A method for constructing and applying a zebrafish nervous system inflammation model.

Abstract

This invention discloses a method for constructing a zebrafish nervous system inflammation model and its applications, belonging to the field of animal neuroinflammation model technology. The method provided by this invention can construct a zebrafish nervous system inflammation model and has the advantages of short modeling time, high success rate, and simplicity. The zebrafish nervous system inflammation model constructed by this invention can be used to develop a drug screening platform for the effects of neuroinflammation, neurodegenerative diseases, and memory impairment, screening for potentially effective drugs by observing behavioral changes in zebrafish. It can also utilize the genetic background of zebrafish to study the molecular mechanisms of memory impairment, providing a theoretical basis for future treatments. It has applications in medicine, drug development, and the screening of natural active ingredients.

Description

TECHNICAL FIELD

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

[0002] Patent CN202111545514.1 discloses a method for constructing a zebrafish memory evaluation model and application thereof, which comprises the following steps: taking fertilized zebrafish larvae to set up normal and model groups, adding dimethyl sulfoxide solution to the normal group, and adding cyclohexane imide solution to the model group; respectively moving into a microplate and opening a behavior analysis system; entering the training stage, cyclically stimulating training 130-150 times according to the stimulation mode of "15s bright-1s dark", and resting for 10-20 min; after repeating 4 training cycles, washing away the drug solution and continuing to illuminate; entering the test stage, cyclically stimulating training 10-20 times according to the stimulation mode of "60s bright-1s dark"; and calculating the memory strength habituation rate. Patent CN202311535431.3 discloses a zebrafish adult fish Alzheimer's disease model, which is induced by sodium silicate exposure, head injection, dark treatment, sodium iodoacetate feeding and ultraviolet irradiation. This multi-factor induced Alzheimer's disease is consistent with the view that Alzheimer's disease is induced by multiple factors in etiology, and can better simulate the characteristics of human Alzheimer's disease.

[0003] At present, the existing technology identifies the change of memory ability through fish behavior analysis, but this technology lacks direct verification of nervous system damage, and a stable damage model has not been formed for the development of next step repair scheme. In addition, the model constructed by the existing technology focuses on 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 mechanism and treatment of other nervous system abnormalities.

[0004] In view of this, the present application is proposed. SUMMARY

[0005] The purpose of the present application is to provide a method for constructing a stable and verifiable zebrafish nervous system inflammation model and application thereof, which is used for screening effective treatment methods for improving or repairing nerve function.

[0006] The present application is implemented as follows:

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

[0008] The zebrafish of more than 3 months old is selected as a modeling object, and is exposed to a solution of at least one chemical selected from bisphenol A, BPG, AlCl3 and a neurotoxic insecticide for more than 30 days; the concentration of the chemical in the solution is maintained relatively constant during the period;

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

[0010] When the chemical is BPG, the concentration of BPG in the BPG solution is 0.2-0.5 mg / L;

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

[0012] When the chemical 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 application further provides a method for constructing a zebrafish nervous system inflammation model, and application of the zebrafish nervous system inflammation model constructed by the method in screening drugs for preventing or treating neuroinflammation.

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

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

[0016] The application has the following advantages:

[0017] The application uses zebrafish with 87% similarity to human genes as a model organism to establish a zebrafish nervous system inflammation model. The method uses bisphenol A, BPG, AlCl3 or a neurotoxic insecticide to expose and culture zebrafish. After detection, the microglial cells of the exposed and cultured zebrafish exhibit activation. The activation of microglial cells refers to the morphological and functional changes of the microglial cells when they are damaged, inflamed or stimulated. It can be seen that after exposure and culture, the zebrafish has a nervous system damage phenotype, causing behavioral disorders in zebrafish. Therefore, the method provided by the application can construct a zebrafish nervous system inflammation model, and has the advantages of short modeling time, high success rate and simplicity.

[0018] The zebrafish nervous system inflammation model constructed by the application can be used for developing a drug screening platform for affecting neural inflammation, neural degenerative diseases and memory disorders, and potential effective drugs can be screened by observing the behavior changes of the zebrafish. In addition, the genetic background of the zebrafish can be used to study the molecular mechanism of memory disorders, thereby providing a theoretical basis for future treatment. The application can be applied in the fields of medicine, drug development and natural active ingredient screening. BRIEF DESCRIPTION OF DRAWINGS

[0019] In order to more clearly illustrate the technical solutions of the embodiments of the application, the drawings needed in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some of the embodiments of the application, and therefore should not be regarded as a limitation on the scope. Other related drawings can also be obtained by those skilled in the art without creative labor.

[0020] Figure 1 The model evaluation technical roadmap is shown in Figure 1.

[0021] Figure 2 The T-maze trajectory heat map is shown in Figure 2.

[0022] Figure 3 The target area residence time and the first target area latency of the zebrafish after 30 days of water environmental exposure of the zebrafish to 0.5 ppm BPA, 0.2 ppm BPG and 0.4 ppm AlCl3 are shown in Figure 3.

[0023] Figure 4 The IBA-1 immunofluorescence staining diagram of the brain of the zebrafish in the BPG group is shown in Figure 4.

[0024] Figure 5 The IBA-1 immunofluorescence staining diagram of the brain of the zebrafish in the AlCl3 group is shown in Figure 5.

[0025] Figure 6 The IBA-1 immunofluorescence staining diagram of the brain of the zebrafish in the BPA group is shown in Figure 6.

[0026] Figure 7 The target area residence time and the first target area latency of the zebrafish after co-exposure of the zebrafish to BPG and PLX3397 are shown in Figure 7.

[0027] Figure 8 The IBA-1 immunofluorescence staining result diagram of the brain of the zebrafish in the PLX3397 group and the co-exposure group of BPG and PLX3397 is shown in Figure 8.

[0028] Figure 9 The colony stimulating factor 1 receptor (CSF1R) Q-PCR experiment result diagram is shown in Figure 9. DETAILED DESCRIPTION

[0029] Reference will now be made in detail to embodiments of the application, one or more examples of which are described hereinbelow. Each example is provided as an explanation and not a limitation of the application. Indeed, it will be apparent to one of ordinary skill in the art that numerous modifications and variations of the present application are possible in light of the above teachings. For example, features described or illustrated as part of one embodiment can be used with another embodiment to yield still a further embodiment.

[0030] In order to construct a zebrafish model of direct damage to the nervous system, the present application exposes zebrafish to bisphenol A, BPG (BPG), AlCl3 and neurotoxic insecticides, and obtains a stable and verifiable zebrafish nervous system inflammation model through cultivation. The development of the model helps to screen effective treatment methods for improving or repairing nerve function.

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

[0032] Zebrafish of 3 months of age or older are exposed to a solution of at least one chemical selected from the group consisting of bisphenol A, BPG (BPG), AlCl3 and neurotoxic insecticides, for more than 30 days; and the concentration of the chemical in the solution is maintained relatively constant during the exposure.

[0033] When the chemical 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, it is possible that the zebrafish will all die due to the excessively high concentration.

[0034] When the chemical is BPG, the concentration of BPG in the BPG solution is 0.2-0.5 mg / L; if the concentration of BPG in the BPG solution is greater than 0.5 mg / L, it is possible that the zebrafish will all die due to the excessively high concentration.

[0035] When the chemical 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, it is possible that the zebrafish will all die due to the excessively high concentration.

[0036] When the chemical is a neurotoxic insecticide, the concentration of the neurotoxic insecticide in the neurotoxic insecticide solution is 0.02-0.1 mg / L. For example, 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] The age of the zebrafish is too small to affect the behavioral observation of the zebrafish, and the movement trajectory of the zebrafish in the maze cannot form a significant difference with the blank group, therefore, the zebrafish of more than 3 months old is selected as the modeling object, which helps to improve the success rate of modeling.

[0038] After the zebrafish is exposed to the water environment for 30 days, the behavioral trajectory of the zebrafish shows obvious changes, the zebrafish model shows a more chaotic distribution in the T-maze trajectory heat map, the time spent in the same target area is reduced, the first target area latency is prolonged, that is, compared with the blank control group, it takes longer time to reach the target area, the memory ability of the zebrafish decreases, and a certain behavioral disorder is shown. After detection, the microglia of the zebrafish after exposure culture shows activation phenomenon, and the activation of microglia refers to the morphological and functional changes of microglia when they are damaged, inflamed or stimulated. It can be seen that after exposure culture, the zebrafish has a nervous system injury phenotype, and the zebrafish has a behavioral disorder. The microglia inhibitor can activate or inhibit the expression of colony stimulating factor (SCF1R) to alleviate the memory disorder caused by nervous system injury. Therefore, the method provided by the present application can construct a zebrafish nervous system inflammation model, and has the advantages of short modeling time, high success rate and simple and convenient.

[0039] The continuous exposure for more than 30 days means that the exposure culture is at least 30 days, for example, 30 days, 32 days, 35 days, 38 days, 40 days, 45 days or 50 days (when the exposure time is greater than 40 days, the exposure concentration can be appropriately reduced to prevent the biological mortality from being too high). If the exposure time is too short, the success rate of modeling may be reduced.

[0040] Under the above exposure concentration, it is helpful to quickly construct a zebrafish with nervous system injury.

[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 BPG in the BPG 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 application, the water is changed every 2-3 days during the chemical exposure period. The exposure solution in the culture medium is refreshed, mixed and the drug concentration is checked daily to ensure that the drug does not decompose and the drug concentration remains substantially constant in the culture water during the drug exposure period.

[0045] In a preferred embodiment of the application, normal feeding is performed daily during the chemical exposure period. For example, normal live feeding of adult shrimps is performed twice a day and shelled adult shrimp feed is fed once a day.

[0046] In a preferred embodiment of the application, the water temperature of the solution of the chemical is 28±0.5℃, 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 application, the method further comprises model evaluation of the zebrafish after exposure culture.

[0048] The model evaluation comprises behavioral analysis, IBA-1 staining of microglia, and colony stimulating factor 1 receptor (CSF1R) activation for comprehensive evaluation to determine whether the modeling is successful.

[0049] The behavioral analysis comprises analysis of the residence time of the zebrafish in the target area and the first target area latency.

[0050] In a preferred embodiment of the application, the neurotoxic insecticide is selected from organophosphorus insecticides, pyrethroid insecticides, carbamates, or thiamethoxam. As long as it can induce neurotoxicity in zebrafish, it can be used, and is not limited to the above examples.

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

[0052] In a preferred embodiment of the application, the pyrethroid insecticide is deltamethrin, fenpropathrin, cypermethrin, or lambda-cyhalothrin.

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

[0054] For example, microglial neuroinflammation.

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

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

[0057] In order to make the objectives, technical solutions and advantages of the embodiments of the present application clearer, the technical solutions in the embodiments of the present application will be described clearly and completely below. If specific conditions are not indicated in the embodiments, the conditions are implemented according to conventional conditions or the conditions suggested by manufacturers. If the manufacturers of reagents or instruments are not indicated, the reagents or instruments are conventional products that can be purchased on the market.

[0058] The features and performances of the present application are further described in detail below in combination with embodiments.

[0059] Embodiment 1

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

[0061] 1. Randomly select 30 three-month-old adult zebrafish as a group for modeling, and perform constant temperature adaptive culture in 5L culture water under certain conditions (water temperature: 28±0.5℃, pH 7.0-8.0, conductivity 500-800 μS / cm, dissolved oxygen 5-8 mg / L) for 7 days. On the 8th day, expose to 0.5 ppm BPA (bisphenol A) prepared by dissolving bisphenol in acetone. Replace the exposure liquid in the fish tank every two days, mix well and detect the drug concentration every day to ensure that the drug concentration in 5L culture water does not change during drug exposure. Continue to culture. During the period, feed the fish with normal brine shrimp live bait twice a day and shell brine shrimp feed once a day. The modeling time lasts for 38 days (i.e. the exposure time is 30 days).

[0062] 2. On the 30th day of exposure (i.e. on the 38th day of culture), the above-mentioned modeled zebrafish are subjected to T-maze training, and their behavior trajectories are analyzed under a behavior analysis instrument, and subsequent model evaluation is performed.

[0063] 3. The model evaluation is comprehensively evaluated by three parts (behavior analysis, microglia IBA-1 staining and colony stimulating factor 1 receptor (CSF1R) activation) to determine whether the modeling is successful (the technical route is shown in Figure 1

[0064] Embodiment 2

[0065] Compared with Embodiment 1, the only difference is that a different chemical substance is used for exposure, and the chemical substance used in the present embodiment is 0.2 ppm BPG (BPG), and the rest of the modeling method is the same.

[0066] Embodiment 3​

[0067] The difference compared with Example 1 is only that the chemical used in exposure is different, this example is 0.4 ppm AlCl3 solution, and the rest of the modeling method is the same.

[0068] Experimental Example 1

[0069] This example tests the memory ability of zebrafish on the damage model constructed in Examples 1-3.

[0070] Figure 2 The results show that after 30 days of water environmental exposure of zebrafish using 0.5 ppm BPA, 0.2 ppm BPG and 0.4 ppm AlCl3, the behavioral trajectory of zebrafish shows obvious changes Figure 2 ), the experimental group shows a more chaotic distribution in the T-maze trajectory heat map, and the time spent in the target area is reduced. The data of T-maze are analyzed as Figure 3 , the number (n=3) is counted, and SPSS is used for statistical difference analysis, and the p value less than 0.05 is considered to have statistical significance difference.

[0071] The results show that: the time spent by zebrafish in the target area and the first target area latency time, the data of the experimental group have significant difference compared with the blank group, the time spent by zebrafish in the target area is significantly shortened, and the first target area latency time is significantly prolonged, especially the effect of AlCl3 and BPA treatment is more obvious, showing certain behavioral disorders.

[0072] Experimental Example 2

[0073] Damage model construction-zebrafish brain tissue nerve damage test.

[0074] Zebrafish microglia is the only immune cell in the central nervous system, which plays an important role in development, maintenance of neural environment homeostasis and immune response of nervous system. Activation of microglia refers to the morphological and functional changes of microglia when they are damaged, inflamed or stimulated by other stimuli. Therefore, this example performs brain IBA-1 fluorescence immunostaining on the zebrafish model (experimental group) constructed by exposure to three chemicals in Examples 1-3 and the blank group, and the results show that after 30 days of water environmental exposure of zebrafish using 0.5 ppm BPA, 0.2 ppm BPG and 0.4 ppm AlCl3, the staining area of zebrafish microglia increases obviously, indicating that microglia shows activation phenomenon (results are shown in Figure 4 、 Figure 5 、 Figure 6 ).

[0075] Experimental Example 3

[0076] This experimental example demonstrates the activation of colony-stimulating factor 1 receptor (CSF1R) in the aforementioned injury model.

[0077] Using the commonly used microglia inhibitor PLX3397, T-maze and novel object experiments were conducted on zebrafish microglia to verify that the use of microglia inhibitors can effectively alleviate microglia activation and behavioral changes induced by BPG exposure. Figure 7 This study verified that microglia-mediated neuroinflammation is the main cause of cognitive impairment in zebrafish.

[0078] It is known that the microglia inhibitor PLX3397 inhibits microglial activation through colony-stimulating factor 1 receptor (CSF1R). In this experiment, by inhibiting CSF1R in zebrafish followed by 30 days of exposure to three drugs, the results showed that co-exposure to BPG and PLX3397 did not induce microglial activation or behavioral changes in zebrafish (results are shown in Figure 1). Figure 8 and Figure 9 ).

[0079] In summary, memory impairment caused by nervous system damage can be alleviated by precisely targeting the colony-stimulating factor 1 receptor (CSF1R) to inhibit zebrafish microglia.

[0080] In summary, various substances can activate or promote the expression of microglia by influencing the expression of colony-stimulating factor 1R (SCF1R), thereby causing neurological damage in zebrafish and leading to behavioral disorders. Therefore, this can be used to screen for drugs to treat neurological damage and to construct responsive zebrafish neurological injury models.

[0081] The zebrafish nervous system inflammation model constructed in this invention can be used to develop a drug screening platform for the effects of neuroinflammation, neurodegenerative diseases, and memory impairment. Potentially effective drugs can be screened by observing behavioral changes in zebrafish. The genetic background of zebrafish can also be utilized to study the molecular mechanisms of memory impairment, providing a theoretical basis for future treatments. It has applications in medicine, drug development, and the screening of natural active ingredients.

[0082] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A method for constructing a microglia-mediated zebrafish nervous system inflammation model, characterized by, It comprises the following steps: Zebrafish over 3 months old are selected as the modeling object, and are exposed to a solution of at least one chemical selected from bisphenol A and BPG for more than 30 days; the concentration of the chemical in the solution is maintained relatively constant during the period; When the chemical is bisphenol A, the concentration of bisphenol A in the bisphenol A solution is 0.5-1.0 mg / L; When the chemical is BPG, the concentration of BPG in the BPG solution is 0.2-0.5 mg / L; The construction method further comprises model evaluation on the zebrafish after exposure culture; the model evaluation comprises comprehensive evaluation of behavior analysis, microglia IBA-1 staining and colony stimulating factor 1 receptor activation to determine whether the modeling is successful.

2. The method for constructing a zebrafish nervous system inflammation model according to claim 1, characterized in that, The water is 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, Normal feeding is performed daily during the chemical exposure period.

4. The method of claim 1, wherein the zebrafish model of nervous system inflammation is constructed by, The water temperature of the chemical solution is 28±0.5℃, the pH is 7.0-8.0, the conductivity is 500-800 μS / cm, and the dissolved oxygen is 5-8 mg / L.

5. Application of the zebrafish nervous system inflammation model constructed by the construction method of the zebrafish nervous system inflammation model according to any one of claims 1-4 in screening drugs for preventing or treating neuroinflammation.

Images