Method for constructing zebra fish fatigue model

Abstract

The invention relates to the technical field of medicines, and provides a method for establishing a zebra fish fatigue model in order to better simulate the fatigue state of modern people. And forcing the zebra fish to continuously move through a magnetic stirring device and generating a fatigue state by noise stimulation so as to establish a fatigue model. The total movement distance, the swimming speed and the swimming track are used as behavior analysis indexes, the lactic acid content is used as a biochemical detection index, the atp2a2b gene expression quantity is used as a gene detection index, and compared with normally-treated zebrafish and zebrafish treated by Chinese traumatic injury pills, the reliability of the model is verified in multiple dimensions. The method is easy and convenient to operate, low in cost and suitable for fatigue mechanism research and anti-fatigue product development.

Description

Technical Field

[0001] The invention relates to a zebrafish-based fatigue model construction method and an evaluation system thereof, which are suitable for studying fatigue mechanisms, screening anti-fatigue drugs and related functional foods. Background Art

[0002] With the accelerated pace of work and increased pressure in life in modern society, fatigue has become a common condition. Fatigue is not only manifested as a decline in function after physical or mental activities, but may also cause a series of physiological and psychological problems, such as decreased immunity, mood swings and sleep disorders, which seriously affect the quality of life and work efficiency. As a model organism, zebrafish has been widely used in the study of fatigue mechanisms because of its advantages such as high homology with human genes (about 87%), short breeding cycle and simple experimental operation. The central nervous system, muscle system and energy metabolism pathways of zebrafish are highly conserved with those of mammals, making it an ideal model for studying fatigue mechanisms.

[0003] There are many methods for modeling zebrafish fatigue models, which are mainly established by continuous exercise, chemical induction or environmental pressure (such as hypoxia). In the existing fatigue modeling methods, no one has used a magnetic stirring device to establish a fatigue model. The magnetic stirring device can force zebrafish to move continuously, and will generate noise interference during the stirring process, which can produce the dual effects of physical and mental fatigue. Therefore, the present invention uses a magnetic stirring device to establish a zebrafish fatigue model. The method is simple and easy to operate, and the model effect is good, which is of great significance to the development of products that improve fatigue. Summary of the invention

[0004] The present invention aims to provide a zebrafish fatigue model and its evaluation method. The zebrafish fatigue model is established by using a magnetic stirring device to force the zebrafish to continue to move and mix noise stimulation to produce a fatigue state. Compared with normal zebrafish, the constructed zebrafish fatigue model has a slower swimming speed, a shorter swimming trajectory, an increased lactic acid content in the body, and energy metabolism-related genes. atp2a2b After treatment with Zhonghua Dieda Pills, it was found that the swimming speed of zebrafish increased, the number of swimming tracks increased, the lactic acid content in the body decreased, and the genes related to energy metabolism decreased. atp2a2b The increased expression level can be applied to the development of anti-fatigue products.

[0005] In order to achieve the above object, the present invention adopts the following technical solutions: A method for constructing a zebrafish fatigue model: using a magnetic stirring device to force the zebrafish to move continuously through alternating cycles of different speeds, and adding noise stimulation during the operation to produce a fatigue state, thereby establishing a fatigue model.

[0006] The magnetic stirring device is an experimental device that uses a magnetic field to drive a stirrer to achieve liquid mixing or heating. It is widely used in laboratories. With its function of mixing solutions, it can accelerate the flow of water and force zebrafish to swim continuously. The rotor of the magnetic stirring device will inevitably produce friction and collision while rotating, making noise. Continuous noise interference can cause physiological and psychological fatigue. Therefore, the magnetic stirring device is used to make zebrafish produce fatigue through the selection and control of the rotation speed, simulating the fatigue problem of modern people.

[0007] Preferably, the rotation speed of the magnetic stirring is controlled at 500-1500 rpm when establishing the fatigue model.

[0008] Preferably, the modeling time is 8-10 h.

[0009] Preferably, the modeling method is to cycle alternately at 500 rpm and 1500 rpm for 8 h.

[0010] Preferably, wild-type AB strain zebrafish at 5-7 dpf are selected for the experiment.

[0011] The present invention detects the swimming speed, swimming trajectory, lactic acid content and atp2a2b The relative expression of genes was used to characterize the degree of fatigue in zebrafish. When compared with the normal control group zebrafish, the swimming speed, swimming trajectory and atp2a2b The relative expression of genes decreased and the lactic acid content increased, indicating that zebrafish were in a state of fatigue. When treated with Zhonghua Dieda Pills, the swimming speed, swimming trajectory and atp2a2b The increase in relative gene expression and the decrease in lactate content indicate that fatigue can be improved by fatigue drugs.

[0012] Preferably, the behavioral analysis method is: using a behavioral analyzer to measure the total movement distance, swimming speed and swimming trajectory of the zebrafish within 1 hour.

[0013] Preferably, the method for determining the content of biochemical indicators is: collecting the whole body of zebrafish and determining the lactic acid content.

[0014] As a preferred method for detecting the relative expression of energy-related genes, the total RNA of zebrafish is extracted and then detected by fluorescence quantitative PCR. gapdh and energy genes atp2a2b Expression, using gapdh As an internal reference for gene expression, atp2a2b The relative expression level.

[0015] The above-mentioned modeling method for inducing zebrafish fatigue is used in detecting the fatigue-improving efficacy of drugs and functional foods.

[0016] Therefore, the present invention has the following beneficial effects: (1) The zebrafish fatigue model established by the magnetic stirring device can be disturbed by noise while exercising continuously, which can more effectively simulate the dual fatigue state of the modern population, both physical and mental. (2) The reliability of the model is fully verified through multi-dimensional evaluation of behavioral, biochemical indicators and gene expression. (3) The established fatigue model can be restored by drugs, which is simple and verifiable, and is suitable for large-scale screening of anti-fatigue drugs or functional foods. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 This is a diagram of the swimming trajectory of a zebrafish.

[0018] Figure 2 This is a graph of zebrafish swimming speed.

[0019] Figure 3 Energy-related genes atp2a2b Schematic diagram of expression levels.

[0020] Figure 4 This is a graph of lactic acid content. DETAILED DESCRIPTION

[0021] The technical solution of the present invention is further described below through specific embodiments.

[0022] Example 1: Determination of the method for establishing a zebrafish fatigue model 1.1 Zebrafish fatigue model established by single speed induction Four groups were set up in the experiment: normal control group, 500 rpm induced group, 1000 rpm induced group, and 1500 rpm induced group.

[0023] The normal control group was randomly selected from wild-type AB strain zebrafish 7 days after fertilization (7 dpf) and placed in a 500 ml beaker, with 50 zebrafish in each beaker and the temperature maintained at 28.5 ℃.

[0024] The 500 rpm induction group was randomly selected from wild-type AB zebrafish 7 days post-fertilization (7 dpf) in a 500 ml beaker, with 50 zebrafish treated in each beaker. The temperature was set at 28.5 °C, the rotor length was 25 mm, the speed was 500 rpm, and the treatment time was 8 h (9:00 am.-5:00 pm.).

[0025] The 1000 rpm induction group was randomly selected from wild-type AB zebrafish 7 days post-fertilization (7 dpf) in a 500 ml beaker, with 50 zebrafish treated in each beaker. The temperature was set at 28.5 °C, the rotor length was 25 mm, the speed was 1000 rpm, and the treatment time was 8 h (9:00 am.-5:00 pm.).

[0026] The 1500 rpm induction group was randomly selected from wild-type AB zebrafish 7 days post-fertilization (7 dpf) in a 500 ml beaker, with 50 zebrafish treated in each beaker. The temperature was set at 28.5 °C, the rotor length was 25 mm, the speed was 1500 rpm, and the treatment time was 8 h (9:00 am.-5:00 pm.).

[0027] After the treatment, 24 zebrafish were randomly drawn from each group and placed in a 96-well plate, with 1 fish per well and 200 μl of liquid added to each well. The movement behavior of zebrafish larvae was monitored for 1 hour using a behavior analyzer under a 28 °C light environment to obtain the total movement distance of zebrafish in 1 hour. SPSS 26.0 software was used for statistical analysis, and the statistical results were expressed as mean ± SE. P ≤ 0.05 indicated statistical significance.

[0028] Table 1 Total movement distance of each group in the zebrafish single speed induced fatigue model

[0029] Compared with the normal control group, *p≤0.05, ***p≤0.001.

[0030] Results: The fatigue model induced by 500 rpm was not successfully established. During the experiment, it was found that forcing it to move at the same speed may cause it to adapt to the speed and drift with the flow and no longer move. This needs to be improved and different speeds should be used to avoid this situation.

[0031] 1.2 Establishment of zebrafish fatigue model by inducing different rotation speeds Four groups were set up in the experiment: normal control group, 500 rpm+1000 rpm induction group, 500 rpm+1500 rpm induction group, and 1000 rpm+1500 rpm induction group.

[0032] The normal control group was randomly selected from wild-type AB strain zebrafish 7 days after fertilization (7 dpf) and placed in a 500 ml beaker, with 50 zebrafish in each beaker and the temperature maintained at 28.5 ℃.

[0033] The 500 rpm+1000 rpm induction group was randomly selected from wild-type AB zebrafish 7 days after fertilization (7 dpf) in a 500 ml beaker, with 50 zebrafish in each beaker. The temperature was set at 28.5 °C, the rotor length was 25 mm, the speed was 500 rpm+1000 rpm for 1 h each, and the alternating cycle was 8 h (9:00 am.-5:00 pm.).

[0034] The 500 rpm+1500 rpm induction group was randomly selected from wild-type AB zebrafish 7 days post-fertilization (dpf) in a 500 ml beaker, with 50 zebrafish in each beaker. The temperature was set at 28.5 °C, the rotor length was 25 mm, the speed was 500 rpm+1500 rpm for 1 h each, and the alternating cycle was 8 h (9:00 am.-5:00 pm.).

[0035] The 1000 rpm+1500 rpm induction group was randomly selected from wild-type AB zebrafish 7 days post-fertilization (dpf) in a 500 ml beaker, with 50 zebrafish in each beaker. The temperature was set at 28.5 °C, the rotor length was 25 mm, the speed was 1000 rpm+1500 rpm for 1 h each, and the alternating cycle was 8 h (9:00 am.-5:00 pm.).

[0036] After the treatment, 24 zebrafish were randomly drawn from each group and placed in a 96-well plate, with 1 fish per well and 200 μl of liquid added to each well. The movement behavior of zebrafish larvae was monitored for 1 hour using a behavior analyzer under a 28 °C light environment to obtain the total movement distance of zebrafish in 1 hour. SPSS 26.0 software was used for statistical analysis, and the statistical results were expressed as mean ± SE. P ≤ 0.05 indicated statistical significance.

[0037] Table 2 Total movement distance of each group in the zebrafish compound speed-induced fatigue model

[0038] Compared with the normal control group, *p≤0.05, ***p≤0.001.

[0039] Results: The total movement distance of each zebrafish group induced by compound speed was significantly reduced compared with the normal control group. Finally, 500 rpm+1500 rpm for 1 h each for 8 h was selected as the method to induce zebrafish fatigue model. This modeling method was further analyzed and tested to determine its reliability.

[0040] Example 2: Evaluation experiment of zebrafish fatigue model 2.1 Experimental groups and treatments: The experiment set up three groups: normal control group, fatigue model group, and drug treatment group.

[0041] The normal control group was randomly selected from wild-type AB strain zebrafish 7 days after fertilization (7 dpf) and placed in a 500 ml beaker, with 50 zebrafish in each beaker and the temperature maintained at 28.5 ℃.

[0042] The fatigue model group randomly selected wild-type AB zebrafish at 7 days post-fertilization (dpf) in a 500 ml beaker, with 50 zebrafish in each beaker. The temperature was set at 28.5 °C, the rotor length was 25 mm, and the speed was set to 500 rpm + 1500 rpm for 1 h each, with a cycle time of 8 h (9:00 am.-5:00 pm.).

[0043] The drug treatment group randomly selected wild-type AB zebrafish 7 days post-fertilization (7 dpf) in a 500 ml beaker, with 50 zebrafish treated in each beaker. The treatment drug Zhonghua Dieda Pills was given to them at a drug treatment concentration of 1000 μg / ml, the temperature was set to 28.5 ℃, the rotor length was 25 mm, and the speed was set to 500 rpm+1500 rpm for 1 h each, with a cycle treatment time of 8 h (9:00 am.-5:00 pm.).

[0044] 2.2 Evaluation of zebrafish fatigue model behavior analysis Randomly take 12 zebrafish from each group in 2.1 and place them in a 24-well plate, 1 fish per well, 2 ml per well, and monitor the movement behavior of zebrafish larvae for 1 hour using a behavior analyzer under a 28 ℃ light environment. The swimming speed of zebrafish in 1 hour was analyzed using the software provided by the behavior analyzer, and the statistical analysis results of this indicator and the swimming trajectory were used to evaluate the fatigue-relieving effect of the sample.

[0045] The results show that: Figure 1 It can be seen that the zebrafish in the fatigue model group swam less than those in the normal control group. The zebrafish swam more after drug treatment, approaching normal levels. Figure 2 It can be seen that the swimming speed of zebrafish in the fatigue model group was significantly reduced compared with the normal control group (p≤0.05), resulting in a fatigue state. The swimming speed of zebrafish after drug treatment increased and the fatigue state was improved.

[0046] 2.3 Evaluation of biochemical indicators of zebrafish fatigue model (lactic acid content) After treatment in 2.1, 100 zebrafish larvae were collected in each group using a 1.5 ml centrifuge tube. The larvae to be collected were washed twice with ultrapure water and then dried. After drying, the tissue weight was accurately weighed. According to the ratio of weight (g): volume (ml) = 1:9, 9 volumes of PBS were added, and mechanical homogenization was performed under ice water bath conditions. The homogenate was centrifuged at 5000×g for 10 minutes, and the supernatant was taken and stored for later use. The lactic acid content was detected according to the steps in the kit instructions, and the absorbance (OD value) of each group was detected at a wavelength of 450 nm using an enzyme marker. The OD value of each sample group was substituted into the standard curve equation to obtain the lactic acid content.

[0047] The results showed that the lactic acid content in the fatigue modeling group was significantly higher than that in the normal control group (p≤0.001), which could promote the production of lactic acid. The lactic acid content in the drug treatment group was significantly lower than that in the fatigue modeling group (p≤0.001), which could promote lactic acid metabolism and improve fatigue. Figure 3 .

[0048] 2.4 Expression of energy-related genes in zebrafish fatigue model After modeling, 60 zebrafish were collected from each group and placed in a 1.5 ml sterile enzyme-free centrifuge tube. The water was dried and 5 parallels were prepared for each group. Total RNA was extracted with trizol reagent and the concentration and purity of RNA were determined with an ultra-micro spectrophotometer. The results showed that the extracted zebrafish RNA was of good quality and could be used for subsequent testing. RNA was reverse transcribed into cDNA using a PCR instrument, and then fluorescent quantitative PCR was performed to detect energy metabolism genes. atp2a2b The primer sequences are as follows:

[0049] The results showed that the fatigue model group was significantly higher than the normal control group. atp2a2b The gene expression level was significantly decreased (p≤0.01) in the drug treatment group compared with the fatigue model group atp2a2b The gene expression level was significantly increased (p ≤ 0.05), the results are shown in Figure 4 .

Claims

1. A method for constructing a zebrafish fatigue model, characterized in that: The steps include: (1) Wild-type AB zebrafish were selected for the experiment. The zebrafish were kept in a state of continuous motion by using a magnetic stirrer to change the rotation speed in a cyclic alternating manner. At the same time, noise stimulation was applied to establish a fatigue model. (2) The fatigue model zebrafish were compared with normal zebrafish. The fatigue model zebrafish showed slower swimming speed, fewer swimming trajectories, higher lactic acid levels in the body, and changes in genes related to energy metabolism. atp2a2b The situation of decreased expression level shows fatigue state; (3) After the fatigue model zebrafish were treated with Zhonghua Dieda Pills, the swimming speed of zebrafish increased, the number of swimming trajectories increased, the lactic acid content in the body decreased, and the genes related to energy metabolism were expressed in atp2a2b The increased expression level indicated that the drug could significantly improve the fatigue state produced by the model.

2. The method according to claim 1, characterized in that The method of magnetic stirring to induce fatigue model is: the rotation speed is 500-1500 rpm, and the alternating cycle is performed for 8 hours.

3. The method according to claim 1, characterized in that The temperature for zebrafish fatigue model establishment and behavioral analysis was 27-29 ℃.

4. The method according to claim 1, characterized in that: Wild-type AB strain zebrafish larvae at 5-7 days post fertilization (dpf) were used for the experiments.

5. The method according to claim 1, characterized in that The rotors used were 20-30 mm long.

6. The method according to claim 1, characterized in that The stocking density of zebrafish for establishing fatigue model was 100-200 fish / L.

7. The method according to claim 1, characterized in that The fatigue model group, normal control group and drug treatment group were all added with the same volume of water and cultured in the same temperature environment.

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