Zebra fish model-based pollutant immunotoxicity comprehensive evaluation method
By employing a multi-level comprehensive evaluation method based on the zebrafish model, combining immune cells, humoral factors, and gene expression, the ITI index is calculated, addressing the shortcomings of traditional detection methods in terms of systematicness and quantification, and achieving accurate assessment of immune damage caused by pollutants.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- SOUTH CHINA INST OF ENVIRONMENTAL SCI MEP
- Filing Date
- 2026-01-29
- Publication Date
- 2026-05-19
AI Technical Summary
Traditional immunotoxicity detection methods often rely on single biochemical or molecular indicators, lacking systematicity and quantification, making it difficult to reveal the degree of immune damage and the comprehensive effects of pollutants.
A multi-level comprehensive evaluation method based on the zebrafish model was adopted. The comprehensive immunotoxicity index (ITI) was calculated and graded by detecting the number of immune cells, the level of humoral immune factors and the expression of immune-related genes in transgenic zebrafish.
It enables the quantification and comprehensive assessment of immune damage from pollutants, improves the sensitivity and repeatability of detection, is suitable for risk studies of low-dose pollutants, and has strong applicability, applicable to the immunotoxicity detection of drugs, pesticides, plastics and other new pollutants.
Smart Images
Figure CN122067602A_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of environmental toxicology and biological detection technology, specifically relating to a comprehensive evaluation method for the immunotoxicity of pollutants based on a zebrafish model. Background Technology
[0002] In recent years, new pollutants (such as drug residues, plastic additives, and flame retardants) have been widely detected in aquatic environments, and long-term exposure can have adverse effects on the immune system of organisms. However, traditional immunotoxicity detection methods mostly rely on single biochemical or molecular indicators, lacking systematicity and quantification, making it difficult to reveal the degree of immune damage and the comprehensive effects of pollutants.
[0003] Zebrafish ( Danio rerio As a transparent, small, rapidly developing model organism with a conserved immune system, zebrafish has significant advantages in environmental toxicology research. By combining multi-level immune response information from zebrafish to establish a comprehensive immunotoxicity evaluation system, the shortcomings of existing technologies such as "dispersed indicators, incomparable results, and lack of quantitative standards" can be overcome, enabling a systematic determination of the immunotoxicity of pollutants.
[0004] In existing immunotoxicity studies, immune damage is often characterized by changes in a single humoral factor or gene expression. However, such detection methods have limited sensitivity and representativeness, and are difficult to accurately reflect the overall response characteristics of the body's immune system.
[0005] Based on this, the present invention proposes a comprehensive evaluation method for the immunotoxicity of pollutants based on a zebrafish model, hoping to solve the problems existing in the prior art. Summary of the Invention
[0006] The purpose of this invention is to address existing problems by providing a comprehensive evaluation method for the immunotoxicity of pollutants based on a zebrafish model.
[0007] This invention is achieved through the following technical solution: A comprehensive evaluation method for the immunotoxicity of pollutants based on a zebrafish model includes the following steps: Step 1: Expose transgenic zebrafish embryos to the contaminants to be tested and culture them to the predetermined developmental stage; Step 2: Detect the number of immune cells in juvenile zebrafish; Step 3: Detect the levels of humoral immune factors in juvenile zebrafish; Step 4: Detect the expression levels of immune-related genes in zebrafish juveniles; Step 5: Based on the test results from Steps 2 to 4, calculate the Integrated Immunotoxicity Index (ITI). Step Six: Classify the immunotoxicity of the pollutants according to the Integrated Immunotoxicity Index (ITI).
[0008] Furthermore, the transgenic zebrafish includes the macrophage fluorescently labeled strain Tg( mpeg1 EGFP) and / or neutrophil fluorescently labeled strain Tg( lyz :DsRed2).
[0009] Furthermore, the humoral immune factors include, but are not limited to, C-reactive protein (CRP), immunoglobulin M (IgM), complement component C3, macrophage inflammatory protein (MIP), and lysozyme activity (LYSO), and at least five of them are selected.
[0010] Furthermore, the immune-related genes include, but are not limited to, those mentioned above. TNF-α , IL-1β , IL-8 , IL-10 , NF-κB And at least 5 types must be selected.
[0011] Furthermore, the calculation of the Integrated Immunotoxicity Index (ITI) includes: Standardize the processing of each immune indicator; The standardized immune index values are multiplied by their corresponding weighting coefficients and summed to obtain the ITI value.
[0012] Furthermore, the weighting coefficients are set according to the immune hierarchy, wherein the weight of the immune cell layer is 0.3-0.5, the weight of the humoral immune layer is 0.2-0.4, and the weight of the gene expression layer is 0.2-0.4.
[0013] Furthermore, the immunotoxicity grading includes: When ITI < 0.2, it is determined that there is no significant immune effect; When 0.2 ≤ ITI < 0.5, it is judged as a mild immune perturbation; When 0.5 ≤ ITI < 0.8, it is considered moderate immune impairment; When ITI ≥ 0.8, it is considered significant immunotoxicity.
[0014] Furthermore, the contaminant includes at least one of pharmaceuticals, pesticides, plastic additives, flame retardants, or other novel contaminants.
[0015] The present invention has the following advantages over the prior art: This invention aims to establish a multi-level, multi-indicator integrated immunotoxicity evaluation system. Combining indicators such as the number of immune cells, humoral immune factors, and the expression of immune-related genes, it constructs a comprehensive immunotoxicity index (ITI) to quantify and comprehensively determine the immune damage caused by pollutants. This provides a scientific basis for the rapid identification and risk assessment of the immunotoxicity of environmental pollutants. Specifically, this is manifested in: (1) Multidimensional integration and systematic evaluation: This method integrates three types of indicators—immune cells, humoral factors, and molecular responses—in the zebrafish model for the first time, so as to achieve a systematic and quantitative evaluation of immunotoxic effects.
[0016] (2) Introduce the Integrated Immunotoxicity Index (ITI): Establish a standardized scoring system that can quantify the degree of immune damage from different pollutants and overcome the limitations of traditional single indicators.
[0017] (3) High sensitivity and strong reproducibility: By using transgenic zebrafish and specific immunoassay technology, weak immune disturbances can be detected in the early development stage, which is suitable for low-dose pollutant risk studies.
[0018] (4) Strong applicability: This method is applicable to the immunotoxicity detection of drugs, pesticides, plastics and other new pollutants, and has broad application prospects. Attached Figure Description
[0019] Figure 1 This is a spectrum of immune damage characteristics. Detailed Implementation
[0020] To further explain the present invention, the following specific embodiments are described.
[0021] Example 1: 1. Detection of immune cell count Transgenic zebrafish strain used: Tg( mpeg1 EGFP (green fluorescent marker for macrophages) and Tg ( lyz :DsRed2 (red fluorescently labeled neutrophils). Under the same conditions as wild-type AB embryos, fertilized eggs (2 hpf) were exposed to different concentrations of the target contaminant (such as the drug amitriptyline (AMI), with concentrations set at 0, 0.06, 0.6, and 6 μg / L) for 5 days. At the 120 hpf juvenile stage, fluorescence microscopy was used for imaging, and cell counting was performed using ImageJ software. At least three replicates were set up for each treatment group, with each replicate containing 12 juvenile fish, and the number of macrophages and neutrophils was counted.
[0022] 2. Humoral immune factor detection The following humoral immune factors were measured using a zebrafish-specific ELISA kit: C-reactive protein (CRP), immunoglobulin M (IgM); complement component C3, macrophage inflammatory protein (MIP); and lysozyme activity (LYSO).
[0023] Biochemical analysis was performed on 240 *Helicobacter pylori* (HPF) larvae. Changes in complement 3 (C3), immunoglobulin M (IgM), MIP (macrophage inflammatory protein), C-reactive protein (CRP), and lysozyme activity (LYSO) levels are classic humoral defense factors of the fish immune system and have also been tested as sensitive biomarkers for chemoimmunization. Changes in complement 3, IgM, CRP, MIP, and LYSO levels are important factors in the fish immune system. Quantitative sandwich enzyme immunoassay was performed using a Fish enzyme-linked immunosorbent assay (ELISA) kit.
[0024] Table 1. Zebrafish ELISA Detection Kit ; The kit should be stored at 2–8°C and left to stand at room temperature for 20 min before use. If the concentrated wash buffer removed from the refrigerator contains crystals, it should be heated in a water bath to completely dissolve the crystals before use. Add the sample, standard, and HRP-labeled detection antibody sequentially to the pre-coated microwells containing ATP, NADH, and NAD. After incubation and thorough washing, use the substrate TMB for color development. TMB is converted to blue by peroxidase, and then to yellow under acidic conditions. The color intensity is positively correlated with the levels of ATP, NADH, and NAD in the sample. Measure the absorbance (OD value) at 450 nm using a microplate reader to calculate the sample activity.
[0025] Collected zebrafish juveniles (120 hpf) samples were mixed with an appropriate amount of physiological saline. Centrifuged at 3000 rpm for 10 min, and the supernatant was collected. 10 μL of the supernatant was added to the sample wells, followed by 40 μL of sample diluent; 50 μL of different concentrations of standards were added to the standard wells. 100 μL of detection antibody-HRP was added to both the standard and sample wells, the membrane was sealed, and incubated at 37°C for 1 h. The liquid was discarded, washing buffer was added, and the membrane was patted dry on paper. This process was repeated 5 times. 50 μL each of substrates A and B were added, and the membrane was incubated at 37°C for 15 min. 50 μL of stop solution was added, and the OD value was measured at 450 nm using a spectrophotometer.
[0026] Example 2: Gene Expression Detection The expression levels of genes related to immune responses were detected by qPCR; as a supplementary indicator, it was used to verify the changing trends of immune cell, mitochondrial function, and humoral immune parameters.
[0027] RNA extraction: RNA was extracted using a total RNA extraction kit, the contents of which are shown in Table 2.
[0028] Table 2 RNA Extraction Kits ; For each 50-100 mg zebrafish juvenile sample, add 1 mL of Trizol lysis buffer and 1-2 appropriately sized steel balls, homogenize thoroughly, and centrifuge at 12000 rpm for 2 min at 4°C. Transfer the supernatant to a new centrifuge tube. Add 200 μL of chloroform, vortex for 15 s, incubate at room temperature for 3 min, and centrifuge at 12000 rpm for 15 min at 4°C. At this point, clear stratification will occur; transfer the supernatant to a new centrifuge tube. Add 0.5 times the volume of anhydrous ethanol, mix well, and transfer the resulting solution and precipitate together to an adsorption column CR3. Centrifuge at 12000 rpm for 30 s at 4°C and discard the waste liquid. Add 500 μL of protein removal solution to the adsorption column CR3, centrifuge at 12000 rpm for 30 s at 4°C, and discard the waste liquid. Add 500 μL of wash buffer to the CR3 adsorption column, centrifuge at 12000 rpm for 30 s at 4℃, discard the waste liquid, repeat the addition of wash buffer once, and discard the waste liquid again. Transfer the adsorption column to a 2 mL collection tube, centrifuge at 12000 rpm for 2 min at 4℃, and then air dry the adsorption column on a clean bench for 5-10 min. Transfer the adsorption column to a new 1.5 mL centrifuge tube with the cap cut off, add 30 μL of RNase-Free ddH2O, incubate at room temperature for 2 min, centrifuge at 12000 rpm for 2 min at 4℃, and repeat the addition of RNase-Free ddH2O and centrifugation once, collecting approximately 60 μL of liquid. Determine the purity and concentration of RNA using a micro spectrophotometer; an OD260 / OD280 ratio of 1.8-2.0 is preferred.
[0029] Reverse transcription of RNA-cDNA: RNA was reverse transcribed using a reverse transcription premixed kit, the products of which are shown in Table 3.
[0030] Table 3 Reverse Transcription Premixed Kit ; Prepare a 10 μL genomic DNA removal reaction system as shown in Table 4. The reaction conditions are: 42℃ for 2 min, then remove the system after cooling to 4℃.
[0031] Table 4 Removal of Genomic DNA ; Prepare a 20 μL reverse transcription reaction system as shown in Table 5. The reaction conditions are: 37℃ for 15 min, followed by 85℃ for 5 sec for reverse transcription. After cooling to 4℃, remove the system and place it on ice for subsequent qRT-PCR reactions.
[0032] Table 5 Reverse transcription reaction ; Primer synthesis: Based on previous literature, the upstream and downstream primer sequences for immune-related genes are shown in Table 6. BGI Genomics Co., Ltd. synthesized all primers. β-actin was used as an internal reference gene.
[0033] Table 6 Immune-related genes ; 4. Calculation and determination of the Immune Toxicity Index (ITI) To achieve comprehensive evaluation across multiple indicators, this invention establishes a comprehensive immunotoxicity index (ITI) evaluation system, standardizing and weighting indicators at different immune levels. By integrating three types of data—immune cell count, humoral immune factors, and gene expression levels—an immune damage profile under pollutant exposure is constructed.
[0034] ① Standardization of indicators: The results of each immune indicator measurement were normalized and calculated using the following formula: ; in, This is a measurement value for a certain exposure group. and These are the minimum and maximum values of the indicator, respectively.
[0035] If the indicator is an inhibitory indicator (a decrease indicates increased toxicity), then the opposite should be taken: ; ② Calculation of comprehensive immunotoxicity index: The Integrated Immunotoxicity Index (ITI) is calculated using the following formula: ; in, The weights of each indicator can be assigned according to the immune hierarchy (e.g., 0.4 for the cellular layer, 0.3 for the humoral layer, and 0.3 for the molecular layer).
[0036] ③ Determination of toxicity level: Immunotoxic effects were graded according to ITI values (Table 7): ; This index can serve as a quantitative indicator of the immunotoxicity of pollutants, providing a unified standard for the immunotoxicity classification of different compounds.
[0037] like Figure 1 Tg( lyz :DsRed2): Transgenic zebrafish with red fluorescently labeled neutrophils. The results showed that the number of neutrophils gradually decreased with increasing AMI concentration, exhibiting a significant immunosuppressive effect.
[0038] Tg( mpeg1 EGFP: Transgenic zebrafish with green fluorescently labeled macrophages. All exposure groups showed a decrease in macrophage numbers, with the high-dose group exhibiting the most significant inhibition, suggesting impaired innate immune defense.
[0039] LYSO (lysozyme): Significantly upregulated in all exposure groups, with the most significant changes observed in the high-concentration group, suggesting possible compensatory activation of the immune system.
[0040] IgM (immunoglobulin M): It shows a dose-dependent increase, which may reflect the activation of antibody-mediated adaptive immunity.
[0041] MIP (macrophage inflammatory protein): significantly increased in the high-dose group, indicating enhanced inflammatory chemotaxis.
[0042] C3 (complement component): The significant upward trend indicates that the complement system activation is involved in immune stimulation.
[0043] CRP (C-reactive protein): It continues to rise with increasing concentration, indicating an enhanced acute inflammatory response.
[0044] L-1β L, a key factor in acute inflammation, was significantly upregulated in all exposed groups, indicating activation of the inflammatory response.
[0045] IL-8 The genes regulating neutrophil chemotaxis were significantly upregulated, which is inconsistent with the decrease in neutrophils, suggesting an imbalance in immune function.
[0046] NF-κB The central regulatory molecule of the inflammatory signaling pathway gradually increases with increasing exposure concentration, indicating that the inflammatory signaling is continuously activated.
[0047] TNF-α The levels of systemic inflammatory marker molecules were significantly elevated, with the highest response observed in the high-dose group, indicating a strong inflammatory immune response.
[0048] IL-10 Anti-inflammatory and inhibitory cytokines were significantly elevated under high concentrations of AMI exposure, suggesting that the body is attempting to regulate an excessive inflammatory response.
[0049] The results suggest that AMI exposure leads to an imbalance in the zebrafish immune system, characterized by "suppressed cellular immunity and excessive activation of humoral and inflammatory signals," indicating a significant risk of immunotoxicity.
[0050] Table 8 ITI values and immunotoxicity effect grading ; Table 8 illustrates that, based on the weighted comprehensive deviation evaluation model, the AMI exposure group exhibited a dose-response immunotoxicity effect. The ITI values were 0.39 (0.06 μg / L), 0.53 (0.6 μg / L), and 0.83 (6 μg / L), respectively. The high-dose group (6 μg / L) reached a significant immunotoxicity level, indicating a marked suppression and dysfunction of the immune system. This index can sensitively reflect the synergistic changes in immune cells and humoral immune factors, and is an effective integrated indicator for assessing the immune risk of environmental pollutants.
[0051] The specific embodiments described above further illustrate the purpose, technical solution, and beneficial effects of the present invention. It should be understood that the above description is only a specific embodiment of the present invention and is not intended to limit the scope of protection of the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.
Claims
1. A comprehensive evaluation method for the immunotoxicity of pollutants based on a zebrafish model, characterized in that, Includes the following steps: Step 1: Expose transgenic zebrafish embryos to the contaminants to be tested and culture them to the predetermined developmental stage; Step 2: Detect the number of immune cells in juvenile zebrafish; Step 3: Detect the levels of humoral immune factors in juvenile zebrafish; Step 4: Detect the expression levels of immune-related genes in zebrafish juveniles; Step 5: Based on the test results from Steps 2 to 4, calculate the Integrated Immunotoxicity Index (ITI). Step Six: Classify the immunotoxicity of the pollutants according to the Integrated Immunotoxicity Index (ITI).
2. The comprehensive evaluation method for the immunotoxicity of pollutants based on the zebrafish model according to claim 1, characterized in that, The transgenic zebrafish includes the macrophage fluorescently labeled strain Tg( mpeg1 EGFP) and / or neutrophil fluorescently labeled strain Tg( lyz :DsRed2).
3. The comprehensive evaluation method for the immunotoxicity of pollutants based on the zebrafish model according to claim 1 or 2, characterized in that, The humoral immune factors include, but are not limited to, C-reactive protein (CRP), immunoglobulin M (IgM), complement component C3, macrophage inflammatory protein (MIP), and lysozyme activity (LYSO), and at least five of them are selected.
4. The comprehensive evaluation method for the immunotoxicity of pollutants based on the zebrafish model according to claim 1 or 2, characterized in that, The immune-related genes include, but are not limited to, those mentioned above. TNF-α , IL-1β , IL-8 , IL-10 , NF-κB And at least 5 types must be selected.
5. The comprehensive evaluation method for the immunotoxicity of pollutants based on the zebrafish model according to claim 1, characterized in that, The calculation of the Integrated Immunotoxicity Index (ITI) includes: Standardize the processing of each immune indicator; The standardized immune index values are multiplied by their corresponding weighting coefficients and summed to obtain the ITI value.
6. The comprehensive evaluation method for the immunotoxicity of pollutants based on the zebrafish model according to claim 5, characterized in that, The weighting coefficients are set according to the immune hierarchy, with the weight of the immune cell layer being 0.3-0.5, the weight of the humoral immune layer being 0.2-0.4, and the weight of the gene expression layer being 0.2-0.
4.
7. The comprehensive evaluation method for the immunotoxicity of pollutants based on the zebrafish model according to claim 1, characterized in that, The immunotoxicity grading includes: When ITI < 0.2, it is determined that there is no significant immune effect; When 0.2 ≤ ITI < 0.5, it is judged as a mild immune perturbation; When 0.5 ≤ ITI < 0.8, it is considered moderate immune impairment; When ITI ≥ 0.8, it is considered significant immunotoxicity.
8. The comprehensive evaluation method for the immunotoxicity of pollutants based on the zebrafish model according to claim 1, characterized in that, The contaminants include at least one of pharmaceuticals, pesticides, plastic additives, flame retardants, or other novel contaminants.