Zebra fish brain toxicity modeling and detection method based on podophyllotoxin and application of zebra fish brain toxicity modeling and detection method

Abstract

The invention relates to the technical field of medical animal models, in particular to a zebrafish brain toxicity modeling and detecting method based on podophyllotoxin and application thereof.The method comprises the steps that S1, 3-day-fertilization (3dpf) wild-type AB-line zebrafish is randomly selected and placed in a beaker, and 30 zebrafish are treated in each cup (experimental group); s2, dissolving the podophyllotoxin in water, carrying out modeling on the podophyllotoxin, setting a normal control group at the same time, setting the volume of each cup to be 20mL, and treating at 28 DEG C for 2 days; s3, performing cognitive function influence analysis on the zebrafish obtained by modeling in the step S2; according to the method, a behavior analyzer is adopted to carry out influence analysis on the cognitive function of the zebra fish, through a cross module experiment, the movement distance proportion of the zebra fish in a blue area is recorded, and the change of the cognitive function is evaluated. The behavioral analysis can provide systematic and comprehensive toxicity data. The influence of podophyllotoxin on a nervous system is evaluated by recording the behavior change of zebra fish in a specific environment, and the comprehensiveness of toxicity research is enhanced.

Description

Technical Field

[0001] The invention relates to the technical field of medical animal models, and in particular to a zebrafish brain toxicity modeling and detection method based on podophyllotoxin and applications thereof. Background Art

[0002] Podophyllotoxin is a natural lignan compound extracted from plants such as Podophyllum spp., which has potent anti-tumor activity. Its main mechanism of action is to inhibit the polymerization of microtubules by binding and stabilizing microtubules, blocking the mitotic phase (M phase) of the cell cycle, and ultimately leading to cell apoptosis. Podophyllotoxin and its derivatives, such as etoposide and teniposide, have been developed as clinical anticancer drugs for the treatment of various cancers such as lung cancer, testicular cancer and leukemia.

[0003] However, the widespread application of podophyllotoxin is limited by its toxic side effects, especially toxic reactions to non-target cells and tissues. Therefore, in-depth research on the toxic mechanism of podophyllotoxin and finding ways to reduce its toxicity are of great significance for its safe and effective clinical application.

[0004] Zebrafish (Danio rerio) is a model organism widely used in biomedical research. Its genome is highly similar to the human genome, with about 70% of its genes homologous to humans. Zebrafish have transparent embryos and larvae, which facilitates real-time observation of the effects of drugs on development and tissue structure. In addition, zebrafish have strong reproductive capacity, short life cycle, simple experimental operation, low cost, suitable for large-scale high-throughput screening, and are an ideal model for drug screening and toxicity research.

[0005] In toxicity studies, zebrafish can be used to evaluate the acute and chronic toxicity, developmental toxicity, neurotoxicity, hepatotoxicity, cardiotoxicity and other types of toxicity of compounds. The zebrafish model can not only provide systematic toxicity data through behavioral analysis, histopathological analysis and other means, but also study the toxicity mechanism of compounds through gene editing, transgenic technology and other methods.

[0006] Although the anti-tumor activity of podophyllotoxin has been widely studied, its toxicity studies are relatively few, especially those on the nervous system and digestive system. Existing toxicity studies mainly rely on in vitro cell experiments and mammalian models, but these methods have the following problems and defects:

[0007] 1. Limited means of toxicity evaluation: Although in vitro cell experiments can provide certain toxicity information, they cannot fully reflect the toxicity characteristics of the compound in the whole organism. Although mammalian models such as mice and rats can provide more comprehensive toxicity data, the experimental cycle is long, the cost is high, and there are ethical issues, which are not suitable for large-scale screening and rapid evaluation.

[0008] 2. High experimental cost and long cycle: Mammalian models are widely used in toxicity studies, but the experimental cycle is long and costly, requiring a lot of time and resources, which is not conducive to high-throughput drug screening.

[0009] 3. Unclear toxicity mechanism: Research on podophyllotoxin toxicity is mostly focused on cytotoxicity, and there is insufficient understanding of the systemic toxic reactions and mechanisms it causes in the whole organism, especially the specific mechanisms of neurotoxicity and intestinal toxicity.

[0010] In view of the above problems, the present invention provides a reliable method for studying podophyllotoxin brain toxicity, and also provides important experimental tools and data support for related toxicity mechanism research. Summary of the invention

[0011] The purpose of the present invention is to provide a zebrafish brain toxicity modeling and detection method based on podophyllotoxin and its application, so as to solve the problems existing in the prior art such as unclear toxicity mechanism, lack of suitable animal models and imperfect detection methods.

[0012] In order to achieve the above technical objectives and the above technical effects, the present invention is implemented through the following technical solutions:

[0013] A zebrafish brain toxicity modeling and detection method based on podophyllotoxin comprises the following steps:

[0014] S1: Wild-type AB zebrafish at 3 days post-fertilization (3 dpf) were randomly selected and placed in beakers, with 30 zebrafish in each beaker (experimental group).

[0015] S2: water-soluble podophyllotoxin was administered to establish the model, and a normal control group was set up at the same time. The capacity of each cup was 20 mL and the treatment was carried out at 28°C for 2 days.

[0016] S3: Analyze the cognitive function of zebrafish obtained by modeling in step S2.

[0017] Furthermore, the concentration of podophyllotoxin in step S2 is 0.140-1.57 μg / mL.

[0018] Further, the cognitive function impact analysis in step S3 specifically includes: each experimental group randomly selects 5 zebrafish and puts them into a "cross" module, the modules are yellow, blue, red and green four areas, each group is set with 6 modules, and the behavior analyzer is used to collect data, and the total movement distance of zebrafish in the blue area within 10 minutes is analyzed as a percentage of the total movement distance of the entire area (referred to as blue proportion), and the statistical analysis results of this indicator are used to evaluate the effect of podophyllotoxin on cognitive function. The statistical processing results are expressed as mean ± SE. SPSS26.0 software was used for statistical analysis, and p < 0.05 indicated that the difference was statistically significant.

[0019] Furthermore, it also includes the determination of MNLC and LC10 of podophyllotoxin on zebrafish, specifically including: randomly selecting wild-type AB strain zebrafish 3 days after fertilization (3dpf) in a 6-well plate, and treating 30 zebrafish in each well (experimental group). Podophyllotoxin was given in water, and a normal control group and a solvent control group (1% DMSO) were set up at the same time, with a capacity of 3mL per well. Treated at 28°C for 2 days, the number of zebrafish deaths in each experimental group was counted every day and removed in time. The best "concentration-mortality" effect curve was drawn using Origin 8.0 statistical software, and the MNLC and LC10 of podophyllotoxin on zebrafish were calculated.

[0020] Furthermore, the podophyllotoxin concentration is: 0.977-3.91 μg / mL.

[0021] In another aspect, the present invention provides the application of the above method in studying the effects of podophyllotoxin on brain tissue and cells.

[0022] Beneficial effects of the present invention:

[0023] The present invention selects 3dpf (3 days after fertilization) wild-type AB strain zebrafish, and 30 zebrafish are treated in each experimental group, and toxicity modeling is performed by administering podophyllotoxin in water. The concentration range of podophyllotoxin is set at 0.140-1.57μg / mL, and the effects of different concentrations on zebrafish brain toxicity are systematically evaluated. The zebrafish genome is highly similar to the human genome, and its transparent embryos and larvae allow the effects of drugs on development and tissue structure to be observed in real time. The zebrafish model provides systematic and comprehensive toxicity data through behavioral analysis, histopathological analysis and other means, which makes up for the shortcomings of in vitro cell experiments and traditional mammalian models, and ensures the comprehensiveness and accuracy of toxicity evaluation.

[0024] The present invention uses a behavioral analyzer to analyze the impact of zebrafish cognitive function. Through the "cross" module experiment, the proportion of zebrafish movement distance in the blue area is recorded to evaluate the changes in cognitive function. Behavioral analysis can provide systematic and comprehensive toxicity data. By recording the behavioral changes of zebrafish in a specific environment, the impact of podophyllotoxin on the nervous system is evaluated, and the comprehensiveness of toxicity research is enhanced.

[0025] The zebrafish model is an ideal tool for high-throughput drug screening and rapid toxicity evaluation due to its short experimental cycle, simple operation and low cost, and the ability to conduct a large number of experiments in a short period of time. Compared with traditional mammalian models, zebrafish has significant time and cost advantages in high-throughput screening.

[0026] The present invention uses Origin 8.0 statistical software to draw the "concentration-mortality" effect curve of podophyllotoxin, calculate its maximum non-lethal concentration (MNLC) and 10% lethal concentration (LC10), and provide fast and accurate toxicity data. By drawing the toxic effect curve, researchers can quickly obtain the toxic effects of podophyllotoxin on zebrafish at different concentrations. This method not only improves the efficiency of toxicity evaluation, but also provides accurate data support for further toxicity research and drug screening.

[0027] The present invention observes and records the conditions of the zebrafish brain and intestines, collects photos of typical toxic organs, and evaluates the toxic effects of podophyllotoxin on the zebrafish intestines and brain at different concentrations. The transparent characteristics of zebrafish and the high similarity of their genomes to humans make it an ideal model for studying brain toxicity. By observing the conditions of the zebrafish brain and intestines through a dissecting microscope, the toxicity manifestations and toxicity incidence can be intuitively recorded, the effects of podophyllotoxin on brain tissue can be systematically evaluated, and its toxicity mechanism can be studied. By setting different concentrations of podophyllotoxin, its effects on zebrafish brain tissue and cognitive function can be systematically evaluated. Short-term toxicity testing can quickly obtain brain toxicity data of podophyllotoxin, providing an experimental basis for in-depth study of its toxicity mechanism. The toxicity manifestations of the zebrafish brain and intestines were observed using a dissecting microscope, typical toxic organ photos were recorded, and the toxicity incidence was analyzed. The transparent characteristics of zebrafish make it an ideal model for studying brain toxicity. By observing the conditions of the zebrafish brain and intestines through a dissecting microscope, the toxicity manifestations and toxicity incidence can be intuitively recorded, providing reliable brain toxicity research data.

[0028] In summary, by using the zebrafish model, the present invention provides a reliable podophyllotoxin brain toxicity modeling and detection method, which can improve the comprehensiveness and accuracy of toxicity evaluation, is suitable for large-scale high-throughput screening and rapid evaluation, and provides important experimental tools and data support for in-depth research on the toxicity mechanism of podophyllotoxin.

[0029] Of course, any product implementing the present invention does not necessarily need to achieve all of the advantages described above at the same time. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings required for describing the embodiments will be briefly introduced below. Obviously, the accompanying drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other accompanying drawings can be obtained based on these accompanying drawings without paying creative work.

[0031] Figure 1 This is a schematic diagram of the “concentration-mortality” curve of podophyllotoxin enteroencephalic toxicity;

[0032] Figure 2 Schematic diagram of intestinal toxicity phenotype after podophyllotoxin treatment in zebrafish; Note: The red dotted box is the zebrafish intestinal analysis area;

[0033] Figure 3 Schematic diagram of the brain toxicity phenotype of zebrafish after podophyllotoxin treatment; Note: The blue dotted box is the zebrafish brain analysis area;

[0034] Figure 4 This is a typical schematic diagram of the behavior trajectory of zebrafish after podophyllotoxin treatment; Note: The blue area of ​​the cross module in the black box is the quantitative area;

[0035] Figure 5 Schematic diagram showing the percentage of the total movement distance of the blue area of ​​zebrafish treated with podophyllotoxin to the total movement distance of the entire area compared with the normal control group; ***p<0.001;

[0036] Figure 6 Schematic diagram of zebrafish intestinal tissue structure; Note: The yellow dotted box is the zebrafish intestinal analysis area;

[0037] Figure 7 This is a typical schematic diagram of the intestinal tissue structure of zebrafish after podophyllotoxin treatment; Note: The yellow dotted box is the zebrafish intestinal analysis area, the red arrow indicates the zebrafish intestinal folds, the white arrow indicates the zebrafish intestinal lumen loss, the black arrow indicates the normal intestinal epithelial cells, and the green arrow indicates the apoptotic cells;

[0038] Figure 8 This is a typical schematic diagram of the zebrafish brain tissue structure after podophyllotoxin treatment; Note: The yellow dotted box is the zebrafish brain analysis area, the black arrows indicate the zebrafish brain cell vacuoles, and the blue arrows indicate the brain apoptotic cells;

[0039] Fig. 9A typical schematic diagram of the fluorescence intensity of apoptotic cells in the zebrafish intestine after podophyllotoxin treatment; Note: The red dotted box is the zebrafish intestinal analysis area, and the green fluorescent particles are apoptotic cells;

[0040] Fig.10 A typical schematic diagram of the fluorescence intensity of apoptotic cells in the zebrafish brain after podophyllotoxin treatment; Note: The yellow dotted box is the zebrafish brain analysis area, and the green fluorescent particles are apoptotic cells;

[0041] Fig.11 Schematic diagram of the comparison of fluorescence intensity of apoptotic cells in the intestine of zebrafish treated with podophyllotoxin and that of the normal control group; *p<0.001;

[0042] Fig.12 Schematic diagram of the comparison of the fluorescence intensity of apoptotic cells in the zebrafish brain after podophyllotoxin treatment and the normal control group; *p<0.001. DETAILED DESCRIPTION

[0043] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0044] Example 1

[0045] The method for modeling and detecting zebrafish brain toxicity based on podophyllotoxin described in this embodiment comprises the following steps:

[0046] S1: Wild-type AB zebrafish at 3 days post-fertilization (3 dpf) were randomly selected and placed in beakers, with 30 zebrafish in each beaker (experimental group).

[0047] S2: water-soluble podophyllotoxin was administered to establish the model, and a normal control group was set up at the same time. The capacity of each cup was 20 mL and the treatment was carried out at 28°C for 2 days.

[0048] S3: Analyze the cognitive function of zebrafish obtained by modeling in step S2.

[0049] In this embodiment, the concentration of podophyllotoxin in step S2 is 0.140-1.57 μg / mL.

[0050] In this embodiment, the cognitive function impact analysis in step S3 specifically includes: each experimental group randomly selects 5 zebrafish and puts them into a "cross" module, and the modules are yellow, blue, red and green four areas, and each group is set with 6 modules, and a behavior analyzer is used to collect data, and the total movement distance of zebrafish in the blue area within 10 minutes is analyzed as a percentage of the total movement distance of the entire area (referred to as blue proportion), and the statistical analysis results of this indicator are used to evaluate the effect of podophyllotoxin on cognitive function. The statistical processing results are expressed by mean ± SE. SPSS26.0 software was used for statistical analysis, and p < 0.05 indicated that the difference was statistically significant.

[0051] In this embodiment, the determination of MNLC and LC10 of podophyllotoxin on zebrafish is also included, specifically including: randomly selecting wild-type AB strain zebrafish 3 days after fertilization (3dpf) in a 6-well plate, and treating 30 zebrafish in each well (experimental group). Podophyllotoxin is given in water, and a normal control group and a solvent control group (1% DMSO) are set up at the same time, with a capacity of 3mL per well. Treat at 28°C for 2 days, and count the number of zebrafish deaths in each experimental group every day and remove them in time. Use Origin 8.0 statistical software to draw the best "concentration-mortality" effect curve, and calculate the MNLC and LC10 of podophyllotoxin on zebrafish.

[0052] In this embodiment, the podophyllotoxin concentration is: 0.977-3.91 μg / mL.

[0053] In another aspect, the present invention provides the application of the above method in studying the effects of podophyllotoxin on brain tissue and cells.

[0054] Example 2

[0055] Testing materials

[0056] Podophyllotoxin, the solvent is DMSO.

[0057] Experimental animals

[0058] Zebrafish were raised in fish farming water at 28°C (water quality: 200 mg of instant sea salt was added to every 1 L of reverse osmosis water, conductivity was 450-550 μS / cm; pH was 6.5-8.5; hardness was 50-100 mg / L CaCO3), bred and provided by our company's fish farming center, the experimental animal use license number is: SYXK (Zhejiang) 2022-0004, and the breeding management complies with the requirements of the international AAALAC certification (certification number: 001458), IACUC ethics review number: IACUC-2023-7407-01.

[0059] Instruments, consumables and reagents

[0060] Dissecting microscope (SZX7, OLYMPUS, Japan); CCD camera (VertA1, Shanghai Tusen Vision Technology Co., Ltd., China); precision electronic balance (CP214, OHAUS, USA); 6-well plate (Zhejiang Bellanbo Biotechnology Co., Ltd., China).

[0061] Methylcellulose (Batch No. C2004046, Shanghai Aladdin Biochemical Technology Co., Ltd., China); dimethyl sulfoxide (DMSO, Batch No. BCCD8942, Sigma, Switzerland; Batch No. I2229063, Shanghai Aladdin Biochemical Technology Co., Ltd., China).

[0062] Detection Methods

[0063] MNLC and LC10 assays

[0064] Wild-type AB strain zebrafish at 3 days post-fertilization (3dpf) were randomly selected in 6-well plates, and 30 zebrafish were treated in each well (experimental group). Podophyllotoxin was given in water (concentrations shown in Table 1), and a normal control group and a solvent control group (1% DMSO) were set up at the same time, with a capacity of 3mL per well. The zebrafish were treated at 28℃ for 2 days, and the number of zebrafish deaths in each experimental group was counted every day and removed in time. The best "concentration-mortality" effect curve was drawn using Origin 8.0 statistical software, and the MNLC and LC10 of podophyllotoxin on zebrafish were calculated.

[0065] Evaluation of entero-brain toxicity

[0066] 3dpf wild-type AB strain zebrafish were randomly selected in a 6-well plate, and 30 zebrafish were treated in each well (experimental group). Water-soluble podophyllotoxin (concentrations shown in Table 2) was given, and a normal control group was set up at the same time, with a volume of 3mL per well. After 2 days of treatment at 28℃, the conditions of the zebrafish brain and intestine were observed and recorded under a dissecting microscope, and photos of typical toxic organs were collected. The intestinal and brain toxicity of podophyllotoxin to zebrafish was evaluated by the incidence of toxicity in the brain and intestine.

[0067] Test results

[0068] MNLC and LC10

[0069] Under the experimental conditions, the maximum non-lethal concentration (MNLC) of podophyllotoxin was 1.26 μg / mL, and the 10% lethal concentration (LC10) was 1.57 μg / mL. Figure 1 .

[0070] Table 1 Results of the sample gut-brain toxicity concentration exploration experiment (n=30)

[0071]

[0072] Evaluation of entero-brain toxicity

[0073] Under the experimental conditions, podophyllotoxin induced a 13% mortality rate at a concentration of 1.57 μg / mL, and induced intestinal toxicity and brain toxicity. The intestinal toxicity was mainly manifested as the loss of intestinal lumen and the reduction of intestines, and the brain toxicity was mainly manifested as the reduction of brain and brain degeneration; at a concentration of 1.26 μg / mL, it induced intestinal toxicity and brain toxicity. The intestinal toxicity was mainly manifested as the loss of intestinal lumen and the reduction of intestines, and the brain toxicity was mainly manifested as the reduction of brain and brain degeneration; at a concentration of 0.420 μL / mL, it induced intestinal toxicity and brain toxicity, which were manifested as the reduction of intestine and brain, respectively; at a concentration of 0.140 μL / mL, it induced intestinal toxicity, which was mainly manifested as the reduction of intestine. See Table 2 for details. Figure 2 and Figure 3 .

[0074] Table 2 Statistics of the incidence of podophyllotoxin enteroencephalopathy (n=30)

[0075]

[0076]

[0077] Note: “-” indicates no obvious abnormality; the numbers in the table indicate: incidence (%).

[0078] Example 3

[0079] The test materials and experimental animals were the same as those in Example 2;

[0080] Instruments, consumables and reagents

[0081] Dissecting microscope (SZX7, OLYMPUS, Japan); CCD camera (VertA1, Shanghai Tusen Vision Technology Co., Ltd., China); precision electronic balance (CP214, OHAUS, USA); behavior analyzer (Zebra Lab 3.22.3.31, ViewPoint, France).

[0082] Dimethyl sulfoxide (DMSO, batch number I2229063, Shanghai Aladdin Biochemical Technology Co., Ltd., China).

[0083] Detection Methods

[0084] Wild-type AB strain zebrafish 3 days post-fertilization (3dpf) were randomly selected and placed in beakers, and 30 zebrafish were treated in each beaker (experimental group). Podophyllotoxin was given in water (concentrations are shown in Table 3), and a normal control group was set up at the same time, with a capacity of 20mL per cup. After 2 days of treatment at 28℃, 5 zebrafish were randomly selected from each experimental group and placed in a "cross" module. The modules were yellow, blue, red and green, and 6 modules were set up in each group. Data were collected using a behavioral analyzer, and the percentage of the total movement distance of zebrafish in the blue area in 10 minutes to the total movement distance of the entire area (referred to as blue proportion) was analyzed. The statistical analysis results of this indicator were used to evaluate the effect of podophyllotoxin on cognitive function. The statistical analysis results were expressed as mean±SE. SPSS26.0 software was used for statistical analysis, and p<0.05 indicated that the difference was statistically significant.

[0085] Test results

[0086] Under the experimental conditions, podophyllotoxin can induce cognitive dysfunction in zebrafish. See Table 3 for details. Figure 4 and Figure 5 .

[0087] Table 3 Experimental results of the effects of samples on cognitive function (n=6)

[0088]

[0089] Compared with the normal control group, *p<0.001;

[0090] Example 4

[0091] Effects on intestinal and brain histopathology

[0092] The test materials and experimental animals were the same as those in Example 2;

[0093] Instruments, consumables and reagents

[0094] Dissecting microscope (SZX7, OLYMPUS, Japan); CCD camera (VertA1, Shanghai Tusen Vision Technology Co., Ltd., China); precision electronic balance (CP214, OHAUS, USA); 6-well plate (Zhejiang Bellanbo Biotechnology Co., Ltd., China); electronic constant temperature stainless steel water bath (HHS-2S, Shanghai Kanglu Instrument Equipment Co., Ltd., China); slicer (KD2258, Jinhua Kedi Instrument Equipment Co., Ltd., China); intelligent electric heating plate (400X280, Tianjin Leyuenag Laboratory Instrument Sales Co., Ltd., China); biological microscope (CX31, OLYMPUS, Japan).

[0095] Dimethyl sulfoxide (DMSO, batch number I2229063, Shanghai Aladdin Biochemical Technology Co., Ltd., China); 4% tissue cell fixative (batch number 20230401, Beijing Solebow Technology Co., Ltd., China); anhydrous ethanol (batch number 20230329, Sinopharm Chemical Reagent Co., Ltd., China); xylene (batch number 20230424, Sinopharm Chemical Reagent Co., Ltd., China); Mayer hematoxylin staining solution (batch number 20220120, Shanghai Yihe Biotechnology Co., Ltd., China); ina); eosin staining solution (batch number 20220120, Shanghai Yihe Biotechnology Co., Ltd., China); hydrochloric acid (batch number 140517, Xilong Chemical Co., Ltd., China); neutral gum (batch number 330A021, Beijing Solebow Technology Co., Ltd., China); high-efficiency section paraffin (melting point 54-56°C, batch number 20230322, Shanghai Huayong Paraffin Co., Ltd., China); high-efficiency section paraffin (melting point 62-64°C, batch number 20221116, Shanghai Huayong Paraffin Co., Ltd., China).

[0096] Detection Methods

[0097] Wild-type AB strain zebrafish at 3 days post-fertilization (3dpf) were randomly selected in 6-well plates, and 30 zebrafish were treated in each well (experimental group). Water-soluble podophyllotoxin (concentrations were 0.140, 0.420, 1.26, and 1.57μg / mL), and a normal control group was set up at the same time, with a capacity of 3mL per well. After 2 days of treatment at 28℃, the zebrafish were fixed with 4% tissue cell fixative, and after a series of dehydration-embedding-sectioning-staining steps, the zebrafish were subjected to histopathological H&E staining analysis. The intestinal toxicity and brain toxicity of the samples to zebrafish were evaluated by intestinal and brain tissue pathological analysis.

[0098] Test results

[0099] Intestinal histopathological analysis

[0100] Under the conditions of this experiment, the zebrafish in the normal control group had many and abundant intestinal folds (indicated by the red arrow), the intestinal epithelial cells were tall columnar, the cell nuclei were long oval (indicated by the black arrow), the cells were evenly distributed and orderly arranged, and they were closely connected to the cilia and mucosa. The zebrafish in the 0.140μg / mL podophyllotoxin concentration group had a normal intestinal cavity, abundant intestinal folds, and normal and neatly arranged intestinal epithelial cells, similar to the normal control group; the zebrafish in the 0.420μg / mL podophyllotoxin concentration group had a smaller intestinal cavity, and a small amount of apoptosis in the epithelial cells (indicated by the green arrow); the zebrafish in the 1.26μg / mL concentration group had a smaller intestinal cavity, and at the same time, the number of intestinal folds decreased significantly, the epithelial cells were disordered, the cell nuclei were condensed and ruptured, and there were many apoptotic cells; the zebrafish in the 1.57μg / mL concentration group had a missing intestinal cavity (indicated by the white arrow), no obvious intestinal folds, abnormal epithelial cell structure and disordered arrangement, reduced cell volume, condensed and ruptured cell nuclei, and a large number of apoptotic cells. Therefore, podophyllotoxin has intestinal toxicity. Figure 6 , 7 shown.

[0101] Brain histopathological analysis

[0102] Under the conditions of this experiment, the zebrafish brain cells in the normal control group had normal structure, large, round nuclei with darker staining, and cells were tightly arranged and evenly distributed. The zebrafish brain cells in the 0.140μg / mL podophyllotoxin concentration group had normal structure and were tightly arranged, similar to the normal control group; the zebrafish brain cells in the 0.420μg / mL concentration group had relatively normal structure, but the brain was significantly smaller; the zebrafish brain in the 1.26μg / mL concentration group was significantly smaller, the cells were damaged and degenerated, the staining depth was uneven, the cells were disorderly arranged, and contained vacuoles (indicated by the black arrow), the cell volume was reduced, the cell nucleus was condensed and ruptured, the cytoplasm was granular, the chromatin was marginalized, and a large number of apoptotic cells were present (indicated by the blue arrow); the zebrafish brain in the 1.57μg / mL concentration group was significantly smaller, the cells were damaged and degenerated, the staining depth was uneven, the cell structure was abnormal, the distribution was uneven, the number of apoptotic cells was large, and contained a large number of vacuoles. Therefore, podophyllotoxin has brain toxicity. Figure 6-8 shown.

[0103] Example 5

[0104] Effects on intestinal and brain cell apoptosis

[0105] The test materials and experimental animals were the same as those in Example 2;

[0106] Instruments, consumables and reagents

[0107] Dissecting microscope (SZX7, OLYMPUS, Japan); CCD camera (VertA1, Shanghai Tusen Vision Technology Co., Ltd., China); precision electronic balance (CP214, OHAUS, USA); 6-well plate (Zhejiang Bellambo Biotechnology Co., Ltd., China); electric focus continuous zoom fluorescence microscope (AZ100, Nikon, Japan).

[0108] Methylcellulose (Batch No. C2004046, Shanghai Aladdin Biochemical Technology Co., Ltd., China); dimethyl sulfoxide (DMSO, Batch No. I2229063, Shanghai Aladdin Biochemical Technology Co., Ltd., China); acridine orange (AO, Batch No. C12894919, Shanghai MacLean Biochemical Technology Co., Ltd., China).

[0109] Detection Methods

[0110] Wild-type AB strain zebrafish at 3 days post-fertilization (3dpf) were randomly selected in a 6-well plate, and 30 zebrafish were treated in each well (experimental group). Water-soluble podophyllotoxin (concentrations shown in Table 4) was given, and a normal control group was set up at the same time, with a volume of 3mL per well. After 2 days of treatment at 28℃, zebrafish in each experimental group were stained with AO light-proof for 30min, washed three times with standard dilution water, and 10 zebrafish were randomly selected from each experimental group and photographed under a fluorescence microscope. The data were analyzed and collected using NIS-Elements D 3.20 advanced image processing software, and the fluorescence intensity of zebrafish intestinal apoptotic cells and brain apoptotic cells was analyzed and statistically analyzed. The statistical analysis results of the above indicators were used to evaluate the effect of podophyllotoxin on intestinal and brain cell apoptosis. The statistical analysis results were expressed as mean±SE. SPSS26.0 software was used for statistical analysis, and p<0.05 indicated that the difference was statistically significant.

[0111] Test results

[0112] Under the experimental conditions, podophyllotoxin can induce intestinal apoptosis and brain apoptosis in zebrafish. See Table 4 for details. Figure 9-12 .

[0113] Table 4 Experimental results of the effects of samples on intestinal and brain cell apoptosis (n=10)

[0114]

[0115] Compared with the normal control group, *p<0.001;

[0116] In summary, the present invention proposes a zebrafish brain toxicity modeling and detection method based on podophyllotoxin and its application. The present invention makes up for the shortcomings of existing toxicity research methods by using a zebrafish model, improves the comprehensiveness and accuracy of toxicity evaluation, is suitable for large-scale high-throughput screening and rapid evaluation, and provides important experimental tools and data support for in-depth research on the toxicity mechanism of podophyllotoxin.

[0117] The preferred embodiments of the present invention disclosed above are only used to help illustrate the present invention. The preferred embodiments do not describe all the details in detail, nor do they limit the invention to the specific implementation methods described. Obviously, many modifications and changes can be made according to the content of this specification. This specification selects and specifically describes these embodiments in order to better explain the principles and practical applications of the present invention, so that those skilled in the art can understand and use the present invention well. The present invention is limited only by the claims and their full scope and equivalents.

Claims

1. A zebrafish brain toxicity modeling and detection method based on podophyllotoxin, characterized in that: S1: Wild-type AB zebrafish 3 days after fertilization were randomly selected and placed in beakers, with 30 zebrafish in each beaker; S2: Water-soluble podophyllotoxin was administered to establish the model, and a normal control group was set up at the same time. The beaker capacity of each group was 20 mL and the treatment was carried out at 28°C for 2 days; S3: Analyze the cognitive function of zebrafish obtained by modeling in step S2.

2. The method for modeling and detecting zebrafish brain toxicity based on podophyllotoxin according to claim 1, characterized in that: The step S2 specifically includes: the concentration of podophyllotoxin is 0.140-1.57 μg / mL.

3. The podophyllotoxin-based zebrafish brain toxicity modeling and detection method according to claim 1, characterized in that: The cognitive function impact analysis in step S3 specifically includes: randomly selecting 5 zebrafish from each experimental group and placing them in a "cross" module, wherein the modules are four areas of yellow, blue, red and green, and each group is provided with 6 modules, collecting data with a behavior analyzer, analyzing the percentage of the total movement distance of the zebrafish in the blue area within 10 minutes to the total movement distance of the entire area, and evaluating the impact of podophyllotoxin on cognitive function with the statistical analysis results of this indicator; the statistical processing results are expressed as mean±SE; and statistical analysis is performed using SPSS26.0 software, and p<0.05 indicates that the difference is statistically significant.

4. The podophyllotoxin-based zebrafish brain toxicity modeling and detection method according to claim 1, characterized in that: Podophyllotoxin also affects zebrafish MNLC and LC 10 The determination specifically includes: randomly selecting wild-type AB strain zebrafish 3 days after fertilization in a 6-well plate, treating 30 zebrafish in each well; administering podophyllotoxin in water, and setting up a normal control group and a solvent control group, with a capacity of 3 mL per well; treating at 28°C for 2 days, counting the number of zebrafish deaths in each experimental group every day and removing them in time; using Origin 8.0 statistical software to draw the best "concentration-mortality" effect curve, and calculating the MNLC and LC of podophyllotoxin on zebrafish 10 .

5. Use of the method according to any one of claims 1 to 4 in the study of the effects of podophyllotoxin on brain tissue and cells.

Images