Zebra fish intestine toxicity modeling and detecting method based on podophyllotoxin and application of zebra fish intestine toxicity modeling and detecting method

Abstract

The invention relates to the technical field of medical animal models, in particular to a zebrafish intestine toxicity modeling and detection method based on podophyllotoxin and application thereof, and the method comprises the following steps: S1, randomly selecting 3-day (3dpf) wild-type AB-line zebrafish after fertilization in a 6-pore plate, and treating 30 zebrafish in each pore (experimental group); s2, dissolving the podophyllotoxin in water, performing modeling, setting a normal control group, setting the volume of each pore to be 3mL, and treating at 28 DEG C for 2 days; and S3, performing intestinal histopathology analysis and intestinal apoptosis analysis on the zebrafish obtained by modeling in the step S2. The method comprises the following steps: Hamp; according to the method, the change of the tissue structure can be clearly observed, and the damage degree of the podophyllotoxin to the intestinal tract and brain tissue of the zebra fish can be intuitively evaluated. AO staining is combined with a fluorescence microscope, so that the apoptosis condition can be quantitatively analyzed; the damage mechanism of the podophyllotoxin to the brain cell structure and function is defined, and the blank of the podophyllotoxin in the aspect of brain toxicity research is filled.

Description

Technical Field

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

[0002] Podophyllotoxin is a natural plant toxin that is mainly found in plants of the genus Podophyllum. As an important cytotoxic drug, podophyllotoxin and its derivatives are widely used in the development of anticancer drugs. However, podophyllotoxin also has significant toxicity and is potentially harmful to normal cells and tissues. Therefore, studying the toxicity mechanism and safe dose of podophyllotoxin is of great significance for its application in medicine.

[0003] At present, the research on podophyllotoxin mainly focuses on the following aspects:

[0004] 1. Anti-cancer mechanism: Podophyllotoxin exerts its anti-cancer effect by inhibiting the polymerization of tubulin and preventing cell division. This mechanism has been widely studied and successfully applied to the treatment of certain cancers.

[0005] 2. Drug development: Podophyllotoxin derivatives such as etoposide and teniposide have been developed and used in clinical practice, showing good anti-tumor effects.

[0006] 3. Toxicity study: Although podophyllotoxin has significant anti-cancer effects, its toxicity limits its clinical application. Studies have shown that podophyllotoxin is toxic to tissues such as the liver, kidneys, and intestines, so further research is needed on its safe dose and toxicity mechanism.

[0007] Although some progress has been made in the research of podophyllotoxin, there are still several problems and defects:

[0008] 1. Unclear toxicity mechanism: The toxicity mechanism of podophyllotoxin on different tissues is not fully understood, especially the toxicity mechanism on intestinal and brain tissues is less studied, which limits its safe application and dosage optimization.

[0009] 2. Lack of suitable animal models: Existing toxicity studies mostly use mammalian models, such as mice and rats, but these models have certain limitations, such as high experimental costs and long cycles. Zebrafish, as an emerging model organism, has advantages such as short growth cycle, low cost and genetic transparency, but its application in podophyllotoxin toxicity research is still insufficient.

[0010] 3. The detection methods are not perfect: Current toxicity detection methods are mostly focused on histopathological analysis, but lack detection of molecular levels such as cell apoptosis, and cannot fully reflect the toxic effects of podophyllotoxin.

[0011] In view of the above problems, the present invention provides a reliable method for studying the enterotoxicity and brain toxicity of podophyllotoxin, and also provides important experimental tools and data support for the study of related toxicity mechanisms. Summary of the invention

[0012] The purpose of the present invention is to provide a zebrafish intestinal toxicity model based on podophyllotoxin and a detection method thereof, 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.

[0013] In order to achieve the above technical objectives and the above technical effects, the present invention is implemented through the following technical solutions:

[0014] A zebrafish enterotoxicity modeling and detection method based on podophyllotoxin comprises the following steps:

[0015] S1: Wild-type AB zebrafish at 3 days post-fertilization (3 dpf) were randomly selected and placed in a 6-well plate, and 30 zebrafish were treated in each well (experimental group).

[0016] S2: water-soluble podophyllotoxin was administered to establish the model, and a normal control group was set up at the same time. The volume of each well was 3 mL and the treatment was carried out at 28°C for 2 days.

[0017] S3: The zebrafish obtained by modeling in step S2 were subjected to intestinal tissue pathology analysis and intestinal cell apoptosis analysis.

[0018] Furthermore, the concentration of podophyllotoxin in step S2 is 0.140-1.57 μg / mL.

[0019] Further, the step S3 midgut histopathological analysis specifically includes: fixing the zebrafish tissue with 4% paraformaldehyde at 4°C overnight to ensure the integrity of the cell structure. After that, dehydration treatment is performed, and the zebrafish sample is gradually dehydrated through a series of increasing concentrations of ethanol, starting with 70% ethanol treatment for 0.5 to 1 hour, and gradually increasing to 100% ethanol, and each step The treatment time is about 15-30 minutes. After dehydration, a 1:1 mixture of xylene and 100% ethanol is used for 20 minutes for transparency, and then two 15-minute 100% xylene treatments are performed. Subsequently, the sample is immersed in soft wax at 60°C and hard wax at 65°C to completely embed the sample in an embedding instrument at 77°C. Use a microtome to cut the fixed and embedded tissue into 5-micron thick slices and attach them to a treated clean slide. Finally, the zebrafish were subjected to histopathological analysis using the hematoxylin-eosin (H&E) staining method. The staining process included hematoxylin staining for 8 minutes and eosin staining for no more than 1.5 minutes. After staining, the tissue sections were fixed in 95% and 100% ethanol, and finally treated with xylene and sealed with neutral resin for preservation.

[0020] Furthermore, the intestinal cell apoptosis analysis in step S3 specifically includes: subjecting the zebrafish to AO light-proof staining for 30 minutes, washing three times with standard dilution water, randomly selecting 10 zebrafish from each experimental group and photographing them under a fluorescence microscope, using NIS-Elements D 3.20 advanced image processing software to analyze and collect data, analyzing and statistically analyzing the fluorescence intensity of zebrafish intestinal apoptotic cells, and using the statistical analysis results of the above indicators to evaluate the effect of podophyllotoxin on intestinal cell apoptosis.

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

[0022] Beneficial effects of the present invention:

[0023] The present invention provides an efficient and reliable toxicity research model. It takes only a few days to a few weeks for zebrafish to grow from fertilization to maturity, so that multiple experimental cycles can be completed in a short period of time, greatly improving the experimental efficiency. This rapid growth characteristic makes zebrafish an ideal choice for acute toxicity experiments.

[0024] Compared with mammalian models, zebrafish are inexpensive to breed and maintain, and experimental costs are significantly reduced. At the same time, zebrafish have strong reproductive capacity and can lay hundreds of eggs at a time, which is conducive to large-scale experimental research. The transparent nature of zebrafish embryos and larvae makes their internal organs clearly visible under a microscope, making it easy to observe the effects of drugs and toxins on various tissues in real time. In particular, the use of fluorescent labeling technology can dynamically monitor cell apoptosis and other biological processes. The zebrafish genome is highly similar to the human genome, and its genomic information has been widely analyzed, so it has a high reference value in toxicity and pharmacology research.

[0025] The present invention conducts an in-depth study of the dose-response relationship by setting a toxic dose gradient; at a low dose (0.140 μg / mL), the zebrafish intestinal and brain tissue structures are basically the same as those of the normal control group, the cells are closely arranged, and no significant toxic effects are observed. This indicates that low concentrations of podophyllotoxin have little effect on zebrafish tissues, providing a reference for determining its safe dose.

[0026] Medium dose (0.420 μg / mL): A small amount of apoptosis occurred in intestinal epithelial cells, and the brain tissue was relatively normal in structure but significantly smaller. At this concentration, toxic reactions were initially observed, indicating that podophyllotoxin had a certain damaging effect on cells at this dose.

[0027] High dose (1.26 μg / mL): The number of intestinal folds decreased significantly, the arrangement of epithelial cells was disordered, and a large number of brain cells showed apoptosis. At this concentration, podophyllotoxin caused significant damage to tissue structure, indicating that its toxicity was evident.

[0028] Extremely high dose (1.57 μg / mL): Intestinal lumen loss, severe tissue disorder, abnormal brain cell structure, and a large number of vacuolar and apoptotic cells. At this concentration, toxicity reaches its peak, significantly affecting the normal function of tissues.

[0029] The present invention conducts a study on podophyllotoxin by combining histopathological analysis with apoptosis analysis; by H&E staining, changes in tissue structure, such as cell arrangement, nuclear morphology and intercellular connections, can be clearly observed. This method can intuitively evaluate the degree of damage of podophyllotoxin to zebrafish intestinal and brain tissues. AO staining combined with fluorescence microscopy can quantitatively analyze cell apoptosis. This technology accurately reflects the degree and distribution of cell apoptosis by detecting fluorescence intensity. Combined with image processing software, a large number of samples can be efficiently analyzed to provide detailed toxicity data. Data statistics are performed by SPSS26.0 software, and mean±SE is used to represent the experimental results to ensure the scientificity and reliability of the data. Significance analysis (p<0.001) shows that the difference between the podophyllotoxin treatment groups with different concentrations and the normal control group is statistically significant, which verifies the significance of the experimental results.

[0030] This invention systematically evaluated the toxicity of podophyllotoxin to zebrafish intestinal tissue for the first time. Through detailed pathological and cell apoptosis analysis, it revealed the effects of different concentrations of podophyllotoxin on intestinal epithelial cells, providing important experimental data and theoretical basis for future intestinal toxicity research. The results showed that podophyllotoxin is not only significantly toxic to intestinal tissue, but also has obvious toxic effects on brain tissue. Through pathological and fluorescence microscopy analysis, the destructive mechanism of podophyllotoxin on brain cell structure and function was clarified, filling the gap in podophyllotoxin research on brain toxicity.

[0031] The zebrafish enterotoxicity modeling and detection method provided by the present invention is not only applicable to the toxicity study of podophyllotoxin, but can also be extended to the toxicity assessment of other drugs and toxins. This method has wide applicability and high experimental efficiency, and provides strong support for drug safety assessment and new drug development.

[0032] 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

[0033] 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.

[0034] Figure 1 Schematic diagram of zebrafish intestinal tissue structure; Note: The yellow dotted box is the zebrafish intestinal analysis area;

[0035] Figure 2 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;

[0036] Figure 3 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;

[0037] Figure 4 A 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;

[0038] Figure 5A 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;

[0039] Figure 6 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;

[0040] Figure 7 Schematic diagram of the comparison of the fluorescence intensity of apoptotic cells in zebrafish brain treated with podophyllotoxin and that in the normal control group; *p<0.001;

[0041] Figure 8 This is a schematic diagram of the “concentration-mortality” curve of podophyllotoxin enteroencephalic toxicity;

[0042] Fig. 9 Schematic diagram of intestinal toxicity phenotype after podophyllotoxin treatment in zebrafish; Note: The red dotted box is the zebrafish intestinal analysis area;

[0043] Fig.10 Schematic diagram of the brain toxicity phenotype of zebrafish after podophyllotoxin treatment; Note: The blue dotted box is the zebrafish brain analysis area;

[0044] Fig.11 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;

[0045] Fig.12 Schematic diagram showing the comparison between 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 and that of the normal control group; ***p<0.001. DETAILED DESCRIPTION

[0046] 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.

[0047] Example 1

[0048] The podophyllotoxin-based zebrafish enterotoxicity modeling and detection method and its application described in this embodiment include the following steps:

[0049] S1: Wild-type AB zebrafish at 3 days post-fertilization (3 dpf) were randomly selected and placed in a 6-well plate, and 30 zebrafish were treated in each well (experimental group).

[0050] S2: water-soluble podophyllotoxin was administered to establish the model, and a normal control group was set up at the same time. The volume of each well was 3 mL and the treatment was carried out at 28°C for 2 days.

[0051] S3: The zebrafish obtained in step S2 were subjected to histopathological analysis and intestinal cell apoptosis analysis.

[0052] In this embodiment, the concentration of podophyllotoxin in step S2 is 0.140-1.57 μg / mL.

[0053] In this embodiment, the histopathological analysis in step S3 specifically includes: fixing the zebrafish tissue with 4% paraformaldehyde at 4°C overnight to ensure the integrity of the cell structure. After that, dehydration treatment is performed, and the zebrafish sample is gradually dehydrated through a series of increasing concentrations of ethanol, starting with 70% ethanol treatment for 0.5 to 1 hour, and gradually increasing to 100% ethanol, and each step treatment time is about 15-30 minutes. After dehydration, a 1:1 mixture of xylene and 100% ethanol is used for 20 minutes for transparency, and then two 15-minute 100% xylene treatments are performed. Subsequently, the sample is immersed in soft wax at 60°C and hard wax at 65°C to completely embed the sample in an embedding instrument at 77°C. Use a microtome to cut the fixed and embedded tissue into 5-micron thick slices and stick them on a treated clean slide. Finally, the zebrafish were subjected to histopathological analysis using the hematoxylin-eosin (H&E) staining method. The staining process included hematoxylin staining for 8 minutes and eosin staining for no more than 1.5 minutes. After staining, the tissue sections were fixed in 95% and 100% ethanol, and finally treated with xylene and sealed with neutral resin for preservation.

[0054] In this embodiment, the intestinal cell apoptosis analysis in step S3 specifically includes: zebrafish are subjected to AO light-proof staining for 30 minutes, and after being washed three times with standard dilution water, 10 zebrafish are randomly selected from each experimental group and photographed under a fluorescence microscope, and NIS-Elements D3.20 advanced image processing software is used to analyze and collect data, and the fluorescence intensity of zebrafish intestinal apoptotic cells is analyzed and counted, and the effect of podophyllotoxin on intestinal cell apoptosis is evaluated by the statistical analysis results of the above indicators.

[0055] On the other hand, the present invention provides the application of the above method in studying the effects of podophyllotoxin on intestinal and brain tissues.

[0056] Example 2

[0057] Effects on intestinal and brain histopathology

[0058] Required Materials

[0059] Podophyllotoxin, the solvent is DMSO.

[0060] Experimental animals

[0061] Zebrafish were kept in aquaculture water at 28°C (water quality: 200 mg instant sea salt was added to 1 L of reverse osmosis water, conductivity was 450-550 μS / cm; pH was 6.5-8.5; hardness was 50-100 mg / L CaCO 3 ), bred and provided by our company's fish farming center, the experimental animal use license number is: SYXK(Zhejiang)2022-0004, the breeding and management complies with the requirements of the international AAALAC certification (certification number: 001458), IACUC ethics review number: IACUC-2023-7407-01.

[0062] Instruments, consumables and reagents

[0063] 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).

[0064] 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).

[0065] Detection Methods

[0066] 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.

[0067] Test results

[0068] Intestinal histopathological analysis

[0069] 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 1 , 2 shown.

[0070] Brain histopathological analysis

[0071] 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 1-3 shown.

[0072] Example 3

[0073] Effects on intestinal and brain cell apoptosis

[0074] The test materials and experimental animals were the same as those in Example 2;

[0075] Instruments, consumables and reagents

[0076] 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).

[0077] 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).

[0078] Detection Methods

[0079] Wild-type AB strain zebrafish at 3 days post-fertilization (3dpf) were randomly selected and placed in a 6-well plate, and 30 zebrafish were treated in each well (experimental group). Water-soluble podophyllotoxin (concentrations shown in Table 1) 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.

[0080] Test results

[0081] Under the experimental conditions, podophyllotoxin can induce intestinal apoptosis and brain apoptosis in zebrafish. See Table 1 for details. Figure 4-7 .

[0082] Table 1 Experimental results of the effects of samples on intestinal and brain cell apoptosis (n=10)

[0083]

[0084] Compared with the normal control group, *p<0.001;

[0085] Example 4

[0086] The test materials and experimental animals were the same as those in Example 2;

[0087] Instruments, consumables and reagents

[0088] 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).

[0089] 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).

[0090] Detection Methods

[0091] MNLC and LC10 assays

[0092] 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.

[0093] Evaluation of entero-brain toxicity

[0094] 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.

[0095] Test results

[0096] MNLC and LC10

[0097] 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 8 .

[0098] Table 2 Results of the sample gut-brain toxicity concentration exploration experiment (n=30)

[0099]

[0100]

[0101] Evaluation of entero-brain toxicity

[0102] 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 3 for details. Fig. 9 and Fig.10 .

[0103] Table 3 Statistics of the incidence of podophyllotoxin enteroencephalopathy (n=30)

[0104]

[0105] Note: “-” indicates no obvious abnormality; the numbers in the table indicate: incidence (%).

[0106] Example 5

[0107] The test materials and experimental animals were the same as those in Example 2;

[0108] Instruments, consumables and reagents

[0109] 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).

[0110] Dimethyl sulfoxide (DMSO, batch number I2229063, Shanghai Aladdin Biochemical Technology Co., Ltd., China).

[0111] Detection Methods

[0112] 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 4), 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 behavior 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.

[0113] Test results

[0114] Under the experimental conditions, podophyllotoxin can induce cognitive dysfunction in zebrafish. See Table 4 for details. Fig.11 and Fig.12 .

[0115] Table 4 Experimental results of the effects of samples on cognitive function (n=6)

[0116]

[0117] Compared with the normal control group, *p<0.001;

[0118] In summary, the present invention proposes a zebrafish enterotoxicity modeling and detection method based on podophyllotoxin and its application. Podophyllotoxin inhibits cell division and causes cell apoptosis by interfering with the polymerization of cell tubulin. This mechanism is not only effective in cancer cells, but also causes toxic reactions in normal cells. The present invention provides a reliable method for studying the enterotoxicity and brain toxicity of podophyllotoxin, and also provides important experimental tools and data support for the study of related toxicity mechanisms.

[0119] 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 enterotoxicity modeling and detection method based on podophyllotoxin, characterized in that: S1: Wild-type AB strain zebrafish 3 days after fertilization were randomly selected and placed in a 6-well plate, with 30 zebrafish treated in each well; S2: water-soluble podophyllotoxin was administered to establish the model, and a normal control group was set up at the same time. The volume of each well was 3 mL and the treatment was carried out at 28°C for 2 days. S3: The zebrafish obtained by modeling in step S2 were subjected to intestinal tissue pathology analysis and intestinal cell apoptosis analysis.

2. The podophyllotoxin-based zebrafish enterotoxicity modeling and detection method according to claim 1, characterized in that: The concentration of podophyllotoxin in step S2 is 0.140-1.57 μg / mL.

3. The podophyllotoxin-based zebrafish enterotoxicity modeling and detection method according to claim 1, characterized in that: The step S3 midgut histopathological analysis specifically includes: fixing the zebrafish tissue with 4% paraformaldehyde at 4°C overnight, and then dehydrating the zebrafish sample through a series of increasing concentrations of ethanol, starting with 70% ethanol treatment for 0.5 to 1 hour, and gradually increasing to 100% ethanol, with each step treatment time of 15-30 minutes; after dehydration, using a 1:1 xylene and 100% ethanol mixture for 20 minutes for transparency, and then performing two 15-minute 100% xylene treatments; then, the sample The samples were completely embedded in an embedding apparatus at 77°C by soaking in soft wax at 60°C and hard wax at 65°C. The fixed and embedded tissues were cut into 5-micron-thick slices using a microtome and mounted on clean treated slides. Finally, the zebrafish were subjected to histopathological analysis using the hematoxylin-eosin staining method. The staining process included hematoxylin staining for 8 minutes and eosin staining for no more than 1.5 minutes. After staining, the tissue sections were fixed in 95% and 100% ethanol, and finally treated with xylene and sealed with a neutral resin for preservation.

4. The podophyllotoxin-based zebrafish enterotoxicity modeling and detection method according to claim 1, characterized in that: The intestinal cell apoptosis analysis in step S3 specifically includes: subjecting the zebrafish to AO light-proof staining for 30 minutes, washing three times with standard diluted water, randomly selecting 10 zebrafish and photographing them under a fluorescence microscope, using NIS-Elements D 3.20 advanced image processing software to analyze and collect data, and analyzing and counting the fluorescence intensity of zebrafish intestinal apoptotic cells.

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

Images