Method for establishing zebra fish myopia model and application thereof

Abstract

The invention relates to the field of drug evaluation methods, and provides a method for establishing a zebra fish myopia model and application thereof in order to solve the problems of high modeling cost, long time consumption and few characterization indexes of an animal myopia model in the prior art, the zebra fish myopia model is established by injecting a zebra fish lumican gene MO into a zebra fish body for induction, and the zebra fish lumican gene MO is used as an animal myopia model. And after modeling, evaluating the myopia prevention and treatment effects of the medicine to be detected through analyzed eyeball shapes, eye cell apoptosis conditions and behavioral change indexes of the zebra fish. The zebrafish myopia model is high in modeling speed and stable in modeling effect, and can be used for rapidly screening medicines with the effects of preventing and treating myopia and researching the action ways of the medicines.

Description

Technical Field

[0001] The present invention relates to the field of drug evaluation methods, and particularly to a method for establishing a zebrafish myopia model and its application. Background Art

[0002] Myopia is caused by an excessive axial length of the eye, which causes the lens and cornea to be unable to focus images on the retina. Myopia has become a major public health problem affecting the eye health of the people in our country, especially teenagers. Although myopia is the most common visual impairment in the world, there are still many unknowns about myopia and its causes and progression.

[0003] Currently, researchers have successfully established myopia models in monkeys, tree shrews, mice, guinea pigs, chickens, etc. There are differences in the eye structures and developmental characteristics of these myopia model animals, so their applications in myopia research are also different. Among them, the eye structure of chickens is quite different from that of humans. Therefore, the research conclusions obtained using chickens as myopia models should be applied with caution in clinical research. The eye structures of tree shrews and monkeys are similar to those of humans, but tree shrews are difficult to obtain, and monkeys have a long experimental period, high cost, poor cooperation, and it is difficult to complete a large sample size, which limits the application of the above animal models. Mice are common experimental animals, and guinea pigs have a docile temperament and slightly higher cooperation compared to other animals. The eye development processes of both are similar to those of humans. However, as typical nocturnal animals, the light reception and feedback of their eyes are different from those of humans. At the same time, during the modeling process, a single eye needs to be physically worn with an eye mask for about 4 - 10 weeks. The model failure rate is high and the time consumption is long. At the same time, the eyes of mice and guinea pigs are small, the optical quality of the refractive medium is poor, and the detection requires high precision of the instrument. After establishing a myopia model with existing model organisms, usually one or 2 - 3 indicators are used to characterize myopia, and it is impossible to explore multiple causes of myopia. Moreover, the modeling time is about 7 days - 6 weeks, which is time-consuming. Summary of the Invention

[0004] In order to overcome the problems of high modeling cost, high failure rate, long time consumption, few characterization indicators, and difficult application of animal myopia models in the prior art, the present invention provides a method for establishing a zebrafish myopia model. This method has a fast modeling speed, stable modeling effect, simple operation, and can be used for myopia research.

[0005] To achieve the above object, the present invention adopts the following technical solutions: In the first aspect, the present invention provides a method for establishing a zebrafish myopia model, and the zebrafish myopia model is induced and established by injecting MO of zebrafish lumican gene into the zebrafish body.

[0006] Zebrafish, like humans, are diurnal tetrachromatic animals with cone photoreceptors sensitive to ultraviolet (UV), blue, green, and red light. Their color vision under bright light conditions is equivalent to the spectral characteristics of normal human vision. The retinas of juvenile and adult zebrafish have many similarities, including both being rich in cone cells and having the same types of photoreceptors. The vision of zebrafish in the first few weeks of life is mainly mediated by cone cells. The developing zebrafish retina expresses opsin 2.5 days after fertilization and has already responded to visual stimuli at 5 dpf. The eyes of juvenile zebrafish account for about half of the head size, making it an excellent model organism for studying visual function and early-onset diseases. In addition, the transparent characteristics of juvenile zebrafish enable the significant observation of the diameter of the retinal pigment epithelium, scleral diameter, and axial length of the eye under an ordinary microscope. Injection of MO into the zebrafish lumican gene can cause excessive dilation of the sclera, leading to myopia.

[0007] Preferably, the zebrafish is a wild-type AB strain zebrafish at the single-cell stage.

[0008] Taking advantage of the in vitro fertilization of zebrafish embryos, a large number of fertilized eggs can be easily and directly obtained. The newly fertilized eggs until their first cleavage occurs, called the zygotic stage, about 40 minutes after fertilization. The zygotic stage only contains one stage, and 1 single cell can be seen, making it easy to divide this stage and control this time period, making the operation of gene MO injection simpler and more accurate.

[0009] Preferably, the injection dose of the zebrafish lumican gene MO is 0.25 - 2 pmol / fish.

[0010] More preferably, the injection dose of the zebrafish lumican gene MO is 2 pmol / fish.

[0011] An injection amount of 0.25 - 2 pmol / fish will not affect the growth of zebrafish and can cause zebrafish to develop myopia. Within this range, the degree of myopia in zebrafish deepens with the increase in the injection amount.

[0012] In a second aspect, the present invention provides the application of the zebrafish myopia model established by the above method in the method for screening drugs with the efficacy of preventing and treating myopia, characterized in that the method comprises the following steps: S1. Randomly group the zebrafish. After establishing the zebrafish myopia model in the sample group, administer the drug to be tested. The normal control group is not specially treated. The model control group establishes the zebrafish myopia model, and the negative control group performs zebrafish MO control injection. S2. Evaluate the efficacy of the drug to be tested in preventing and treating myopia by analyzing the eye ball morphology, apoptosis of eye cells, and behavioral change indexes of the zebrafish in the above experimental groups.

[0013] The present invention evaluates the improvement of myopia in zebrafish through multiple indicators such as the morphology of the eyeball, the apoptosis of ocular cells, and behavioral changes, and can explore the ways for the drug to be tested to exert the effects of preventing and treating myopia. Myopia is comprehensively affected by genetic, environmental factors, and bad eye-using habits, and is divided into simple myopia (no pathological changes in the fundus) and pathological myopia according to the disease course. In addition to poor distant vision, pathological myopia also shows fundus changes, including visual fatigue (photophobia, dry eye, eye soreness and distension), exo / eso-strabismus (weakening of the convergence function of the extraocular muscles, double vision), eyeball changes (scleral diameter, axial length of the eye, etc.), color vision abnormalities (blue, yellow, and red color vision abnormalities), light perception abnormalities (weakening of night vision), floaters, etc. At present, there are few drugs that can directly improve the physical morphology of the eyeball. Most drugs cannot change the eyeball morphology but can prevent or improve myopia by alleviating visual fatigue, improving light perception and color vision. In the prior art, the evaluation indicators of the myopia model are all concentrated in the eyeball change module, and the number of preventive and / or therapeutic drugs involved and the reference mechanisms are extremely limited. Therefore, this patent expands the evaluation indicators of the myopia model, comprehensively explores the ways for the drug to be tested to exert the effects of preventing and treating myopia, and reduces false negative results.

[0014] Preferably, in S1, the sample group is given the drug to be tested by water solution at 2 - 5 dpf after establishing the zebrafish myopia model.

[0015] Preferably, in S1, the sample group is cultured to 5 - 6 dpf after establishing the zebrafish myopia model for index detection.

[0016] More preferably, in S1, the sample group is cultured for 5 dpf after establishing the zebrafish myopia model for index detection.

[0017] At 5 - 6 days after injection for modeling, the scleral diameter of the zebrafish is significantly different from that of the normal control group, and there is no obvious difference in the myopia degree of the zebrafish at 5 days and 6 days. Therefore, considering the time cost, index detection is carried out at 5 days after modeling.

[0018] Preferably, the eyeball morphology indicators in S2 include scleral diameter, the ratio of the diameter of the retinal pigment epithelium to the scleral diameter, axial length of the eye, and ocular area; The apoptosis of ocular cells includes the apoptosis of retinal cells and the apoptosis of lens cells; The behavioral changes include color preference changes and light and dark stimulation behaviors.

[0019] Myopia can lead to changes in the shape of the eyeball, such as the sclera becoming larger and thinner, the eye axis becoming longer, etc. The changes in the shape of the zebrafish eyeball can be observed and photographed under a dissection microscope, and the specific values can be obtained after the collected images are converted by image processing software; pathological myopia can cause apoptosis of retinal cells and lens cells, and the apoptosis of these two types of cells can be observed under a fluorescence microscope after AO staining of zebrafish; myopia can also cause changes in the behavior of zebrafish. Normal zebrafish prefer blue. When zebrafish become myopic, their preference for blue decreases, their motor ability decreases, and their perception of light and dark stimuli decreases.

[0020] Preferably, in S2, the evaluation is also carried out by analyzing the gene expression indexes of mmp-2, tgf-β, timp-2a and col1a1a.

[0021] The genes of mmp-2, tgf-β, timp-2a and col1a1a are genes related to type I collagen fibers. Type I collagen fibers are related to the thickness and elasticity of the sclera. The sclera is located at the outermost 2 / 3 of the eyeball wall and is a dense fibrous connective tissue, which determines the size and shape of the eyeball to a certain extent, especially the length of the eye axis. Therefore, the gene expression of mmp-2, tgf-β, timp-2a and col1a1a can also characterize the myopia situation.

[0022] Preferably, the detection method for the change in color preference is as follows: The four-arm regions of the cross module are painted yellow, blue, red and green respectively. The zebrafish are placed in the center of the cross module, and data are collected by a behavior instrument to analyze the percentage of the total movement distance of the zebrafish in the blue region in the total movement distance of the whole region.

[0023] Preferably, the detection method for the light and dark stimulation behavior is as follows: The zebrafish are placed in a container, and the zebrafish are given light-dark cycle stimulation. Data are collected by a behavior instrument to analyze the average movement speed per minute of the zebrafish under each light-dark cycle stimulation.

[0024] The container can be a microplate, with 1 zebrafish placed in each well. The tested zebrafish do not interfere with each other's movements, and data of multiple zebrafish can be obtained simultaneously.

[0025] Therefore, the present invention has the following beneficial effects: (1) A zebrafish myopia model is established, with a fast modeling speed, stable modeling effect and simple operation; (2) The evaluation indexes of the pathological myopia model are innovated and expanded; (3) Drugs with the efficacy of preventing and treating myopia can be quickly screened, and their action pathways can be studied. Specific embodiments

[0026] The following further describes the present invention in combination with specific implementation methods.

[0027] Example 1 Establishment of zebrafish myopia model: (1) Confirmation of the optimal dose of lumican gene MO injection in zebrafish: The index is the mortality rate at different doses; Method: Randomly select wild-type AB strain zebrafish at the single-cell stage into 6-well plates, with 30 tails in each well. Inject lumican-MO at doses of 0.25, 0.5, 1, 2, and 4 pmol / tail. At the same time, set up a normal control group and an MO negative control group, and the volume of each well is 3 mL. After treatment at 28°C for 5 days, observe and calculate the mortality rate of zebrafish to determine the optimal detection dose of lumican-MO for normal zebrafish. The results are shown in Table 1.

[0028] Table 1 The results show that the injection volume of 0.25 - 2 pmol / tail does not affect the growth of zebrafish and can cause zebrafish to develop myopia. Within this range, the degree of myopia in zebrafish deepens with the increase of the injection volume. The optimal dose of lumican gene MO injection is 2 pmol / tail.

[0029] (2) Optimization of the optimal fish stage: The index is the change in scleral diameter; Method: Randomly select wild-type AB strain zebrafish at the single-cell stage into 6-well plates, with 30 tails in each well. Inject lumican-MO at a dose of 2 pmol / tail. At the same time, set up a normal control group and an MO negative control group, and the volume of each well is 3 mL. Set up four parallel experiments. When treated at 28°C for 3 - 6 days respectively, randomly select 10 zebrafish from each experimental group and place them under a dissection microscope to take pictures and save the pictures. Use NIS-Elements D 3.20 advanced image processing software to analyze and collect data, and analyze the scleral diameter of the zebrafish eyes. Evaluate whether lumican-MO induces myopia in zebrafish based on the statistical analysis results of this index. The statistical processing results are expressed as mean ± SE. Use SPSS 26.0 software for statistical analysis. p < 0.05 indicates that the difference is statistically significant. The results are shown in Table 2.

[0030] Table 2 Compared with the MO control group, **p < 0.01.

[0031] The results showed that the scleral diameters of the zebrafish in the lumican-MO 2 pmol / tail group were significantly increased at 5 days and 6 days after the single-cell stage, indicating successful induction of the myopia model. There was no significant difference in the degree of myopia (scleral diameter) of zebrafish between these two experimental stages. Therefore, considering the time cost, the optimal stage for using zebrafish in the myopia model is 5 days after the single-cell stage.

[0032] (III) Multidimensional index characterization of the myopia model (1) Ocular morphological indexes Method: Randomly select wild-type AB strain zebrafish at the single-cell stage and place 30 tails in each well of a 6-well plate. Inject a dose of lumican-MO at 2 pmol / tail. At the same time, set up a normal control group and an MO negative control group, with a volume of 3 mL per well. When treated at 28 °C for 5 days, randomly select 10 zebrafish from each experimental group and place them under a dissection microscope to take and save pictures. Use NIS-Elements D 3.20 advanced image processing software to analyze and collect data, and analyze the ratio of the diameter of the retinal pigment epithelium to the scleral diameter, the axial length of the eye, and the ocular area of the zebrafish. Evaluate the ocular changes in myopic zebrafish based on the above index results. The statistical processing results are expressed as mean ± SE. Use SPSS 26.0 software for statistical analysis. p < 0.05 indicates that the difference is statistically significant. The results are shown in Table 3.

[0033] Table 3 Compared with the MO control group, *p < 0.05, ***p < 0.001 The results showed that after treating zebrafish with lumican-MO at 2 pmol / tail for 5 days, myopic zebrafish showed a significant decrease in the ratio of the diameter of the retinal pigment epithelium to the scleral diameter, a significant increase in the axial length of the eye, and a significant decrease in the ocular area.

[0034] (2) Ocular cell apoptosis situation Method: Randomly select wild-type AB strain zebrafish at the single-cell stage and place 30 tails in each well of a 6-well plate. Inject a dose of lumican-MO at 2 pmol / tail. At the same time, set up a normal control group and an MO negative control group, with a volume of 3 mL per well. When treated at 28 °C for 5 days, perform AO staining on the zebrafish. After staining, randomly select 10 zebrafish from each group and place them under a fluorescence microscope to take and save pictures. Use NIS-Elements D 3.20 advanced image processing software to analyze and collect data, and analyze the fluorescence intensity of retinal cell apoptosis and the fluorescence intensity of lens cell apoptosis in zebrafish. Evaluate the ocular changes in myopic zebrafish based on the above index results. The statistical processing results are expressed as mean ± SE. Use SPSS 26.0 software for statistical analysis. p < 0.05 indicates that the difference is statistically significant. The results are shown in Table 4.

[0035] Table 4 Compared with the MO control group, **p < 0.01, ***p < 0.001 The results showed that after treating zebrafish with 2 pmol / tail of lumican - MO for 5 days, the apoptotic fluorescence intensity of retinal and lens cells in myopic zebrafish was significantly enhanced.

[0036] (3) Behavioral change method: Randomly select wild - type AB - strain zebrafish at the single - cell stage and place them in a 6 - well plate, with 30 tails in each well. Inject 2 pmol / tail of lumican - MO. At the same time, set up a normal control group and an MO negative control group, with a volume of 3 mL per well. Set up 2 parallel experiments. When treating at 28 °C for 5 days, (1) Randomly select 5 zebrafish from each experimental group and place them in a cross - shaped module. The module is divided into four regions: yellow, blue, red, and green. Place 6 modules in each group. Use a behavior instrument to collect data and analyze the percentage (%) of the total movement distance of zebrafish in the blue region accounting for the total movement distance of the entire region; (2) Randomly select 10 zebrafish from each experimental group and transfer them to a 96 - well plate, 1 tail per well. Use a behavior instrument to collect data and analyze the average movement speed per minute of zebrafish under each light - dark cycle stimulation. Evaluate the behavioral changes of myopic zebrafish based on the above index results. The statistical processing results are expressed as mean ± SE. Use SPSS 26.0 software for statistical analysis. p < 0.05 indicates that the difference is statistically significant. The results are shown in Table 5.

[0037] Table 5 Compared with the MO control group, *p < 0.05 The results showed that after treating zebrafish with 2 pmol / tail of lumican - MO for 5 days, myopic zebrafish showed a weakened preference selectivity for blue and a decreased sensitivity to light - dark stimuli.

[0038] (4) Method for detecting gene expression changes: Randomly select wild-type AB zebrafish at the single-cell stage and place them in a 6-well plate, with 30 tails in each well. Inject lumican-MO at a dose of 2 pmol / tail. At the same time, set up a normal control group and a MO negative control group, with a volume of 3 mL per well. Set up 3 parallel experiments. When treated at 28°C for 5 days, collect zebrafish samples, extract total RNA, and then synthesize cDNA according to the instructions of the first-strand cDNA synthesis kit. Detect the expression of β-actin, lumican, mmp-2, tgf-β, timp-2a, and col1a1a genes by q-PCR. Use β-actin as the internal reference for gene expression and calculate the relative expression levels of other target genes. Evaluate the gene changes in myopic zebrafish based on the results of the above target genes. The statistical treatment results are expressed as mean ± SE. Use SPSS 26.0 software for statistical analysis. p < 0.05 indicates that the difference is statistically significant. The results are shown in Table 6.

[0039] Table 6 Compared with the MO control group, *p < 0.05, **p < 0.01, ***p < 0.001 The results showed that after treating zebrafish with lumican-MO at a dose of 2 pmol / tail for 5 days, the myopic zebrafish showed significant downregulation of lumican, tgf-β, timp-2a, and col1a1a genes, and significant upregulation of the mmp-2 gene.

[0040] Example 2 Verification of positive drug 1 using the zebrafish myopia model: (1) Method for confirming the optimal detection dose of lutein ester nanoemulsion: Randomly select wild-type AB zebrafish at the single-cell stage and place them in a 6-well plate, with 30 tails in each well. Except for the normal control group, the others are injected with lumican-MO at a dose of 2 pmol / tail to establish a zebrafish myopia model. After treatment at 28°C for 2 days, administer lutein ester nanoemulsion at concentrations of 0.488, 0.977, 1.95, 3.91, and 7.81 μL / mL by water solution. At the same time, set up a normal control group, a MO negative control group, and a model control group, with a volume of 3 mL per well. After continuing to treat at 28°C for 3 days, the experiment ends, and the maximum detection concentration for evaluating the preventive effect of lutein ester nanoemulsion on myopia is measured. The results are shown in Table 7.

[0041] Table 7 The results showed that the maximum detection concentration of lutein ester nanoemulsion for preventing myopia in the model established in the present invention is 1.95 μL / mL.

[0042] (2) Method for improving the phenotype of myopic zebrafish by lutein ester nanoemulsion: Randomly select wild-type AB zebrafish at the single-cell stage and place them in a 6-well plate, with 30 tails in each well. Except for the normal control group, the rest are injected with lumican-MO at a dose of 2 pmol / tail to establish a zebrafish myopia model. After treatment at 28°C for 2 days, lutein ester nanoemulsion is given by water solution at concentrations of 0.488, 0.977, and 1.95 μL / mL. At the same time, a normal control group, an MO negative control group, and a model control group are set up, and the volume of each well is 3 mL. The experiment ends after continuing to treat at 28°C for 3 days. Randomly select 10 zebrafish from each experimental group and place them under a dissection microscope to take pictures and save the images. Use NIS-Elements D 3.20 advanced image processing software to analyze and collect data, and analyze the retinal pigment epithelium diameter / scleral diameter, fluorescence intensity of retinal cell apoptosis, fluorescence intensity of lens cell apoptosis, and the proportion of the blue area of the zebrafish. The preventive effect of lutein ester nanoemulsion on myopia is evaluated based on the statistical analysis results of this index. The statistical processing results are expressed as mean±SE. Use SPSS 26.0 software for statistical analysis. p<0.05 indicates that the difference is statistically significant. The results are shown in Table 8.

[0043] Table 8 Compared with the model control group, *p<0.05, **p<0.01, ***p<0.001.

[0044] The results show that lutein ester nanoemulsion has the effect of preventing myopia, which is specifically manifested as significantly increasing the ratio of retinal pigment epithelium diameter / scleral diameter in myopic model zebrafish, improving retinal and lens cell apoptosis, and enhancing the preference of zebrafish for blue.

[0045] Example 3 Verification of positive drug 2 using a zebrafish myopia model: (1) Method for confirming the optimal detection dose of atropine hydrochloride: Randomly select wild-type AB zebrafish at the single-cell stage and place them in a 6-well plate, with 30 tails in each well. Except for the normal control group, the rest are injected with lumican-MO at a dose of 2 pmol / tail to establish a zebrafish myopia model. After treatment at 28°C for 2 days, atropine hydrochloride is given by water solution at concentrations of 250, 500, 1000, 2000, and 4000 μg / mL. At the same time, a normal control group, an MO negative control group, and a model control group are set up, and the volume of each well is 3 mL. The experiment ends after continuing to treat at 28°C for 3 days, and the maximum detection concentration for evaluating the preventive effect of atropine hydrochloride on myopia is measured. The results are shown in Table 9.

[0046] Table 9 The results showed that the maximum detection concentration of atropine hydrochloride for preventing myopia in the model established in the present invention was 2000 μg / mL.

[0047] (2) Method for the effect of atropine hydrochloride on improving the phenotype of myopic zebrafish: Wild-type AB zebrafish at the single-cell stage were randomly selected and placed in a 6-well plate, with 30 tails treated in each well. Except for the normal control group, the rest were injected with lumican-MO at a dose of 2 pmol / tail to establish a zebrafish myopia model. After treatment at 28 °C for 2 days, atropine hydrochloride at concentrations of 500, 1000, and 2000 μg / mL was administered by water solution. At the same time, a normal control group, an MO negative control group, and a model control group were set up, and the volume of each well was 3 mL. The experiment ended after continued treatment at 28 °C for 3 days. Ten zebrafish were randomly selected from each experimental group and placed under a dissection microscope to take and save pictures. The NIS-Elements D 3.20 advanced image processing software was used to analyze and collect data, and the diameter ratio of the retinal pigment epithelium to the sclera diameter, the fluorescence intensity of retinal cell apoptosis, and the gene expression levels of type I collagen fibers (mmp-2, tgf-β, timp-2a, col1a1a) of zebrafish were analyzed. The statistical analysis results of these indicators were used to evaluate the efficacy of atropine hydrochloride in preventing myopia. The statistical processing results were expressed as mean ± SE. SPSS 26.0 software was used for statistical analysis. p < 0.05 indicated that the difference was statistically significant, and the results are shown in Table 10.

[0048] Table 10 Compared with the model control group, **p < 0.01, ***p < 0.001.

[0049] The results showed that atropine hydrochloride had the efficacy of preventing myopia. Among them, 2000 μg / mL of atropine hydrochloride could significantly increase the ratio of the diameter of the retinal pigment epithelium to the sclera diameter of myopic model zebrafish, improve retinal cell apoptosis, down-regulate the relative expression level of the mmp-2 gene, and up-regulate the relative expression levels of the tgf-β, timp-2a, and col1a1a genes; 500 - 1000 μg / mL of atropine hydrochloride had no obvious effect on increasing the ratio of the diameter of the retinal pigment epithelium to the sclera diameter, but had obvious effects on improving retinal cell apoptosis, down-regulating the relative expression level of the mmp-2 gene, and up-regulating the relative expression levels of the tgf-β, timp-2a, and col1a1a genes.

Claims

1. A method for establishing a zebrafish myopia model, characterized in that, The zebrafish myopia model is induced and established by injecting the lumican gene MO into the zebrafish body.

2. The method for establishing a zebrafish myopia model according to claim 1, characterized in that, The zebrafish is a wild-type AB strain zebrafish at the single-cell stage.

3. The method for establishing a zebrafish myopia model according to claim 1 or 2, characterized in that, The zebrafish lumican The injection dose of gene MO is 0.25 - 2 pmol per fish.

4. Use of the zebrafish myopia model established by the method according to any one of claims 1-3 in a method for screening drugs with the efficacy of preventing and treating myopia, characterized in that, The method includes the following steps: S1. Randomly divide the zebrafish into groups. After establishing a zebrafish myopia model in the sample group, administer the drug to be tested. The normal control group has no special treatment. The model control group establishes a zebrafish myopia model, and the negative control group conducts zebrafish MO control injection. S2. Evaluate the preventive and therapeutic effects of the drug to be tested by analyzing the eye ball morphology, ocular cell apoptosis, and behavioral change indexes of the zebrafish in the above experimental groups.

5. The application according to claim 4, characterized in that, In S1, the drug to be tested is administered by water solution to the sample group at 2 - 5 dpf after establishing the zebrafish myopia model.

6. The application according to claim 4, characterized in that, In S1, index detection is performed on the sample group at 5 - 6 dpf after establishing the zebrafish myopia model.

7. The application according to claim 4 or 5 or 6, characterized in that, The eye ball morphology indexes in S2 include scleral diameter, ratio of retinal pigment epithelium diameter to scleral diameter, eye axis length, and ocular area. The ocular cell apoptosis includes retinal cell apoptosis and lens cell apoptosis. The behavioral changes include color preference changes and light-dark stimulation behaviors.

8. The application according to claim 7, wherein In S2, evaluation is also performed by analyzing mmp-2, tgf-β, timp-2a and col1a1a gene expression indicators.

9. The application according to claim 7, characterized in that, The detection method for the color preference change is as follows: Paint the four arm areas of the cross module yellow, blue, red, and green respectively. Place the zebrafish at the center of the cross module, collect data using a behavior instrument, and analyze the percentage of the total movement distance of the zebrafish in the blue area in the total movement distance of the entire area.

10. The application according to claim 7, characterized in that, The detection method for the light-dark stimulation behavior is as follows: Place the zebrafish in a container, give the zebrafish light-dark cycle stimulation, collect data using a behavior instrument, and analyze the average movement speed per minute of the zebrafish under each light-dark cycle stimulation.

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