culture. In the dark culture environment, the bees were cultured for 96 hours (4 days). During the last 24 hours of the last 4 days of dark culture, the bees' sleep and activity images were collected in the culture dish using a night vision camera in the incubator. After the above image acquisition was completed, the images were analyzed using the Ethovision XT17 animal behavior analysis system to statistically analyze the bees' sleep duration, number of sleeps, sleep latency, and activity status and activity level every 30 minutes.
[0178] 5. Experimental Results
[0179] The results are as follows Figure 22-26As shown, compared with the control group, there were no significant changes in any indicators in the methanol control group. The sleep duration and number of sleeps in the modafinil-induced group and the caffeine-induced group were significantly reduced, the sleep latency was significantly prolonged, the activity state was more active in both the light and dark periods, and the overall activity level was significantly increased. Compared with the modafinil group, the sleep duration and number of sleeps in the caffeine-induced group were significantly reduced, the sleep latency was significantly prolonged, the activity state was more active in both the light and dark periods, and the overall activity level was significantly increased.
[0180] The above results indicate that the disease efficacy of the photoperiod alternation combined with caffeine-induced sleep rhythm disruption model in bees provided in this application is significantly superior to that of modafinil. Specifically, compared to modafinil-induced disruption, the caffeine-induced sleep rhythm disruption model in bees exhibits more pronounced disease efficacy, including reduced sleep duration, reduced sleep frequency, prolonged sleep latency, increased activity levels, and increased activity levels. In the figure, * indicates p < 0.05; ** indicates p < 0.01; *** indicates p < 0.001; **** indicates p < 0.0001.
[0181] The results of the above embodiments show that the method for establishing a bee sleep rhythm disorder model is particularly applicable to bees, especially honeybees such as honeybees and bumblebees. For different bee species, their specific experimental conditions need to be fine-tuned to achieve better modeling results.
[0182] The above description is merely a preferred embodiment of this application and is not intended to limit the application in any other way. Any person skilled in the art may make changes or modifications to the disclosed technical content to create equivalent embodiments. However, any simple modifications, equivalent changes, and modifications made to the above embodiments based on the technical essence of this application without departing from the scope of the technical solution of this application shall still fall within the protection scope of this application.
Claims
1. A method for establishing a bee sleep rhythm disorder model, characterized in that, It includes the following steps: Animal selection: Mature bees were selected as subjects for establishing a bee sleep rhythm disorder model; Preparation of caffeine sucrose solution: Caffeine is dissolved in a sterile sucrose solution to prepare a caffeine sucrose solution; Model establishment: The mature bees were cultured under alternating light and dark conditions while being fed the caffeine-sucrose solution to establish a bee sleep rhythm disorder model; The bee sleep rhythm damage model is a bacterial bee sleep rhythm damage model. In the animal selection step, intestinal bacterial solution is prepared using the intestinal contents of normal bees. Then, bees that have been cultured in the dark until they emerge are fed a mixed solution containing the intestinal bacterial solution. Subsequently, they are cultured again to obtain mature bees.
2. A method for establishing a bee sleep rhythm disorder model, characterized in that, It includes the following steps: Animal selection: Mature bees were selected as subjects for establishing a bee sleep rhythm disorder model; Preparation of caffeine sucrose solution: Caffeine is dissolved in a sterile sucrose solution to prepare a caffeine sucrose solution; Model establishment: The mature bees were cultured under alternating light and dark conditions while being fed the caffeine-sucrose solution to establish a bee sleep rhythm disorder model; The bee sleep rhythm damage model is a sterile bee sleep rhythm damage model. In the animal selection step, bees that have been cultured in the dark until they emerge are fed a mixed solution containing sterile pollen, sterile sucrose solution and sterile buffer solution, and then recultured to obtain mature bees.
3. The method according to claim 1, characterized in that, The mixed solution also includes sterile pollen, sterile sucrose solution, and sterile buffer solution.
4. The method according to claim 1, characterized in that, Intestinal flora were prepared using the intestinal contents of more than 20 normal bees.
5. The method according to claims 1 and 2, characterized in that, The time period for the light-dark alternation is light:dark = 8-16h:16-8h. Preferably, the time period for the light-dark alternation is light:dark = 12h:12h.
6. The method according to claims 1 and 2, characterized in that, The illumination intensity during the alternating light and dark light cycle is 500-12000 lux. Preferably, the illumination intensity during the alternating light and dark light cycle is 2500-6000 lux.
7. The method according to claims 1 and 2, wherein the concentration of caffeine in the caffeine sucrose solution is 20 mg / L to 2000 mg / L; Preferably, the concentration of caffeine in the caffeine sucrose solution is 200 mg / L to 1000 mg / L.
8. The method according to claims 1 and 2, characterized in that, The re-culturing time is 1 to 13 days; Preferably, the reculturing time is 3 to 8 days.
9. The method according to claims 1 and 2, characterized in that, During the reculturing process, a sterile sucrose solution was fed.
10. The method according to any one of claims 1 to 9, characterized in that, The concentration of the sterile sucrose solution is 40 w / v%~60 w / v%. Preferably, the concentration of the sterile sucrose solution is 50% (w / v).
11. The method according to any one of claims 1 to 10, characterized in that, The sterile sucrose solution was sterilized by filtration using a filter membrane with a diameter of 0.22 μm.
12. The method according to claims 1 to 10, characterized in that, The sterilization conditions for the sterile pollen are: sterilization with an electron beam intensity of 20-40 kGy for 3-5 hours.
13. The method according to claims 1 to 10, characterized in that, The sterile buffer solution is a buffer solution used to maintain the activity of bee gut bacteria; Preferably, the sterile buffer solution is sterile PBS buffer.
14. The method according to any one of claims 1 to 10, characterized in that, The mature bees are 2 to 15 days old; Preferably, the mature bee is a 3- to 9-day-old bee.
15. The method according to any one of claims 1 to 10, characterized in that, The mature bee in question is a bee belonging to the family Apidae.
16. The method according to claim 15, characterized in that, The mature bee mentioned is either a honeybee or a bumblebee.
17. The method for establishing a circadian rhythm disorder model of bees according to any one of claims 1 to 16 is used in screening methods or pharmaceutical compositions for the prevention or treatment of circadian rhythm disorder-related diseases.
18. The application according to claim 17, wherein the pharmaceutical composition comprises a chemical agent and / or a biological agent.
19. The application of the method for establishing the sleep rhythm disorder model of fungi in any one of claims 1 to 16 in the study of the disease mechanisms related to sleep rhythm disorder.