A method for constructing a chemical-induced zebrafish eczema model and application thereof

Abstract

This invention relates to the field of zebrafish model technology, and discloses a method for constructing and applying a chemically induced zebrafish eczema model. The invention constructs a zebrafish eczema model by inducing sensitization and challenge in zebrafish after egg hatching. A two-stage induction method is used to simulate the sensitization and challenge phases of the eczema induction mechanism. Successful model construction is indicated by the repetitive behavioral characteristics observed after both inductions. This invention also reveals that under specific chemical inducer doses, zebrafish exhibit aggregation on the sidewalls of culture dishes and rubbing against the walls, a behavior corresponding to the skin itching symptoms induced by eczema inflammation, which can be directly observed with the naked eye. This model is highly efficient and predictive, and can be used to evaluate the efficacy of drugs, health foods, and other foods in improving eczema.

Description

Technical Field

[0001] This invention relates to the field of zebrafish model technology, and in particular to a method for constructing and applying a chemically induced zebrafish eczema model. Background Technology

[0002] Eczema is a common skin disease characterized by impaired skin barrier function, chronic inflammation, and immune imbalance. Clinical manifestations include dry skin, erythema, papules, vesicles, erosions, and intense itching. The most common types are atopic dermatitis (CD) and contact dermatitis (AD), both of which can present with alternating acute and chronic phases. Epidemiologically, the incidence of eczema is rising globally, with a prevalence of 10-20% in children and approximately 2-10% in adults, significantly impacting quality of life and the social healthcare burden. Its pathology is influenced by both genetic factors (barrier genes such as filaggrin) and environmental factors and contact with allergens. Treatment needs remain unmet (long-term safety / efficacy, drug resistance, and impaired patient quality of life).

[0003] Animal models have been widely used to study skin immune responses, barrier repair mechanisms, and anti-inflammatory drug screening. Current techniques often use chemical inducers to establish animal evaluation models of atopic dermatitis and contact dermatitis. However, a research gap exists between drug mechanisms and drug screening: many novel small molecule / biologic agents require high-throughput, in vivo screening and rapid imaging validation; while traditional mouse models are mature, their cost, throughput, and subsequent microscopic dynamic observation are limited, thus requiring complementary models to accelerate drug discovery and mechanism studies. With the increasing demand for high-throughput drug screening and mechanism imaging, zebrafish (Daniorerio), due to its transparent skin, conserved immune system, ease of gene manipulation, and imaging observation, is gradually becoming a potential model for studying inflammation, allergies, and barrier repair mechanisms. Compared to mammalian models, zebrafish allow for real-time visualization of inflammatory cell migration, pro-inflammatory factor expression, and the dynamic effects of compounds on skin immune responses, providing a new technical pathway for eczema mechanism and intervention research. For example, the technical solution disclosed in publication number CN113355385B uses zebrafish embryos that have not yet hatched at 24-36 hpf as model animals. Chemical drugs are administered to the zebrafish embryos to establish an evaluation model. The difference in the number of times the embryos roll before and after drug administration is used to evaluate whether the drug is irritating, and this can be used to evaluate the inflammatory response caused by the stimulus. However, the stimulation process in this technical solution is inconsistent with the pathogenesis of eczema. The pathogenesis of eczema includes two states: the sensitization phase and the provocation phase. These two states give eczema its recurrent nature, therefore this solution cannot accurately construct an eczema model.

[0004] 2,4-Dinitrochlorobenzene (DNCB) and 2,4-dinitrofluorobenzene (DNFB) are among the most commonly used chemical haptens in the study of contact allergies and atopic dermatitis. They have been used in numerous mouse / rat and in vitro studies to stably induce skin lesions, inflammatory cell infiltration, and cytokine changes similar to certain human AD / ACD pathologies, thus demonstrating high applicability in comparing therapeutic efficacy and mechanisms. Small-molecule electrophilic haptens such as DNCB and DNFB can covalently bind to epidermal proteins, forming neoantigenic epitopes, activating dendritic cells, and inducing T-cell-mediated immune responses, thereby reproducing eczematous inflammatory processes in animals.

[0005] Zebrafish combine the physiological relevance of in vivo studies with the technical convenience of in vitro studies, giving them a unique advantage in eczema research. Their application will contribute to a deeper understanding of the molecular mechanisms of eczema and accelerate the development of related treatment strategies. Whether DNCB / DNFB can induce eczema in zebrafish has not yet been reported. We urgently need to develop a simple and rapid eczema detection model as an important strategy for managing and preventing dermatitis and other inflammatory conditions. Summary of the Invention

[0006] This invention is the first to construct a zebrafish eczema model induced by DNCB and DNFB, aiming to explore phenomena similar to acute skin diseases such as contact dermatitis, atopic dermatitis, contact allergy, and eczema, providing an effective and rapid detection method for drugs, health foods, and food-based eczema treatments. This invention uses chemical inducers to sensitize and / or challenge zebrafish after egg hatching, respectively simulating the sensitization and challenge phases of the eczema induction mechanism. This model boasts high throughput, low cost, and strong predictive power.

[0007] The specific technical solution of this invention is as follows: A method for constructing a chemically induced zebrafish eczema model includes the following steps: sensitizing zebrafish with a chemical inducer; wherein the zebrafish are post-egg zebrafish, and the chemical inducer is 2,4-dinitrochlorobenzene (DNCB) or 2,4-dinitrofluorobenzene (DNFB), wherein the concentration of DNCB is 0.75~2.25 μg / mL, and the concentration of DNFB is 0.0391~0.625 μg / mL.

[0008] As a preferred embodiment, a method for constructing a chemically induced zebrafish eczema model includes the following steps: sensitizing zebrafish with a chemical inducer, and then stimulating the sensitized zebrafish with a chemical inducer; wherein the zebrafish are post-egg zebrafish, the chemical inducer is DNCB or DNFB, the concentration of DNCB is 0.75~2.25 μg / mL, and the concentration of DNFB is 0.0391~0.625 μg / mL.

[0009] Small-molecule electrophilic haptens such as DNCB and DNFB can covalently bind to epidermal proteins to form neoantigenic epitopes, activate dendritic cells, and induce T-cell-mediated immune responses, thus reproducing eczema-like inflammatory processes in animals. This invention uses DNCB or DNFB as chemical inducers to sensitize or challenge zebrafish, both of which produce pruritus-like behavioral responses, indicating the successful establishment of a zebrafish eczema model.

[0010] The pathogenic mechanism of induced eczema mainly includes two stages: the sensitization phase and the provocation phase. ① During the sensitization phase, DNFB enters the body through the skin, binds to autologous proteins to form neoantigens, and activates epidermal dendritic cells (Langerhans cells). These cells present the antigen to regional lymph nodes, triggering a T cell (especially Th1 / Th2) response. ② During the provocation phase, repeated exposure to DNFB leads to the rapid recruitment of memory T cells to the local skin, inducing an inflammatory response. In this model, T cell-mediated inflammation leads to the release of a large number of inflammatory factors (such as TNF-α, IL-1β, IL-6, IL-4, and TSLP), which in turn activate neutrophils, mast cells, eosinophils, etc., to participate in the response, resulting in clinical manifestations such as redness, swelling, itching, and edema. Therefore, adopting a two-stage induction approach—first using sensitization induction to induce the sensitization phase, and then using provocation induction to induce the provocation phase—better simulates the induction mechanism of eczema.

[0011] This invention, in exploring models using chemical inducers, discovered that zebrafish activity significantly increases with increasing inducer dosage. At a certain dosage, zebrafish aggregate on the sidewalls of cell culture plates and exhibit a "wall-rubbing" phenomenon. Furthermore, tests were conducted to determine if the zebrafish had entered an inflammatory response, revealing that an inflammatory response had already occurred. This result indicates that the aggregation and rubbing of zebrafish on the sidewalls of cell culture plates signifies an inflammatory response. This phenomenon is highly similar to the pruritus caused by eczema. Therefore, observing these behavioral characteristics in zebrafish can determine whether an inflammatory response has occurred. These behavioral characteristics can be directly observed with the naked eye, making modeling simpler, faster, and more efficient.

[0012] Preferably, the concentration of DNFB is 0.312~0.625 μg / mL. When DNFB is used as a chemical inducer, a concentration of 0.312~0.625 μg / mL is more preferred, as the behavior of zebrafish induced by eczema is more pronounced.

[0013] DNCB is the preferred chemical inducer. Using DNCB allows for more precise determination of the effective dosage for drug modeling, and because DNCB is less toxic than DNFB, it offers some protection for researchers. Therefore, DNCB is more preferred as the chemical inducer for constructing zebrafish eczema models.

[0014] Preferably, the concentration of the chemical inducer used for challenge induction is higher than that used for sensitization induction. Induction methods with a higher challenge induction concentration than sensitization induction concentration result in more pronounced itching behavior and inflammatory responses in zebrafish.

[0015] Preferably, the sensitization induction time is 24~48h.

[0016] Preferably, the sensitization induction time is 21-42 hours and the stimulation induction time is 3-6 hours.

[0017] The zebrafish eczema model provided by this invention has high modeling efficiency, with an induction time of only 24-48 hours. Compared with mouse and rat models (7 days of adaptation, more than 14 days of modeling, and more than 21 days of drug administration), the modeling cycle is significantly shortened.

[0018] Preferably, the zebrafish has a strength of 3-7 dpf. More preferably, the zebrafish has a strength of 4-5 dpf.

[0019] Preferably, the zebrafish used are wild-type AB strain zebrafish or transgenic neutrophil-positive fluorescent zebrafish (MPX). Among them, the transgenic neutrophil-positive fluorescent zebrafish can be analyzed under a fluorescence microscope to determine the number of neutrophils, which is convenient for detecting this indicator.

[0020] Another objective of this invention is to provide an application of the chemically induced zebrafish eczema model constructed by the above-described method in product efficacy evaluation.

[0021] The solution specifically includes the following steps: (1) Set up sample groups: Place zebrafish in standard dilution water containing the product and treat them using the method for constructing a chemically induced zebrafish eczema model to prepare sample groups; (2) Set up a model control group. Place zebrafish in standard dilution water and treat them using the method for constructing a chemically induced zebrafish eczema model to make a model control group; (3) Set up a normal control group: zebrafish were placed in standard dilution water to prepare a normal control group; (4) Evaluation tests were conducted on zebrafish in the sample group, model control group and normal control group.

[0022] Preferably, the evaluation tests include one or more of the following: pruritus behavior, trypsin content, and neutrophil count.

[0023] Preferably, the pruritus behavior includes one or more of the following: rapid movement distance, total movement distance, outer circle movement ratio, and rapid movement speed.

[0024] Preferably, zebrafish are placed in a 24-well or 96-well plate for the total movement distance, rapid movement distance, and rapid movement speed evaluation tests, while zebrafish are placed in a 24-well plate for the outer ring movement proportion evaluation test. Replacing the 24-well plate with a 96-well plate for the total movement distance, rapid movement distance, or rapid movement speed evaluation tests can increase the flux and complete the experimental measurements more efficiently and quickly. Furthermore, experiments have shown that zebrafish can also rub against the well walls in a 96-well plate.

[0025] Preferably, the product includes pharmaceuticals, health foods, or food products, and the product is suitable for contact dermatitis, atopic dermatitis, contact allergies, or eczema.

[0026] Compared with the prior art, this application has the following technical effects: (1) The present invention uses chemical inducers DNCB and DNFB to induce sensitization, stimulation, or two-stage induction of sensitization and stimulation in zebrafish after egg breakage, to simulate the sensitization and stimulation phases of the eczema induction mechanism. The repeated inflammatory response produced during the induction process indicates that the model was successfully constructed. The model can accurately characterize eczema symptoms. In particular, the two-stage induction is more consistent with the pathogenesis of eczema, and the model has strong predictive ability. (2) Under specific chemical inducer doses, zebrafish will exhibit the behavior of aggregating on the side wall of cell culture plate and rubbing against the wall. When this behavior occurs, the zebrafish has already developed an inflammatory response. Therefore, it is possible to directly observe whether the zebrafish exhibits the above-mentioned behavior to determine whether the zebrafish has developed an inflammatory response, making the modeling operation simpler and faster. (3) Compared with mouse and rat models (long modeling cycle, 7 days of adaptation, more than 14 days of modeling, and more than 21 days of drug administration), the zebrafish eczema model provided by this invention has high modeling efficiency (the drug administration cycle is within 120 hours), and zebrafish has low cost and zebrafish model detection method is relatively simple. (4) This invention can be used to evaluate the efficacy of drugs, health foods and foods in improving eczema. Attached Figure Description

[0027] Figure 1 Phenotypic diagram of the maximum detectable concentration mortality phenomenon in zebrafish in Example 1, used to explore model conditions.

[0028] Figure 2 This is a schematic diagram of the outer circle range for the outer circle movement ratio indicator.

[0029] Figure 3 To explore the model conditions, Example 1 shows the trajectory of the outer circle movement of zebrafish after sensitization and induction.

[0030] Figure 4 Example 1: Trajectory diagram of the proportion of outer circle movement of zebrafish after sensitization and stimulation induction to explore model conditions.

[0031] Figure 5 Phenotypic diagram of the maximum detectable concentration mortality phenomenon in zebrafish in Example 2, used to explore model conditions.

[0032] Figure 6 To explore the model conditions, Example 2 shows the total movement distance trajectory of zebrafish after sensitization and induction.

[0033] Figure 7 Example 2: Trajectory diagram of the total movement distance of zebrafish after sensitization and stimulation induction to explore model conditions.

[0034] Figure 8 The diagram shows the overall movement trajectory of the zebrafish in Example 1.

[0035] Figure 9 This is a typical diagram of neutrophil count in zebrafish using Example 1.

[0036] Figure 10 The graph shows the trypsin content in zebrafish, as shown in Example 1.

[0037] Figure 11 The diagram shows the overall movement trajectory of the zebrafish in Example 2.

[0038] Figure 12 This is a typical diagram of neutrophil count in zebrafish, as shown in Example 2. Detailed Implementation

[0039] To better understand the content of this invention, further explanation is provided below with reference to specific embodiments.

[0040] The instruments used in the following examples and applications mainly include: dissecting microscope (SZX7); precision electronic balance (CP214); behavior analyzer (Hunter Lab HT-XW-2D-12); motorized focusing continuous zoom fluorescence microscope (AZ100).

[0041] Model Condition Exploration Example 1 (Chemical Inducer: DNFB): (1) Determination of the maximum detectable concentration of DNFB Wild-type AB strain zebrafish (4 dpf) were randomly selected and placed in 6-well plates, with 30 zebrafish treated in each well. DNFB was dissolved in DMSO at concentrations of 0.156, 0.312, 0.625, 1.25, and 2.50 μg / mL. A normal control group was also included. The volume of each well was 3 mL. After treatment at 28℃ for 1 day, the maximum detectable concentration (MTC) of DNFB was determined based on the number of zebrafish deaths and toxicity.

[0042] The test results are shown in Table 1 and Figure 1 : Table 1. Maximum detectable concentration of DNFB (n=30) As shown in Table 1 and Figure 1 As shown, when the concentration of DNFB reached 1.25 and 2.50 μg / mL, 100% mortality occurred in zebrafish. Therefore, under the conditions of this experiment, the MTC of DNFB, the modeling agent for eczema, was 0.625 μg / mL.

[0043] (2) Determination of DNFB sensitization-induced eczema Wild-type AB strain zebrafish at 4 dpf were randomly selected and placed in 6-well plates, with 30 zebrafish treated in each well (experimental group). The groups were: normal control group, and groups treated with DNFB at concentrations of 0.0195, 0.0391, 0.0781, 0.156, 0.312, and 0.625 μg / mL, with continuous stimulation for 24 h. The normal control group received no treatment. After the above stimulation was continued for 4–5 dpf, at the experimental endpoint, 10 zebrafish from each group were randomly selected and placed in 24-well plates. These plates were then placed in a zebrafish behavior analyzer, and data were collected using analysis software to analyze the proportion of zebrafish moving in the outer ring. Statistical results are expressed as mean ± SE. Statistical analysis was performed using SPSS software, and p < 0.05 was considered statistically significant.

[0044] The outer ring is a circular area with a hole radius of 3.26~5.6mm, such as... Figure 2 As shown, the test results are shown in Table 2 and Figure 3 : Table 2 Results of DNFB sensitization-induced eczema (n=10) Compared with the normal control group, **p < 0.01, ***p < 0.001 Zebrafish exhibit rubbing behavior against the zebrafish wall while scratching, thus the outer ring movement corresponds to symptoms of eczema. The proportion of movement in the outer ring can be used to determine whether zebrafish have eczema. (See Table 2 and...) Figure 3 As shown, when DNFB was at concentrations of 0.0391, 0.0781, 0.156, 0.312 and 0.625 μg / mL, a significant increase in the proportion of outer-circle movement in zebrafish was observed, indicating that DNFB could induce eczema behavior in zebrafish under these conditions. The eczema model was not significantly induced at a concentration of 0.0195 μg / mL.

[0045] (3) Determination of DNFB sensitization-induced and challenge-induced (two-stage induction) eczema Wild-type AB strain zebrafish, 4 days post-fertilization (dpf), were randomly selected and placed in 6-well plates, with 30 zebrafish treated in each well (experimental group). Groups were set as follows: normal control group, DNFB sensitization induction (0.625 μg / mL) + challenge induction (0.312 μg / mL), and DNFB sensitization induction (0.312 μg / mL) + challenge induction (0.625 μg / mL). Sensitization induction lasted 21 hours, followed by challenge induction for 3 hours. The normal control group received no treatment. All stimuli were applied for 4–5 days post-fertilization (dpf). At the experimental endpoint, 10 zebrafish from each group were randomly selected and placed in 24-well plates. These plates were then placed in a zebrafish behavior analyzer, and data were collected using analysis software. The percentage of zebrafish exhibiting outer-circle movement was analyzed. Statistical results are expressed as mean ± SE. Statistical analysis was performed using SPSS software; p < 0.05 was considered statistically significant.

[0046] The test results are shown in Table 3 and Figure 4 : Table 3 Results of DNFB sensitization-induced and challenge-induced eczema (n=10) Compared with the normal control group, ***p < 0.001 Compared with DNFB single-stage stimulation of 0.625 μg / mL + two-stage stimulation of 0.312 μg / mL, ### p < 0.001 As shown in Table 3 and Figure 3 As shown, both DNFB sensitization induction (0.625 μg / mL) followed by challenge induction (0.312 μg / mL) and challenge induction (0.625 μg / mL) induced eczema; the eczema induced by DNFB sensitization induction (0.312 μg / mL) followed by challenge induction (0.625 μg / mL) was more pronounced. Comparing the results in Tables 3 and 2, the two induction methods resulted in more severe outer-circumferential movement than a single stimulation induced by sensitization, and the results are more consistent with the mechanistic tendency for symptom development.

[0047] Model Condition Exploration Example 2 (Chemical Inducer: DNCB): (1) Determination of the maximum detectable concentration of DNCB Wild-type AB strain zebrafish, 4 days post-fertilization (dpf), were randomly selected and enclosed in 6-well plates, with 30 zebrafish treated in each well (experimental group). DNCB was dissolved in DMSO at concentrations of 0.750, 1.50, 3.00, 6.00, and 12.0 μg / mL. A normal control group was also included, with a volume of 3 mL per well. After treatment at 28°C for one day, the MTC of DNCB was determined based on the number of zebrafish deaths and toxicity levels.

[0048] The test results are shown in Table 4 and Figure 5 : Table 4. Maximum detectable concentration of DNCB (n=30) As shown in Table 4 and Figure 5 As shown, when the concentration of DNCB reached 6.00 and 12.0 μg / mL, 100% mortality occurred in the zebrafish. When the concentration of DNCB reached 3.00 μg / mL, the zebrafish were in poor condition, specifically exhibiting weakened behavioral abilities. Therefore, under the conditions of this experiment, the MTC of DNCB, the modeling agent for the eczema model, was 1.50 μg / mL.

[0049] (2) Determination of DNCB sensitization-induced eczema Wild-type AB strain zebrafish, 4 days post-fertilization (dpf), were randomly selected and placed in 6-well plates, with 30 zebrafish treated in each well (experimental group). Groups were set as follows: normal control group, and DNCB groups with concentrations of 0.375, 0.750, 1.50, 2.25, and 3.00 μg / mL, with continuous stimulation for 24 hours. The normal control group received no treatment. After 4–5 dpf, at the experimental endpoint, 10 zebrafish from each group were randomly selected and placed in a 96-well plate. These plates were then placed in a zebrafish behavior analyzer, and data were collected using analysis software to analyze the total movement distance, rapid movement distance, and rapid movement speed of the zebrafish. Statistical results are expressed as mean ± SE. Statistical analysis was performed using SPSS software, with p < 0.05 indicating statistical significance.

[0050] The test results are shown in Table 5 and Figure 6 : Table 5 Results of DNCB sensitization-induced eczema (n=10) Compared with the normal control group, *p<0.05, **p < 0.01 As shown in Table 5 and Figure 6 When DNCB was at concentrations of 0.750, 1.50, and 2.25 μg / mL, zebrafish showed a significant increase in total movement distance, indicating that DNCB could induce eczema behavior in zebrafish under these conditions. The eczema model was not significantly induced at concentrations of 0.375 and 3.00 μg / mL.

[0051] (3) Determination of DNCB sensitization-induced and challenge-induced eczema Wild-type AB strain zebrafish, 4 days post-fertilization (dpf), were randomly selected and placed in 6-well plates, with 30 zebrafish treated in each well (experimental group). The groups were: normal control group, DNCB sensitization induction group (0.750 μg / mL + challenge induction group (0.375 μg / mL), DNCB sensitization induction group (0.750 μg / mL + challenge induction group (1.50 μg / mL), and DNCB sensitization induction group (0.750 μg / mL + challenge induction group (2.25 μg / mL)). Sensitization induction lasted 21 hours, followed by challenge induction for 3 hours. The normal control group received no treatment. After 4–5 dpf, at the experimental endpoint, 10 zebrafish from each group were randomly selected and placed in a 96-well plate. The plate was then placed in a zebrafish behavior analyzer, and data were collected using analysis software. The total movement distance, rapid movement distance, and rapid movement speed of the zebrafish were analyzed. Statistical results are expressed as mean ± SE. Statistical analysis was performed using SPSS software; p < 0.05 was considered statistically significant.

[0052] The test results are shown in Table 6 and Figure 7 : Table 6 Results of DNCB sensitization-induced and provocation-induced eczema (n=10) Compared with the normal control group, *p < 0.05, **p < 0.01, ***p < 0.001 As shown in Table 6 and Figure 7 Continuing the sensitization and provocation phases of eczema, a two-stage induction model was used on DNCB. Results showed that eczema was induced in all three groups: DNCB sensitization induction (0.750 μg / mL + provocation induction (0.375 μg / mL)), DNCB sensitization induction (0.750 μg / mL + provocation induction (1.50 μg / mL)), and DNCB sensitization induction (0.750 μg / mL + provocation induction (2.25 μg / mL)). The provocation induction concentration was significantly higher than the sensitization induction concentration, resulting in more significant increases in total movement distance, rapid movement distance, and rapid movement speed, similar to the results of the two-stage DNFB induction in Table 3. Similarly, compared to the results of single sensitization induction in Table 5, the outer-circle movements were more severe in the two-stage induction, better reflecting the mechanistic tendency for symptom development.

[0053] In addition, in Example 2 of the DNCB model condition exploration, when measuring the total motion distance, the 24-well plate was replaced with a 96-well plate, which can increase the throughput and complete the experimental measurement more efficiently and quickly. Furthermore, the experiment found that the 96-well plate can also exhibit zebrafish rubbing against the plate.

[0054] The relevant parameters for model construction were determined through conditional exploration experiments, and the model was constructed based on these parameters.

[0055] When detecting the number of neutrophils in chemically induced zebrafish, the zebrafish used in Examples 1-6 and Comparative Examples 1-4 were 4dpf transgenic green fluorescent neutrophil zebrafish (MPX). For other detection indicators, such as trypsin content, 4dpf wild-type AB strain zebrafish were sufficient.

[0056] Example 1 A method for constructing a chemically induced zebrafish eczema model includes the following steps: (1) Dissolve DNFB in dimethyl sulfoxide (DMSO), and then take a small amount and dilute it in standard dilution water to prepare a chemical inducer (concentration of 0.312 μg / mL). (2) Add 3 mL of chemical inducer to a 6-well plate, and then select 30 zebrafish (4 dpf) after egg breakage and place them in a 6-well plate for sensitization induction; (3) Add 3 mL of chemical inducer to a 6-well plate, then wash the sensitized zebrafish and add them to the 6-well plate for stimulation induction; observe the behavioral characteristics of the zebrafish. Both sensitization and stimulation induction produce the behavior characteristics of zebrafish gathering and rubbing against the side wall of the 6-well plate, which means that the chemical-induced zebrafish eczema model is successfully modeled.

[0057] Example 2 A method for constructing a chemically induced zebrafish eczema model includes the following steps: (1) Dissolve DNFB in DMSO, and then take a small amount to dilute in standard water to prepare chemical inducer 1 (DNFB concentration of 0.312 μg / mL) and chemical inducer 2 (DNFB concentration of 0.625 μg / mL). (2) Add 3 mL of chemical inducer 1 to a 6-well plate, and then select 30 zebrafish (4dpf) after egg breakage and place them in a 6-well plate for sensitization induction; (3) Add 3 mL of chemical inducer 2 to a 6-well plate, then wash the sensitized zebrafish and add them to the 6-well plate for stimulation induction; observe the behavioral characteristics of the zebrafish. Both sensitization and stimulation induction produce the behavior characteristics of zebrafish gathering and rubbing against the side wall of the 6-well plate, which means that the chemically induced zebrafish eczema model is successfully modeled.

[0058] Example 3 A method for constructing a chemically induced zebrafish eczema model includes the following steps: (1) Dissolve DNFB in DMSO, and then take a small amount to dilute in standard water to prepare chemical inducer 1 (DNFB concentration of 0.312 μg / mL) and chemical inducer 2 (DNFB concentration of 0.781 μg / mL). (2) Add 3 mL of chemical inducer 1 to a 6-well plate, and then select 30 zebrafish (4dpf) after egg breakage and place them in a 6-well plate for sensitization induction, and observe the behavioral characteristics of the zebrafish. (3) Add 3 mL of chemical inducer 2 to a 6-well plate, then wash the sensitized zebrafish and add them to the 6-well plate for stimulation and induction, and observe the behavioral characteristics of the zebrafish. Both sensitization and stimulation induction produce the behavior characteristics of zebrafish gathering and rubbing against the side wall of the 6-well plate, which means that the chemical-induced zebrafish eczema model is successfully modeled.

[0059] Example 4 A method for constructing a chemically induced zebrafish eczema model includes the following steps: (1) Dissolve DNCB in DMSO, then take a small amount and dilute it in standard dilution water to prepare chemical inducer 1 (DNCB concentration of 0.750 μg / mL) and chemical inducer 2 (DNCB concentration of 0.375 μg / mL). (2) Add 3 mL of chemical inducer 1 to a 6-well plate, and then select 30 zebrafish (4dpf) after egg breakage and place them in a 6-well plate for sensitization induction, and observe the behavioral characteristics of the zebrafish. (3) Add 3 mL of chemical inducer 2 to a 6-well plate, then wash the sensitized zebrafish and add them to the 6-well plate for stimulation and induction, and observe the behavioral characteristics of the zebrafish. Both sensitization and stimulation induction produce the behavior characteristics of zebrafish gathering and rubbing against the side wall of the 6-well plate, which means that the chemical-induced zebrafish eczema model is successfully modeled.

[0060] Example 5 A method for constructing a chemically induced zebrafish eczema model includes the following steps: (1) Dissolve DNCB in DMSO, then take a small amount and dilute it in standard dilution water to prepare chemical inducer 1 (DNCB concentration of 0.75 μg / mL) and chemical inducer 2 (DNCB concentration of 1.5 μg / mL). (2) Add 3 mL of chemical inducer 1 to a 6-well plate, and then select 30 zebrafish (4dpf) after egg breakage and place them in a 6-well plate for sensitization induction, and observe the behavioral characteristics of the zebrafish. (3) Add 3 mL of chemical inducer 2 to a 6-well plate, then wash the sensitized zebrafish and add them to the 6-well plate for stimulation and induction, and observe the behavioral characteristics of the zebrafish. Both sensitization and stimulation induction produce the behavior characteristics of zebrafish gathering and rubbing against the side wall of the 6-well plate, which means that the chemical-induced zebrafish eczema model is successfully modeled.

[0061] Example 6 A method for constructing a chemically induced zebrafish eczema model includes the following steps: (1) Dissolve DNCB in DMSO, then take a small amount and dilute it in standard dilution water to prepare chemical inducer 1 (DNCB concentration of 0.75 μg / mL) and chemical inducer 2 (DNCB concentration of 2.25 μg / mL). (2) Add 3 mL of chemical inducer 1 to a 6-well plate, select 30 zebrafish (4dpf) after egg hatching and place them in a 6-well plate for sensitization induction, and observe the behavioral characteristics of the zebrafish; (3) Add 3 mL of chemical inducer 2 to a 6-well plate, then wash the sensitized zebrafish and add them to the 6-well plate for stimulation and induction, and observe the behavioral characteristics of the zebrafish. Both sensitization and stimulation induction produce the behavior characteristics of zebrafish gathering and rubbing against the side wall of the 6-well plate, which means that the chemical-induced zebrafish eczema model is successfully modeled.

[0062] Comparative Example 1 The difference between Comparative Example 1 and Example 2 is that DMSO of equal mass was used instead of the chemical inducing agent, while all other conditions were the same as in Example 2.

[0063] Comparative Example 2 The difference between Comparative Example 2 and Example 6 is that DMSO of equal mass was used instead of the chemical inducing agent, while all other conditions were the same as in Example 6.

[0064] Comparative Example 3 The difference between Comparative Example 3 and Example 1 is that no stimulation induction was performed.

[0065] The specific steps are as follows: (1) Dissolve DNFB in DMSO, then take a small amount and dilute it in standard dilution water to prepare a chemical inducer (concentration of 0.312 μg / mL). (2) Add 3 mL of chemical inducer to a 6-well plate, and then select 30 zebrafish (4dpf) after egg breakage and place them in the 6-well plate for sensitization induction; observe the behavioral characteristics of zebrafish. The behavior characteristics of zebrafish that are sensitized and induced to gather and rub against the side wall of the 6-well plate are the successful modeling of chemically induced zebrafish eczema.

[0066] Comparative Example 4 The difference between Comparative Example 4 and Example 4 is that no excitation induction was performed.

[0067] The specific steps are as follows: (1) Dissolve DNCB in DMSO, then take a small amount and dilute it in standard dilution water to prepare a chemical inducer (concentration of 0.312 μg / mL). (2) Add 3 mL of chemical inducer to a 6-well plate, and then select 30 zebrafish (4dpf) after egg breakage and place them in the 6-well plate for sensitization induction; observe the behavioral characteristics of zebrafish. The behavior characteristics of zebrafish that are sensitized and induced to gather and rub against the side wall of the 6-well plate are the successful modeling of chemically induced zebrafish eczema.

[0068] The number of neutrophils and the content of trypsin in zebrafish chemically induced in Examples 1-6 and Comparative Examples 1-4 were determined. Ten zebrafish were randomly selected and placed under a zebrafish fluorescence microscope. Data were collected using analysis software, and the number of neutrophils in the zebrafish was analyzed. The content of trypsin was tested using a zebrafish trypsin kit. Statistical results are expressed as mean ± SE. Statistical analysis was performed using SPSS software, and p < 0.05 indicated statistical significance. The test results are shown in Table 7. Table 7 Test results of Examples 1-6 and Comparative Examples 1-4 (n=10) Compared with Comparative Example 1, Examples 1-3 showed **p < 0.01 and ***p < 0.001; Examples 4-6 showed ***p < 0.001 compared with Comparative Example 1; Comparative Example 3 showed *p < 0.05 compared with Examples 1-3; Comparative Example 4 showed **p < 0.01 compared with Examples 4-6. As shown in Table 7, Examples 1-3 use DNFB as a chemical inducer, Examples 4-6 use DNCB as a chemical inducer, and Comparative Examples 1-2 use an equal amount of DMSO as a chemical inducer. Analysis of the results of Examples 1-6 and Comparative Examples 1-2 shows that using DNFB or DNCB as chemical inducers for sensitization and challenge induction significantly increases the number of neutrophils and the content of trypsin in zebrafish. This indicates that the two-stage stimulation of sensitization and challenge induction using DNFB or DNCB as chemical inducers can induce an inflammatory response in zebrafish.

[0069] Comparative Examples 3-4 demonstrate techniques for sensitization induction using DNFB or DNCB. Following sensitization with DNFB or DNCB, zebrafish also exhibited significant inflammatory responses. Comparing the results of Examples 1, 4, and Comparative Examples 3-4, it was found that the inflammatory responses in zebrafish after both sensitization and provocation stimulation in Examples 1 and 4 were highly significant. These results indicate that the two stimulation stages of sensitization and provocation stimulation better simulate the sensitization and provocation phases of zebrafish eczema, resulting in a more significant inflammatory response.

[0070] Comparing Examples 1-3 or Examples 4-6, it is evident that under certain dosage conditions of chemical inducers, the inflammatory response in zebrafish is more pronounced when the concentration of the chemical inducer used for challenge induction is higher than that used for sensitization induction. Therefore, using an induction method with a higher concentration for challenge induction than for sensitization induction is more beneficial for constructing a zebrafish eczema model.

[0071] The following section explains the uses of the zebrafish eczema model in conjunction with product efficacy evaluation.

[0072] Application Example 1 (1) Evaluation of the efficacy of loratadine in improving eczema - pruritus behavior S1. Wild-type AB strain zebrafish (4 dpf) were randomly selected and placed in 6-well plates, with 30 zebrafish treated in each well. Three wells were designated as sample groups, receiving loratadine at concentrations of 93.8, 187.5, and 375 ng / mL, respectively, labeled as sample group A, sample group B, and sample group C. A normal control group and a model control group were also set up, with a volume of 3 mL per well. Except for the normal control group, which was treated with DMSO for 21 h and 3 h, all other experimental groups were treated with 0.75 μg / mL DNCB (DNCB was dissolved in DMSO, and a small amount was then added to standard dilution water for zebrafish, with a final DNCB concentration of 0.75 μg / mL) at 28℃ for 21 h. Afterward, the zebrafish were washed and treated with 2.25 μg / mL DNCB for 3 h to establish a zebrafish eczema model. S2. At the experimental endpoint, 10 zebrafish were randomly selected from each group and placed in a 96-well plate. The plate was then placed in a zebrafish behavior analyzer, and data was collected using analysis software. The distance of the zebrafish's rapid movement was analyzed, and the statistical analysis results of this index were used to evaluate the efficacy of loratadine in improving eczema.

[0073] (2) Evaluation of the efficacy of loratadine in improving eczema - neutrophil count S1. Four-day-first-flush (dpf) transgenic neutrophil-positive green fluorescent zebrafish (MPX) were randomly selected and placed in 6-well plates, with 30 zebrafish treated in each well. Three wells were designated as sample groups, receiving loratadine at concentrations of 93.8, 187.5, and 375 ng / mL, respectively, and labeled as sample group A, sample group B, and sample group C. A normal control group and a model control group were also included, with a volume of 3 mL per well. Except for the normal control group, which was treated with DMSO for 21 h and 3 h, all other experimental groups were treated with 0.75 μg / mL DNCB at 28℃ for 21 h. Following this, the zebrafish were washed and treated with 2.25 μg / mL DNCB for 3 h to establish a zebrafish eczema model. S2. At the experimental endpoint, 10 zebrafish were randomly selected from each group and placed under a zebrafish fluorescence microscope. Data were collected using analysis software, and the number of neutrophils in the zebrafish was analyzed. The statistical analysis results of this indicator were used to evaluate the efficacy of loratadine in improving eczema.

[0074] (3) Evaluation of the efficacy of loratadine in improving eczema - trypsin content S1. Wild-type AB strain zebrafish (4 dpf) were randomly selected and placed in 6-well plates, with 30 zebrafish treated in each well. Three wells were designated as sample groups, receiving loratadine at concentrations of 93.8, 187.5, and 375 ng / mL, respectively, and labeled as sample group A, sample group B, and sample group C. A normal control group and a model control group were also set up, with a volume of 3 mL per well. Except for the normal control group, which was treated with DMSO for 21 h and 3 h, all other experimental groups were treated with 0.75 μg / mL DNCB at 28℃ for 21 h. Following this, the zebrafish were washed and treated with 2.25 μg / mL DNCB for 3 h to establish a zebrafish eczema model. S2. At the experimental endpoint, zebrafish from each group were collected, and the overall zebrafish trypsin content was determined using a zebrafish trypsin kit. The statistical analysis results of this index were used to evaluate the efficacy of loratadine in improving eczema.

[0075] The experimental results evaluating the efficacy of loratadine in improving eczema are shown in Table 8 and... Figures 8-10 Statistical results are expressed as mean ± SE. Statistical analysis was performed using SPSS software, and p < 0.05 was considered statistically significant.

[0076] Table 8. Experimental results evaluating the efficacy of loratadine in improving eczema (n=10) Compared with the model control group, ***p < 0.001 Movement distance reflects the degree of itching felt by zebrafish. The higher the degree of itching, the greater the total movement distance of the zebrafish. Therefore, by testing the trypsin content, neutrophil count, and total movement distance of zebrafish, the condition of zebrafish with eczema can be accurately reflected.

[0077] As shown in Table 8 and Figure 8-10 As shown, the total movement distance, trypsin content, and neutrophil content were significantly increased in the model control group compared with the normal control group. This result indicates that the model control group produced a strong eczema reaction, indicating that the zebrafish eczema model was successfully constructed.

[0078] Compared with the model control group, sample group A showed no significant changes in total running distance, trypsin content, and neutrophil content. This result indicates that loratadine at a dose of 93.8 ng / mL could not significantly improve zebrafish eczema. Compared with the model control group, sample groups B and C showed significantly reduced total running distance, trypsin content, and neutrophil content. Compared with the normal control group, sample groups B and C showed no significant changes in these parameters. This result indicates that loratadine at doses of 187.5 ng / mL and above can significantly alleviate zebrafish eczema symptoms and restore them to a normal state.

[0079] In summary, loratadine is effective in improving eczema, and administration of a certain dose can restore zebrafish to a normal state. Therefore, the chemically induced zebrafish eczema model can be used to evaluate the efficacy of loratadine.

[0080] Application Example 2 (1) Evaluation of the efficacy of prednisone in improving eczema - pruritus behavior S1. Wild-type AB strain zebrafish (4 dpf) were randomly selected and placed in 6-well plates, with 30 zebrafish treated in each well. Three wells were designated as sample groups, treated with prednisone at concentrations of 7.50, 15.0, and 30.0 μg / mL, respectively, and labeled as sample group A, sample group B, and sample group C. A normal control group and a model control group were also set up, with a volume of 3 mL per well. Except for the normal control group, which was treated with DMSO for 21 h and 3 h, all other experimental groups were treated with 0.75 μg / mL DNCB at 28℃ for 21 h. The zebrafish were then washed and treated with 2.25 μg / mL DNCB for 3 h to establish a zebrafish eczema model. S2. At the experimental endpoint, 10 zebrafish were randomly selected from each group and placed in a 96-well plate. The plate was then placed in a zebrafish behavior analyzer, and data was collected using analysis software. The distance of the zebrafish's rapid movement was analyzed, and the statistical analysis results of this indicator were used to evaluate the efficacy of prednisone in improving eczema.

[0081] (2) Evaluation of the efficacy of prednisone in improving eczema - neutrophil count S1. Four-day-first-fly (dpf) transgenic neutrophil-positive green fluorescent zebrafish (MPX) were randomly selected and placed in 6-well plates, with 30 zebrafish treated in each well. Three wells were designated as sample groups, receiving prednisone at concentrations of 7.50, 15.0, and 30.0 μg / mL, respectively, and labeled as sample group A, sample group B, and sample group C. A normal control group and a model control group were also included, with a volume of 3 mL per well. Except for the normal control group, which was treated with DMSO for 21 h and 3 h, all other experimental groups were treated with 0.75 μg / mL DNCB at 28℃ for 21 h. Following this, the zebrafish were washed and treated with 2.25 μg / mL DNCB for 3 h to establish a zebrafish eczema model. S2. At the experimental endpoint, 10 zebrafish were randomly selected from each group and placed under a zebrafish fluorescence microscope. Data were collected using analysis software, and the number of neutrophils in the zebrafish was analyzed. The statistical analysis results of this indicator were used to evaluate the efficacy of prednisone in improving eczema.

[0082] The experimental results evaluating the efficacy of prednisone in improving eczema are shown in Table 9 and Figure 11 , Figure 12 Statistical results are expressed as mean ± SE. Statistical analysis was performed using SPSS software, and p < 0.05 was considered statistically significant.

[0083] Table 9. Experimental results evaluating the efficacy of prednisone in improving eczema (n=10) Compared with the model control group, *p<0.05, ***p < 0.001 As shown in Table 9, Figure 11 and Figure 12 As shown, the total movement distance and neutrophil content were significantly higher in the model control group compared with the normal control group. This result indicates that the model control group produced a strong eczema reaction, indicating that the zebrafish eczema model was successfully constructed.

[0084] Compared with the model control group, sample group A showed no significant changes in total running distance and neutrophil content, indicating that a prednisone dose of 7.5 μg / mL did not significantly improve zebrafish eczema. Sample group B showed significantly decreased total running distance and neutrophil content compared with the model control group, but significantly increased total running distance and neutrophil content compared with the normal control group. This indicates that a prednisone dose of 15 μg / mL could significantly alleviate zebrafish eczema, but could not restore the zebrafish to a normal state. Sample group C showed significantly decreased total running distance and neutrophil content compared with the model control group, but no significant changes compared with the normal control group. This indicates that a prednisone dose of 30 μg / mL could significantly alleviate zebrafish eczema and restore the zebrafish to a normal state.

[0085] In summary, prednisone has the effect of improving eczema, and administering a certain dose can restore zebrafish to a normal state. Therefore, the chemically induced zebrafish eczema model can be used to evaluate the efficacy of prednisone.

[0086] Application Example 3 Evaluation of the efficacy of dexamethasone in improving eczema - pruritus behavior S1. Wild-type AB strain zebrafish (4 dpf) were randomly selected and placed in 6-well plates, with 30 zebrafish treated in each well. Three wells were designated as sample groups, treated with dexamethasone at concentrations of 50, 100, and 200 μM, respectively, and labeled as sample group A, sample group B, and sample group C. A normal control group and a model control group were also set up, with a volume of 3 mL per well. Except for the normal control group, which was treated with DMSO for 21 h and 3 h, all other experimental groups were treated with 0.312 μg / mL DNFB at 28℃ for 21 h. The zebrafish were then washed and treated with 0.625 μg / mL DNFB for 3 h to establish a zebrafish eczema model. S2. At the experimental endpoint, 10 zebrafish were randomly selected from each group and placed in a 24-well plate. The plate was then placed in a zebrafish behavior analyzer, and data was collected using analysis software. The percentage of zebrafish moving in the outer circle was analyzed, and the statistical analysis results of this indicator were used to evaluate the efficacy of prednisone in improving eczema.

[0087] The results of the experiment evaluating the efficacy of dexamethasone in improving eczema are shown in Table 10. Statistical results are expressed as mean ± SE. Statistical analysis was performed using SPSS software, and p < 0.05 indicated statistical significance.

[0088] Table 10. Experimental results evaluating the efficacy of dexamethasone in improving eczema (n=10) Compared with the model control group, *p<0.05, **p < 0.01 As shown in Table 10, the proportion of outer circle movement was significantly higher in the model control group compared with the normal control group. This result indicates that the model control group produced a strong eczema reaction, indicating that the zebrafish eczema model was successfully constructed.

[0089] Compared with the model control group, the proportion of outer ring movement in sample group A showed no significant change, indicating that a dose of dexamethasone at 50 μM could not significantly improve zebrafish eczema. Compared with the model control group, the proportion of outer ring movement in sample group B was significantly decreased; compared with the normal control group, the proportion of outer ring movement in sample group B was significantly increased. This indicates that a dose of prednisone at 100 μM could significantly alleviate zebrafish eczema, but could not restore the zebrafish to a normal state. Compared with the model control group, the proportion of outer ring movement in sample group C was significantly decreased; compared with the normal control group, the proportion of outer ring movement in sample group C was not significantly changed. This indicates that a dose of dexamethasone at 200 μM could significantly alleviate zebrafish eczema and restore the zebrafish to a normal state.

[0090] In summary, dexamethasone is effective in improving eczema, and administering a certain dose can restore zebrafish to a normal state. Therefore, the chemically induced zebrafish eczema model can be used to evaluate the efficacy of dexamethasone.

Claims

1. A method for constructing a chemically induced zebrafish eczema model, characterized by, Includes the following steps: Zebrafish were sensitized using a chemical inducer; the zebrafish were post-egg zebrafish, and the chemical inducer was 2,4-dinitrofluorobenzene or 2,4-dinitrochlorobenzene, with the concentration of 2,4-dinitrofluorobenzene being 0.0391~0.625 μg / mL and the concentration of 2,4-dinitrochlorobenzene being 0.75~2.25 μg / mL.

2. A method for constructing a chemical-induced eczema model of zebrafish, characterized in that, Includes the following steps: Zebrafish were sensitized using a chemical inducer, and then the sensitized zebrafish were challenged using the same chemical inducer. The zebrafish were post-egg zebrafish. The chemical inducer was 2,4-dinitrofluorobenzene or 2,4-dinitrochlorobenzene, with the concentration of 2,4-dinitrofluorobenzene ranging from 0.0391 to 0.625 μg / mL and the concentration of 2,4-dinitrochlorobenzene ranging from 0.75 to 2.25 μg / mL.

3. The method of construction according to claim 2, wherein, The concentration of the chemical inducer used for stimulation induction is higher than that used for sensitization induction.

4. The method of construction of claim 1 wherein, The sensitization induction time is 24~48h.

5. The construction method according to claim 2 or 3, characterized in that, The sensitization induction time is 21-42 hours, and the challenge induction time is 3-6 hours.

6. The use of the chemical-induced zebrafish eczema model constructed according to the construction method of any one of claims 1-5 in product efficacy evaluation. Includes the following steps: (1) Set up sample groups: Place zebrafish in standard dilution water containing the product and treat them using the method for constructing a chemically induced zebrafish eczema model to prepare sample groups; (2) Set up a model control group. Place zebrafish in standard dilution water and treat them using the method for constructing a chemically induced zebrafish eczema model to make a model control group; (3) Set up a normal control group: zebrafish were placed in standard dilution water to prepare a normal control group; (4) Evaluation tests were conducted on zebrafish in the sample group, model control group and normal control group.

7. Use according to claim 6, characterized in that, Evaluation tests include one or more of the following: pruritus behavior, trypsin levels, and neutrophil count.

8. Use according to claim 7, characterized in that, The pruritus behavior includes one or more of the following: total movement distance, rapid movement distance, rapid movement speed, and the proportion of outer circle movement.

9. Use according to claim 8, characterized in that, When evaluating the total movement distance, rapid movement distance, and rapid movement speed, zebrafish were placed on a 24-well or 96-well plate. When evaluating the proportion of movement in the outer ring, zebrafish were placed on a 24-well plate.

10. The use according to claim 6, characterized in that The products include pharmaceuticals, health foods, or food products, and are suitable for contact dermatitis, atopic dermatitis, contact allergies, or eczema.

Images