Method for researching influence mechanism of gastrodin on anxiety behavior after sleep deprivation
By studying the effect of Gastrodiatin on anxiety-like behavior in mice in sleep deprivation models, it reveals its mechanism of action, solves the problem of Gastrodiatin action mechanism in the existing technology, provides a new perspective on clinical application, and ensures the reliability of the research results through rigorous experimental design.
Patent Information
- Application Number
- CN202510293834.4
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-03-13
- Publication Date
- 2025-06-17
AI Technical Summary
The prior art has not yet fully understood the mechanism of action of Gastrodiatin in anxiety-like behavior after sleep deprivation, especially in the regulation of autophagy-related proteins.
By selecting 32 ICR male mice, a sleep deprivation model was established using the modified multi-platform water environment method, the effect of Gastrodiatin on anxiety-like behavior in mice was observed, and the expression level of autophagy-related proteins was started to reveal its mechanism of action.
The effect of Gastrodiatin on anxiety-like behavior in mice after sleep deprivation was systematically explored for the first time, revealing its mechanism of action, providing a new perspective for the clinical application of Gastrodiatin, and ensuring the accuracy and reliability of the research results through rigorous experimental design.
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Abstract
Description
Technical Field
[0001] The present invention relates to the technical fields of pharmacology and behavioral psychology, and particularly to a research method for the mechanism of the effect of gastrodin on anxiety-like behaviors after sleep deprivation. Background Art
[0002] Sleep deprivation is a common phenomenon widely existing in modern society. Due to factors such as work pressure and living habits, many people cannot get enough sleep. Long-term sleep deprivation can have serious impacts on an individual's physical and mental health, including decreased cognitive function, mood swings, weakened immunity, etc. In recent years, more and more studies have shown that sleep deprivation may also trigger or exacerbate mental disorders such as anxiety, seriously affecting people's quality of life and social functions.
[0003] Gastrodin is an active ingredient extracted from the traditional Chinese medicine Gastrodia elata, and has various pharmacological effects such as sedation and anti-anxiety. Previous studies have shown that gastrodin can exert its pharmacological effects by regulating neurotransmitters and reducing oxidative stress, etc., but its specific mechanism of action has not been fully clarified. Especially its mechanism of action in anxiety-like behaviors caused by sleep deprivation still needs further in-depth study. Summary of the Invention
[0004] The purpose of the present invention is to solve the drawbacks existing in the prior art, and to propose a research method for the mechanism of the effect of gastrodin on anxiety-like behaviors after sleep deprivation.
[0005] To achieve the above purpose, the present invention adopts the following technical scheme:
[0006] A research method for the mechanism of the effect of gastrodin on anxiety-like behaviors after sleep deprivation, including: selecting 32 male ICR mice, establishing a sleep deprivation model by the improved multi-platform water environment method, and observing the expression level of autophagy-related proteins in the mice of the model group on the anxiety-like behaviors of the mice;
[0007] The specific steps include:
[0008] S1: Experimental preparation, reagent preparation, and reagent preparation includes the preparation of 0.9% normal saline, the preparation of SDS-PAGE electrophoresis solution, the preparation of transfer buffer, the preparation of Tris Buffered Saline with Tween-20 buffer, and the preparation of 5x rapid blocking solution;
[0009] S2: Grouping of experimental animals, randomly dividing 32 male ICR mice into 4 groups by the random number method: blank control group, gastrodin control group, sleep deprivation group, and gastrodin intervention group; the sleep deprivation group and the gastrodin intervention group establish a mouse sleep deprivation model by the improved multi-platform water environment method, and the blank control group and the gastrodin control group are normally raised without deprivation;
[0010] S3: Establish a sleep deprivation model;
[0011] S4: Conduct a sleep deprivation experiment and administer drugs;
[0012] S5: Conduct an open field test on mice;
[0013] S6: Conduct an elevated plus maze test on mice;
[0014] S7: Experimental statistics.
[0015] Preferably, in the preparation of the reagent, the SDS-PAGE electrophoresis buffer is prepared as follows: Tris powder: 3.03 g, sodium dodecyl sulfate: 1 g, glycine powder: 14.4 g;
[0016] Precisely weigh the above materials using an electronic balance, add 800 mL of primary pure water, stir with a magnetic suspension instrument for 10 minutes. After complete dissolution, continue to add primary pure water to make up the volume to 1000 mL.
[0017] Preferably, in the preparation of the reagent, the transfer buffer is prepared as follows: glycine powder: 14.4 g, Tris powder: 3.3 g, methanol: 200 mL;
[0018] Precisely weigh the above materials using an electronic balance, add 800 mL of primary pure water, stir with a magnetic suspension instrument for 10 minutes. After complete dissolution, continue to add 200 mL of methanol to make up the volume to 1000 mL, and store it at 4 °C for later use after preparation.
[0019] Preferably, in the preparation of the reagent, the Tris Buffered Saline with Tween-20 buffer is prepared as follows: Tris powder: 6.6 g, Nacl powder: 17.6 g, Tween-20: 2 mL;
[0020] Precisely weigh the above materials using an electronic balance, fully dissolve them in 1000 ml of primary pure water, then use a pipette to aspirate 2 ml of Tween-20, stir with a magnetic suspension instrument for 10 minutes. After complete dissolution, add primary pure water to make up the volume to 2000 mL, and store it at room temperature after preparation.
[0021] Preferably, in the preparation of the reagent, the 5x rapid blocking solution is prepared as follows: rapid blocking solution: 3 mL, TBST buffer: 12 mL;
[0022] Use a pipette to aspirate 3 mL of the rapid blocking solution into a blocking box, then aspirate 12 mL of the TBST buffer again, and place it on a shaker to gently shake and mix evenly. Store it at 4 °C for later use after preparation.
[0023] Preferably, in step S3, a sleep deprivation animal model is established by using the improved multi-platform water environment method. The mice are placed in a platform sleep deprivation chamber, which contains 20 small cylinders with a diameter of 2.5 cm and a height of 5 cm. The platform is 1 cm above the water level. The mice can move freely on each platform, but cannot straddle adjacent platforms. The water temperature is maintained at 20 ± 2 °C. Each group of mice can freely obtain food and drinking water on the mesh top, and the water in the water tank is changed regularly every day.
[0024] Preferably, in step S4, the sleep deprivation experiment and drug administration are as follows:
[0025] S41: Sleep deprivation: After 1 week of adaptive feeding of the mice, the mice in the gastrodin control group and the gastrodin intervention group were given gastrodin intragastric pretreatment for 7 days before modeling; at the same time, the blank control group and the sleep deprivation group were given intragastric administration of the same dose of normal saline for 7 consecutive days; before modeling, the sleep deprivation group and the gastrodin intervention group were adapted to the deprivation chamber for 2 hours for 3 consecutive days; sleep deprivation started at 8:00 in the morning on the first day after the end of adaptation and continued for 5 consecutive days. The blank control group and the gastrodin control group were not subjected to sleep deprivation.
[0026] S42: Gastrodin intragastric administration: After using a high-precision electronic analytical balance, 100 mg of gastrodin was dissolved in 10 ml of normal saline, and the administration dose was 100 mg / kg based on the body weight of the mice; the drug was formulated into a solution or suspension and injected into the stomach through a catheter with a syringe.
[0027] Preferably, in step S42, when performing intragastric administration, specifically:
[0028] With the right hand, lift the mouse's tail, first place the mouse on the experimental table, gently pull the tail backward. When the mouse grabs the tabletop with its front paws and stops moving, use the thumb and index finger of the left hand to pinch the skin of the head and neck under the mouse's ears to control the mouse's head so that it cannot move left and right. Then, use the middle finger, ring finger, little finger and the palm to gently clamp the back skin, straighten the mouse's head and neck. Hold the intragastric needle in the right hand and place it into the mouse's mouth from the left corner of the mouth, gently press the tongue surface, and then enter the esophagus along the upper palate; when the intragastric needle enters the esophagus, keep the mouse's body perpendicular to the ground, and then push the liquid medicine. After the liquid medicine is injected, gently pull out the intragastric needle.
[0029] Preferably, in step S5, 7 days before the start of the experiment, the experiment operator should stroke the mice for 5 minutes every day to make the mice familiar with the experimenter's smell; after the sleep deprivation ends, first place the mice in the experimental operation room to adapt for 1 hour to reduce the influence of external environmental factors on the mice.
[0030] The open field reaction chamber uses a square box; a square area with a side length of 30 cm is set as the central field at the center position of the test area, and the other areas are used as the peripheral fields; each mouse is placed in the central area of this area, allowing the mouse to freely explore in the open field box for 5 minutes, and the activities of the experimental animals are recorded with a overhead video tracking system; record the moving speed, movement trajectory, staying time and total movement distance of the mouse entering the central area; after the experiment of each mouse is completed, clean the container with ethanol.
[0031] Preferably: in the step S6, the mouse conducts an EPM experiment to measure anxiety-related behaviors; the standard elevated plus maze device is located 70 cm above the ground; the experimental animal is gently taken out of the breeding cage and placed in the central area of the elevated plus maze, ensuring that the back is facing the closed arm, and the activities of the experimental animal freely exploring in the device for 5 minutes are recorded by video monitoring and videotaped; after the recording is completed, put it back into the breeding cage, and clean the feces and urine with 75% alcohol. Wait for the device to dry before conducting the next experiment;
[0032] Record the experimental indicators:
[0033] Open arm time: That is, the time the mouse stays in the open arm. Taking the time when both front paws of the mouse enter the open arm from the central area as the standard and starting to record the time, and the time stops when both front paws completely withdraw from the open arm, which means the completion of this entry; accumulate the residence time in the open arm within 5 minutes to obtain the time of entering the open arm;
[0034] Open arm times: That is, the number of times the mouse stays in the open arm. Taking the time when both front paws of the mouse enter the open arm from the central area as the standard and starting to record the time, and when both front paws completely withdraw from the open arm, it means the completion of this entry, which is recorded as one time;
[0035] Closed arm time: That is, the time the mouse stays in the closed arm. Taking the time when both front paws of the mouse enter the closed arm from the central area as the standard and starting to record the time, and the time stops when both front paws completely withdraw from the closed arm, which means the completion of this entry; accumulate the residence time in the closed arm within 5 minutes to obtain the time of entering the closed arm;
[0036] Closed arm times: That is, the number of times the mouse stays in the closed arm. Taking the time when both front paws of the mouse enter the closed arm from the central area as the standard and starting to record the time, if both front paws completely withdraw from the closed arm, it means the end of this entry, which is recorded as one time;
[0037] The beneficial effects of the present invention are as follows:
[0038] 1. The present invention systematically explores the effect of gastrodin on the anxiety-like behavior of sleep-deprived mice for the first time, and reveals its mechanism of action from the perspective of autophagy-related proteins, providing a new perspective for the clinical application of gastrodin.
[0039] 2. The present invention successfully established a mouse sleep deprivation model by improving the multi-platform water environment method. This method is simple and easy to implement, highly reproducible, and suitable for large-scale experimental research.
[0040] 3. The present invention not only focuses on the improvement effect of gastrodin on anxiety-like behaviors, but also deeply explores its influence on the expression of autophagy-related proteins, providing more comprehensive information for understanding the pharmacological mechanism of gastrodin.
[0041] 4. The experimental design of the present invention is rigorous, including multiple links such as the setting of control groups, random grouping, and behavioral tests, ensuring the accuracy and reliability of the experimental results; the research results are of great significance for the development of new anti-anxiety drugs, helping to relieve anxiety symptoms caused by sleep deprivation and improve people's quality of life. BRIEF DESCRIPTION OF THE DRAWINGS
[0042] Figure 1 The trajectory and heat map of mice in the open field experiment of the present invention;
[0043] Figure 2 The schematic diagram of the time in the central area, moving distance, and moving speed of mice in the open field experiment of the present invention;
[0044] Figure 3 The trajectory and heat map of mice in the elevated plus maze experiment of the present invention;
[0045] Figure 4 The schematic diagram of the number of times mice enter the open arms, the time entering the open arms, and the number of times entering the closed arms in the elevated plus maze experiment of the present invention;
[0046] Figure 5 The HE staining map of the CA3 region of the hippocampus of mice in the present invention;
[0047] Figure 6 The schematic diagram of the proportion of damaged cells in the CA3 region of the hippocampus of mice in the present invention;
[0048] Figure 7 The schematic diagram of the Beclin-1 protein content in the hippocampus of mice in the present invention;
[0049] Figure 8 The schematic diagram of the quantification of Beclin-1 protein content in the hippocampus of mice in the present invention;
[0050] Figure 9 The schematic diagram of the LC3B and P62 protein contents in the hippocampus of mice in the present invention;
[0051] Figure 10 The schematic diagram of the quantification of LC3B and P62 protein contents in the hippocampus of mice in the present invention. DETAILED DESCRIPTION OF THE INVENTION
[0052] The technical solution of the present invention will be further described in detail below in conjunction with specific embodiments.
[0053] Example 1:
[0054] A research method on the mechanism of the effect of gastrodin on anxiety-like behaviors after sleep deprivation. Thirty-two male ICR mice were selected and a sleep deprivation model was established using the "modified multi-platform water environment method" to observe the effect of the expression level of autophagy-related proteins in the model group of mice on their anxiety-like behaviors.
[0055] Specifically as follows:
[0056] 1.1 Experimental materials
[0057] 1.1.1 Experimental animals
[0058] The experimental animals were 32 male ICR mice at 6 weeks old of SPF grade, with a body weight of 20 ± 2 g, purchased from Chongqing Ensville Biotechnology Co., Ltd. The mice were raised under standard laboratory conditions: the room temperature was maintained at 22 ± 2 °C, the environment was quiet, the relative humidity was 50 ± 5%, and the strict 12-hour light-dark rhythm (8:00 am / 8:00 pm) was followed to ensure that the mice could drink water and eat freely. All experimental procedures related to the mice were approved by the Animal Protection and Utilization Committee of Chongqing Medical University.
[0059] 1.1.2 Main experimental instruments
[0060]
[0061]
[0062] 1.1.3 Reagents required for the experiment
[0063]
[0064]
[0065] 1.1.4 Preparation of important reagents
[0066] (1) 0.9% normal saline:
[0067] Sodium chloride powder: 9 g
[0068] Primary pure water: 1000 ml
[0069] Fold the weighing paper and place it on the electronic balance. Accurately weigh 9 g of sodium chloride powder, fully dissolve it in 800 ml of primary pure water. After using a glass stirring rod to dissolve it fully, continue to add primary pure water to make the volume up to 1 L. After preparation, store it in a refrigerator at 4 °C for standby.
[0070] (2) Preparation of SDS-PAGE electrophoresis solution:
[0071] Tris powder: 3.03 g
[0072] Sodium dodecyl sulfate (SDS): 1 g
[0073] Glycine powder: 14.4 g
[0074] Type I pure water: 1000 mL
[0075] Use an electronic balance to accurately weigh the above powders, add 800 mL of Type I pure water, stir with a magnetic suspension mixer for 10 minutes. After complete dissolution, continue to add Type I pure water to make up to 1000 mL. Use it immediately after preparation.
[0076] (3) Preparation of transfer buffer:
[0077] Glycine powder: 14.4 g
[0078] Tris powder: 3.3 g
[0079] Methanol: 200 mL
[0080] Type I pure water: 1000 mL
[0081] Use an electronic balance to accurately weigh the above powders, add 800 mL of Type I pure water, stir with a magnetic suspension mixer for 10 minutes. After complete dissolution, continue to add 200 mL of methanol and make up to 1000 mL. Store it at 4 °C for later use after preparation.
[0082] (4) Preparation of Tris Buffered Saline with Tween-20 (TBST) buffer:
[0083] Tris powder: 6.6 g
[0084] Nacl powder: 17.6 g
[0085] Tween-20: 2 mL
[0086] Type I pure water: 2000 mL
[0087] Use an electronic balance to accurately weigh the above powders, dissolve them completely in 1000 ml of Type I pure water, then use a pipette to aspirate 2 ml of Tween-20, stir with a magnetic suspension mixer for 10 minutes. After complete dissolution, add Type I pure water to make up to 2000 mL. Store it at room temperature after preparation.
[0088] (5) Preparation of 5x rapid blocking solution:
[0089] Rapid blocking solution: 3 mL
[0090] TBST buffer: 12 mL
[0091] Use a pipette to aspirate 3 mL of rapid blocking solution into a blocking box, then aspirate 12 mL of TBST buffer again, place it on a shaker and gently shake to mix well. Prepare and store it for later use at 4°C.
[0092] 1.2 Experimental methods
[0093] 1.2.1 Grouping of experimental animals
[0094] Before the experiment, the mice were adaptively raised for 1 week. Male ICR mice were divided into 4 groups by the random number method: blank control group (CON group), gastrodin control group (GAS group), sleep deprivation group (SD group), and gastrodin intervention group (GAS+SD group). The sleep deprivation group and the gastrodin intervention group used the "modified multi-platform water environment method" to establish a mouse sleep deprivation model. The blank control group and the gastrodin control group were normally raised without deprivation.
[0095] 1.2.2 Establishment of sleep deprivation model
[0096] The "modified multi-platform water environment method" was used to establish a sleep deprivation animal model. The mice were placed in a platform sleep deprivation box (52 cm × 36 cm × 20 cm), which contained 20 small cylinders with a diameter of 2.5 cm and a height of 5 cm. The platform was 1 cm above the water level. The mice could move freely on each platform but could not cross over to adjacent platforms. The water temperature was maintained at about 20 ± 2°C. Each group of mice could freely obtain food and drinking water on the mesh top, and the water in the water tank was changed regularly every day. Since the mice's whole body skeletal muscles relax when they enter the rapid eye movement sleep period (REM) during sleep and they will fall into the water, due to the mice's fear of water, sleep deprivation was caused.
[0097] 1.2.3 Sleep deprivation experiment and drug administration
[0098] (1) Sleep deprivation: After 1 week of adaptive feeding of the mice, the mice in the GAS group and the GAS+SD group were pretreated with intragastric administration of gastrodin for 7 days before modeling; at the same time, the CON group and the SD group were given intragastric administration of the same dose of normal saline for 7 consecutive days. The intragastric administration started at 9:00 every morning. Before modeling, the SD group and the GAS+SD group were adapted to the deprivation box for 2 hours (10:00 - 12:00) for 3 consecutive days. Sleep deprivation started at 8:00 in the morning on the first day after the adaptation ended and continued for 5 consecutive days. The CON group and the GAS group were not subjected to sleep deprivation.
[0099] (2) Gastric gavage with gastrodin: After weighing 100 mg of gastrodin using a high-precision electronic analytical balance, it was dissolved in 10 ml of physiological saline, and the dosage was 100 mg / kg based on the body weight of the mice. Gastrodin is a highly water-soluble phenolic glycoside sedative with poor lipid solubility but good oral bioavailability. The gastric gavage method involves preparing the drug as a solution or suspension and injecting it into the stomach through a catheter with a syringe. To ensure the accuracy of the drug dosage, the gastric gavage method for mice was used in this experiment: When operating, hold the mouse's tail with the right hand, first place the mouse on the experimental table, gently pull the tail backward, and when the mouse grabs the tabletop with its front paws and stops moving, use the thumb and index finger of the left hand to pinch the skin of the mouse's head and neck under the ears to control the mouse's head and prevent it from moving left and right. Then, use the middle finger, ring finger, little finger, and palm to gently hold the back skin, straighten the mouse's head and neck, and hold the gastric gavage needle (the gastric gavage needle is 4.5 cm long, about 0.8 mm in diameter, and the needle tip is smooth) with the right hand and carefully place it into the mouse's mouth from the left corner of the mouth, gently press the tongue surface, and then insert it into the esophagus along the upper palate. After the gastric gavage needle enters the esophagus, keep the mouse's body perpendicular to the ground, and then push the liquid medicine. After the liquid medicine is injected, gently pull out the gastric gavage needle. Identification of successful injection of the liquid medicine into the mouse's stomach: The injection of the liquid medicine is smooth without resistance, and the mouse shows no coughing or violent reactions. During the whole operation process, the operation method should not be rough, and the movements should be as gentle as possible. If the gastric gavage needle enters the trachea or there is a feeling of resistance, it should be withdrawn and reinserted.
[0100] 1.2.4 Open-field test for mice
[0101] The open-field test is often used to evaluate the mental state of mice. Seven days before the start of the experiment, the experiment operator should stroke the mice for 5 minutes every day to make the mice familiar with the experimenter's smell. After sleep deprivation, the mice should be placed in the experimental operation room for 1 hour to adapt to reduce the influence of external environmental factors on the mice. The open-field reaction chamber consists of a 50x50x40 cm square box. A square area with a side length of 30 cm is defined as the central field in the center of the test area, and the other areas are defined as the peripheral fields. Place each mouse in the central area of this region and let the mouse freely explore in the open-field box for 5 minutes, and use a overhead video tracking system to record the activities of the experimental animals. Record the moving speed, movement trajectory, staying time, and total movement distance of the mice entering the central area. After each mouse's experiment, clean the container with ethanol to avoid leaving a smell that affects the activities of the next mouse.
[0102] 1.2.5 Elevated plus-maze test for mice
[0103] Mice were subjected to the EPM experiment to measure anxiety-related behaviors. The standard elevated plus-maze apparatus was located 70 cm above the ground. The experimental animals were gently taken out of the breeding cage and placed in the central area of the elevated plus-maze, ensuring that their backs were towards the closed arms. Video monitoring was used to record the activities of the experimental animals freely exploring in the apparatus for 5 minutes. After the recording was completed, the animals were returned to the breeding cage, and the feces and urine were cleaned with 75% alcohol. After the apparatus was dried, the next experiment was carried out. Record the experimental indicators (with the entry of the first two paws as the standard):
[0104] Open-arm time (s): This is the time that the mouse stays in the open arm. It is based on the standard that both front paws of the mouse enter the open arm from the central area and the timing starts. The timing stops when both front paws completely withdraw from the open arm, indicating the completion of this entry. The cumulative residence time in the open arm within 5 minutes is obtained to get the open-arm time (s).
[0105] Open-arm entries (n): This is the number of times the mouse stays in the open arm. It is based on the standard that both front paws of the mouse enter the open arm from the central area and the timing starts. When both front paws completely withdraw from the open arm, it indicates the completion of this entry, which is recorded as one time.
[0106] Closed-arm time (s): This is the time that the mouse stays in the closed arm. It is based on the standard that both front paws of the mouse enter the closed arm from the central area and the timing starts. The timing stops when both front paws completely withdraw from the closed arm, indicating the completion of this entry. The cumulative residence time in the closed arm within 5 minutes is obtained to get the closed-arm time (s).
[0107] Closed-arm entries (n): This is the number of times the mouse stays in the closed arm. It is based on the standard that both front paws of the mouse enter the closed arm from the central area and the timing starts. If both front paws completely withdraw from the closed arm, it indicates the end of this entry, which is recorded as one time.
[0108] 1.2.6 Statistical methods
[0109] GraphPad Prism version 9.5 software (Graph Pad Software, San Diego, CA) and SPSS version 26.0 software were used for statistical analysis. The data were expressed as mean ± standard deviation (±). One-way analysis of variance (one-way ANOVA) was used for comparing the data between groups. When the variances were homogeneous, the LSD method was used for pairwise comparison between groups, and P < 0.05 indicated that the data had statistical differences. When the variances were heterogeneous, Tamhane's T2 method was used for pairwise comparison between groups, and P < 0.05 indicated that the data had statistical differences.
[0110] As described above, it is only the preferred specific implementation manner of the present invention, but the protection scope of the present invention is not limited thereto. Any person skilled in the art within the technical scope disclosed by the present invention, according to the technical solution and inventive concept of the present invention, makes equivalent replacements or changes, and should be covered within the protection scope of the present invention.
Claims
1. A method for studying the mechanism of effect of gastrodin on anxiety-like behavior after sleep deprivation, characterized in that: include: ICR male mice were selected to establish a sleep deprivation model using the modified multi-platform water environment method, and the effect of the expression level of autophagy-related proteins in the model group on the anxiety-like behavior of mice was observed; The specific steps include: S1: Experimental preparation, reagent preparation, including 0.9% saline, SDS-PAGE electrophoresis solution, transfer buffer, Tris Buffered Saline with Tween-20 buffer, and 5x fast blocking solution; S2: Experimental animal grouping: 32 male ICR mice were randomly divided into 4 groups: blank control group, gastrodin control group, sleep deprivation group, and gastrodin intervention group. The sleep deprivation model of mice was established in the sleep deprivation group and gastrodin intervention group using the modified multi-platform water environment method. The blank control group and gastrodin control group were raised normally without deprivation. S3: Establish a sleep deprivation model; S4: sleep deprivation experiment and drug administration; S5: open field test for mice; S6: Elevated plus maze test for mice; S7: Experimental statistics.
2. The research method of the effect mechanism of gastrodin on anxiety-like behavior after sleep deprivation according to claim 1, characterized in that: In the reagent preparation, the SDS-PAGE electrophoresis solution is prepared as follows: Tris powder: 3.03 g, sodium dodecyl sulfate: 1 g, glycine powder: 14.4 g; Use an electronic balance to accurately weigh the above materials, add 800 mL of first-grade pure water, stir with a magnetic mixer for 10 minutes, and continue to add first-grade pure water to make up to 1000 mL after full dissolution.
3. The research method of the effect mechanism of gastrodin on anxiety-like behavior after sleep deprivation according to claim 2, characterized in that: In the reagent preparation, the transfer buffer is prepared as follows: glycine powder: 14.4 g, Tris powder: 3.3 g, methanol: 200 mL; Use an electronic balance to accurately weigh the above materials, add 800 mL of first-grade pure water, stir with a magnetic mixer for 10 minutes, and add 200 mL of methanol to make the volume up to 1000 mL after full dissolution. After preparation, store at 4 degrees for later use.
4. The method for studying the mechanism of the effect of gastrodin on anxiety-like behavior after sleep deprivation according to claim 3, characterized in that: In the reagent preparation, the Tris Buffered Saline with Tween-20 buffer is prepared as follows: Tris powder: 6.6 g, Nacl powder: 17.6 g, Tween-20: 2 mL; Use an electronic balance to accurately weigh the above materials, fully dissolve them in 1000 ml of first-grade pure water, then use a pipette to aspirate 2 ml of Tween-20, stir with a magnetic suspension instrument for 10 minutes, and after fully dissolved, add first-grade pure water to make up to 2000 ml. After preparation, store at room temperature.
5. The method for studying the mechanism of the effect of gastrodin on anxiety-like behavior after sleep deprivation according to claim 4, characterized in that: In the reagent preparation, the 5x rapid blocking solution is prepared as follows: rapid blocking solution: 3 mL, TBST buffer: 12 mL; Use a pipette to aspirate 3 mL of rapid blocking solution into the sealing box, and then aspirate 12 mL of TBST buffer solution. Place the solution on a shaker and gently shake to mix. Store the solution at 4 degrees for later use.
6. The method for studying the mechanism of effect of gastrodin on anxiety-like behavior after sleep deprivation according to claim 1, characterized in that: In S3, a sleep deprivation animal model was established using a modified multi-platform water environment method, and mice were placed in a platform sleep deprivation box, which contained 20 small cylinders with a diameter of 2.5 cm and a height of 5 cm, and the platform was 1 cm above the water level; the mice moved freely on each platform but could not cross over adjacent platforms; the water temperature was maintained at 20±2°C; each group of mice had free access to food and drinking water on the top of the net, and the water in the water tank was replaced regularly every day.
7. The method for studying the mechanism of the effect of gastrodin on anxiety-like behavior after sleep deprivation according to claim 1, characterized in that: In S4, the sleep deprivation experiment and drug administration are as follows: S41: Sleep deprivation: After 1 week of adaptive feeding of mice, the mice in the gastrodin control group and the gastrodin intervention group were given gastrodin gavage for 7 days before modeling; at the same time, the blank control group and the sleep deprivation group were given the same dose of normal saline gavage for 7 consecutive days; before modeling, the sleep deprivation group and the gastrodin intervention group were adapted in the deprivation box for 2 hours for 3 consecutive days; sleep deprivation started at 8:00 am on the first day after the end of adaptation, and sleep deprivation lasted for 5 consecutive days. The blank control group and the gastrodin control group were not given sleep deprivation; S42: Intragastric administration of gastrodin: 100 mg of gastrodin was weighed using a high-precision electronic analytical balance and dissolved in 10 ml of normal saline. The dosage was 100 mg / kg based on the body weight of the mouse. The drug was prepared into a solution or suspension and injected into the stomach via a catheter using a syringe.
8. The method for studying the mechanism of effect of gastrodin on anxiety-like behavior after sleep deprivation according to claim 1, characterized in that: In said S42, when gavage is performed, specifically: Lift the mouse's tail with your right hand, place the mouse on the experimental table first, and gently pull its tail back. When the mouse's forelimbs grab the table and do not move, pinch the skin of the head and neck under the ears of the mouse with the thumb and index finger of your left hand to control the mouse's head so that it cannot move left and right. Then carefully pinch the skin on the back with your middle finger, ring finger, little finger and palm, straighten the mouse's head and neck, hold the gavage needle in your right hand and put it into the mouth from the left corner of the mouse's mouth, gently press the tongue surface, and then enter the esophagus along the upper palate; after the gavage needle enters the esophagus, keep the mouse's body vertical to the ground, and then push the drug solution in. When the drug solution is injected, gently pull out the gavage needle.
9. The method for studying the mechanism of effect of gastrodin on anxiety-like behavior after sleep deprivation according to claim 1, characterized in that: In S5, the experimenter should stroke the mice for 5 minutes every day 7 days before the experiment to make the mice familiar with the experimenter's smell; after the end of sleep deprivation, the mice should be placed in the experimental operation room for 1 hour to adapt to the experiment to reduce the impact of external environmental factors on the mice; The open field reaction box adopts a square box; a square area with a side length of 30 cm is set in the center of the test area as the central venue, and other areas are used as peripheral venues; each mouse is placed in the central area of the area, and the mouse is allowed to freely explore in the open field box for 5 minutes, and the activities of the experimental animals are recorded with an overhead video tracking system; the movement speed, movement trajectory, residence time and total movement distance of the mouse entering the central area are recorded; the container is cleaned with ethanol after the experiment of each mouse.
10. The method for studying the mechanism of effect of gastrodin on anxiety-like behavior after sleep deprivation according to claim 1, characterized in that: In S6, mice were subjected to an EPM experiment to measure anxiety-related behaviors; a standard elevated plus maze apparatus was located 70 cm above the ground; the experimental animals were gently taken out of the cage and placed in the central area of the elevated plus maze, ensuring that the back was facing the closed arms, and the experimental animals were monitored by video and recorded for 5 minutes of free exploration in the apparatus; after the recording was completed, the mice were returned to the cage, and feces and urine were cleaned with 75% alcohol, and the next experiment was carried out after the apparatus was dry; Record experimental indicators: Open arm time: refers to the time the mouse stays in the open arm. The time starts when both front paws of the mouse enter the open arm from the central area. The time recording stops when both front paws completely withdraw from the open arm. The time of entering the open arm is accumulated within 5 minutes to get the time of entering the open arm. Open arm times: refers to the number of times the mouse stayed in the open arm. The time was recorded when both front paws of the mouse entered the open arm from the central area. The entry was completed when both front paws completely withdrew from the open arm and was recorded as one time. Closed arm time: refers to the time the mouse stays in the closed arm. The time starts when both front paws of the mouse enter the closed arm from the central area. The entry is completed when both front paws are completely withdrawn from the closed arm, and the time recording stops. The time of entering the closed arm is accumulated within 5 minutes to get the time of entering the closed arm. The number of closed arms is the number of times the mouse stays in the closed arm. The time is recorded when both front paws of the mouse enter the closed arm from the central area. If both front paws are completely withdrawn from the closed arm, it means that the entry is over and is recorded as one time.