Construction method and application of zebrafish nervous system inflammation model

Abstract

The invention discloses a construction method and application of a zebrafish nervous system inflammation model, and relates to the technical field of animal neuroinflammation models. According to the method provided by the invention, the zebra fish nervous system inflammation model can be constructed, and the method has the advantages of short modeling time, high success rate, simplicity and convenience. The zebrafish nervous system inflammation model constructed by the invention can be used for developing a drug screening platform which influences neuroinflammation, neurodegenerative diseases and dysmnesia, and potential effective drugs are screened by observing behavior changes of zebrafish. The genetic background of zebra fish can be utilized to research the molecular mechanism of dysmnesia, and a theoretical basis is provided for future treatment. The method can be applied to the fields of medicine, medicine development, natural active ingredient screening and the like.

Description

Technical Field

[0001] The present invention relates to the technical field of animal neuroinflammation models, and in particular to a method for constructing a zebrafish nervous system inflammation model and an application thereof. Background Art

[0002] Patent CN202111545514.1 discloses a method for constructing a zebrafish memory evaluation model and its application, including the following steps: taking fertilized zebrafish fry and setting up a normal group and a model group respectively, adding dimethyl sulfoxide solution to the normal group and cycloheximide solution to the model group; transferring them into microplates respectively and turning on the behavior analysis system; entering the training phase, cyclically stimulating and training for 130 to 150 times according to the stimulation mode of "15s light-1s dark", and resting for 10 to 20 minutes; after repeating 4 training cycles, washing off the drug solution and continuing illumination; entering the testing phase, cyclically stimulating and training for 10 to 20 times according to the stimulation mode of "60s light-1s dark"; calculating the memory intensity habituation rate. Patent CN202311535431.3 discloses an Alzheimer's disease model for adult zebrafish. The zebrafish Alzheimer's disease model is induced by sodium silicate exposure + head injection + darkness treatment + sodium iodoacetate feeding + ultraviolet light irradiation. This multi-factor induced Alzheimer's disease is consistent with the etiology of Alzheimer's disease being induced by multiple factors, and can better simulate the characteristics of human Alzheimer's disease.

[0003] Currently, existing technologies can identify changes in memory ability through fish behavioral analysis, but this technology lacks verification of direct damage to the nervous system and has not formed a stable damage model for the development of the next repair plan. In addition, the model that the existing technology focuses on is the Alzheimer's disease model, but whether fish can develop Alzheimer's disease is still under discussion; and Alzheimer's disease is only one of many nervous system diseases or abnormalities, and has a unique pathogenesis. Therefore, the animal model of Alzheimer's disease cannot be used to study the mechanisms and treatments of other nervous system abnormalities.

[0004] In view of this, the present invention is proposed. Summary of the invention

[0005] The purpose of the present invention is to provide a method for constructing a stable and verifiable zebrafish nervous system inflammation model and its application for screening effective therapeutic means for improving or repairing neural function.

[0006] The present invention is achieved in that:

[0007] In a first aspect, the present invention provides a method for constructing a zebrafish nervous system inflammation model, which comprises the following steps:

[0008] Zebrafish aged 3 months and above were selected as modeling subjects and exposed to a solution of at least one chemical selected from the following: bisphenol A, 1,3-diphosphoglyceric acid (BPG), AlCl3 and neurotoxic pesticides for more than 30 days; during which the concentration of the chemical in the solution was kept relatively unchanged;

[0009] When the chemical substance is bisphenol A, the concentration of bisphenol A in the bisphenol A solution is 0.5-1.0 mg / L;

[0010] When the chemical substance is 1,3-diphosphoglyceric acid, the concentration of 1,3-diphosphoglyceric acid in the 1,3-diphosphoglyceric acid solution is 0.2-0.5 mg / L;

[0011] When the chemical substance is AlCl3, the concentration of AlCl3 in the AlCl3 solution is 0.4-1.0 mg / L;

[0012] When the chemical substance is a neurotoxic insecticide, the concentration of the neurotoxic insecticide in the neurotoxic insecticide solution is 0.02-0.1 mg / L.

[0013] In a second aspect, the present invention also provides a method for constructing a zebrafish nervous system inflammation model and the use of the constructed zebrafish nervous system inflammation model in screening drugs for preventing or treating neuroinflammation.

[0014] In a third aspect, the present invention also provides a method for constructing a zebrafish nervous system inflammation model and the use of the constructed zebrafish nervous system inflammation model in screening drugs for preventing or treating neurodegenerative diseases.

[0015] In a preferred embodiment of the present invention, the neurodegenerative disease is selected from Alzheimer's disease, Parkinson's disease, depression, cerebral stroke, postoperative neurological complications, amyotrophic lateral sclerosis or multiple sclerosis.

[0016] The present invention has the following beneficial effects:

[0017] The present invention uses zebrafish, which has 87% similarity with human genes, as a model organism to establish a zebrafish nervous system inflammation model. The method uses bisphenol A, 1,3-diphosphoglyceric acid (BPG), AlCl3 or neurotoxic pesticides to expose and culture zebrafish. After testing, the zebrafish microglia after exposure and culture show activation, and the activation of microglia refers to their morphological and functional changes when they are damaged, inflamed or other stimuli. It can be seen that after exposure and culture, zebrafish have a nervous system injury phenotype, which causes behavioral disorders in zebrafish. Therefore, the method provided by the present invention can construct a zebrafish nervous system inflammation model, and has the advantages of short modeling time, high success rate and simplicity and convenience.

[0018] The zebrafish nervous system inflammation model constructed by the present invention can be used to develop a drug screening platform for neuroinflammation, neurodegenerative diseases, and memory impairment, and to screen potential effective drugs by observing the behavioral changes of zebrafish. The genetic background of zebrafish can also be used to study the molecular mechanism of memory impairment, providing a theoretical basis for future treatment. It can be applied in the fields of medicine, drug development, and natural active ingredient screening. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for use in the embodiments are briefly introduced below. It should be understood that the following drawings only show certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other related drawings can be obtained based on these drawings without creative work.

[0020] Figure 1 Evaluate technology roadmaps for models;

[0021] Figure 2 is the heat map of T-maze trajectories;

[0022] Figure 3 This is a statistical graph showing the time zebrafish spend in the target area and the first target area latency time after being exposed to 0.5ppm BPA, 0.2ppm BPG and 0.4ppm AlCl3 in water for 30 days;

[0023] Figure 4 This is the immunofluorescence staining of IBA-1 in zebrafish brain in the BPG group;

[0024] Figure 5 This is the immunofluorescence staining of IBA-1 in zebrafish brain in the AlCl3 group;

[0025] Figure 6 This is the immunofluorescence staining of IBA-1 in zebrafish brain in the BPA group;

[0026] Figure 7 This is a statistical graph showing the time zebrafish spend in the target area and the first latency time in the target area after co-exposure to BPG and PLX3397;

[0027] Figure 8 The results of immunofluorescence staining of IBA-1 in zebrafish brain after PLX3397 group and co-exposure of BPG and PLX3397;

[0028] Fig. 9 This is a graph showing the results of a colony stimulating factor 1 receptor (CSF1R) Q-PCR experiment. DETAILED DESCRIPTION

[0029] References to embodiments of the present invention will now be provided in detail, one or more examples of which are described below. Each example is provided as an explanation rather than a limitation of the present invention. In fact, it will be apparent to those skilled in the art that various modifications and variations may be made to the present invention without departing from the scope or spirit of the present invention. For example, a feature illustrated or described as part of one embodiment may be used in another embodiment to produce a further embodiment.

[0030] In order to construct a zebrafish model of direct damage to the nervous system, the inventors exposed zebrafish to bisphenol A, 1,3-diphosphoglycerate (BPG), AlCl3 and neurotoxic pesticides. After cultivation, a stable and verifiable zebrafish nervous system inflammation model was obtained. The development of this model will help screen effective therapeutic methods for improving or repairing neural function.

[0031] In a first aspect, the present invention provides a method for constructing a zebrafish nervous system inflammation model, which comprises the following steps:

[0032] Zebrafish aged 3 months and above were selected as modeling subjects and exposed to a solution of at least one chemical selected from the following: bisphenol A, 1,3-diphosphoglyceric acid (BPG), AlCl3 and neurotoxic pesticides for more than 30 days; during which the concentration of the chemical in the solution was kept relatively unchanged;

[0033] When the chemical substance is bisphenol A, the concentration of bisphenol A in the bisphenol A solution is 0.5-1.0 mg / L; if the concentration of bisphenol A in the bisphenol A solution is greater than 1.0 mg / L, all zebrafish may die due to excessive concentration.

[0034] When the chemical substance is 1,3-diphosphoglyceric acid, the concentration of 1,3-diphosphoglyceric acid in the 1,3-diphosphoglyceric acid solution is 0.2-0.5 mg / L; if the concentration of 1,3-diphosphoglyceric acid in the 1,3-diphosphoglyceric acid solution is greater than 0.5 mg / L, all zebrafish may die due to excessive concentration.

[0035] When the chemical substance is AlCl3, the concentration of AlCl3 in the AlCl3 solution is 0.4-1.0 mg / L; if the concentration of AlCl3 in the AlCl3 solution is greater than 1.0 mg / L, all zebrafish may die due to excessive concentration.

[0036] When the chemical substance is a neurotoxic insecticide, the concentration of the neurotoxic insecticide in the neurotoxic insecticide solution is 0.02-0.1 mg / L, such as 0.02 mg / L, 0.03 mg / L, 0.04 mg / L, 0.05 mg / L, 0.06 mg / L, 0.07 mg / L, 0.08 mg / L, 0.09 mg / L or 0.1 mg / L.

[0037] If the age of zebrafish is too young, the behavioral observation of zebrafish will be affected. The movement trajectory of zebrafish in the maze cannot be significantly different from that of the blank group. Therefore, the present invention selects zebrafish over 3 months old as modeling objects, which helps to improve the success rate of modeling.

[0038] After 30 days of water environment exposure to zebrafish, the zebrafish behavioral trajectory showed obvious changes, and the zebrafish model showed a more messy distribution in the T-maze trajectory heat map, and the time spent in the same target area was reduced, and the first target area latency was extended, that is, compared with the blank control group, it took longer time to reach the target area, and the zebrafish memory ability decreased, showing certain behavioral disorders. After testing, the zebrafish microglia after exposure culture showed activation phenomenon, and the activation of microglia refers to their morphological and functional changes when they are damaged, inflammatory or other stimuli. It can be seen that after exposure culture, zebrafish has a nervous system injury phenotype, and zebrafish produces behavioral disorders. Microglia inhibitors can activate or inhibit the expression of microglia by inhibiting the expression of colony stimulating factor (SCF1R), alleviating the memory disorder caused by nervous system injury. Therefore, the method provided by the present invention can construct a zebrafish nervous system inflammation model, and has the advantages of short modeling time, high success rate and simple and convenient.

[0039] Continuous exposure for more than 30 days means: exposure culture for at least 30 days, such as exposure culture for 30 days, 32 days, 35 days, 38 days, 40 days, 45 days or 50 days (when the exposure time is more than 40 days, the exposure concentration can be appropriately reduced to prevent excessive biological mortality). If the exposure time is too short, the success rate of modeling may be reduced.

[0040] At the above exposure concentrations, it helps to quickly build up nervous system damage in zebrafish.

[0041] The concentration of bisphenol A in the bisphenol A solution is, for example, 0.5 mg / L, 0.6 mg / L, 0.7 mg / L, 0.8 mg / L, 0.9 mg / L, or 1.0 mg / L.

[0042] The concentration of 1,3-diphosphoglyceric acid in the 1,3-diphosphoglyceric acid solution is, for example, 0.2 mg / L, 0.3 mg / L, 0.4 mg / L, or 0.5 mg / L.

[0043] The concentration of AlCl3 in the AlCl3 solution is, for example, 0.4 mg / L, 0.5 mg / L, 0.6 mg / L, 0.7 mg / L, 0.8 mg / L, 0.9 mg / L or 1.0 mg / L.

[0044] In a preferred embodiment of the present invention, the water is changed every 2-3 days during the exposure period of the chemical substance. The exposure solution in the culture medium is renewed, mixed and the drug concentration is tested daily to ensure that the drug in the culture water is not decomposed and the drug concentration remains substantially unchanged during the drug exposure period.

[0045] In a preferred embodiment of the present invention, normal feeding is performed daily during the exposure to the chemical substance, for example, normal live brine shrimp bait is fed twice a day, and shelled brine shrimp feed is fed once a day.

[0046] In a preferred embodiment of the present invention, the water temperature of the solution of the chemical substance is 28±0.5°C, the pH is 7.0-8.0, the conductivity is 500-800 μS / cm, and the dissolved oxygen is 5-8 mg / L.

[0047] In a preferred embodiment of the present invention, the construction method further comprises performing a model evaluation on zebrafish after exposure culture.

[0048] Model evaluation included behavioral analysis, microglial IBA-1 staining, and colony stimulating factor 1 receptor (CSF1R) activation to determine whether the model was successful.

[0049] Behavioral analysis included analysis of the time zebrafish stayed in the target area and the first latency time to the target area.

[0050] In a preferred embodiment of the present invention, the neurotoxic insecticide is selected from organophosphorus insecticides, pyrethroid insecticides, carbamates or thiamethoxam. Any insecticide that can induce neurotoxicity in zebrafish is acceptable, and is not limited to the above examples.

[0051] In a preferred embodiment of the present invention, the organophosphorus insecticide is trichlorfon, phoxim or triazophos.

[0052] In a preferred embodiment of the present invention, the pyrethroid insecticide is deltamethrin, cypermethrin, cypermethrin or flucythrinate.

[0053] In a second aspect, the present invention also provides a method for constructing a zebrafish nervous system inflammation model and the use of the constructed zebrafish nervous system inflammation model in screening drugs for preventing or treating neuroinflammation.

[0054] For example, microglial neuroinflammation.

[0055] In a third aspect, the present invention also provides a method for constructing a zebrafish nervous system inflammation model and the use of the constructed zebrafish nervous system inflammation model in screening drugs for preventing or treating neurodegenerative diseases.

[0056] In a preferred embodiment of the present invention, the neurodegenerative disease is selected from Alzheimer's disease, Parkinson's disease, depression, cerebral stroke, postoperative neurological complications, amyotrophic lateral sclerosis or multiple sclerosis.

[0057] In order to make the purpose, technical scheme and advantages of the embodiments of the present invention clearer, the technical scheme in the embodiments of the present invention will be described clearly and completely below. If the specific conditions are not specified in the embodiments, they are carried out according to conventional conditions or conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be purchased commercially.

[0058] The features and performance of the present invention are further described in detail below in conjunction with the embodiments.

[0059] Example 1

[0060] This embodiment provides a method for constructing a zebrafish nervous system inflammation model.

[0061] 1. 30 3-month-old adult zebrafish were randomly selected as a group for modeling. They were cultured in 5L culture water under certain conditions (water temperature: 28±0.5℃, pH 7.0-8.0, conductivity 500-800μS / cm, dissolved oxygen 5-8mg / L) for 7 days at constant temperature. On the 8th day, they were exposed to 0.5ppm BPA (bisphenol A), in which bisphenol was dissolved in acetone. The exposure solution in the fish tank was updated by changing the water every two days, mixed and tested daily to ensure that the drug in the 5L culture water was not decomposed and the drug concentration remained unchanged during the drug exposure period, and the culture was continued. During this period, normal live brine shrimp were fed twice a day and shelled brine shrimp feed was fed once. The modeling time lasted until the 38th day (i.e., the exposure time was 30 days).

[0062] 2. When the exposure time is 30 days (i.e., cultured to 38 days), the above-mentioned model zebrafish are trained in T-maze, their behavioral trajectories are analyzed under a behavioral analyzer, and subsequent model evaluation is performed.

[0063] 3. Model evaluation uses three parts (behavioral analysis, microglial IBA-1 staining and colony stimulating factor 1 receptor (CSF1R) activation) for comprehensive evaluation to determine whether the model is successful (technical route such as Figure 1 shown).

[0064] Example 2

[0065] Compared with Example 1, the only difference is that the chemical substance used for exposure is different, which is 0.2 ppm BPG (1,3-diphosphoglyceric acid), and the rest of the modeling methods are the same.

[0066] Example 3

[0067] Compared with Example 1, the only difference is that the chemical substance used during exposure is different, which is 0.4 ppm AlCl3 solution in this example, and the rest of the modeling methods are the same.

[0068] Experimental Example 1

[0069] In this example, the memory ability of zebrafish was tested on the injury model constructed in Examples 1-3.

[0070] Figure 2 The results showed that after 30 days of exposure to 0.5ppm BPA, 0.2ppm BPG and 0.4ppm AlCl3 in water environment, the behavioral trajectory of zebrafish showed obvious changes ( Figure 2 ), the experimental group showed a more chaotic distribution in the T-maze trajectory heat map, and the time spent in the target area was reduced. The data of the T-maze were analyzed as follows Figure 3 The numbers of the samples were counted (n=3) and analyzed by SPSS. A p value less than 0.05 was considered to be statistically significant.

[0071] The results showed that the zebrafish's residence time in the target area and the first target area latency time were significantly different from those in the blank group. The zebrafish's residence time in the target area was significantly shortened, and the first target area latency time was significantly prolonged, especially the AlCl3 and BPA treatments had more obvious effects, showing certain behavioral disorders.

[0072] Experimental Example 2

[0073] Injury model construction-neural injury test in zebrafish brain tissue.

[0074] Zebrafish microglia are the only immune cells in the central nervous system. They play an important role in development, maintaining the homeostasis of the neural environment, and the immune response of the nervous system. The activation of microglia refers to the changes in their morphology and function when they are damaged, inflamed, or otherwise stimulated. Therefore, this example performed brain IBA-1 fluorescent immunostaining on the zebrafish model (experimental group) constructed by exposure to the three chemical substances in Examples 1-3 and the blank group. The results showed that after the zebrafish were exposed to a water environment of 0.5ppm BPA, 0.2ppm BPG, and 0.4ppm AlCl3 for 30 days, the zebrafish microglia staining area increased significantly, indicating that the microglia showed activation (see results). Figure 4 , Figure 5 , Figure 6 ).

[0075] Experimental Example 3

[0076] In this experimental example, the colony stimulating factor 1 receptor (CSF1R) activation test was performed on the above injury model.

[0077] Using the commonly used microglia inhibitor PLX3397, we inhibited zebrafish microglia and conducted T-maze and novel object experiments, verifying that the use of microglia inhibitors can effectively alleviate microglia activation and behavioral changes caused by BPG exposure ( Figure 7 ), verifying that microglia-mediated neuroinflammation is the main cause of cognitive impairment in zebrafish.

[0078] It is known that the microglial inhibitor PLX3397 inhibits microglial activation through colony stimulating factor 1 receptor (CSF1R). In this experiment, the colony stimulating factor 1 receptor (CSF1R) of zebrafish was inhibited and then three drugs were exposed for 30 days. The results showed that co-exposure of BPG and PLX3397 did not cause microglial activation and behavioral changes in zebrafish (results are shown in Figure 8 and Fig. 9 ).

[0079] In summary, memory impairment caused by nervous system damage can be alleviated by inhibiting zebrafish microglia through precise targeting of colony stimulating factor 1 receptor (CSF1R).

[0080] In summary, various substances can activate or promote the expression of microglia by affecting the expression of colony stimulating factor (SCF1R), thereby causing damage to the nervous system of zebrafish and causing behavioral disorders in zebrafish. Therefore, based on this, drugs for treating nerve damage can be screened and a responsive zebrafish nerve damage model can be constructed.

[0081] The zebrafish nervous system inflammation model constructed by the present invention can be used to develop a drug screening platform for neuroinflammation, neurodegenerative diseases, and memory impairment, and to screen potential effective drugs by observing the behavioral changes of zebrafish. The genetic background of zebrafish can also be used to study the molecular mechanism of memory impairment, providing a theoretical basis for future treatment. It can be applied in the fields of medicine, drug development, and natural active ingredient screening.

[0082] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A method for constructing a zebrafish nervous system inflammation model, characterized in that: It includes the following steps: Zebrafish aged 3 months or older were selected as modeling subjects and exposed to a solution of at least one chemical selected from the following: bisphenol A, 1,3-diphosphoglyceric acid (BPG), AlCl3 and neurotoxic pesticides for more than 30 days; during which the concentration of the chemical in the solution was maintained relatively unchanged; When the chemical substance is bisphenol A, the concentration of bisphenol A in the bisphenol A solution is 0.5-1.0 mg / L; When the chemical substance is 1,3-diphosphoglyceric acid, the concentration of 1,3-diphosphoglyceric acid in the 1,3-diphosphoglyceric acid solution is 0.2-0.5 mg / L; When the chemical substance is AlCl3, the concentration of AlCl3 in the AlCl3 solution is 0.4-1.0 mg / L; When the chemical substance is a neurotoxic insecticide, the concentration of the neurotoxic insecticide in the neurotoxic insecticide solution is 0.02-0.1 mg / L.

2. The method for constructing a zebrafish nervous system inflammation model according to claim 1, characterized in that: The water was changed every 2-3 days during the chemical exposure period.

3. The method for constructing a zebrafish nervous system inflammation model according to claim 1, characterized in that: The animals were fed normally daily during the chemical exposure period.

4. The method for constructing a zebrafish nervous system inflammation model according to claim 1, characterized in that: The water temperature of the solution of the chemical substance is 28±0.5°C, the pH is 7.0-8.0, the conductivity is 500-800 μS / cm, and the dissolved oxygen is 5-8 mg / L.

5. The method for constructing a zebrafish nervous system inflammation model according to claim 4, characterized in that: The construction method also includes conducting model evaluation on zebrafish after exposure culture.

6. The method for constructing a zebrafish nervous system inflammation model according to claim 1, characterized in that: The neurotoxic insecticide is selected from organophosphorus insecticides, pyrethroid insecticides, carbamates or thiamethoxam.

7. The method for constructing a zebrafish nervous system inflammation model according to claim 6, characterized in that: The organophosphorus insecticide is trichlorfon, phoxim or triazophos.

8. The method for constructing a zebrafish nervous system inflammation model according to claim 6, characterized in that: The pyrethroid insecticide is deltamethrin, cypermethrin, cypermethrin or cyfluthrin.

9. Use of the zebrafish nervous system inflammation model constructed by the method for constructing a zebrafish nervous system inflammation model according to any one of claims 1 to 8 in screening drugs for preventing or treating neuroinflammation.

10. Use of the zebrafish nervous system inflammation model constructed by the method for constructing a zebrafish nervous system inflammation model according to any one of claims 1 to 8 in screening drugs for preventing or treating neurodegenerative diseases; Preferably, the neurodegenerative disease is selected from Alzheimer's disease, Parkinson's disease, depression, cerebral stroke, postoperative neurological complications, amyotrophic lateral sclerosis or multiple sclerosis.

Images