Establishment method of epilepsy animal model and medicine obtained by screening of epilepsy animal model and used for treating epilepsy

Abstract

The invention relates to the technical field of experimental animal model construction methods and anti-environmental factor related epilepsy drug screening, in particular to an epilepsy animal model construction method and a drug obtained through model screening and used for treating epilepsy. The invention discloses a construction method of an epilepsy animal model. The epilepsy animal model is a zebra fish model of epilepsy caused by permethrin exposure. The zebrafish embryo is soaked in permethrin environment for a certain time, and the epilepsy animal model can be obtained. The model can be used for researching the effect of environmental factors in the occurrence and development of epilepsy. The medicine screened by the animal model comprises at least one of topiramate, sodium valproate, carbamazepine, methocarbamol, flecainib acetate, SYM2206, D-AP5, CNQX and baclofen. According to the technical scheme, the technical problem that an epilepsy animal model related to environmental factors is lacked in the prior art can be solved, and the method has ideal application and popularization value.

Description

Technical Field

[0001] The present invention relates to the field of experimental animal model construction method and the technical field of anti-environmental factor related epilepsy drug screening, and specifically to a method for constructing an epilepsy animal model and a drug for treating epilepsy obtained by screening the model. Background Art

[0002] Epilepsy is a chronic neurological disease characterized by abnormal diffuse discharges in localized or widespread areas of the brain. It usually manifests as recurrent and uncontrollable seizures, which have serious cognitive, social and psychological effects on patients. Epilepsy is one of the most common and serious brain diseases worldwide, affecting an estimated 65 million people. The incidence and prevalence of epilepsy are higher in children and the elderly, especially infants under 1 year old and the elderly over 50 years old. Epilepsy rarely occurs alone: ​​more than 50% of epilepsy patients have comorbidities such as depression, anxiety and autism. In clinical settings, the prevalence of anxiety in patients with epilepsy is approximately 30%-35% in children and 11%-50% in adults. Pathophysiologically, epilepsy is the result of an imbalance between excitatory and inhibitory neural activity in neural circuits, usually resulting from excessive excitation or weakened inhibition. Epilepsy can be caused by a variety of brain pathologies, including developmental malformations, brain trauma, tumors, and bacterial and parasitic infections. The cause of approximately 60% of epilepsy cases is still unknown, and further research on the pathogenesis of epilepsy is needed to better develop drugs to treat epilepsy.

[0003] Animal models of epilepsy are important tools for studying the pathogenesis of epilepsy, drug screening, and treatment strategies. These models are designed to simulate specific aspects of human epilepsy, although they cannot fully replicate the full complexity of human diseases. For example, pentylenetetrazol is used to enhance excitatory neurotransmission, resulting in excessive excitation and discharge of neurons. The prepared animal model of epilepsy is suitable for preliminary screening of anti-epileptic drugs. Human tonic-clonic epileptic seizures are simulated by electrical stimulation, and then an electric shock model of epilepsy is prepared. Fever seizures in young animals are induced by high temperature environment, and febrile seizures in human children are simulated to prepare a febrile seizure model of epilepsy. Epilepsy is a complex neurological disease, and its pathogenesis involves multiple factors such as genetic factors, abnormal brain structure, and metabolic disorders. In recent years, more and more studies have shown that environmental factors also play an important role in the occurrence and development of epilepsy. There is still a lack of animal models of epilepsy related to environmental factors in the prior art, which in turn affects the screening and research of related drugs. Summary of the invention

[0004] The purpose of the present invention is to provide a method for constructing an epilepsy animal model to solve the technical problem of the lack of an epilepsy animal model related to environmental factors in the prior art.

[0005] To achieve the above purpose, the technical solution adopted by the present invention is as follows:

[0006] A method for constructing an epilepsy animal model, wherein the epilepsy animal model is a zebrafish model of epilepsy caused by permethrin exposure; 3hpf zebrafish embryos are immersed in an environment containing 100-500μg / L permethrin until 3dpf-5dpf; or 3hpf zebrafish embryos are immersed in an environment containing 50μg / L permethrin until 3dpf-6 months.

[0007] Furthermore, the drug is used to treat epilepsy caused by permethrin exposure; the zebrafish used to construct the epilepsy animal model is a wild-type zebrafish or a transgenic zebrafish; the epilepsy animal model exhibits the characteristics of convulsions, nystagmus, neuronal damage and reduced neuronal proliferation.

[0008] Furthermore, 3hpf zebrafish embryos were immersed in an environment containing 500 μg / L permethrin, and then the drugs to be screened were added at 3hpf-2dpf, and culture was continued until 3dpf-5dpf.

[0009] Alternatively, a reagent for knocking out the scn8aa gene is injected into zebrafish embryos at the one-cell stage to obtain scn8aa gene knockout zebrafish; 3hpf scn8aa gene knockout zebrafish embryos are immersed in an environment containing 500 μg / L permethrin and cultured until 3dpf-5dpf.

[0010] Furthermore, the drug comprises at least one of topiramate, sodium valproate and carbamazepine; preferably, it comprises sodium valproate or carbamazepine; further preferably, it comprises carbamazepine.

[0011] Furthermore, the working concentrations of topiramate, sodium valproate and carbamazepine were 50 μg / mL, 150 μg / mL and 50 mg / L, respectively.

[0012] Further, the drug includes at least one of methocarbamol, flecainide acetate and SYM2206;

[0013] Or it includes a reagent for knocking out the scn8aa gene; the reagent includes a ribonucleoprotein complex formed by gRNA1 and Cas9 protein, and a ribonucleoprotein complex formed by gRNA2 and Cas9 protein; gRNA1 is composed of tracrRNA and crRNA with a sequence of SEQ ID NO.1, and gRNA2 is composed of tracrRNA and crRNA with a sequence of SEQ ID NO.2; the reagent is used for injection at the single-cell stage of wild-type zebrafish embryos.

[0014] Further, the drug is a composition formed by methocarbamol and flecainide acetate, or a composition formed by methocarbamol and SYM2206, or a composition formed by flecainide acetate and SYM2206, or a composition formed by methocarbamol, flecainide acetate and SYM2206;

[0015] Preferably, it is a combination of methocarbamol and SYM2206, or a combination of flecainide acetate and SYM2206, or a combination of methocarbamol, flecainide acetate and SYM2206.

[0016] Furthermore, the dosage ratio of methocarbamol, flecainide acetate and SYM2206 was 25:10:1.

[0017] Furthermore, the drug comprises at least one of D-AP5, CNQX and baclofen;

[0018] Preferably, it is D-AP5, or CNQX, or baclofen, or a combination of D-AP5 and CNQX.

[0019] Furthermore, the working concentrations of D-AP5, CNQX and baclofen when used alone were 20 μg / mL, 4 μg / mL and 50 μg / mL, respectively; when D-AP5 and CNQX were used in combination, the dosage ratio was 20:4.

[0020] The technical principle and beneficial effects of this technical solution are:

[0021] In this study, we observed that zebrafish exposed to environmental concentrations of permethrin exhibited a phenotype or susceptibility similar to generalized seizures of epilepsy, and based on the results of behavioral, electrophysiological, and neuropathological assessments, zebrafish exposed to permethrin almost completely displayed the basic features of epilepsy. Therefore, permethrin can be used as a tool to induce epilepsy, and zebrafish exposed to permethrin can be used as a model organism in epilepsy research, that is, an animal model of epilepsy caused by environmental exposure. Nystagmus-like phenomena were observed in samples exposed to more than 50 μg / L permethrin. This symptom is similar to the nystagmus observed in some epilepsy patients. However, this eye movement phenomenon was not found in zebrafish treated with other chemicals, including pentylenetetrazol (PTZ), a drug commonly used to model epilepsy in animal models. We believe that this nystagmus phenotype can serve as an ideal indicator for studying epilepsy and facilitate large-scale screening of specific anti-epileptic drugs.

[0022] Adding anti-epileptic drugs topiramate (TPR), sodium valproate (VPA) and carbamazepine (CBZ) after permethrin exposure can relieve the convulsions of the embryo, while adding anti-epileptic drug ethosuximide (ESX) has limited effect. It shows that there are particularities and differences in the mechanisms and required treatments for epilepsy caused by permethrin exposure and epilepsy caused by other inducements. The inventor analyzed that the reason may be that TPR, VPA and CBZ reduce glutamate levels and increase GABA levels, while ESX increases glutamate levels and reduces GABA levels. Due to the differences in the mechanisms of action of the four drugs, there are differences in their therapeutic effects on epilepsy caused by permethrin exposure. Among them, the effect of CBZ is particularly excellent, followed by VPA and TRP, and ESX is even worse than the permethrin alone treatment group in some indicators.

[0023] In addition, specific inhibitors against Nav1.4 and Nav1.5 reduced the frequency of tail twitches and eye movements in zebrafish treated with permethrin, suggesting that permethrin exposure may affect the intensity of muscle contractions, similar to tonic and clonic seizures accompanied by muscle twitches. However, the number of neurons partially increased when Nav1.6 was inhibited instead of Nav1.4 or Nav1.5. These results suggest that sodium channels have multiple functions in neurons. Nav1.6 may be the main target of permethrin in epilepsy, and knocking out the subunit gene of Nav1.6 can alleviate the epileptic symptoms caused by permethrin. The use of methocarbamol, flecainide acetate and SYM2206, targeting Nav1.4, Nav1.5 and Nav1.6 respectively, can achieve the effect of alleviating epilepsy caused by permethrin exposure, which is specifically reflected in the reduction of twitch frequency and eye movement frequency. In addition, for epilepsy caused by permethrin exposure, glutamate receptor inhibitors D-AP5 and CNQX, and GABA receptor agonist baclofen can alleviate tail twitching and eye rotation caused by permethrin exposure.

[0024] Therefore, using the zebrafish model of epilepsy caused by permethrin exposure established in this protocol, a large number of drugs that can effectively act on epilepsy caused by permethrin exposure can be screened. Epilepsy caused by permethrin exposure is different from epilepsy caused by other causes in terms of treatment methods and pathological mechanisms, so it is necessary to adopt a specific drug screening method. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1Results of the experiment on the acute permethrin exposure in Example 1 that induces epilepsy-like convulsions and neuronal hyperexcitability in zebrafish embryos (a: representative motion trajectory tracking of permethrin-exposed embryos, stage 0 S0: no swimming activity, stage 1 S1: increased swimming activity), stage 2 S2: whole-body convulsions, the trajectory is blindly scored; b: percentage of embryos with motion trajectory tracking in a, n=24 per group; c: schematic diagram of zebrafish installation for recording convulsions; d and e,: quantitative histograms of oscillation frequency and maximum swing angle of the tail of 3dpf embryos, n=5 per group; f: GCaMP6s in the 3dpf midbrain after permethrin exposure + Representative confocal time-lapse imaging of Ca signals. The enlarged image of the circled area is shown in the upper right corner of each panel. Scale bar 20 μm; g: Ca signal during 300 imaging cycles. 2+ Representative trajectories of sparks; h: GCaMP6s + Quantitative histogram of signal fluctuation range, DMSO: n = 50 from 5 fish, 20 μg / L: n = 44 from 4 fish, 50 μg / L: n = 42 from 5 fish, 100 μg / L: n = 54 from 5 fish, 500 μg / L: n = 16 from 5 fish; data are expressed as mean ± standard error, ***P < 0.001; ****P < 0.0001; ns, no significant difference, statistical analysis was performed using unpaired one-tailed Student's t test).

[0026] Figure 2 The results of the study on the damage of chlormethrin exposure to the locomotor ability and neuronal composition of zebrafish larvae in Example 1 (a: representative heat map of spontaneous swimming of 5dpf zebrafish larvae; b: quantitative histogram of eye rotation frequency of 5dpf larvae after chlormethrin exposure, DMSO: n=7, 20μg / L: n=9, 50μg / L: n=10, 100μg / L: n=8, 500μg / L: n=9; c: swimming distance was analyzed in segments at 1-minute intervals, DMSO: n=18, 20μg / L: n=12, 50μg / L: n=18, 100μg / L: n=6, 500μg / L: n=18; d: GFAP fluorescence images of each group, n=8 in each group, scale bar: 20μm; e and f: NEUN in the midbrain of 5dpf larvae + Fluorescence images and quantitative histograms of cells, DMSO: n = 10, 50 μg / L: n = 6, 500 μg / L: n = 6, scale bar: 20 μm; g and h: islet-GFP in the brain of 5 dpf larvae + Fluorescence images and quantitative histograms of cells, DMSO: n = 5, 50 μg / L: n = 9, 500 μg / L: n = 9, scale bar: 20 μm; i and j: VGlut2a in the brain of 5 dpf larvae +Fluorescence images and quantitative histograms of cells, n=7 per group), scale bar: 20 μm; data are expressed as mean ± standard error, *P<0.05, **P<0.01; ***P<0.001, ****P<0.0001, statistical analysis was performed using unpaired one-tailed Student's t test).

[0027] Figure 3 The experimental study results of Example 1 that chronic permethrin exposure causes anxiety-like symptoms, gliosis and neuronal loss in adult zebrafish (a: representative heat map of adult zebrafish in the open field test; b, c: quantitative histograms of the residence time and travel distance of adult zebrafish in the control group and permethrin exposure group in the open field test, b: DMSO: n = 10, 50μg / L: n = 8; c: central area: DMSO: n = 11, 50μg / L: n = 8, peripheral area: DMSO: n = 13, 50μg / L: n = 6; d: representative heat map of adult zebrafish swimming in the light / dark preference test, with the dark chamber on the left and the light chamber on the right; e and f: quantitative histograms of the total travel distance and average speed of adult zebrafish in the light zone and the dark zone, DMSO: n = 20, 50μg / L: n = 8; g: representative heat map of adult zebrafish in the social preference test Swimming trajectories and heat maps; h and i: Quantitative histograms of the residence time and travel distance of adult zebrafish in area 1 and area 2, h: DMSO: n = 16, 50 μg / L: n = 8; i: area 1: DMSO: n = 14, 50 μg / L: n = 7, area 2: DMSO: n = 16, 50 μg / L: n = 8; j: Red fluorescence image of GFAP in the cross section of the midbrain of 8-month-old adult zebrafish, n = 3, scale bar 200 μm; k: Red fluorescence image of NEUN in the cross section of the optic tectum area of ​​8-month-old adult zebrafish, the left panel is an enlarged image of the boxed area on the right, n = 3, scale bar 50 μm; data are expressed as mean ± standard error; *P < 0.05, ***P < 0.001, ****P < 0.0001, ns, no significant difference; statistical analysis was performed using unpaired one-tailed Student's t test).

[0028] Figure 4Experimental results of RNA sequencing analysis and anti-epileptic drug screening in Example 2 (a: In the PCA diagram, the control group and the permethrin-treated group are completely distinguished by different colors, C: control group 0.1% DMSO, T1: 50 μg / L permethrin-treated group, T2: 500 μg / L permethrin-treated group; b: Venn diagram showing the overlap of differentially expressed genes (DEGs) between the groups; c: GO and KEGG terms (upregulated) co-enriched in the 50 μg / L and 500 μg / L permethrin-treated groups compared with the control group; d: Schematic timeline of the behavioral tracking experiment after permethrin and drug treatment; e: Quantitative histogram of the total swimming distance of 3 dpf embryos after permethrin or permethrin combined with four drugs treatment, DMSO: n=22, PM: n=2 8, PM+ESX: n=27, PM+TPR: n=28, PM+VPA: n=28, PM+CBZ: n=26; f and g: Quantitative histograms of twitch frequency and eye rotation frequency of 5dpf juveniles in the control group, permethrin-treated group, and permethrin plus carbamazepine-treated group; f: DMSO: n=12, PM: n=13, PM+CBZ: n=8; g, DMSO: n=10, PM: n=13, PM+CBZ: n=8; h: GCaMP6s in the control group, permethrin-treated group, and permethrin plus carbamazepine-treated group + Representative confocal time-lapse imaging of the signal, the upper right corner of each panel is a magnified image of the circled area, scale bar 20 μm; i and j: neurod1-GFP in the midbrain of larval fish in the control group, permethrin-treated group, carbamazepine-treated group, and permethrin plus carbamazepine-treated group + Fluorescence images and quantitative histograms of signals, DMSO: n = 10, PM: n = 14, CBZ: n = 16, PM + CBZ: n = 13, scale bar 20 μm; data are expressed as mean ± standard error; *P < 0.05, **P < 0.01; ***P < 0.001, ****P < 0.0001; ns, no significant difference; statistical analysis was performed using unpaired one-tailed Student's t test).

[0029] Figure 5The experimental study results of Example 3 on the induction of epilepsy-like symptoms by permethrin through voltage-gated sodium channels (VGSCs) (a: quantitative histogram of the total swimming distance of the control group and the veratridine-treated group, n=24 for each group; b: quantitative histogram of the percentage of the control group and the veratridine-treated group in the motion tracking system, n=24 for each group; c: heat map of the transcription level of voltage-gated sodium channels (VGSCs) in the transcriptome data; d: qRT-PCR results of the transcription level of VGSCs genes in the control group and permethrin-treated larvae, β-actin was used as an internal control, and each point represents an independent experiment; e: quantitative histogram of the twitch frequency of the control group, permethrin-treated group, and permethrin plus VGSCs inhibitor-treated group, n=10 for each group; f and g: neurod1-GFP in the midbrain of the control group, permethrin-treated group, SYM2206-treated group, and permethrin plus SYM2206-treated group larvae + Fluorescence images and quantitative histograms of signals, DMSO: n = 9, PM: n = 7, SYM2206: n = 8, PM + SYM2206: n = 8), scale bar: 20 μm; h: Fluorescence images of Scn8a in the midbrain of 5 dpf larvae in the control group and permethrin-treated group, the bottom panel is an enlarged image of the boxed area in the top panel, DMSO: n = 10, 50 μg / L PM: n = 9, 500 μg / L PM: n = 10, scale bar: 20 μm; i: Fluorescence images of Scn8a (red) in cross-sections of the brain of 8-month-old zebrafish in the control group and permethrin-treated group. The right panel is an enlarged image of the boxed area, n = 3 in each group, scale bar: 200 μm; j and k: wild type (WT) and scn8aa crispant Quantitative histogram of twitch frequency and eye rotation frequency of juvenile fish after permethrin exposure, WT+DMSO: n=11, WT+PM: n=8, scn8aa crispant +DMSO: n=10,scn8aa crispant +PM: n=14), data are expressed as mean ± standard error, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001, ns, no significant difference, statistical analysis was performed using unpaired one-tailed Student's t test).

[0030] Figure 6 Schematic diagram of the dosing regimen of methocarbamol, flecainide acetate and SYM2206 in Example 3.

[0031] Figure 7 The figure is a statistical graph of the twitch frequency and eye movement frequency of methocarbamol, flecainide acetate and SYM2206 administered alone in Example 3.

[0032] Figure 8The experimental study results of Example 4 on the increase of glutamate levels caused by permethrin exposure (a: heat map of glutamate receptor gene transcription levels in transcriptome data; b: qRT-PCR results of glutamate receptor and transporter gene transcription levels in juvenile fish of the control group, permethrin treatment group (PM), carbamazepine treatment group (CBZ) and permethrin plus carbamazepine treatment group, each point represents an independent experiment, β-actin is used as an internal control; c: qRT-PCR results of glutamate receptor and transporter gene transcription levels in the brain of adult zebrafish in the control group and permethrin treatment group, each point represents an independent experiment, β-actin is used as an internal control; d and e: glutamate concentrations in the brain of 5 dpf juvenile fish and 8-month-old adult zebrafish under permethrin exposure, d: n = 7 per group, from 500 fish, e: n = 4 per group, from 3 fish; f: 3 dpf control group and veratridine treatment group glutamate receptor and transporter gene transcription levels β-actin was used as an internal control, and each point represents an independent experiment; g and h: Quantitative histograms of twitch frequency and eye rotation frequency of 5 dpf control group, permethrin-treated group (PM) and permethrin plus glutamate receptor inhibitor-treated group, DMSO: n = 10, PM: n = 8, PM + D-AP5: n = 9, PM + CNQX: n = 8, PM + D-AP5 + CNQX: n = 11; i and j: Quantitative histograms of twitch frequency and eye rotation frequency of 5 dpf control group, permethrin-treated group and permethrin plus baclofen-treated group, DMSO: n = 10, PM: n = 8, PM + baclofen: n = 9); Data are expressed as mean ± standard error; *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, ns, no significant difference; Statistical analysis was performed using unpaired one-tailed Student's t-test).

[0033] Fig. 9 Schematic diagram of the dosing regimen of D-AP5, CNQX and baclofen in Example 4.

[0034] Fig.10 scn8aa of Example 5 crispant Western blot images of the expression levels of Scn8a in .

[0035] Fig.11 scn8aa of Example 5 crispant The moving distance and speed test results. DETAILED DESCRIPTION

[0036] The following is further described in detail through specific implementation methods:

[0037] Example 1: Zebrafish epilepsy model

[0038] Zebrafish were raised and maintained under standard laboratory conditions. Wild-type (WT) AB strains and the following transgenic strains were used: Tg(neurod1:eGFP), Tg(HuC:GCaMP6s), and Tg(gad1b:mcherry). The above experimental animals are conventional experimental animals and can be obtained commercially. The transgenic strains Tg(islet:GFP) and Tg(vglut2a;GUL4-UAS:GFP) were donated by South China University of Technology. More specifically, the Tg(neurod1:eGFP) strain uses the promoter of the neurod1 gene to drive the expression of green fluorescent protein (eGFP). The Tg(HuC:GCaMP6s) strain uses the promoter of the HuC (also known as Dlx5a) gene to drive the expression of the green fluorescent protein-based calcium indicator GCaMP6s. The Tg(gad1b:mcherry) strain uses the promoter of the gad1b gene to drive the expression of red fluorescent protein (mCherry). The Tg(islet:GFP) strain uses the promoter of the islet gene to drive the expression of green fluorescent protein (GFP). The Tg(vglut2a; GUL4-UAS:GFP) strain uses the promoter of the vglut2a gene to drive the expression of GAL4, which in turn drives the expression of GFP downstream of the UAS sequence.

[0039] In order to inhibit the development of endogenous pigments, embryos were treated with 0.003% 1-phenyl-2-thiourea (PTU; Sigma, #103-85-5) starting from 12 hours post fertilization (hpf). For acute zebrafish exposure experiments, zebrafish embryos at 3hpf (Hours PostFertilization, i.e. hours after fertilization) were immersed in egg water (egg water, a conventional medium for culturing zebrafish in the prior art, which can be obtained by commercial means) containing chemicals, and immersed to 3dpf or 5dpf (Days Post Fertilization) according to experimental requirements. For chronic exposure experiments, zebrafish were exposed to 50μg / L of permethrin (Permethrin, Target Mol, #52645-53-1) starting from 3hpf. These solutions were replaced every two weeks, and any dead fish were removed immediately.

[0040] Embryonic twitch and nystagmus analysis: Larvae were embedded in 1% (w / v) low melting point agarose (Thermo Fisher Scientific, #16520050) at 38°C with the dorsal surface facing up. After the agarose solidified, the agarose below the embryo's yolk sac was carefully removed with a scalpel, ensuring that the embryo was not damaged. This allowed the upper part of the yolk sac to be fixed in the agarose while the lower part remained mobile in the water. Each embryo was videotaped for 3 minutes under a microscope (Nikon, SMZ18). The frequency of nystagmus and tail twitches was recorded manually. The tail swing was referenced to the central axis of the body, and a swing was recorded when the tail left the central axis and returned. The maximum swing amplitude (°) was defined as the sum of the angles between the farthest position reached by the tail swing and the central axis.

[0041] Zebrafish locomotor behavior tests: Locomotor behavior tests were performed as previously reported. All locomotor behavior tests for embryos and larvae were performed in 24-well plates. For adult zebrafish, spontaneous behavior, open field test, and light-dark preference test were performed in a box with dimensions of 17.5 cm × 10.5 cm × 9.5 cm (length × width × depth). The apparatus used for social preference detection was a transparent PP tank with dimensions of 21 cm × 6 cm × 10 cm. The tank was divided into two compartments: a smaller one of 7 cm × 6 cm × 10 cm and a larger one of 14 cm × 6 cm × 10 cm. The movement of zebrafish was captured by a Danio Vision observation chamber system (Noldus IT, The Netherlands), and the video was analyzed by EthoVision XT software. The bout behavior of zebrafish was classified by two observers who were unaware of the phenotype of the larvae as grade 0 (S0, no movement, baseline activity), grade 1 (slightly increased movement), and grade 2 (significantly increased movement, bout behavior).

[0042] Neuronal calcium imaging: In vivo time-lapse imaging was performed in Tg(HuC:GCaMP6s) transgenic zebrafish according to previously reported methods. Briefly, the GCaMP6s fluorescence signal in the midbrain region was manually selected as the region of interest (ROI). The fluorescence intensity of each ROI at each time point was calculated as (F-F0) / F0. F0 is the average fluorescence intensity value of 50 ROIs in the control group (n=5). GCaMP6s + The fluctuation amplitude of the signal is defined as (ΔF / F0)max-(ΔF / F0)min.

[0043] (1) Zebrafish epilepsy model with acute permethrin exposure

[0044] (1.1) Permethrin exposure caused whirlpool-like swimming, clonic convulsions, and enhanced neuronal activity in zebrafish embryos in a dose-dependent manner

[0045] In the acute exposure experiment, fertilized zebrafish embryos were exposed to 20, 50, 100, and 500 μg / L permethrin, which are concentrations that reflect the permethrin concentrations in environmental water (0.05–811 μg / L). We first assessed the responses of 3-day postfertilization (dpf) embryos after 70 hours of exposure. Most 3 dpf control embryos were immobile. In contrast, permethrin-treated embryos, especially in the 500 μg / L group, showed a sudden increase in swimming activity accompanied by a loss of direction and balance, resulting in rapid vortex-like swimming. These embryos often fell to one side, collapsed, and remained motionless for 1–3 seconds. During the unbalanced swimming bursts, their tails curled up extensively; this movement resembled the clonic twitches induced by pentylenetetrazol (PTZ) treatment. We graded these behaviors based on the results of previous studies. Most (95.65%) control embryos showed limited swimming activity at stage 0 (S0), and only 4.35% of embryos showed slight movement at stage 1 (S1). However, more than 12% of 20 μg / L permethrin-treated embryos showed swimming activity at the S1 stage. This swimming activity increased with increasing permethrin concentrations. Notably, more than 93% of embryos in the 500 μg / L treatment group swam at either the S1 (23.26%) or S2 (69.77%) stages. Figure 1 We designed an assay to determine the frequency and amplitude of tail twitches by embedding the upper body of the embryo (including the head and yolk sac) in 1% agarose gel, while the lower body remains free to move in water ( Figure 1 c). In 3dpf embryos treated with 500μg / L permethrin, 10.60±0.40 enhanced symmetrical swings were observed per minute, with each interval lasting from 8 to 20 seconds ( Figure 1 d). The maximum tail swing amplitude during twitching was 103.30±10.27°; the tail sometimes even curled up and touched the yolk sac ( Figure 1 e) However, this action was rarely observed in the control group or the groups treated with 20, 50 or 100 μg / L permethrin.

[0046] The abnormally enhanced motility seen after permethrin exposure prompted us to examine neuronal activity by imaging calcium sparks in Tg(HuC:GCaMP6s) embryos. GCaMP6s signals were recorded for 9 min (300 cycles) in the optic tectum. + Regular low-frequency spontaneous calcium activity was observed in the cells. However, as the concentration of permethrin increased, the frequency and amplitude of signal fluctuations in the treatment group gradually increased, as shown in the fluorescence images and ΔF / F0 trajectories ( Figure 1 f, g). GCaMP6s after exposure to 20, 50, 100 and 500 μg / L permethrin +The fluctuation amplitudes of the signals were 1.62, 2.48, 2.80 and 4.52 times that of the control group ( Figure 1 h). These results indicate that permethrin exposure induced neuronal excitability in a dose-dependent manner.

[0047] (1.2) Prolonged permethrin exposure causes additional nystagmus and response disturbances in zebrafish larvae

[0048] As embryos develop into larvae, zebrafish develop additional movements. Therefore, we further investigated the effects of permethrin treatment on 5 dpf zebrafish larvae. Video recordings showed that control larvae swam smoothly and continuously at this stage. However, larvae exposed to 500 μg / L permethrin collapsed after twitching, lost posture, and were unable to control direction, showing a significantly reduced average speed and range of motion ( Figure 2 a). More frequent twitches (16.00 ± 0.98 times / min) and enhanced tail swing amplitude (119.10 ± 5.58°) were observed in 5dpf larvae, which were more obvious than those in 3dpf embryos. Interestingly, we detected rapid and repetitive eye movements in larvae exposed to 50μg / L permethrin. In one bout of movement, both eyes of the larvae turned simultaneously in one direction for 6 to 10 seconds and then turned to the other direction. The intensity of the eye movements increased with increasing permethrin concentrations ( Figure 2 b). 5dpf control juveniles occasionally showed eye rotations, with an average frequency of 0.43±0.30 times / min. However, the eye rotation frequencies of the 20, 50, 100, and 500 μg / L permethrin-treated groups increased to 1.33±0.47, 1.50±0.45, 3.38±0.38, and 9.89±0.68 times / min, respectively ( Figure 2 b). This phenomenon mirrors the nystagmus (quick, horizontal, and conjugated) observed in clinical cases of epilepsy but was not observed in larvae treated with 1 mg / mL PTZ. Meanwhile, larvae exposed to 20 μg / L permethrin exhibited subtle body twitches. The body twitches became more pronounced as the permethrin concentration increased. However, this phenotype was not observed in larvae of the control group.

[0049] Zebrafish larvae begin to sense environmental changes, including light, at 5 dpf. Therefore, we performed a photoperiodic stimulation test. Control larvae increased their movements during light-dark transitions, especially during the dark phase ( Figure 2 c). However, the movement distance and speed of juveniles exposed to 100 and 500 μg / L permethrin under light-dark cycle stimulation were significantly decreased compared with the control group and other treatment groups ( Figure 2c). It is noteworthy that the movement distance and speed of 500 μg / L permethrin-treated larvae decreased by 53.78% in the light cycle, and by 88.11% and 76.23% in the dark cycle, respectively. These results indicate that the behavior of 5 dpf permethrin-treated larvae was similar to that of 3 dpf larvae, but the deterioration was greater.

[0050] Reduced neurogenesis and gliosis are common features of epilepsy models. We examined multiple cell types, including neurons and glial cells. Glial fibrillary acidic protein (GFAP) signaling was significantly increased after permethrin exposure ( Figure 2 d), while olig2-DsRed + The number of oligodendrocytes was comparable to that of the control group. + and NEUN + The number of neuronal cells decreased in a dose-dependent manner after permethrin exposure ( Figure 2 e,f). Among them, islet + The signal of cranial neurons decreases ( Figure 2 g,h). However, vglut2a + The number of excitatory glutamatergic neurons increased after permethrin exposure, whereas the number of gad1b-mCherry+GABAergic neurons did not change significantly ( Figure 2 i,j). In addition, PCNA + The number of cells was significantly reduced. However, acridine orange + (AO + ) signals did not change significantly after permethrin exposure compared with controls. Taken together, these data suggest that permethrin exposure impairs neuronal organization and proliferation but has limited effects on apoptosis.

[0051] (2) Zebrafish epilepsy model with chronic permethrin exposure

[0052] (2.1) Chronic permethrin exposure causes anxiety-like symptoms in adult zebrafish

[0053] When juveniles were continuously exposed to permethrin, juveniles in the 100 and 500 μg / L groups gradually died starting at 8 dpf (8 days after fertilization) and all died before 12 dpf. However, 22% of the 50 μg / L permethrin-exposed samples survived to adulthood, allowing us to explore the effects of chronic low-dose permethrin exposure. After 6 months of exposure (50 μg / L permethrin), the spontaneous movements of the surviving zebrafish were comparable to those of the control group, including the motor responses to the unfamiliar environment that we assessed in the open field test. However, compared with the control group, zebrafish in the permethrin-exposed group tended to move closer to the walls and away from the central area ( Figure 3a, b). The distance parameter showed that the movement of zebrafish in the exposure group decreased in the central area and increased by 37.67% in the peripheral area ( Figure 3 c) Increased haptotaxis in the peripheral areas indicates anxiety after chronic permethrin exposure.

[0054] (2.2) Light / dark preference and social preference test

[0055] Light / dark preference and social preference tests are effective methods for assessing anxiety-like behaviors. Adult zebrafish naturally prefer dark conditions (scototropism) and avoid light (phototropism). In fact, chronic permethrin exposure significantly increases the time spent in the dark area ( Figure 3 d, e). Statistical results showed that exposure to 50 μg / L permethrin resulted in an increase in movement distance and speed in the dark area by 49.77% and 33.52%, respectively ( Figure 3 e,f), which is an indicator of anxiety-like behavior. The total movement distance and average speed of zebrafish treated with permethrin increased by 27.46% and 27.36%, respectively, compared with the control group. In the social preference test, zebrafish in the permethrin-treated group spent more time in area 2 than in area 1, while the total movement distance and average speed remained unchanged. Compared with the control group, the residence time and movement distance of adult zebrafish exposed to permethrin in area 2 increased by 16.32 times and 19.35 times, respectively ( Figure 3 h,i). We further examined glial cells and neurons. Immunofluorescence staining results showed that GFAP in the brain of adult zebrafish treated with permethrin was significantly higher than that in the control group. + The signal increased significantly, while NEUN + The signal is significantly reduced ( Figure 3 j,k). These results are consistent with those observed in zebrafish larvae. Taken together, chronic permethrin exposure leads to anxiety-like behaviors by exacerbating dark-movement and wall-touching behaviors, reducing exploratory abilities, and impairing sociability. These results are consistent with the comorbid anxiety observed in adult patients with epilepsy (11-50%).

[0056] Example 2: Drug screening based on epilepsy model

[0057] Samples of the control group and the 50 and 500 μg / L permethrin-treated groups were extracted from the head tissues of 5 dpf (5 days after fertilization) zebrafish, and RNA sequencing was performed to elucidate the molecular mechanism after permethrin exposure. The results of principal component analysis (PCA) showed that the nine samples could be divided into three different groups ( Figure 4 a). We found that in the 50 μg / L group, there were 2338 differentially expressed genes (DEGs), of which 1469 were upregulated and 869 were downregulated; in the 500 μg / L group, there were 5025 DEGs, of which 2756 were upregulated and 2269 were downregulated ( Figure 4 b). Biological processes (BPs) and molecular functions (MFs) in the Gene Ontology (GO) database showed that transmembrane transport (GO:0055085) was significantly enriched in the 50 and 500 μg / L permethrin treatment groups compared with the control group ( Figure 4 c). Among the differences in transmembrane transport, sodium ion transport (GO:0006814) and intracellular sodium ion homeostasis (GO:0006883) were particularly significant ( Figure 4 c). The downregulated GO terms were mainly related to cell division and brain and eye development. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis results showed that the upregulated terms were mainly related to metabolic pathways, especially amino acid metabolism ( Figure 4 c).

[0058] Based on the model of this protocol, we tested four drugs: ethosuximide (ESX), topiramate (TPR), sodium valproate (VPA) and carbamazepine (CBZ). The information of the above four drugs is: Topiramate (Topiramate, Target Mol, #97240-79-4, 50μg / mL), sodium valproate (Valproic acid sodium salt, Target Mol, #1069-66-5, 150μg / mL), ethosuximide (Ethosuximide, Target Mol, #77-67-8, 1mg / mL), carbamazepine (Carbamazepine, MCE, #298-46-4, 50mg / L). We pre-treated zebrafish embryos (3hpf) with 500μg / L permethrin and maintained this concentration in the egg water until 3dpf. Then, the above four drugs were added at 2dpf for treatment until 3dpf. The above doses are the recommended doses of the four drugs in previous studies. We assessed embryonic motility at 3 dpf (3 days post fertilization) Figure 4 d). Compared with the group treated with permethrin alone, the movement distance was reduced by 46.11%, 82.67%, and 90.89% after the addition of TPR, VPA, and CBZ, respectively. However, the effect of ESX was limited ( Figure 4 e). CBZ treatment significantly promoted the recovery of permethrin-treated zebrafish to normal phenotypes, including convulsions in larvae ( Figure 4 f) Eye movement ( Figure 4 g), neuronal activity ( Figure 4 h) and neurod1 + Cell count ( Figure 4 i,j). The levels of some symptoms returned to those of the control group ( Figure 4 fj). TPR, VPA, and especially CBZ, but not ESX, showed significant effects in alleviating the phenotype induced by permethrin.

[0059] Example 3: Inhibitors targeting sodium channels alleviate epilepsy-like symptoms induced by permethrin exposure

[0060] Fertilized zebrafish embryos (3 hpf) were exposed to 6 and 12 μg / mL of the sodium channel activator veratridine to confirm the activation of sodium channels in the epilepsy-like symptoms induced by permethrin treatment. Embryos 3 days after treatment showed rapid vortex-like circular swimming and brief clonic-like twitching behaviors. After veratridine treatment, the movement distance of embryos was significantly increased ( Figure 5 a). In the 6μg / mL veratridine group, 45.83% of embryos showed S2 stage movement, and this proportion increased to 91.67% when the dose was increased to 12μg / mL ( Figure 5 b). In contrast, the movement of the control group was very limited. In addition, the GCaMP6s in the veratridine-treated group were significantly increased compared to the control group. + The frequency and amplitude of the cell fluctuations were significantly higher. The behavior of the treated embryos was similar to that of embryos cultured in permethrin, suggesting that permethrin plays a key role in epilepsy-like symptoms by activating sodium ion channels.

[0061] Voltage-gated sodium channels (VGSCs) are the main channels for sodium ions to permeate cells, thereby supporting the excitability of neurons. In zebrafish, VGSCs include scn1laa / scn1lab, scn4aa / scn4ab, scn5laa / scn5lab, and scn8aa / scn8ab, encoding Nav1.1, Nav1.4, Nav1.5, and Nav1.6 channels, respectively. The heat map of RNA-seq and qRT-PCR results showed that the expression of scn4aa, scn4ab, scn5laa, scn5lab, scn8aa, and scn8ab was upregulated in zebrafish after permethrin treatment ( Figure 5 c,d).

[0062] Therefore, we used methocarbamol, flecainide acetate, and SYM2206 to target Nav1.4, Nav1.5, and Nav1.6, respectively. The specific drug dosage information is as follows: SYM2206 (Target Mol, #173952-44-8, 1 μg / mL), flecainide acetate (Target Mol, #54143-56-5, 10 μg / mL), methocarbamol (Target Mol, #532-03-6, 25 μg / mL), dosing regimen: When 3 hpf embryos were exposed to permethrin (500 μg / L), the three drugs (concentrations as described above) were added at the same time to observe the symptoms of the embryos at 5 dpf. The specific dosing regimen can be found in Figure 6When permethrin and Nav1.4, Nav1.5, and Nav1.6 inhibitors were given simultaneously, the twitch frequency decreased by 34.53%, 60.67%, and 51.14%, respectively, compared with the group treated with permethrin alone; the eye movement frequency decreased by 20.63%, 37.30%, and 27.59%, respectively (experimental results in Figure 7 ). Figure 7 The specific data are: twitch frequency (left, times / minute): DMSO: 0.50±0.15, PM (500μg / L): 14.62±0.87, PM+Nav1.4-i (methocarbamol): 9.57±1.49, PM+Nav1.5-i (flecainide acetate): 5.75±0.49, PM+Nav1.6-i (SYM2206): 7.14±0.67; eye movement frequency (right, times / minute): DMSO: 1.40±0.31; PM (500μg / L): 16.38±1.15, PM+Nav1.4-i: 13.00±1.21, PM+Nav1.5-i: 10.27±0.97, PM+Nav1.6-i: 11.86±0.94. No inhibitor of Nav1.4, Nav1.5, or Nav1.6 was found to have an absolute advantage in alleviating epileptic symptoms caused by permethrin. In terms of reducing the frequency of convulsions and eye movements, Nav1.5-i (flecainide acetate) showed a slightly better effect. In addition, after the addition of these chemical inhibitors, a similar decrease in the frequency and amplitude of calcium signal fluctuations in neurons was observed.

[0063] Furthermore, the combination of different sodium channel inhibitors had a more pronounced effect during permethrin exposure than any single sodium channel inhibitor ( Figure 5 e). This suggests that permethrin interacts with multiple sodium channels simultaneously. Notably, when the Nav1.6 inhibitor was added to the combination treatment, the twitching frequency of larvae was significantly lower than that of the combination treatment with only Nav1.4 and Nav1.5 inhibitors ( Figure 5 e). More specifically, Figure 5The experimental data of e are (twitch frequency, times / minute): DMSO: 0±0.00, PM (500μg / L): 15.58±0.84, PM+Nav(1.4+1.5)-i: 7.32±0.84, PM+Nav(1.4+1.6)-i: 3.54±0.17, PM+Nav(1.5+1.6)-i: 3.24±0.31, PM+Nav(1.4+1.5+1.6)-i: 2.24±0.36. Compared with the above three inhibitors used alone, although the effect of Nav1.6-i (SYM2206) alone is not as good as Nav1.5-i (flecainide acetate), Nav1.6-i (SYM2206) combined with other types of inhibitors showed a very significant effect of reducing twitch frequency. The effect of Nav1.6-i (SYM2206) combined with Nav1.4-i (methocarbamol) (reduction of 77%) is much better than that of Nav1.5-i (flecainide acetate) combined with Nav1.4-i (methocarbamol) (reduction of 53%). Nav1.6-i (SYM2206) combined with other inhibitors can also achieve the ideal effect of reducing the frequency of convulsions. This shows that in order to alleviate the epileptic symptoms caused by permethrin exposure, the combination of Nav1.6-i (SYM2206) and other types of voltage-gated sodium channel inhibitors is the best choice, rather than the combination of random voltage-gated sodium channel inhibitors to achieve the ideal effect.

[0064] In addition, methocarbamol and flecainide acetate alone had no significant effect on the number of neuronal cells. However, when methocarbamol and flecainide acetate were used in combination, the number of neuronal cells was even lower than that of the group treated with permethrin alone. In contrast, the application of Nav1.6 inhibitor (SYM2206) partially (11.73%) restored the number of neuronal cells in the permethrin-treated group ( Figure 5 f,g). This shows that the Nav1.6 inhibitor (SYM2206) can not only relieve typical symptoms of epilepsy, but also avoid the decrease in the number of neurons caused by permethrin treatment to a certain extent. Methocarbamol and flecainide acetate do not have the above functions when used alone or in combination. The effect of the Nav1.6 inhibitor (SYM2206) was not expected by the inventors before the experiment.

[0065] Taken together, these findings suggest that permethrin induces epilepsy-like symptoms in embryos by activating sodium channels, in which Nav1.6 may play a major role.

[0066] Example 4: Effects of glutamate receptor inhibitors on epilepsy symptoms in zebrafish

[0067] Epilepsy is characterized by an imbalance between excitatory and inhibitory neurotransmitters in neural circuits. Glutamate and GABA are key excitatory and inhibitory neurotransmitters, respectively. After permethrin exposure, VGlut2A + The number of glutamatergic neurons increased significantly ( Figure 2 i,j). GO and KEGG analysis results showed that permethrin treatment upregulated the transport and metabolism of glutamate ( Figure 4 c). Heat map and qRT-PCR data showed that the expression levels of glutamate receptor and transporter genes (including grin1a, grin1b, gria4a, grinaa, grik1a, grid1a, vglut1, vglut2a, and vglut2b) were significantly increased after permethrin exposure ( Figure 8 ac). Consistently, glutamate concentrations in larval and adult zebrafish treated with permethrin increased by 23.36% and 24.16%, respectively, compared with those in the control group ( Figure 8 d,e). However, when embryos were exposed to permethrin and carbamazepine simultaneously, the expression levels of glutamate receptor and transporter genes were significantly reduced ( Figure 8 b). Application of the sodium channel activator veratridine increased the expression levels of glutamate receptor and transporter genes compared with the control group ( Figure 8 f) These findings suggest that permethrin exposure induces an increase in glutamate levels via sodium channels.

[0068] To investigate the key role of glutamate in epilepsy-like symptoms induced by permethrin exposure, zebrafish embryos were treated with glutamate receptor inhibitors D-AP5 (NMDA receptor antagonist, 20 μg / mL) and CNQX (AMPA and kainate receptor antagonist, 4 μg / mL). 3hpf embryos were exposed to permethrin (500 μg / L) and two glutamate receptor inhibitors were added at the same time, and treated until 5dpf. The specific dosing schedule is shown in Fig. 9 . Specific drug dosage information: D-AP5 (Target Mol, #79055-68-8, 20μg / mL), CNQX (Target Mol, 541#115066-14-3, 4μg / mL). When D-AP5 and CNQX were used in combination with permethrin, the frequency of tail twitches and eye rotations was reduced by approximately 20% to 50%, respectively. When the two inhibitors were used in combination, this effect was more pronounced compared to the group using permethrin alone ( Figure 8 g,h), suggesting that the overproduction of glutamate induced by permethrin exposure plays a key role in epileptic symptoms.

[0069] Since epilepsy is characterized by an imbalance between excitatory and inhibitory activity in neural circuits, we wondered whether these syndromes could be alleviated by increasing the levels of inhibitory neurotransmitters. To this end, we used the GABA receptor agonist baclofen, dosage information: Baclofen (Target Mol, #1134-47-0, 50 μg / mL). It was added simultaneously with permethrin exposure at 3 hpf and treated until 5 dpf. Compared with the group treated with permethrin alone, the frequency of tail twitches and eye movements was reduced by 59.60% and 51.17%, respectively ( Figure 8 i,j). Taken together, these results suggest that epilepsy-like symptoms in permethrin-exposed zebrafish are caused by glutamate and that balancing excitatory and inhibitory neurotransmitters can mitigate these effects.

[0070] Example 5: scn8aa gene knockout can alleviate epilepsy symptoms induced by permethrin exposure

[0071] The scn8aa gene (Gene bank, Gene ID: 58152) of zebrafish was knocked down using Crispant technology (refer to the literature method: Bek, JW et al. Lrp Mutant and Crispant Zebrafish Faithfully Model Human Osteoporosis, Establishing the Zebrafish as a Platformfor CRISPR-Based Functional Screening of Osteoporosis Candidate Genes. Journal of Bone and Mineral Research 36, 1749–1764 (2020)), that is, the Cas9 protein and gRNA mixture (RNP) were injected into zebrafish embryos using CRISPR / Cas9 technology. In short, gRNA consists of a transcriptionally activated CRISPR-RNA (tracrRNA) and a specifically targeted CRISPR-RNA (crRNA). The two crRNA sequences of the scn8aa gene are:

[0072] Target1:5′-GGACGCACAGGCAAATGCAA-3′ (SEQ ID NO.1);

[0073] Target2: 5′-ATTGCTGAGAGTGTTCAAGT-3′ (SEQ ID NO. 2).

[0074] These sequences were designed and ordered by Integrated DNA Technologies (https: / / eu.idtdna.com). The final gRNA duplex was mixed with 200 μM of each crRNA and tracrRNA (IDT, #1072532), heated to 95°C for 5 minutes, and stored at -20°C to form two gRNAs. Subsequently, the two gRNAs were mixed with Cas9 protein (IDT, #1081058) at a ratio of 800 pg: 800 pg, incubated at 37°C for 5 minutes to form ribonucleoprotein complexes (RNPs), and then the two RNPs were mixed in equal volumes of 1:1 and microinjected into wild-type zebrafish embryos at the one-cell stage at a volume of 1 nL. The obtained scn8aa gene knockout zebrafish were treated with permethrin (500 μg / L permethrin was added at 3 hpf) until 3 dpf, and then the expression level of related proteins, movement distance and speed were tested and statistically analyzed; when the drug treatment lasted until 5 dpf, the twitch frequency and eye movement frequency were tested and statistically analyzed.

[0075] Compared with the control group, the expression levels of scn8aa and scn8ab were significantly increased in embryos treated with permethrin ( Figure 5 d). Immunofluorescence staining results showed that the expression level of Scn8a was significantly upregulated in zebrafish embryos (larvae) acutely exposed to permethrin and adult zebrafish chronically exposed to permethrin compared with the control group ( Figure 5 h,i), indicating that permethrin has a certain effect on Nav1.6. Therefore, we used the Crispant method to knock out the scn8aa gene (encoding a subunit of the Nav1.6 channel). Western blotting results showed that scn8aa crispant The expression level of Scn8a in the Fig.10 ). Compared with the control group, scn8aa crispant There were no abnormalities in appearance and no embryonic twitching was observed. In contrast, in embryos treated with 500 μg / L permethrin from 4 to 5 dpf, scn8aa crispant After scn8aa knockout, twitch frequency and eye movement decreased by 75.50% and 55.90%, respectively ( Figure 5 j, k). In addition, after scn8aa knockout, the frequency and amplitude of calcium signal fluctuations in neurons in the permethrin-treated group were significantly reduced, and the moving distance and speed were reduced by 60.71% and 64.85%, respectively ( Fig.11These findings suggest that the Nav1.6 channel plays a comprehensive role in the epilepsy-like symptoms induced by permethrin exposure, and knocking out the scn8aa gene, a subunit of the Nav1.6 channel, can alleviate the epilepsy-like symptoms induced by permethrin exposure.

[0076] The above is only an embodiment of the present invention, and the common knowledge such as the known specific structure and characteristics in the scheme is not described in detail here. It should be pointed out that for those skilled in the art, several deformations and improvements can be made without departing from the structure of the present invention, which should also be regarded as the protection scope of the present invention, and these will not affect the effect of the implementation of the present invention and the practicality of the patent. The scope of protection required by this application shall be based on the content of its claims, and the specific implementation methods and other records in the specification can be used to interpret the content of the claims.

Claims

1. A method for constructing an epilepsy animal model, characterized in that: The epilepsy animal model is a zebrafish model of epilepsy caused by permethrin exposure; 3hpf zebrafish embryos are immersed in an environment containing 100-500μg / L permethrin until 3dpf-5dpf; or 3hpf zebrafish embryos are immersed in an environment containing 50μg / L permethrin until 3dpf-6 months.

2. The drug obtained by screening the epilepsy animal model formed by the construction method of claim 1, characterized in that: The drug is used to treat epilepsy caused by permethrin exposure; the zebrafish used to construct the epilepsy animal model is a wild-type zebrafish or a transgenic zebrafish; the epilepsy animal model exhibits the characteristics of convulsion, nystagmus, neuronal damage and reduced neuronal proliferation.

3. The drug according to claim 2, characterized in that: The epilepsy animal model is prepared as follows: immersing 3hpf zebrafish embryos in an environment containing 500 μg / L permethrin, then adding the drug to be screened at 3hpf-2dpf, and continuing to culture until 3dpf-5dpf.

4. The drug according to claim 3, characterized in that: The invention comprises at least one of topiramate, sodium valproate and carbamazepine; preferably, comprises sodium valproate or carbamazepine; further preferably, comprises carbamazepine.

5. The drug according to claim 4, characterized in that: The working concentrations of topiramate, sodium valproate, and carbamazepine were 50 μg / mL, 150 μg / mL, and 50 mg / L, respectively.

6. The drug according to claim 3, characterized in that: including at least one of methocarbamol, flecainide acetate and SYM2206; Or it includes a reagent for knocking out the scn8aa gene; the reagent includes a ribonucleoprotein complex formed by gRNA1 and Cas9 protein, and a ribonucleoprotein complex formed by gRNA2 and Cas9 protein; gRNA1 is composed of tracrRNA and crRNA with a sequence of SEQ ID NO.1, and gRNA2 is composed of tracrRNA and crRNA with a sequence of SEQ ID NO.2; the reagent is used for injection at the single-cell stage of wild-type zebrafish embryos.

7. The drug according to claim 6, characterized in that: It is a composition formed by methocarbamol and flecainide acetate, or a composition formed by methocarbamol and SYM2206, or a composition formed by flecainide acetate and SYM2206, or a composition formed by methocarbamol, flecainide acetate and SYM2206; Preferably, it is a combination of methocarbamol and SYM2206, or a combination of flecainide acetate and SYM2206, or a combination of methocarbamol, flecainide acetate and SYM2206.

8. The drug according to claim 6, characterized in that: The dosage ratio of methocarbamol, flecainide acetate and SYM2206 was 25:10:

1.

9. The drug according to claim 3, characterized in that: It includes at least one of D-AP5, CNQX and baclofen; Preferably, it is D-AP5, or CNQX, or baclofen, or a combination of D-AP5 and CNQX.

10. The drug according to claim 9, characterized in that: The working concentrations of D-AP5, CNQX and baclofen when used alone were 20 μg / mL, 4 μg / mL and 50 μg / mL, respectively; the dosage ratio of D-AP5 and CNQX when used in combination was 20:4.

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