A method for establishing a psoriasis model of zebrafish and application thereof

Abstract

This invention specifically relates to a method for establishing and applying a zebrafish psoriasis model. The technical solution of this invention utilizes the characteristics of zebrafish—short developmental cycle, ease of rearing, and convenient live observation—effectively shortening the experimental cycle and reducing modeling and screening costs. Simultaneously, this invention establishes standardized induction conditions and quantitative evaluation indicators, improving the stability of disease model construction and the accuracy of drug efficacy evaluation. It provides a live model tool that conforms to the 3R principles of animal protection for research on the pathogenesis of psoriasis and high-throughput preclinical screening of related drugs.

Description

Technical Field

[0001] This invention relates to a method for establishing and applying a zebrafish psoriasis model, belonging to the field of biomedical experimental animal model technology. Background Technology

[0002] Psoriasis is a common and relapsing chronic inflammatory skin disease, characterized by abnormal epidermal apoptosis, oxidative stress imbalance, and extensive infiltration of immune cells. With the accelerated pace of modern life and environmental changes, the incidence of psoriasis is increasing year by year. Current clinical treatments remain limited, and the disease is prone to relapse. Therefore, elucidating the pathogenesis of psoriasis and developing highly effective and safe novel targeted drugs are urgent clinical needs.

[0003] Animal models play a crucial role in preclinical research and drug screening for psoriasis. Currently, the most commonly used model is the imiquimod (IMQ)-induced mouse psoriasis model, but this model has several significant limitations: (1) There are deviations in pathological simulation: The IMQ mouse model can only simulate acute innate immune response and cannot truly replicate the chronic adaptive immunopathological process of human psoriasis.

[0004] (2) The experiment has a long cycle, high cost and ethical restrictions: the breeding and raising of mice has a long cycle, high hardware investment, and the use of a large number of mammals for large-scale initial screening faces complex ethical review, which does not comply with the 3R principle of animal experiments (replace, reduce, optimize).

[0005] (3) Limited live observation: Mouse skin is opaque, making it difficult to observe microscopic pathological processes in real time and dynamically in a live state. Usually, animals need to be sacrificed for section staining, which is cumbersome and lacks continuous data.

[0006] Zebrafish (Danio rerio), as an emerging model organism, possesses natural advantages for constructing skin disease models. It shares 87% genetic homology with humans, and its oxidative stress and inflammation regulation pathways are highly conserved. Zebrafish have an extremely short developmental cycle; within 48 hours of fertilization, they can form a complete epidermal structure containing core components such as keratinocytes and immune cells. Furthermore, zebrafish embryos and juveniles are completely transparent, making them ideal for high-throughput drug screening and in vivo dynamic monitoring when combined with fluorescent labeling technology.

[0007] Despite the great potential shown by zebrafish, there is currently no mature and standardized zebrafish psoriasis model in this field, and existing exploratory studies have the following significant limitations: (1) Lack of clear construction parameters and standards: The induction conditions in existing studies are too broad and the parameters are unclear. Imiquimod has certain toxicity, and subtle changes in induction concentration and exposure time can easily lead to a surge in mortality or phenotypic loss in zebrafish, resulting in poor reproducibility and unstable mortality in existing models, making it impossible to stably replicate the core pathological phenotype of psoriasis.

[0008] (2) The evaluation system is too simple and lacks a single morphological observation: the existing exploration methods rely on single morphological observation and lack quantitative evaluation indicators for multiple dimensions such as epidermal apoptosis, oxidative stress and inflammatory factors, which makes it impossible to accurately verify the effectiveness of the model and the effect of drug intervention.

[0009] (3) Insufficient practicality: Due to the lack of a unified standard in the construction process and the difficulty in balancing the contradiction between "significant pathological features" and "high survival rate", the existing methods simply cannot meet the needs of high-throughput drug screening and large-scale mechanism research in modern new drug development.

[0010] In summary, how to find precise induction conditions, establish a standardized, stable zebrafish psoriasis model with a multi-dimensional quantitative evaluation system, and use it as an efficient alternative to mouse models is a technical challenge that urgently needs to be overcome in this field. Summary of the Invention

[0011] To address the aforementioned problems, this invention provides a method for establishing and applying a zebrafish psoriasis model.

[0012] The technical solution of the present invention is as follows: In a first aspect, the present invention provides a method for establishing a zebrafish psoriasis model, comprising the following steps: (1) Obtain normally developing zebrafish embryos; (2) The zebrafish embryos were placed in an embryo culture medium containing an inducing drug for exposure culture; The zebrafish embryos were those 24-72 hours after fertilization; The inducing agent includes imiquimod; The final concentration of imiquimod in the embryo culture medium is 1 μM-10 μM; The exposure culture temperature is 25℃-31℃, and the exposure culture time is 12h-48h.

[0013] This application provides a method for establishing a zebrafish psoriasis model and its application system. The technical solution of this invention utilizes the characteristics of zebrafish—short development cycle, ease of rearing, and convenient live observation—effectively shortening the experimental cycle and reducing modeling and screening costs. Simultaneously, this invention establishes standardized induction conditions and quantitative evaluation indicators, improving the stability of disease model construction and the accuracy of drug efficacy evaluation. It provides a live model tool that conforms to the 3R principles of animal protection for the study of psoriasis pathogenesis and high-throughput preclinical screening of related drugs.

[0014] Preferably, the zebrafish embryo is an embryo that is 44-52 hours after fertilization.

[0015] Preferably, the final concentration of imiquimod in the embryo culture medium is 4 μM-7 μM.

[0016] Preferably, the exposure culture time is 20h-28h.

[0017] Preferably, the temperature of the exposure culture is 27.5℃-28.5℃, and the exposure culture is carried out under light-protected conditions.

[0018] Preferably, the embryo culture medium further includes an organic solvent; Preferably, the organic solvent is dimethyl sulfoxide, and its volume fraction in the embryo culture medium is 0.05%-0.15%.

[0019] Preferably, in the step of obtaining normally developed zebrafish embryos, the screening criteria for zebrafish embryos include: intact body segments, well extended tail, no morphological deformities, and normal heartbeat and blood circulation.

[0020] Secondly, the present invention provides the application of the zebrafish psoriasis model obtained by the method described in the first aspect in psoriasis drug screening, psoriasis pathological mechanism research, or psoriasis treatment target verification.

[0021] Utilizing the transparent physiological characteristic of zebrafish juveniles, and combining acridine orange (AO) staining, ROS fluorescent probes, and qRT-PCR detection techniques, a multi-dimensional quantitative evaluation system was constructed, encompassing epidermal cell apoptosis levels, oxidative stress status, and inflammatory gene expression. This system enables real-time observation and quantitative analysis of disease phenotypes and drug intervention effects in vivo, reducing cumbersome steps such as traditional tissue sectioning and improving the objectivity and screening efficiency of data collection.

[0022] Preferably, in the screening of psoriasis drugs or the validation of psoriasis treatment targets, the efficacy of the drug or the effectiveness of the target is evaluated by detecting at least one of the following quantitative indicators: 1) The number of apoptotic cells in the epidermal region; 2) Oxidative stress-activated oxygen levels in the epidermal region; 3) Expression levels of psoriasis-related inflammatory factor mRNAs.

[0023] Preferably, the psoriasis-associated inflammatory factors are selected from any one or a combination of at least two of TNF-α, IL-1β and IL-6.

[0024] Compared with the prior art, the present invention has the following technical effects: This invention utilizes the short developmental cycle, ease of rearing, and convenient live observation characteristics of zebrafish, effectively shortening the experimental cycle and reducing modeling and screening costs. Simultaneously, this invention establishes standardized induction conditions and quantitative evaluation indicators, improving the stability of disease model construction and the accuracy of drug efficacy evaluation. It provides a live model tool that conforms to the 3R principles of animal protection for the study of psoriasis pathogenesis and high-throughput preclinical screening of related drugs. Attached Figure Description

[0025] Figure 1 Fluorescence staining image of reactive oxygen species (ROS) levels in the epidermis of zebrafish, which is the blank control group; Figure 2 Fluorescent staining image of reactive oxygen species (ROS) levels in the epidermis of zebrafish in the model group; Figure 3 A statistical analysis of reactive oxygen species (ROS) levels in the epidermis of zebrafish in the model group and the blank control group; Figure 4 Statistical graph of tumor necrosis factor-α (TNF-α) expression levels in zebrafish from the model group and the blank control group; Figure 5 A statistical graph showing the expression levels of interleukin-1β (IL-1β) in zebrafish from the model group and the blank control group; Figure 6 The graph shows the expression of interleukin-6 (IL-6) in zebrafish from the model group and the blank control group. Detailed Implementation

[0026] The principles and features of the present invention are described below with reference to the accompanying drawings. The examples given are only for explaining the present invention and are not intended to limit the scope of the present invention.

[0027] The experimental materials for this invention were obtained from the following sources: (1) Laboratory animals: Wild-type AB lineage zebrafish, sourced from Qingdao Benyue Biotechnology Co., Ltd.

[0028] (2) Main reagents: Imiquimod, purchased from Shanghai Maclean Biotechnology Co., Ltd., product number: I838239; Embryo culture medium, E3 embryo medium (5 mM NaCl, 0.17 mM KCl, 0.33 mM CaCl2, 0.33 mM MgSO4); Dimethyl sulfoxide (DMSO), purchased from Beijing Solarbio Technology Co., Ltd. Acridine Orange (AO), purchased from Shanghai Yuanye Biotechnology Co., Ltd.; DCFH-DA probe, purchased from Beijing Solarbio Technology Co., Ltd. RNA extraction kit, purchased from Nanjing Novizan Biotechnology Co., Ltd., product number: A112-1-1; The reverse transcription kit was purchased from Toyobo (Shanghai) Biotechnology Co., Ltd., catalog number: FSQ-201; The real-time PCR kit was purchased from Toyobo (Shanghai) Biotechnology Co., Ltd., catalog number: QPK-201.

[0029] (3) Main instruments and software: Zebrafish standard aquaculture system, purchased from Qingdao Jinshui Marine Biological Equipment Co., Ltd., model: Zebrafish Aquaculture System-5; Stereo microscope, purchased from Anhui Naike Vision Technology Co., Ltd., model: JSZ6; Fluorescence microscope, purchased from Anhui Naike Vision Technology Co., Ltd., model: NSZ818; The quantitative PCR instrument, model FQD-96A, was purchased from Hangzhou Borui Technology Co., Ltd. ImageJ image analysis software, ImageJ v1.54p version Example 1 (1) Zebrafish rearing and embryo collection Healthy wild-type AB-type zebrafish were selected and housed in a standard aquaculture system. The following conditions were maintained: water temperature 28.0±0.5℃, pH 7.2-7.5, dissolved oxygen ≥6mg / L, photoperiod of 14h light / 10h dark, and feeding with brine shrimp larvae twice daily to maintain clean and stable water quality. After 1-2 months of rearing until sexual maturity, female to male zebrafish were paired at a 1:2 ratio and placed in a breeding tank. The following morning, fertilized eggs were collected, washed three times with standard embryo culture medium, and unfertilized eggs and deformed embryos were removed. The eggs were then placed in petri dishes and statically cultured in a 28℃ incubator.

[0030] (2) Screening of zebrafish embryos Once the embryos have developed to 48 hpf (48 hours after fertilization), normally developing embryos are selected under a stereomicroscope: zebrafish embryos with intact body segments, well-extended tails, no morphological deformities, and normal heartbeat and blood circulation are selected as experimental subjects, and individuals with delayed development and abnormal morphology are removed to ensure the uniformity of the experimental samples.

[0031] (3) Exploration of induced drug concentration and exposure period The optimal induction conditions were determined by exploring the concentration and time gradients of the psoriasis-inducing drug imiquimod. (3.1) Concentration gradient exploration: 48 hpf zebrafish embryos were randomly divided into a control group and an experimental group. The control group was given embryo culture medium containing 0.1% DMSO, while the experimental groups were given embryo culture medium containing 1 μM, 2 μM, 4 μM, 5 μM, 7 μM, and 10 μM of inducing drugs, respectively. Each group had three replicates, with 30 embryos per replicate, and the embryos were cultured at 28℃. After 24 h of exposure, the results showed that the embryo survival rate was ≥80% within the 4-7 μM range, consistent with the pathological characteristics of psoriasis. Further narrowing the concentration gradient to 4 μM, 5 μM, and 6 μM for repeated experiments, the results showed that at a concentration of 5 μM, the embryo survival rate reached over 95%, and the levels of epidermal apoptosis and oxidative stress were stable and closest to the pathological state of human psoriasis, thus determining this as the optimal exposure concentration.

[0032] (3.2) Exploration of the exposure period: Using a fixed induction concentration of 5 μM, embryos in the control and experimental groups were exposed for 12 h, 24 h, 36 h, and 48 h, respectively (3 replicates per group, 30 embryos per replicate). The results showed that the phenotype was not obvious after 12 h of exposure; after 24 h of exposure, the number of epidermal apoptotic cells and the ROS level reached their peak and stabilized, and the survival rate met the target; after exposure for more than 24 h, the mortality rate increased significantly and excessive damage occurred. Therefore, the optimal exposure time was determined to be 24 h.

[0033] Example 2 (1) Zebrafish rearing and embryo collection: AB line zebrafish were reared in a standard culture system at 28.0±0.5℃, pH 7.2-7.5, 14h light / 10h darkness. They were paired and bred in a 1:2 ratio of males and females. Fertilized eggs were collected, washed three times with embryo culture medium, and unfertilized eggs were removed.

[0034] (2) Embryo screening: After the embryos develop to 48 hpf, normal and malformed embryos are screened under a stereomicroscope. 90 embryos are selected for each group of experiments (3 replicates, 30 embryos per replicate).

[0035] (3) Model induction: A blank control group, a solvent control group (0.1% DMSO) and a model group were set up. The model group was added with embryo culture medium containing 5 μM imiquimod, and the control group was added with the corresponding culture medium. The models were cultured at 28℃ in the dark for 24 h.

[0036] (4) Model evaluation: ①AO staining: Embryos were stained with 10 μg / mL AO for 15 min and observed under a fluorescence microscope. The number of apoptotic cells in the epidermis was counted using ImageJ. The number of apoptotic cells in the model group was significantly increased compared with that in the control group (P<0.05). ②ROS detection: 10μM DCFH-DA probe was incubated at 37℃ in the dark for 30 min, and the fluorescence intensity was detected by fluorescence microscopy. The ROS level in the model group was more than 40% higher than that in the control group (P<0.05). ③qRT-PCR: The expression of TNF-α, IL-1β and IL-6 mRNA was detected. The expression of TNF-α, IL-1β and IL-6 was upregulated by 2.3 times, 2.5 times and 1.8 times respectively in the model group compared with the control group (P<0.05).

[0037] The results showed that this method successfully constructed a zebrafish psoriasis model, which conformed to the core pathological characteristics of psoriasis and exhibited good stability and reproducibility.

[0038] Example 3 To verify the effectiveness of the zebrafish model for psoriasis and ensure that the model conforms to the core pathological characteristics of psoriasis, this embodiment provides a comprehensive evaluation method for the zebrafish model, which specifically includes the following detection steps: (1) AO staining to count the number of apoptotic cells in the epidermis: Zebrafish embryos of the model group and the blank control group after induction were placed in acridine orange (AO) staining solution with a final concentration of 10 μg / mL and incubated in the dark for 15 min; excess staining solution was removed by washing with PBS buffer; epidermal images were observed and acquired under a stereofluorescence microscope, and the number of apoptotic cells (i.e., cells emitting green fluorescence) in the epidermal region was counted using image processing software (such as ImageJ). The test results showed that the number of apoptotic cells in the model group was higher than that in the control group, and the difference was statistically significant (P<0.05).

[0039] (2) Detection of oxidative stress level using ROS probe: 2',7'-dichlorofluorescein diacetate (DCFH-DA) was used as a fluorescent probe for reactive oxygen species (ROS). Embryos from the model group and control group were placed in a DCFH-DA probe solution with a final concentration of 10 μM and incubated at 37°C in the dark for 30 min, followed by washing twice with PBS. Images were acquired under a fluorescence microscope, and the fluorescence intensity in the epidermal region was detected. Quantitative analysis was performed using image processing software. The test results showed that the ROS fluorescence intensity in the model group was higher than that in the control group (P<0.05), indicating that the model exhibited an oxidative stress imbalance.

[0040] (3) Detection of mRNA expression levels of inflammatory factors: The mRNA expression levels of psoriasis-related inflammatory factors, including TNF-α, IL-1β, and IL-6, were detected using qRT-PCR. The test results showed that, compared with the control group, the expression levels of the above-mentioned inflammatory factors in the model group were significantly upregulated (P<0.05), indicating that the model has the characteristics of immune inflammatory infiltration, which is consistent with the pathological mechanism of immune dysregulation in psoriasis.

[0041] Combined with appendix Figure 1-6 As shown, to verify the effectiveness of the model described in this invention in drug screening, methotrexate at a final concentration of 10 μM was used to intervene in the model. The test results showed that the methotrexate group reduced the number of apoptotic cells and ROS levels in the epidermis of the model zebrafish; and the mRNA expression levels of inflammatory factors TNF-α, IL-1β, and IL-6 were downregulated by 1.5-fold, 1.2-fold, and 1.3-fold, respectively, compared to the model group (P<0.05). These results confirm that the model described in this invention has a good phenotypic and molecular response to clinical therapeutic drugs.

[0042] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for establishing a zebrafish psoriasis model, characterized in that, Includes the following steps: (1) Obtain normally developing zebrafish embryos; (2) The zebrafish embryos were placed in an embryo culture medium containing an inducing drug for exposure culture; The zebrafish embryos were those 24-72 hours after fertilization; The inducing agent includes imiquimod; The final concentration of imiquimod in the embryo culture medium is 1 μM-10 μM; The exposure culture temperature is 25℃-31℃, and the exposure culture time is 12h-48h.

2. The method for establishing according to claim 1, characterized in that, The zebrafish embryos were those 44-52 hours after fertilization.

3. The method for establishing according to claim 1, characterized in that, The final concentration of imiquimod in the embryo culture medium is 4 μM-7 μM.

4. The method for establishing according to claim 1, characterized in that, The exposure and culture time is 20h-28h.

5. The method for establishing according to claim 1, characterized in that, The exposure culture was conducted at a temperature of 27.5℃-28.5℃, and the exposure culture was carried out under light-protected conditions.

6. The method for establishing according to any one of claims 1 to 5, characterized in that, The embryo culture medium also includes organic solvents; The organic solvent is dimethyl sulfoxide, and its volume fraction in the embryo culture medium is 0.05%-0.15%.

7. The method for establishing according to any one of claims 1 to 5, characterized in that, The selection criteria for obtaining normally developed zebrafish embryos include: intact body segments, well-extended tail, no morphological deformities, and normal heartbeat and blood circulation.

8. The application of the zebrafish psoriasis model obtained by the method described in any one of claims 1 to 7 in psoriasis drug screening, psoriasis pathological mechanism research, or psoriasis therapeutic target verification.

9. The application according to claim 8, characterized in that, In the screening of psoriasis drugs or the validation of psoriasis treatment targets, the efficacy of the drug or the effectiveness of the target is evaluated by detecting at least one of the following quantitative indicators: 1) The number of apoptotic cells in the epidermal region; 2) Oxidative stress-activated oxygen levels in the epidermal region; 3) Expression levels of psoriasis-related inflammatory factor mRNAs.

10. The application according to claim 9, characterized in that, The psoriasis-related inflammatory factors are selected from any one or a combination of at least two of TNF-α, IL-1β and IL-6.

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