Modeling method for zebra fish intestinal brain axis toxicity based on podophyllotoxin

Abstract

The invention belongs to the field of basic medicine, and particularly relates to a podophyllotoxin-based zebrafish intestinal brain axis toxicity modeling method which comprises the following steps: step 1, exposure treatment of zebrafish: selecting wild type AB-line zebrafish which is fertilized for 3 days, and treating for 2 days by dissolving podophyllotoxin in water; step 2, multi-omics joint detection: (1) extracting zebra fish intestinal tract and brain contents, performing 16S rRNA sequencing, quantitative metabolite detection and transcriptomics detection, and identifying substances; and 3, performing correlation analysis on the toxicity of the intestinal brain axis: performing correlation analysis on the differential flora, the differential metabolite and the differential expression gene identified in the step 2, and constructing an intestinal flora-metabolite-brain gene regulatory network. The invention aims to solve the problems that podophyllotoxin toxicity research in the prior art is limited to single organ evaluation, and an internal association mechanism among intestinal injury, dysbacteriosis, metabolic disorder and brain toxicity induced by podophyllotoxin is not revealed from an intestinal brain axis level system.

Description

Technical Field

[0001] This invention relates to the field of basic medicine, and in particular to a method for modeling the toxicity of zebrafish intestinal-brain axis based on podophyllotoxin. Background Technology

[0002] The gut-brain axis (GBA) is a bidirectional regulatory network between the gut and the central nervous system, mediated by neural, endocrine, immune, and gut microbiota mechanisms. Gut microbiota and their metabolites are key signaling molecules within this axis. Gut toxicity induced by toxins or drugs can disrupt gut microbiota balance and metabolite profiles, thereby mediating structural and functional damage to the brain. Therefore, systematically studying the toxic effects of exogenous substances at the gut-brain axis level is an important trend for comprehensively assessing their in vivo safety and elucidating their systemic toxicological mechanisms.

[0003] Podophyllotoxin is a natural aryltetrahydronaphthalene lignan compound derived from plants, possessing significant antitumor activity. Its derivatives, such as etoposide and teniposide, have been widely used in clinical cancer treatment. However, podophyllotoxin itself exhibits strong cytotoxicity against normal tissues, especially rapidly proliferating intestinal epithelial cells and neurons, leading to side effects such as intestinal mucosal damage, apoptosis, intestinal flora imbalance, and cognitive impairment, severely limiting its clinical application. Furthermore, podophyllotoxin can enter the environment through medical wastewater and other routes, posing a potential risk to aquatic ecosystems. Currently, toxicological studies on podophyllotoxin mainly focus on assessing damage to single organs; no research has systematically elucidated the systemic toxicity mechanism at the gut-brain axis level, revealing the correlation between intestinal damage, flora imbalance, metabolic disorders, and encephalopathy.

[0004] Regarding models for gut-brain axis toxicity studies, existing technologies include the following approaches: First, disease models based on mammals (such as mice and rats), such as models of Alzheimer's disease, Parkinson's disease, and irritable bowel syndrome related to the gut-brain axis. These models are primarily used to simulate the pathological characteristics of the gut-brain axis in specific diseases, and are not designed to evaluate the gut-brain axis toxicity of exogenous compounds. They also suffer from drawbacks such as long experimental cycles, high costs, numerous ethical restrictions, and difficulty in high-throughput screening. Second, single-organ toxicity evaluation models based on zebrafish, such as CN119199087A "A Method for Establishing and Detecting Zebrafish Brain Toxicity Based on Podophyllotoxin and Its Application" and CN119199088A "A Method for Establishing and Detecting Zebrafish Intestinal Toxicity Based on Podophyllotoxin and Its Application." These models utilize zebrafish to construct intestinal and brain toxicity models of podophyllotoxin, respectively, and evaluate them using histopathological methods (such as H&E staining), apoptosis (such as AO staining), and behavioral methods (such as cross-module analysis). However, these methods only target a single organ and fail to integrate multi-dimensional indicators such as gut microbiota, metabolic pathways, and gene expression. They cannot elucidate the gut-brain axis linkage mechanism by which podophyllotoxin disrupts gut microbiota and its metabolism, thereby causing neurological dysfunction. Summary of the Invention

[0005] The purpose of this invention is to provide a modeling method for zebrafish gut-brain axis toxicity based on podophyllotoxin. In view of the fact that the existing technology for podophyllotoxin toxicity research is limited to single organ assessment and lacks a systematic understanding of the intrinsic link between podophyllotoxin-induced intestinal damage, dysbiosis, metabolic disorders and brain toxicity at the gut-brain axis level.

[0006] To address the aforementioned technical problems, the present invention provides a method for establishing zebrafish intestinal-brain axis toxicity based on podophyllotoxin, implemented as follows: A method for modeling zebrafish intestinal-brain axis toxicity based on podophyllotoxin includes the following steps: Step 1, zebrafish exposure treatment: Select wild-type AB strain zebrafish that are 3 days after fertilization and treat them with podophyllotoxin water-soluble for 2 days; Step 2, Multi-omics Joint Detection: (1) Extract zebrafish intestinal contents, perform 16S rRNA sequencing, and identify differential bacterial communities; (2) Extract zebrafish brain tissue, perform metabolomics detection, and identify differential metabolites; (3) Extract zebrafish brain tissue and perform transcriptomics sequencing or qPCR detection to identify differentially expressed genes; Step 3: Correlation analysis of gut-brain axis toxicity: Correlation analysis was performed on the differentially expressed bacteria, metabolites, and genes identified in step 2 to construct a regulatory network of gut microbiota-metabolites-brain genes and evaluate the podophyllotoxin-induced gut-brain axis toxicity.

[0007] Optionally, the differential flora mentioned in step 2 (1) includes Acinetobacter and / or Hyaluronic Acids; The correlation analysis described in step 3 includes: performing a correlation analysis between the abundance changes of Acinetobacter and / or Hyaluronic Acids and the content changes of differential metabolites in brain tissue.

[0008] Optionally, the differential metabolites mentioned in step 2 (2) include metabolites in the purine metabolism pathway; the metabolites in the purine metabolism pathway are selected from one or more of adenosine, inosine, quinolinic acid, and oxalic acid; The correlation analysis described in step 3 includes: performing a correlation analysis between the changes in the content of the purine metabolites and the expression levels of the differentially expressed genes identified in step 2 (3).

[0009] Optionally, after step 1, cognitive function behavioral tests on the zebrafish may also be performed. The behavioral test is preferably a cross-shaped module analysis, which records the percentage of the zebrafish's movement distance in the blue area to evaluate the degree of cognitive impairment.

[0010] Optionally, the differentially expressed genes mentioned in step 2 (3) include: Synaptic function-related genes: selected from one or more of gad1b, drd2b, and grin3b; Inflammation-related genes: selected from one or more of IL-6, IL-1β, TNF-α, c3a.1, and c1qc; Purine metabolism-related genes: selected from one or more of p2rx4b and p2ry8; The correlation analysis described in step 3 includes: performing a correlation analysis between the expression levels of the differentially expressed genes and the behavioral detection results of zebrafish in step 1.

[0011] Optionally, after step 1, the process may also include simultaneous histopathological examination and / or apoptosis detection of the zebrafish's intestinal and brain tissues; The histopathological examination was performed by H&E staining, and the number of intestinal folds, intestinal lumen morphology, degree of epithelial cell disorder, brain cell morphology, number of vacuoles, and neuronal density were observed simultaneously. The apoptosis detection was performed using AO staining in the dark, with simultaneous quantification of the fluorescence intensity of apoptotic cells in the intestine and brain.

[0012] Optionally, the concentration of podophyllotoxin in step 1 is 0.140-1.57 μg / mL; The concentration gradient was set to 0.140 μg / mL, 0.420 μg / mL, 1.26 μg / mL, and 1.57 μg / mL; The processing temperature was 28°C, and at least three biological replicates were set up for each concentration group.

[0013] Optionally, the metabolomics assay described in step 2(2) is performed using an LC-MS / MS platform; Metabolites were extracted using the HM Meta 700 high-throughput quantitative metabolite detection kit and detected using a Waters ACQUITY UPLC system combined with a SCIEX QTRAP 6500 PLUS mass spectrometer.

[0014] Optionally, the transcriptomics sequencing described in step 2 (3) is RNA-seq, and PE150 sequencing is performed using the DNBSEQ-G400 platform; The screening criteria for differentially expressed genes were |log2(fold change)|≥1, q≤0.05; qPCR validation was performed using Gapdh as an internal reference gene, employing 2... -△△CT The relative expression level is calculated using this method.

[0015] Application of the zebrafish intestinal-brain axis toxicity modeling method based on podophyllotoxin in the preparation of kits for evaluating the intestinal-brain axis toxicity of podophyllotoxin or its derivatives.

[0016] The present invention has the following beneficial effects: 1. For the first time, the complete molecular regulatory pathway of podophyllotoxin gut-brain axis toxicity has been revealed. This invention, by integrating 16S rRNA sequencing of gut microbiota, metabolomics, and transcriptomics analysis of brain tissue, elucidates for the first time a complete gut-brain axis toxicity regulatory pathway induced by podophyllotoxin: "Gut microbiota dysbiosis (enrichment of Acinetobacter and Hyaluronic Acids) leads to purine metabolism disorders (decreased adenosine / inosine, accumulation of quinolinic acid / oxalate), thereby activating neuroinflammatory activity (upregulation of inflammatory factors such as IL-6 and TNF-α), ultimately resulting in synaptic dysfunction (downregulation of synaptic genes such as gad1b and drd2b), leading to cognitive and behavioral impairment." Compared to existing technologies that can only evaluate the enterotoxicity or neurotoxicity of podophyllotoxin separately, this invention reveals for the first time, from a systems biology perspective, the intrinsic link between gut and brain toxicity, filling the gap in research on the entero-brain axis toxicity of podophyllotoxin and providing crucial molecular evidence for elucidating its toxic mechanism.

[0017] 2. Construct a multi-dimensional integrated standardized evaluation system for gut-brain axis toxicity. This invention establishes a joint detection system covering five dimensions: gut microbiota, metabolism, genes, pathology, and behavior. It enables: gut microbiota structure and function analysis (16S rRNA sequencing + PICRUSt2 functional prediction), brain tissue metabolite profile and pathway analysis (LC-MS / MS targeted / non-targeted metabolomics), brain tissue transcriptome analysis and qPCR validation (RNA-seq + core gene validation), simultaneous detection of pathological damage in gut and brain tissue (H&E staining), simultaneous quantification of cell apoptosis in gut and brain tissue (AO staining), and cognitive function behavioral evaluation (cross-module analysis).

[0018] This system overcomes the limitations of existing technologies that rely on single detection indicators, achieving for the first time simultaneous modeling, multidimensional detection, and correlation analysis of gut-brain axis toxicity, significantly improving the systematicness, accuracy, and scientific rigor of toxicity evaluation. Furthermore, this invention clarifies standardized modeling parameters for 3 dpf AB strain zebrafish, a concentration gradient of 0.140-1.57 μg / mL, and treatment at 28℃ for 2 days, establishing a repeatable and scalable standard operating procedure, providing technical specifications for the gut-brain axis toxicity evaluation of toxins / drugs.

[0019] 3. It combines the dual advantages of high-throughput screening and in-depth mechanism analysis. This invention uses zebrafish as an experimental model, giving full play to its advantages of strong reproductive capacity, transparent embryos, short experimental cycle, and low cost: the experimental cycle is only 2 days, which is more than 90% shorter than that of mammalian models (which usually take several weeks to several months); the experimental cost is only 1 / 10 of that of mammalian models; a single experiment can process hundreds of zebrafish; it supports large-scale concentration gradient settings and multi-omics sample collection; it supports high-throughput formats such as 96-well plates, which are suitable for rapid screening of compound libraries.

[0020] 4. Clarify the dose-response pattern of podophyllotoxin intestinal-brain axis toxicity. This invention systematically revealed the dose-response relationship of podophyllotoxin's gut-brain axis toxicity by setting four concentration gradients: 0.140 μg / mL, 0.420 μg / mL, 1.26 μg / mL, and 1.57 μg / mL. Low concentration (0.140 μg / mL) induced mild gut microbiota disturbance but did not cause significant brain function damage; medium to high concentrations (0.420-1.26 μg / mL) induced significant changes in gut microbiota structure, purine metabolite disorder, neuroinflammatory activation, and downregulation of synaptic function genes; extremely high concentration (1.57 μg / mL) led to severe pathological damage to gut and brain tissues, apoptosis, and cognitive impairment.

[0021] This dose-response relationship provides crucial data support for determining the safe clinical dose of podophyllotoxin, identifying toxicity thresholds, and optimizing the structure of its derivatives, and can effectively guide the control of toxicity risks in its clinical application.

[0022] 5. Key regulatory factors and biomarkers for identifying gut-brain axis toxicity This invention, through multi-omics association analysis, identifies for the first time key regulatory factors and candidate biomarkers of podophyllotoxin gut-brain axis toxicity. Microbial biomarkers: Acinetobacter ( Acinetobacter ), Bacillus spp. ( Perlucidibaca Changes in abundance are significantly correlated with the severity of encephalopathy; metabolic markers: decreased adenosine and inosine (neuroprotective metabolites), and accumulated quinolinic acid and oxalic acid (neurotoxic metabolites) can serve as early warning indicators of gut-brain axis toxicity; gene markers: downregulation of synaptic functional genes (gad1b, drd2b, grin3b) and upregulation of inflammatory genes (IL-6, IL-1β, TNF-α) can serve as molecular indicators of brain function damage.

[0023] The identification of these markers provides a simple and sensitive detection target for rapid toxicity screening, clinical toxicity monitoring, and environmental risk assessment of podophyllotoxin compounds. Attached Figure Description

[0024] Figure 1 This is the "concentration-mortality" curve of podophyllotoxin enteroencephalopathy provided by the present invention; Figure 2 This refers to the percentage of the total movement distance of the blue area in zebrafish after treatment with different concentrations of podophyllotoxin provided by this invention, ***p < 0.001; Figure 3 This invention presents the intestinal and cerebral toxicity phenotypes of zebrafish treated with different concentrations of podophyllotoxin; Note: The area within the red dashed box represents the zebrafish intestinal and brain analysis regions. Figure 4 These are typical images of zebrafish brain and intestinal tissue structures after treatment with different concentrations of podophyllotoxin provided by this invention. Note: The area within the yellow dashed box is the zebrafish intestinal analysis area, the red arrow indicates the zebrafish intestinal folds, and the white arrow indicates the absence of the zebrafish intestinal lumen. Figure 5 This is a typical fluorescence intensity diagram of apoptotic cells in zebrafish brains after treatment with different concentrations of podophyllotoxin provided by this invention. Note: The area within the yellow dashed box is the zebrafish brain analysis region, and the green fluorescent particles are apoptotic cells; compared with the control group, ***p< 0.001; Figure 6 This invention provides a Venn diagram of the ASV distribution between the PPT group and the control group. Figure 7 This invention provides an α-diversity analysis of gut microbiota between the PPT group and the control group. Figure 8 This invention provides a PCoA analysis of gut microbiota between the PPT group and the control group. Figure 9 This refers to the relative abundance of bacterial phyla between the PPT group and the control group provided by this invention; Figure 10 This refers to the relative abundance of bacterial genera between the PPT group and the control group provided by this invention; Figure 11 This invention provides a multi-level species hierarchical tree diagram (from phylum to genus) of the gut microbiota of zebrafish in the PPT group and the control group. Figure 12 This is a bar chart showing the LDA discrimination of the gut microbiota of zebrafish in the PPT group and the control group provided by this invention (LDA > 4, p < 0.05). Figure 13 This is a PCoA analysis chart of the PPT group and the control group provided by the present invention; Figure 14 This is a bar chart showing the differential metabolite expression between the PPT group and the control group provided by this invention; Figure 15 This invention provides a volcano diagram of the differential metabolite distribution between the PPT group and the control group. Figure 16 This invention provides a heatmap of hierarchical clustering analysis of differential metabolites between the PPT group and the control group. Figure 17 This is a bubble diagram of the KEGG enrichment analysis of differential metabolites between the PPT group and the control group provided by the present invention. Figure 18 This is a bar chart of key differential metabolites provided by the present invention; Figure 19 This is a PCoA analysis chart of the PPT group and the control group provided by the present invention; Figure 20 This is a bar chart showing the differential gene expression between the PPT group and the control group provided by the present invention; Figure 21 This is a volcano diagram of differentially expressed genes between the PPT group and the control group provided by the present invention; Figure 22 This is a GO analysis bar chart of differentially expressed genes between the PPT group and the control group provided by this invention; Figure 23 This is a bubble chart of KEGG enrichment analysis of differentially expressed genes between the PPT group and the control group provided by this invention. Figure 24 This is a gene network diagram of 5 key KEGG pathways provided by the present invention. Red represents upregulated expression and blue represents downregulated expression. Figure 25 This is a heatmap of five key genes in the KEGG pathway provided by this invention; Figure 26These are the PCR verification results provided by this invention. Compared with the control group, ** p < 0.01, *** p < 0.001; Figure 27 This invention provides a heatmap showing the correlation between key differential metabolites and differentially expressed genes. Figure 28 This invention provides a heatmap showing the correlation between key differential metabolites and differential bacterial genera. Figure 29 This is a schematic diagram of the differential bacterial genera, key differential metabolites, and differential gene mechanisms provided by the present invention; Detailed Implementation

[0025] To make the objectives, technical solutions, and advantages of this invention clearer, the following embodiments provide a more detailed description of the invention. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the scope of the invention.

[0026] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0027] This invention uses wild-type AB strain zebrafish as an experimental model. Based on the gut-brain axis toxicity effect of podophyllotoxin, a standardized system of "simultaneous modeling - multi-omics joint detection - correlation analysis" is constructed. This system integrates multi-dimensional indicators such as gut microbiota, metabolomics, transcriptomics, histopathology, apoptosis, and behavior to clarify the molecular regulatory pathway of podophyllotoxin gut-brain axis toxicity, as detailed below: Example 1: Experimental Materials and Methods 1.1 Preparation of Experimental Materials Experimental animals: Wild-type AB strain zebrafish were selected 3 days after fertilization (3 dpf), healthy and without deformities, and raised in standard fish tank water at 28℃ (conductivity 450~550 μS / cm, pH 6.5~8.5, hardness 50~100 mg / L CaCO3). They were acclimatized for 24 hours before the experiment.

[0028] Experimental reagents: podophyllotoxin (solvent: DMSO), 4% paraformaldehyde fixative, hematoxylin-eosin (H&E) staining solution, acridine orange (AO) staining solution, anhydrous ethanol, xylene, neutral resin, and reagents related to 16S rRNA sequencing, metabolomics (LC-MS / MS), transcriptomics (RNA-seq), and qPCR.

[0029] Experimental instruments: precision electronic balance, 6-well plates / beakers, dissecting microscope, fluorescence microscope, tissue slicer, zebrafish behavior analyzer (Zebra Lab 3.22.3.31), NIS-Elements D 3.20 image processing software, SPSS 26.0 statistical software, Origin 8.0 software, and high-throughput sequencing platform (DNBSEQ-G400).

[0030] 1.2 Experimental Grouping and Modeling Treatment Grouping: Zebrafish were randomly divided into a normal control group, a solvent control group (1% DMSO), and a podophyllotoxin concentration gradient group, with concentration gradients of 0.140 μg / mL, 0.420 μg / mL, 1.26 μg / mL, and 1.57 μg / mL. Six-well plates were used for pathological / apoptotic / microbial / omics detection (30 fish per well), and beakers were used for behavioral detection (30 fish per beaker). Each group had three biological replicates.

[0031] Modeling treatment: Podophyllotoxin was added to each concentration group using a water-soluble administration method. The normal control group was given only standard fish tank water, while the solvent control group was given fish tank water with 1% DMSO. The liquid volume per well / cup was 3 mL (6-well plate) and 20 mL (beaker), respectively. All groups were placed in a 28℃ constant temperature incubator for 2 days, during which normal dissolved oxygen was maintained and the culture medium was not changed.

[0032] 1.3, 10% lethal concentration (LC50) 10 ) and Maximum Non-Lethal Concentration (MNLC) Based on concentration-mortality data, a dose-response model was fitted to determine the 10% lethal concentration (LC50) of podophyllotoxin in zebrafish. 10 The concentration-mortality rate was 1.57 μg / mL, the maximum non-lethal concentration (MNLC) was 1.26 μg / mL, and the correlation coefficient (R²) was 1. The corresponding concentration-mortality curve is shown below. Figure 1 As shown. Based on the LC determined above. 10 Based on the MNLC values, four concentrations of 0.140, 0.420, 1.26, and 1.57 μg / mL were selected for subsequent histopathological experiments.

[0033] Example 2: Detection of zebrafish intestinal-brain axis toxicity based on podophyllotoxin 2.1 Podophyllotoxin-induced behavioral disorders and gut-brain developmental abnormalities To assess the neurotoxicity of different concentrations of podophyllotoxin, behavioral tests were first conducted. 2.8: Cognitive Function Behavioral Testing (Cross-Module Analysis) After the modeling was completed, five zebrafish were randomly selected from each group and placed into a cross-shaped module containing four regions: yellow, blue, red, and green. Each group had six modules. The behavior analyzer collects the movement trajectory of zebrafish within 10 minutes, calculates the proportion of movement distance in the blue area, and evaluates the degree of cognitive dysfunction. By linking behavioral data with brain pathology, apoptosis, and gene expression results, a neurotoxicity correlation chain of "pathology-gene-behavior" is constructed.

[0034] The results of the cross maze color preference experiment showed that, compared with the control group, zebrafish exposed to 1.26 μg / mL and 1.57 μg / mL podophyllotoxin had a significantly reduced proportion of total swimming distance in the blue area in a dose-dependent manner. Figure 2 This indicates impaired cognitive function. Furthermore, exposure to 1.57 μg / mL podophyllotoxin resulted in a 13% mortality rate. At concentrations of 1.26 μg / mL and 1.57 μg / mL, zebrafish exhibited both enterotoxicity and neurotoxicity: enterotoxicity manifested as intestinal shrinkage and intestinal contraction; neurotoxicity manifested as brain shrinkage and brain degeneration. At a concentration of 0.420 μg / mL, similar intestinal and brain abnormalities were observed, including intestinal narrowing and brain volume reduction. At the lowest tested concentration of 0.140 μg / mL, only enterotoxicity was detected, primarily manifested as intestinal volume reduction (…). Figure 3 These results indicate that podophyllotoxin exposure disrupts the development of zebrafish gut and brain tissue and significantly impairs their neurological function.

[0035] 2.2: Podophyllotoxin-induced histopathological changes in zebrafish brain and intestinal tissues After modeling, 10 zebrafish were randomly selected from each group and fixed overnight with 4% paraformaldehyde at 4°C. After dehydration with gradient ethanol, clearing with xylene, and embedding in soft / hard wax, the sections were cut into 5μm thick continuous sections and mounted on glass slides. Stain with hematoxylin for 8 min, eosin for ≤1.5 min, fix with graded ethanol, clear with xylene, and mount with neutral resin; The pathological damage of the intestine (number of folds, morphology of the intestinal lumen, degree of disorder in epithelial cell arrangement) and the brain (morphology of brain cells, number of vacuoles, density of neuronal arrangement) was observed and quantitatively analyzed under an optical microscope, and the correlation between intestinal and brain pathological damage was simultaneously correlated.

[0036] Hematoxylin-eosin (H&E) staining, such as Figure 4Regarding brain tissue, the zebrafish brain cells in the control group had normal morphology, with large, round, and deeply stained nuclei, and the cells were densely packed and evenly distributed. The brain cells in the 0.140 μg / mL podophyllotoxin group had normal structure and were densely packed, similar to the control group. The brain cells in the 0.420 μg / mL group had relatively intact morphology, but the brain volume was significantly reduced. The brain in the 1.26 μg / mL group was significantly reduced in size, with obvious cell degeneration, uneven staining, disordered arrangement, and vacuolar structures. The brain in the 1.57 μg / mL group was even more severely reduced in size, with extensive cell degeneration, uneven nuclear staining, abnormal morphology, irregular distribution, and a large number of vacuoles. Regarding intestinal tissue, the control group of zebrafish exhibited well-developed intestinal lumen with abundant mucosal folds, and epithelial cells tightly connected to villi and the mucosal layer. The 0.140 μg / mL group showed intestinal morphology similar to the control group, with preserved folded structures. The 0.420 μg / mL group showed a reduced intestinal lumen. The 1.26 μg / mL group showed a narrowed intestinal lumen and a significant decrease in the number of mucosal folds. The 1.57 μg / mL group showed complete loss of the intestinal lumen and loss of tissue folds. These histological results confirm that podophyllotoxin exposure induces dose-dependent pathological damage to the brain and intestinal tissues of zebrafish, consistent with earlier assessments of behavioral disturbances and organ toxicity.

[0037] 2.3: Effects of podophyllotoxin exposure on apoptosis in zebrafish brain and intestinal tissue cells After the modeling was completed, 10 zebrafish were randomly selected from each group, stained with AO staining solution in the dark for 30 minutes, and washed 3 times with standard dilution water. Apoptotic cells (green fluorescence) in the intestine and brain were observed and photographed under a fluorescence microscope. The fluorescence intensity of apoptotic cells in the intestine and brain was quantitatively analyzed using NIS-Elements D3.20 software. Statistical analysis was performed to assess the correlation between intestinal and brain cell apoptosis intensity and to evaluate the intestinal-brain apoptosis association effect induced by podophyllotoxin.

[0038] The results are as follows Figure 5 As shown, the apoptosis levels in brain and intestinal tissues in the 0.140 μg / mL and 0.420 μg / mL concentration groups were not significantly different from those in the control group; however, in the 1.26 μg / mL and 1.57 μg / mL concentration groups, the degree of apoptosis in both brain and intestinal tissues was significantly increased.

[0039] 2.4: Effects of podophyllotoxin on gut microbiota diversity and functional pathways in zebrafish After the modeling was completed, zebrafish from the maximum non-lethal concentration (MNLC=1.26μg / mL) group and the normal control group were selected, with 6 samples from each group, and genomic DNA of gut microbiota was extracted. The V4 region of the 16S rRNA gene was amplified (primers 515F / 806R), a sequencing library was constructed, and sequencing was performed using the DNBSEQ-G400 platform; Bioinformatics analysis: α-diversity (Chao1, ACE, Shannon, Simpson index) assesses gut microbiota richness and diversity; β-diversity (PCoA analysis) assesses gut microbiota structural differences; LEfSe analysis screens differentially enriched gut microbiota (LDA>4, p<0.05); PICRUSt2 predicts gut microbiota functional pathways (focusing on gut-brain axis-related pathways such as purine metabolism).

[0040] Clustering of amplicon sequence variants (ASVs) based on 97% sequence similarity, Venn diagrams show that there are a total of 632 ASVs in the podophyllotoxin group and the control group, with 104 ASVs specific to the podophyllotoxin group and 72 ASVs specific to the control group. Figure 6 Alpha diversity analysis showed that podophyllotoxin exposure significantly reduced gut microbiota richness (decreased ACE and Chao indices); compared with the control group, the podophyllotoxin group showed a significant decrease in the Simpson diversity index and a significant increase in the Shannon diversity index—the Shannon index integrates species richness and evenness, and its increase indicates enrichment of gut microbiota or the environment in zebrafish, with a more even distribution of different species; the Simpson index mainly reflects the dominance of dominant groups, and its decrease indicates a reduction in the relative abundance of dominant species, which may mean increased community diversity and structural homogenization. Figure 7 Furthermore, principal coordinate analysis (PCoA) based on the unweighted UniFrac distance matrix showed a significant separation between the podophyllotoxin group and the control group. Figure 8 This indicates that podophyllotoxin significantly altered the overall composition of the gut microbiota. To identify the specific bacterial groups affected, taxonomic analysis was performed at the phylum and genus levels: at the phylum level, compared to the control group, the podophyllotoxin group showed a decrease in the relative abundance of Proteobacteria and an increase in the relative abundance of Firmicutes, Bacteroidetes, and Actinobacteriota. Figure 9 At the genus level, the relative abundance of *Aeromonas*, *Perlucidibaca*, *Methyloversatilis*, *Bifidobacterium*, *Sphaerotilus*, and *Acinetobacter* changed significantly. Figure 10 Linear discriminant analysis of effect size (LEfSe) further identified differentially enriched groups between the two groups (LDA>4, p<0.05). Figure 11 A total of 30 differentially expressed ASVs were screened, of which 8 were enriched in the control group (mainly from Acidicapsa and Aeromonas), and 22 were enriched in the podophyllotoxin group (mainly from Acinetobacter and Amycosis). Figure 12 Based on KEGG functional annotation and abundance data, a total of 112 pathways were found to be significantly different, among which the purine metabolism pathway was significantly downregulated in the podophyllotoxin group.

[0041] 2.5: Podophyllotoxin exposure induces changes in metabolic pathways in zebrafish Zebrafish brain tissue was collected from section 2.2, with 6 samples per group. Metabolites were extracted using the HM Meta 700 high-throughput quantitative metabolite detection kit. LC-MS / MS analysis (Waters ACQUITY UPLC system + SCIEX QTRAP 6500 PLUS mass spectrometer), data processing using Skyline software, and differential metabolites were identified (screening criteria: VIP>1, q<0.05). KEGG pathway enrichment analysis was used to identify differentially expressed metabolites, with a focus on purine metabolism pathways (key metabolites such as adenosine, inosine, quinolinic acid, and oxalic acid), and to correlate them with gut microbiota function prediction results.

[0042] Principal component analysis (PCA) showed a significant separation in the metabolite profiles between the podophyllotoxin group and the control group. Figure 13 This indicates that podophyllotoxin exposure significantly disrupts metabolic homeostasis. Based on the screening criteria of variable importance projection (VIP) > 1 and q value < 0.05, a total of 79 significantly differentially expressed metabolites were identified, of which 41 were upregulated and 39 were downregulated. Figure 14-15 The differential metabolite heatmap clearly shows that there are different metabolic patterns between the two groups. Figure 16 Pathway enrichment analysis of differentially metabolites showed significant enrichment in the ABC transporter, purine metabolism, and phenylalanine metabolism pathways. Figure 17 It is noteworthy that previous studies have confirmed the crucial role of purine metabolism in the nervous system, and its disruption is closely associated with various neurodegenerative diseases and neurotoxic reactions. This is consistent with the results of gut microbiota function prediction (both showing significant disruption of purine metabolic pathways). Specifically, compared with the control group, the podophyllotoxin exposure group showed a significant downregulation of key intermediates of purine metabolism (such as adenosine, guanosine, inosine, and 5-aminoimidazole-4-carboxamide), while oxalate was significantly upregulated. Figure 18 Furthermore, the neurotoxic metabolite quinolinic acid was also significantly elevated after podophyllotoxin exposure. Abnormal accumulation of these metabolites may disrupt neurotransmitter balance, ultimately leading to neurotoxic damage.

[0043] 2.6: Transcriptomic analysis reveals changes in gene expression profiles induced by podophyllotoxin. Zebrafish brain tissue from section 2.2 was collected, total RNA was extracted, an mRNA sequencing library was constructed, and the mRNA was sequenced using the DNBSEQ-G400 platform PE150. Differentially expressed genes (DEGs) screening (criteria: |log2 (fold change)|≥1, q≤0.05), GO / KEGG enrichment analysis (focusing on synaptic function, inflammatory response, and purine metabolism-related pathways); qPCR was used to validate core genes: synaptic function-related genes (gad1b, drd2b, grin3b), inflammation-related genes (IL-6, IL-1β, TNF-α, c3a.1, c1qc), and purine metabolism-related genes (p2rx4b, p2ry8). Gapdh was used as an internal reference gene, and the relative expression levels were calculated using the 2-ΔΔCT method.

[0044] Principal coordinate analysis (PCoA) showed a significant separation between the two groups on principal component 1 (59.87%) and principal component 2 (22.69%). Figure 19 This indicates that podophyllotoxin exposure significantly altered gene expression patterns. Using DESeq2 software, with a threshold of |log2-fold change| ≥ 1 and a false detection rate (FDR) ≤ 0.05, a total of 3974 differentially expressed genes (DEGs) were identified, of which 2126 were downregulated and 1848 were upregulated. Figure 20 ), differentially expressed gene volcano diagram as shown Figure 21 As shown. Functional enrichment analysis of differentially expressed genes: Gene Ontology (GO) enrichment showed significant enrichment of 22 biological process (BP) terms, with the top three being complement activation, axonoderm-dynein complex assembly, and synaptic structure or activity regulation; in terms of cellular components (CC), 31 categories were significantly enriched, with the plasma membrane, synapse, and extracellular regions being the most prominent; in terms of molecular function (MF), four terms were significantly enriched, including calcium-dependent phospholipid binding, adenylate cyclase inhibition of G protein-coupled glutamate receptor activity, calcium ion binding, and G protein-coupled receptor binding (…). Figure 22 Pathway enrichment analysis by the Kyoto Encyclopedia of Genes and Genomes (KEGG) revealed significant enrichment of 14 signaling pathways, many of which are closely related to neuronal and synaptic dysfunction and inflammatory responses. These include synaptic function-related pathways (synaptic vesicle circulation, glutamatergic synapses, γ-aminobutyric acid synapses, and dopaminergic synapses) and inflammation-related pathways (especially complement and coagulation cascade pathways). Figure 23 ).

[0045] 2.7 Network Analysis and qPCR Validation of Key Regulatory Pathways To further elucidate the key regulatory mechanisms, five significantly enriched KEGG pathway-related genes were introduced into Cytoscape to construct an interaction network. Figure 24 ), and identified core genes, including synaptic function-related genes (γ-aminobutyric acid synapses: gad1a / gad1b, gad2; dopaminergic synapses: drd2a / drd2b, th; synaptic vesicle cycle: slc17a6b; glutamatergic synapses: grin3b) and complement cascade-related genes (c1qc, c3a.1) Figure 25 Given that both 16S rRNA sequencing and metabolomics analysis showed significant disruption of the purine metabolism pathway, the expression of key genes in the synaptic and complement pathways, as well as purine metabolism-related genes (p2rx4b, p2ry8, p2ry13), was detected by qPCR. Furthermore, to verify the effect of podophyllotoxin exposure on neuroinflammatory responses, the expression of pro-inflammatory cytokines IL-1β, IL-6, and TNF-α was analyzed. The results showed that, compared with the control group, the expression of inflammation-related genes (c1qc, c3a.1, TNF-α, IL-1β, IL-6) was significantly increased in the podophyllotoxin group, while the expression of synaptic function-related genes (gad1a / gad1b, gad2, drd2a / drd2b, th, slc17a6b, grin3b) and purine metabolism-related genes (p2rx4b, p2ry8, p2ry13) was significantly downregulated. Figure 26 These results further support the transcriptomics data, reinforcing the research conclusions at the molecular level.

[0046] Example 3: Correlation analysis and statistical processing of zebrafish intestinal-brain axis toxicity based on podophyllotoxin 3.1 Multidimensional Correlation Analysis: Microbiota-metabolic association: Analyze the correlation between differentially expressed microbiota (such as Acinetobacter and Perlucidibaca) and differentially expressed metabolites (such as adenosine and quinolinic acid); Metabolic-gene association: Analysis of the correlation between purine metabolites and the expression of synaptic function / inflammatory genes; Gut-brain association: Analyze the correlation between intestinal pathology / apoptosis and brain pathology / apoptosis / behavioral indicators, and construct a complete regulatory link of "intestinal damage-dysbiosis-metabolic disorder-encephaltoxicity".

[0047] Analysis of differentially metabolized substances in the purine metabolism pathway and key genes in synaptic and inflammation-related pathways showed that the expression levels of drd2b, grin2bb, gad1a, and gad1b were positively correlated with guanosine and inosine levels, and negatively correlated with quinolinic acid and oxalate levels. Figure 27Subsequently, the correlation between differentially metabolized purine metabolism pathways and genus-level differential gut microbiota was assessed. The results showed that *Acinetobacter* and *Bacillus* were negatively correlated with adenosine, guanosine, inosine, and 5-aminoimidazole-4-carboxamide, and positively correlated with oxalic acid and quinolinic acid. Figure 28 ).

[0048] Correlation analysis showed that podophyllotoxin exposure significantly altered the abundance of Acinetobacter in the gut microbiota of zebrafish. This change was closely related to purine metabolism disorders and abnormal accumulation of neurotoxic metabolites in brain tissue—a decrease in neuroprotective metabolites (adenosine, inosine, guanosine) and an increase in neurotoxic metabolites (oxalate, quinolinic acid), suggesting a potential link between metabolic disorders and neurotoxicity. Simultaneously, abnormal expression of purine metabolism receptors was accompanied by upregulation of pro-inflammatory cytokines (TNF-α, IL-1β, IL-6) and genes related to complement and coagulation cascades, suggesting a possible neuroinflammatory response. These inflammatory markers were associated with altered expression of synaptic function-related genes (involving dopaminergic, γ-aminobutyric, glutamatergic synapses and synaptic vesicle circulation), potentially leading to synaptic dysfunction. Figure 29 The above findings suggest that gut microbiota dysbiosis (especially changes in Acinetobacter) is associated with alterations in brain metabolic pathways and inflammatory signal transduction, and may further be linked to changes in the expression of synaptic function-related genes, collectively supporting the involvement of the gut-microbiota-brain axis in podophyllotoxin-induced neurotoxic outcomes.

[0049] It should be noted that: In Examples 2 and 3, statistical analysis was performed using SPSS 26.0 software, and the results are expressed as mean ± SE. The comparison between groups was performed using t-test or two-way ANOVA, with p < 0.05 indicating statistical significance and p < 0.001 indicating extremely significant difference; Origin 8.0 was used to plot concentration-effect curves, correlation heatmaps, etc.

[0050] 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, and improvements 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 modeling zebrafish intestinal-brain axis toxicity based on podophyllotoxin, characterized in that, Includes the following steps: Step 1, zebrafish exposure treatment: Select wild-type AB strain zebrafish that are 3 days after fertilization and treat them with podophyllotoxin water-soluble for 2 days; Step 2, Multi-omics Joint Detection: (1) Extract zebrafish intestinal contents, perform 16S rRNA sequencing, and identify differential bacterial communities; (2) Extract zebrafish brain tissue, perform metabolomics detection, and identify differential metabolites; (3) Extract zebrafish brain tissue and perform transcriptomics sequencing or qPCR detection to identify differentially expressed genes; Step 3: Correlation analysis of gut-brain axis toxicity: Correlation analysis was performed on the differentially expressed bacteria, metabolites, and genes identified in step 2 to construct a regulatory network of gut microbiota-metabolites-brain genes and evaluate the podophyllotoxin-induced gut-brain axis toxicity.

2. The method according to claim 1, characterized in that, The differential flora mentioned in step 2(1) includes Acinetobacter and / or Hyaluronic Acids; The correlation analysis described in step 3 includes: performing a correlation analysis between the abundance changes of Acinetobacter and / or Hyaluronic Acids and the content changes of differential metabolites in brain tissue.

3. The method according to claim 1 or 2, characterized in that, The differential metabolites mentioned in step 2 (2) include metabolites in the purine metabolism pathway; the metabolites in the purine metabolism pathway are selected from one or more of adenosine, inosine, quinolinic acid, and oxalic acid; The correlation analysis described in step 3 includes: performing a correlation analysis between the changes in the content of the purine metabolites and the expression levels of the differentially expressed genes identified in step 2 (3).

4. The method according to claim 3, characterized in that, After step 1, the process also includes cognitive function behavioral testing of the zebrafish; The behavioral test is preferably a cross-shaped module analysis, which records the percentage of the zebrafish's movement distance in the blue area to evaluate the degree of cognitive impairment.

5. The method according to claim 4, characterized in that, The differentially expressed genes mentioned in step 2 (3) include: Synaptic function-related genes: selected from one or more of gad1b, drd2b, and grin3b; Inflammation-related genes: selected from one or more of IL-6, IL-1β, TNF-α, c3a.1, and c1qc; Purine metabolism-related genes: selected from one or more of p2rx4b and p2ry8; The correlation analysis described in step 3 includes: performing a correlation analysis between the expression levels of the differentially expressed genes and the behavioral detection results of zebrafish in step 1.

6. The method according to claim 5, characterized in that, After step 1, the process also includes simultaneous histopathological examination and / or apoptosis detection of the zebrafish's intestinal and brain tissues; The histopathological examination was performed by H&E staining, and the number of intestinal folds, intestinal lumen morphology, degree of epithelial cell disorder, brain cell morphology, number of vacuoles, and neuronal density were observed simultaneously. The apoptosis detection was performed using AO staining in the dark, with simultaneous quantification of the fluorescence intensity of apoptotic cells in the intestine and brain.

7. The method according to claim 6, characterized in that, The concentration of podophyllotoxin mentioned in step 1 is 0.140-1.57 μg / mL; The concentration gradient was set to 0.140 μg / mL, 0.420 μg / mL, 1.26 μg / mL, and 1.57 μg / mL; The processing temperature was 28°C, and at least three biological replicates were set up for each concentration group.

8. The method according to claim 7, characterized in that, The metabolomics assay described in step 2(2) was performed using an LC-MS / MS platform; Metabolites were extracted using the HM Meta 700 high-throughput quantitative metabolite detection kit and detected using a Waters ACQUITYUPLC system combined with a SCIEX QTRAP 6500 PLUS mass spectrometer.

9. The method according to claim 8, characterized in that, The transcriptomics sequencing described in step 2 (3) is RNA-seq, and PE150 sequencing is performed using the DNBSEQ-G400 platform; The screening criteria for differentially expressed genes were |log2(fold change)|≥1, q≤0.05; qPCR validation was performed using Gapdh as an internal reference gene, employing 2... -△△CT The relative expression level is calculated using this method.

10. Use of the method of any one of claims 1-9 in the preparation of a kit for evaluating the intestinal-brain axis toxicity of podophyllotoxin or its derivatives.

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