Evaluation method for comprehensively analyzing immune induction effect of plasticizer by using caenorhabditis elegans

Abstract

The invention relates to the technical field of chemical toxicology, in particular to an evaluation method for comprehensively analyzing the immune induction effect of a plasticizer by using caenorhabditis elegans, according to the evaluation method, a mother worm breeding mode is changed to replace a lysis solution to synchronize nematodes, experimental interference is reduced, the result accuracy is improved, monitoring indexes are newly increased, intestinal injuries and in-vivo bacterial indexes are increased, and the evaluation method is suitable for comprehensive analysis of the immune induction effect of the plasticizer. The evaluation is more accurate and reliable by combining oxidative stress indexes and performing two-dimensional comprehensive evaluation from an intestinal immune defense line and oxidative stress, and by drawing a multi-index monitoring shape graph, adopting a comprehensive biomarker response index analysis method and associating immune-related representative indexes, the evaluation accuracy is improved. According to the method, the change of each index and the immunity intensity on the caenorhabditis elegans under different exposure conditions are intuitively presented, the immune defense effect of the caenorhabditis elegans under the stress of the plasticizer can be quickly and effectively evaluated without transcriptome and metabonomics, and efficient evaluation is realized.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of chemical toxicology, and particularly relates to a method for evaluating the immunization effect induced by plasticizers by using Caenorhabditis elegans. BACKGROUND

[0002] Phthalates are widely used plasticizers in life, and are widely present in environmental media (water, soil, air dust) and various consumer products. Their continuous release and accumulation constitute a serious environmental pollution problem. Moreover, such chemical substances can enter the organism through food chain enrichment or direct contact, causing multiple health damages such as endocrine disruption, reproductive development toxicity, and neurotoxicity to various organisms, and have clear ecological and health risks. Modern medical research has found that the immune system can recognize all exogenous antigens and produce responses under normal circumstances, and produces immune tolerance to self cells and molecules. Immunity is the core defense mechanism against pathogens, and studying the immune mechanism under pollutant exposure helps us to better provide effective defense strategies against external pollutants. In the face of the universal exposure and potential threat of plasticizers, it is particularly crucial to deeply study how they affect the immune system function of organisms. It is necessary to reveal the internal path of the immune disorder induced by pollutants, to explore the immune toxicity mechanism of plasticizers, to evaluate their overall health risks, and to find potential intervention targets.

[0003] Caenorhabditis elegans is a transparent free-living organism that lives in nature and feeds on bacteria, belonging to the Rhabditidae genus of the Rhabditis genus. It has the advantages of low experimental cost, simple feeding environment, transparent whole body for observation, and rapid generation replacement. The innate immune system of Caenorhabditis elegans is highly conserved (such as the p38MAPK pathway and the insulin signaling pathway), and the homology of key immune effector genes with humans is more than 60%, which is an ideal model for studying the immune toxicity mechanism of exogenous pollutants. The immune suppression, oxidative stress, and changes in anti-infection ability of nematodes caused by food chain or direct contact can characterize the ecological health risks under pollutant stress. Among them, the abnormality of immune defense function is the core indication of systemic damage induced by pollutants, and its dynamic change can directly reveal the immune toxicity potential of plasticizers.

[0004] Although the current evaluation index of Caenorhabditis elegans immune toxicity is relatively mature, the detection of the immune index of Caenorhabditis elegans at present mostly relies on traditional molecular biology or morphological methods, which cannot simultaneously detect multiple immune indexes of Caenorhabditis elegans with high efficiency, has low detection efficiency, a complicated and time-consuming process, and extremely low throughput, consumes a large amount of time and manpower, and the obtained immune index data are independent of each other, which leads to insufficient comprehensiveness of data analysis, resulting in deviation in the evaluation of the immune effect of plasticizers and low reliability of the results. SUMMARY

[0005] The application aims to provide a method for evaluating the immune effect induced by plasticizers by using C. elegans, and aims to solve the problem of low detection efficiency of the immune indicators of C. elegans.

[0006] To achieve the above-mentioned purpose, the application provides a method for evaluating the immune effect induced by plasticizers by using C. elegans, comprising the following steps:

[0007] Prepare a culture medium of uracil-deficient E. coli (OP50) and culture it as the food of the nematodes;

[0008] Prepare a NGM solid culture medium for the growth of C. elegans, add OP50 bacterial liquid to the culture medium, and culture after coating for standby use;

[0009] Prepare plasticizer poisoning liquid with gradient concentrations, and add it to the flat plate culture medium;

[0010] Transfer C. elegans synchronized to the L4 stage to the poisoning flat plate for culture;

[0011] Determine the median life span, intestinal damage, bacterial content in the body, active oxygen content and antioxidant enzyme activity of the nematodes after poisoning respectively;

[0012] Analyze multiple detection indicators by using the integrated biomarker response (IBR) index to evaluate the immune effect intensity of the plasticizers on the nematodes.

[0013] In the step of "preparing a NGM solid culture medium for the growth of C. elegans, adding OP50 bacterial liquid to the culture medium, and culturing after coating for standby use", the OD value of the bacterial liquid is 0.8-1.

[0014] In the step of "transferring C. elegans synchronized to the L4 stage to the poisoning flat plate for culture", the step specifically comprises: placing the nematodes after cryopreservation and recovery on the culture medium with coated flat plate, and culturing at 20-25 DEG C in the dark for 60-84 h; removing the 15-20 oogenesis nematodes on the flat plate after oogenesis, collecting the nematode eggs with M9 buffer, culturing and hatching to the L1 stage, and then moving to the NGM culture medium containing OP50, and culturing for 48-60 h to the L4 stage; the M9 buffer comprises 0.75 g of potassium dihydrogen phosphate, 3.75 g of dodecahydrate disodium hydrogen phosphate, and 1.25 g of sodium chloride, which are sterilized at high temperature, and then 250 microliters of 1M magnesium sulfate is added after the temperature is reduced, and the mixture is stored at 4 DEG C.

[0015] In the "determining the median lifespan of nematodes after being infected, intestinal damage, bacterial content in vivo, active oxygen content and antioxidant enzyme activity", the determination of the median lifespan of nematodes after being infected specifically includes: picking 100-300 L4 stage nematodes in the culture and moving them to the control plate and the experimental plate, recording the number of surviving nematodes every day, and moving the dead nematodes away until the median lifespan of nematodes is reached when half of the nematodes die, and calculating the inhibition rate of the experimental group.

[0016] In the "determining the median lifespan of nematodes after being infected, intestinal damage, bacterial content in vivo, active oxygen content and antioxidant enzyme activity", the determination of the intestinal damage of nematodes after being infected specifically includes: picking 100-300 L4 stage nematodes and moving them to the control plate and the experimental plate, after 72-168 h of culture, determining the intestinal damage of nematodes by the bright blue method, mixing the infected nematodes with concentrated E. coli OP50 (OD value of 1) and 10% bright blue dye, staining for 3 hours, then washing with K solution until the solution is colorless, moving to the NGM culture plate containing OP50 and culturing for 45 min, then washing with K solution for 2-3 times until it is sterile, moving to the 2% agarose pad, taking a bright field image under a microscope, calculating the intestinal damage coefficient by ImageJ, and calculating the inhibition rate of the experimental group.

[0017] In the "determining the median lifespan of nematodes after being infected, intestinal damage, bacterial content in vivo, active oxygen content and antioxidant enzyme activity", the determination of the bacterial content in vivo of nematodes after being infected specifically includes: picking 100-300 L4 stage nematodes and moving them to the control plate and the experimental plate, after 72-168 h of culture, selecting one nematode, crushing it in 0.1-0.15 mL M9 buffer under ice bath conditions using a cell ultrasonic crusher, taking 50 μL of the nematode homogenate stock solution, diluting it 400 times, taking 50 μL of the diluted nematode stock solution with a pipette, transferring it to the NGM culture medium, and incubating at 30-37°C for 10-14 h, counting the number of bacteria on the plate, and calculating the inhibition rate of the experimental group.

[0018] In the "determining the median lifespan of nematodes after being infected, intestinal damage, bacterial content in vivo, active oxygen content and antioxidant enzyme activity", the determination of the active oxygen content of nematodes after being infected specifically includes: picking 100-300 L4 stage nematodes and moving them to the control plate and the experimental plate, after 72-168 h of culture, washing with M9 buffer and grinding, taking the supernatant as the crude enzyme solution and storing it in the dark, detecting the active oxygen of the crude enzyme solution of the control group and the experimental group using the DCFH-DA (2,7-dichlorofluorescein diacetate) probe, and calculating the inhibition rate of the experimental group.

[0019] In "respectively determining the median lifespan of nematodes after exposure, intestinal damage, in vivo bacterial content, active oxygen content and antioxidant enzyme activity", the determination of the antioxidant enzyme activity of nematodes after exposure specifically includes: measuring the total protein (TP) content, total superoxide dismutase (SOD) activity, catalase (CAT) activity, and glutathione peroxidase (GSH-PX) activity of the crude enzyme solution using a kit, and calculating the inhibition rate of the experimental group.

[0020] The evaluation method for comprehensively analyzing the immune effect induced by plasticizers by using C. elegans according to the present application changes the lysate into a mother worm breeding mode, reduces the interference of the conventional lysate on the experiment, and the experimental results are more accurate. The intestinal damage and in vivo bacterial indicators are added to the monitoring indicators for comprehensive evaluation, and compared with the traditional toxicity and oxidative stress indicator evaluation, the present application combines the intestinal related indicators and the oxidative stress related indicators from the immune defense line of C. elegans, so that the evaluation results are more accurate and reliable. By drawing a shape diagram of the monitoring results of multiple indicators, the comprehensive biomarker response index analysis method is used to associate the results of multiple representative indicators related to immunity, and the changes between the detection indicators and the immune effect intensity of C. elegans under different exposure conditions are directly and visually displayed. Without doing transcriptome and metabolomics on C. elegans, the immune defense effect of C. elegans under plasticizer stress can be quickly and effectively evaluated, thereby solving the problem of low detection efficiency of the immune indicators of C. elegans. BRIEF DESCRIPTION OF DRAWINGS

[0021] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the following will briefly introduce the drawings needed to be used in the embodiments or prior art description. Obviously, the drawings in the following description are only some embodiments of the present application, and those skilled in the art can also obtain other drawings according to these drawings without creative labor.

[0022] Figure 1 The figure is a median lifespan inhibition rate diagram of C. elegans under different concentrations of DEHP exposure.

[0023] Figure 2 The figure is an intestinal damage inhibition rate diagram of C. elegans under different concentrations of DEHP exposure.

[0024] Figure 3 The figure is a bacterial content inhibition rate diagram of C. elegans under different concentrations of DEHP exposure.

[0025] Figure 4 The figure is a TP activity inhibition rate diagram of C. elegans under different concentrations of DEHP exposure.

[0026] Figure 5The ROS activity inhibition rate chart of C. elegans under different concentrations of DEHP exposure.

[0027] Figure 6 The SOD activity inhibition rate chart of C. elegans under different concentrations of DEHP exposure.

[0028] Figure 7 The CAT activity inhibition rate chart of C. elegans under different concentrations of DEHP exposure.

[0029] Figure 8 The GSH-Px activity inhibition rate chart of C. elegans under different concentrations of DEHP exposure.

[0030] Figure 9 The IBR analysis schematic diagram of C. elegans under different concentrations of DEHP exposure.

[0031] Figure 10 The flow chart of the evaluation method for comprehensively analyzing the immune effect induced by plasticizers by using C. elegans provided by the present application. DETAILED DESCRIPTION

[0032] Embodiments of the present application are described in detail below, examples of which are shown in the accompanying drawings, wherein the same or similar reference numerals represent the same or similar elements or elements having the same or similar functions throughout. The embodiments described below by referring to the drawings are exemplary and are intended to explain the present application, and cannot be understood as a limitation of the present application.

[0033] Referring to Figures 1 to 10 , the present application provides an evaluation method for comprehensively analyzing the immune effect induced by plasticizers by using C. elegans, comprising the following steps:

[0034] S1: preparing and culturing uracil-deficient E. coli (OP50) medium as nematode food;

[0035] Specifically, the LB solid medium and LB liquid medium required for OP50 are sterilized at high temperature and high pressure for 30 min and then reserved. In the clean bench, a small amount of glycerol bacteria is taken with a inoculating loop and plated on the solid medium, and after 12-14 h of culture at 37℃, a single colony is picked and cultured in LB liquid medium at 37℃ and 180 rpm for 12-14 h, which is used as nematode food and reserved in a refrigerator at 4℃. 1 g of tryptone, 0.5 g of yeast extract, 0.5 g of sodium chloride, 1.5 g of agar, and ultrapure water are used to make up to 100 mL, and the pH is adjusted to 7.0. The solid LB medium is prepared by high-temperature and high-pressure sterilization for 20-30 min, and the liquid LB medium is prepared by adding 1 g of tryptone, 0.5 g of yeast extract, 0.5 g of sodium chloride, and ultrapure water to make up to 100 mL, and adjusting the pH to 7.0.

[0036] S2 prepare NGM solid medium for C. elegans growth, and add OP50 bacterial liquid to the medium, and culture after coating.

[0037] The OD value of the bacterial liquid is 0.8-1.

[0038] Specifically, NGM solid medium for C. elegans growth is prepared, and after high-temperature and high-pressure sterilization for 30 min, the medium is poured in a super-clean workbench, 20-50 μl of OP50 bacterial liquid is added to the medium, and the plate is coated and cultured at 37°C for 10-14 h before ultraviolet sterilization for standby. Sodium chloride 3.0 g, tryptone 2.5 g, agar 17 g, and ultrapure water 950-1050 mL are used for high-temperature and high-pressure sterilization for 20-30 min, and then 0.8-1.2 ml of 1M magnesium sulfate, 0.8-1.2 ml of 1M calcium chloride, 22-28 ml of 1M potassium phosphate buffer, and 0.8-1.2 ml of 5 mg / L cholesterol are added to the super-clean workbench, and then mixed thoroughly, and the plate is poured and cooled, and ultraviolet sterilized for 1-2 h before storage in a refrigerator at 4°C.

[0039] The C. elegans after cryopreservation is placed on the coated medium, and cultured in the dark at 20-25°C for 60-84 h; 15-20 egg-laying C. elegans are picked on the plate to lay eggs, and then moved back, and the eggs are collected with M9 buffer, and cultured at 20-25°C to hatch to the L1 stage, and then moved to NGM medium containing OP50, and cultured for 48-60 h to the L4 stage, and the M9 buffer is prepared by using 0.75 g of potassium dihydrogen phosphate, 3.75 g of dodecahydrate disodium hydrogen phosphate, and 1.25 g of sodium chloride, and after high-temperature sterilization, 250 μL of 1M magnesium sulfate is added after the temperature is reduced, and stored at 4°C.

[0040] S3 prepare plasticizer poisoning liquid with gradient concentration, and add to the plate medium;

[0041] Specifically, DEHP poisoning liquid is prepared according to gradient concentration, and 0.6-0.9 ml of the poisoning liquid is added to the surface of the plate with a pipette gun, and then dried in a super-clean workbench before storage at 4°C in the dark.

[0042] S4 move C. elegans synchronized to the L4 stage to the poisoning plate for culture;

[0043] Specifically, the C. elegans after cryopreservation is placed on the coated medium, and cultured in the dark at 20-25°C for 60-84 h. 15-20 egg-laying C. elegans are picked on the plate to lay eggs, and then moved back, and the eggs are collected with M9 buffer, and cultured at 20-25°C to hatch to the L1 stage, and then moved to NGM medium containing OP50, and cultured for 48-60 h to the L4 stage.

[0044] S5 respectively determine the median life span of nematodes after exposure, intestinal damage, bacterial content in vivo, reactive oxygen content and antioxidant enzyme activity;

[0045] The determination of the median life span of nematodes after exposure specifically includes: picking 100-300 L4 stage nematodes in the medium respectively to the control plate and the experimental plate, recording the number of surviving nematodes every day, and moving the dead nematodes to the half of the nematode death time as the median life span of the nematodes, and calculating the inhibition rate of the experimental group. The M9 buffer uses 0.75 g of potassium dihydrogen phosphate, 3.75 g of dodecahydrate disodium phosphate, 1.25 g of sodium chloride, and after high-temperature sterilization, 250 μL of 1M magnesium sulfate is added after the temperature is reduced, and it is stored at 4°C.

[0046] The determination of intestinal damage of nematodes after exposure specifically includes: picking 100-300 L4 stage nematodes respectively to the control plate and the experimental plate, culturing for 72-168 h, and then determining the intestinal damage of nematodes by the brilliant blue method. The nematodes after exposure are mixed with concentrated E. coli OP50 (OD value is 1) and 10% brilliant blue dye, dyed for 3 hours, then washed with K solution until the solution is colorless, transferred to an NGM culture plate containing OP50 and cultured for 45 min, then washed with K solution for 2-3 times until sterile, transferred to a 2% agarose pad, photographed under a microscope, and the intestinal damage coefficient is calculated by ImageJ, and the inhibition rate of the experimental group is calculated.

[0047] 2% agarose pad uses 2 g of agarose, 100 mL of ultrapure water, and a microwave oven at high heat for 1-2 min. After melting, the first drop is added to a glass slide with a dropper, and another glass slide is pressed. After cooling, the upper glass slide is removed for standby.

[0048] The death standard of nematodes is that the nematodes are lightly touched three times with a platinum wire nematode needle, and the nematodes are dead if they do not respond.

[0049] K solution uses 0.5-2.5 g of sodium chloride, 0.68-1.68 g of potassium chloride, and 450-550 mL of ultrapure water, which is sterilized at high temperature and high pressure for 20-30 min for standby.

[0050] The determination of bacterial content in nematodes after exposure specifically includes: picking 100-300 L4 stage nematodes respectively to the control plate and the experimental plate, culturing for 72-168 h, selecting 1 in 0.1-0.15 mL of M9 buffer, and completing the crushing under ice bath conditions with a cell ultrasonic crusher. 50 μL of nematode homogenate stock solution is taken, and 400-fold dilution is performed. 50 μL of diluted nematode stock solution is taken with a pipette gun, transferred to NGM medium, and coated, cultured at 30-37°C for 10-14 h, the number of bacteria on the plate is calculated, and the inhibition rate of the experimental group is calculated.

[0051] The determination of the active oxygen content of the infected nematodes specifically includes: 100-300 L4 nematodes are picked and moved to the control plate and the experimental plate respectively, and after 72-168 h of culture, they are washed with M9 buffer and grinded, the supernatant is taken as the crude enzyme solution and stored in the dark, the DCFH-DA (2,7-dichlorofluorescein diacetate) probe is used to detect the active oxygen of the crude enzyme solution of the control group and the experimental group, and the inhibition rate of the experimental group is calculated.

[0052] Specifically, the median lifespan of the infected C. elegans: 100-300 L4 nematodes are picked and moved to the control plate and the experimental plate respectively, the number of surviving nematodes is recorded every day, and the median lifespan of the nematodes is calculated when the number of dead nematodes is half, and the inhibition rate of the experimental group is calculated.

[0053] Intestinal injury determination of the infected C. elegans: 100-300 L4 nematodes are picked and moved to the control plate and the experimental plate respectively, and after 72-168 h of culture, the intestinal injury of the nematodes is determined by the brilliant blue method. The infected nematodes are mixed with concentrated E. coli OP50 (OD value is 1) and 10% brilliant blue dye, dyed for 3 hours, then washed with K solution until the solution is colorless, transferred to the NGM culture plate containing OP50 and cultured for 45 min, then washed with K solution for 2-3 times until sterile, transferred to a 2% agarose pad, photographed under a microscope, and the intestinal injury coefficient is calculated by Image J, and the inhibition rate of the experimental group is calculated.

[0054] In vivo bacteria determination of the infected C. elegans: 100-300 L4 nematodes are picked and moved to the control plate and the experimental plate respectively, and after 72-168 h of culture, 1 nematode is selected and broken in 0.1-0.15 mL M9 buffer under ice bath condition by using a cell ultrasonic disrupter, 50 μL of the nematode homogenate stock solution is taken and diluted by 400 times. 50 μL of the diluted nematode stock solution is taken by using a pipette, transferred to the NGM culture medium, and cultured at 30-37°C for 10-14 h, the number of bacteria on the plate is calculated, and the inhibition rate of the experimental group is calculated.

[0055] Active oxygen (ROS) content determination of the infected C. elegans: 100-300 L4 nematodes are picked and moved to the control plate and the experimental plate respectively, and after 72-168 h of culture, they are washed with M9 buffer and grinded, the supernatant is taken as the crude enzyme solution and stored in the dark. The DCFH-DA (2,7-dichlorofluorescein diacetate) probe is used to detect the active oxygen of the crude enzyme solution of the control group and the experimental group, and the inhibition rate of the experimental group is calculated.

[0056] Antioxidant enzyme content determination of the infected C. elegans: the crude enzyme solution is used to measure the total protein (TP) content, the activity of total superoxide dismutase (SOD), the activity of catalase (CAT), and the activity of glutathione peroxidase (GSH-PX) by using a kit, and the inhibition rate of the experimental group is calculated.

[0057] The selected nematodes are evenly divided into three plates as parallel plates;

[0058] The median lifespan of the exposed C. elegans is determined, and the inhibition rate is calculated according to the following formula:

[0059]

[0060] The inhibition rate in intestinal injury, bacterial content in vivo, reactive oxygen content and antioxidant enzyme activity is determined according to the following formula:

[0061]

[0062] S6 uses the integrated biomarker response index (IBR) to analyze multiple detection indexes to evaluate the immune effect intensity of the plasticizer on the nematodes.

[0063] Specifically, the results of the multiple detection indexes of the different concentrations of the exposure solution are analyzed by the integrated biomarker response index (IBR), the distribution of each index is displayed by calculating the star graph, the IBR value of each concentration gradient is obtained by calculating the sum of the areas of the triangles formed by the radial lines of adjacent biomarkers, and the immune effect intensity of the nematodes under the exposure of DEHP and AMP can be quickly evaluated without gene detection by comparing the results of the multiple detection indexes and the IBR.

[0064] Embodiment 1 specifically includes the following steps:

[0065] Step 1, culture of uracil-deficient E. coli (OP50): prepare the LB solid medium and LB liquid medium required for OP50, and sterilize them at high temperature and high pressure for 30 min. In the clean bench, use a loop to dip a small amount of glycerol bacteria on the solid medium for plate streaking, and culture at 37℃ for 12-14h, then pick single colonies in 10mL of LB liquid medium, and culture at 37℃ and 180rpm for 12h, as nematode food, which is stored at 4℃.

[0066] Step 2, preparation of NGM solid medium for growth of C. elegans, sterilize at high temperature and high pressure for 30 min, then pour the plate in the clean bench, and sterilize under ultraviolet light for 1-2h, add 30μL of OP50 bacterial solution to the medium, and culture at 37℃ for 12h after plate coating, then sterilize under ultraviolet light for 1h for standby.

[0067] Step 3, sterile K liquid preparation: 0.75 g of potassium dihydrogen phosphate, 3.75 g of dodecahydrate disodium hydrogen phosphate, 1.25 g of sodium chloride, high temperature sterilization, after the temperature decreases, add 250 μL of 1M magnesium sulfate, 4℃ cold storage; M9 buffer preparation: using 0.75 g of potassium dihydrogen phosphate, 3.75 g of dodecahydrate disodium hydrogen phosphate, 1.25 g of sodium chloride, high temperature sterilization, after the temperature decreases, add 250 μL of 1M magnesium sulfate, 4℃ cold storage.

[0068] Step 4, exposure method: according to the gradient concentration preparation phthalate (2-ethylhexyl) ester (DEHP) exposure liquid, using volume content not more than 0.1% of dimethyl sulfoxide (DMSO) as a cosolvent, with K liquid as solvent, the concentration gradient is 0 mg / L, 0.1 mg / L, 1 mg / L, 10 mg / L, 100 mg / L DEHP exposure liquid, and add 0.9 ml exposure liquid on the surface of the plate with a pipette gun to cover the surface of OP50 colony, dry in a clean bench, and store at 4℃ in the dark.

[0069] Step 5, culture and synchronization of Caenorhabditis elegans: the frozen recovered nematodes were placed on the coated plate, and cultured at 20-25℃ in the dark for 60-84h to the pregnant stage. 15-20 pairs of nematodes were picked on the plate to lay eggs, then moved back to the original plate, and the eggs were collected with M9 buffer and washed 2-3 times to remove OP50. The eggs were cultured at 20-25℃ with shaking until L1 stage, then moved to NGM medium containing OP50, and cultured for 48-60h to L4 stage.

[0070] Step 6, median lifespan of nematodes exposed to DEHP: 150 L4 stage nematodes in step 5 were picked and moved to control and experimental plates, respectively, and divided into three groups in parallel. The number of surviving nematodes was recorded every day, and the dead nematodes that did not respond to the platinum wire picking needle were removed. The number of days when half of the nematodes died was the median lifespan of the nematodes. The median lifespan inhibition rate of the experimental group compared with the control group was 2.77%, 8.30%, 16.49%, and 20.58%.

[0071] Step 7, determination of intestinal damage of nematodes exposed to DEHP: 100-300 L4 stage nematodes in step 5 were picked and moved to control and experimental plates, respectively, and cultured for 72-168h. The nematodes were mixed with concentrated E. coli OP50 (OD value of 1) and 10% brilliant blue dye after exposure, and stained for 3h. The solution was washed with K liquid until it was colorless, then transferred to NGM culture plate containing OP50 and cultured for 45min. The solution was washed with K liquid 2-3 times until it was sterile, then transferred to a 2% agarose pad. The intestinal damage coefficient was calculated using Image J, and the intestinal damage of the experimental group compared with the control group was increased by 34.64%, 37.49%, 56.26%, and 124.44%.

[0072] Step 8, in vivo bacterial assay of the infected C. elegans: 100-300 L4 stage nematodes in step 5 were respectively picked and moved to the control plate and the experimental plate, and cultured for 72-168 h. Then, one was selected, broken in 0.1-0.15 mL M9 buffer under ice bath condition by using a cell ultrasonic disruptor, 50 μL of the nematode homogenate stock solution was taken, and 400-fold dilution was performed. 50 μL of the diluted nematode stock solution was taken by using a pipette, transferred to the NGM medium, and cultured at 30-37 °C for 10-14 h. The number of bacteria on the plate was calculated, and the in vivo bacterial content of the experimental group was increased by 162.5%, 31.25%, -25%, and -75% compared with the control group.

[0073] Step 9, ROS content determination of the infected C. elegans: 100-300 L4 stage nematodes in step 5 were respectively picked and moved to the control plate and the experimental plate, and cultured for 72-168 h. Then, they were washed with M9 buffer and ground, and the supernatant was taken as the crude enzyme solution and stored in the dark. DCFH-DA (2,7-dichlorofluorescein diacetate) probe was used to detect the ROS of the crude enzyme solution of the control group and the experimental group, and the ROS content of the experimental group was increased by 138.47%, 155.60%, 230.84%, and 268.99% compared with the control group.

[0074] Step 10, antioxidant enzyme content determination of the infected C. elegans: the crude enzyme solution in step 9 was used to measure the total protein (TP) content, total superoxide dismutase (SOD) activity, catalase (CAT) activity, and glutathione peroxidase (GSH-PX) activity by using a kit. The TP content of the experimental group was increased by 42.86%, 57.14%, 92.86%, and 50% compared with the control group; the SOD was increased by 42.50%, 64.88%, 33.99%, and 94.93%; the CAT was increased by -29.65%, -41.36%, -53.03%, and -26.74%; and the GSH-Px was increased by 115.82%, 55.90%, 13.28%, and -64.23%.

[0075] Step 11, the results of various detection indexes of different concentrations of the infected solution were analyzed by using the integrated biomarker response (IBR), the distribution of each index was displayed by calculating the star graph, the IBR value of each concentration gradient was obtained by calculating the sum of the areas of the triangles formed by the adjacent biomarkers, and the comparison of the results of various detection indexes and IBR can quickly evaluate the immune effect intensity of the nematodes under the exposure of DEHP and AMP without gene detection.

[0076] In this step, the integrated biomarker response index (IBR) values of the monitored multiple detection indexes are calculated according to the IBR calculation method in the paper Integrated biomarker response: A useful tool for ecological risk assessment by Benoit Beliaeff and Thierry Burgeot published in 2002.

[0077] First, the average values of each index of 0.1 mg / L, 1 mg / L, 10 mg / L, and 100 mg / L are calculated as Xi, and the total average values of each index at all concentrations are calculated as m, and the standard deviations are calculated as s.

[0078] The average values Xi are normalized according to the calculation formula to obtain Yi, and the formula is:

[0079]

[0080] If the monitored biomarker increases, it indicates that the stress effect is enhanced, and the leader Z i = Y i If the monitored biomarker decreases, it indicates that the stress effect is enhanced, and the leader Z i = -Y i The score of the biomarker at each concentration is B i = Z i + |Min|, where |Min| is the absolute value of the minimum value of the normalized data Zi of the biomarker at all concentrations, which is 1.26, 1.31, 1.16, 0.68, 0.96, 0.80, 0.97, 0.92, 1.28, and 1.25, respectively.

[0081] The B i values are plotted in the origin software according to the star chart, and the size of the B i value of each biomarker at a certain concentration is represented by the length of the radial line in the star chart, and the IBR value of a certain station is obtained by calculating the area of the star chart (i.e. the sum of the areas of the triangles Ai formed by the adjacent biomarker radial lines in the chart, Ai is positive), and the calculation formula is:

[0082]

[0083] Where n is the number of biomarkers selected; α is the angle between two adjacent radiation lines; B n+1 =B1.

[0084] The calculation results showed that the comprehensive biomarker response index (IBR) values ​​for four exposure concentrations of 0.1 mg / L, 1 mg / L, 10 mg / L, and 100 mg / L were 0.1552, 0.0773, 0.0986, and 0.1570, respectively. Among them, the IBR value was the highest at the 100 mg / L exposure concentration, followed by the IBR value at the 0.1 mg / L exposure concentration. This indicates that the immune response of *C. elegans* is strongest when exposed to high concentrations of pollutants. Combining different indicators, it can be analyzed that the toxicity of DEHP increases with increasing concentration. The star plot shows that under the low concentration stress of 0.1 mg / L, the immune response of nematodes is mainly in the gut and even in vivo microbiota metabolism. As the DEHP exposure concentration increases, the immune focus shifts to the body's oxidative stress defense.

[0085] Beneficial effects:

[0086] First, by changing the method of synchronously lysing nematodes with lysate to selecting mother nematodes for reproduction, the interference of conventional lysate on the experiment is reduced, and the experimental results are more accurate.

[0087] Second, by adding intestinal damage and in vivo bacterial indicators to the monitoring indicators for comprehensive evaluation, this invention achieves a combination of intestinal-related indicators and oxidative stress-related indicators from the nematode immune defense line compared to traditional toxicity and oxidative stress indicator evaluations, making the evaluation results more accurate and reliable.

[0088] Third, by drawing shape diagrams of monitoring results for multiple indicators, and using a comprehensive biomarker response index analysis method, the results of multiple representative immune-related indicators are linked together. This method can intuitively and vividly display the changes between various detection indicators under different exposure conditions and the intensity of immune effects on *C. elegans*. Without transcriptomics and metabolomics studies on *C. elegans*, the immune defense function in *C. elegans* under plasticizer stress can be rapidly and effectively evaluated.

[0089] The above-disclosed embodiments are merely preferred embodiments of the evaluation method for plasticizer-induced immune responses using *C. elegans*, and should not be construed as limiting the scope of the invention. Those skilled in the art will understand that implementing all or part of the above embodiments and making equivalent changes according to the claims of the invention are still within the scope of the invention.

Claims

1. A method for evaluating plasticizer-induced immunological effects using a comprehensive analysis of Caenorhabditis elegans, characterized by, The method comprises the following steps: Preparation of uracil-deficient E. coli (OP50) culture medium and cultivation as nematode food; Preparation of NGM solid medium for C. elegans growth, and addition of OP50 bacterial liquid to the medium, coating and then culturing for standby; Preparation of plasticizer exposure liquid with gradient concentrations, and addition to the plate medium; Transfer of C. elegans synchronized to L4 stage to the exposure plate for cultivation; Measurement of median lifespan, intestinal injury, bacterial content, active oxygen content and antioxidant enzyme activity of the nematodes after exposure; IBR (Integrated Biomarker Response) analysis of multiple detection indexes to evaluate the immune effect intensity of the plasticizer on the nematodes.

2. The method for evaluating the immunological effects induced by plasticizers using C. elegans according to claim 1, wherein the C. elegans is C. elegans strain CL3035. In the step of "preparation of NGM solid medium for C. elegans growth, and addition of OP50 bacterial liquid to the medium, coating and then culturing for standby", the OD value of the bacterial liquid is 0.8-1. ​ 3. The method for evaluating the immunological effects induced by plasticizers using C. elegans according to claim 1, wherein the C. elegans is C. elegans strain CL3035. In the step of "transfer of C. elegans synchronized to L4 stage to the exposure plate for cultivation", the steps specifically include: placing the nematodes after cryopreservation and recovery on the coated plate medium, and cultivating at 20-25°C in the dark for 60-84h; picking 15-20 egg-producing nematodes on the plate to produce eggs, and then moving back; collecting the nematode eggs with M9 buffer, and cultivating at 20-25°C to hatch to L1 stage, and then moving to NGM medium containing OP50, and cultivating for 48-60h to L4 stage; the M9 buffer uses 0.75g potassium dihydrogen phosphate, 3.75g disodium hydrogen phosphate dodecahydrate, 1.25g sodium chloride, and 250μL 1M magnesium sulfate after high-temperature sterilization, and then adding 250μL 1M magnesium sulfate after the temperature is reduced, and storing at 4°C. ​ 4. The method for evaluating the immunological effects induced by plasticizers using C. elegans according to claim 1, wherein the C. elegans is C. elegans strain CL3035. In the step of "measurement of median lifespan, intestinal injury, bacterial content, active oxygen content and antioxidant enzyme activity of the nematodes after exposure", the measurement of the median lifespan of the nematodes after exposure specifically includes: picking 100-300 L4 stage nematodes in the step of "measurement of median lifespan, intestinal injury, bacterial content, active oxygen content and antioxidant enzyme activity of the nematodes after exposure" and moving to the control plate and the experimental plate, recording the number of surviving nematodes every day, and moving the dead nematodes away, and the median lifespan of the nematodes is when half of the nematodes die, and the inhibition rate of the experimental group is calculated. ​ 5. The method for evaluating the immunological effects induced by plasticizers using C. elegans according to claim 1, wherein the C. elegans is C. elegans strain CL3035. In the step of "measurement of median lifespan, intestinal injury, bacterial content, active oxygen content and antioxidant enzyme activity of the nematodes after exposure", the measurement of intestinal injury of the nematodes after exposure specifically includes: picking 100-300 L4 stage nematodes and moving to the control plate and the experimental plate, cultivating for 72-168h, and then measuring the intestinal injury of the nematodes by the brilliant blue method, mixing the nematodes after exposure with concentrated E. coli OP50 (OD value is 1) and 10% brilliant blue dye, dyeing for 3h, washing with K liquid until the solution is colorless, moving to NGM culture plate containing OP50 and cultivating for 45min, washing with K liquid for 2-3 times until sterile, moving to a 2% agarose pad, taking a microscope bright field image, calculating the intestinal injury coefficient by ImageJ, and calculating the inhibition rate of the experimental group. ​ 6. The method for evaluating the immunological effects induced by plasticizers using C. elegans according to claim 1, wherein the C. elegans is C. elegans strain CL3035. In the "respectively determining the median life span of nematodes after being contaminated, intestinal damage, in vivo bacterial content, active oxygen content and antioxidant enzyme activity", the determination of in vivo bacterial content of nematodes after being contaminated specifically includes: 100-300 L4 stage nematodes are respectively picked and moved to control plates and experimental plates, and after being cultured for 72-168 h, one is selected in 0.1-0.15 mL M9 buffer, and is broken by using a cell ultrasonic crusher under ice bath conditions, 50 μL of nematode homogenate stock solution is absorbed, and is diluted by 400 times, 50 μL of the diluted nematode stock solution is absorbed by using a pipette gun, is transferred to NGM culture medium, and is coated, and is cultured at 30-37 °C for 10-14 h, the number of bacteria on the plate is calculated, and the inhibition rate of the experimental group is calculated. ​ 7. The method for evaluating the immunological effects induced by plasticizers using C. elegans according to claim 6, wherein the C. elegans is C. elegans strain CL3035. In the "respectively determining the median life span of nematodes after being contaminated, intestinal damage, in vivo bacterial content, active oxygen content and antioxidant enzyme activity", the determination of active oxygen content of nematodes after being contaminated specifically includes: 100-300 L4 stage nematodes are respectively picked and moved to control plates and experimental plates, and after being cultured for 72-168 h, are washed with M9 buffer and are ground, the supernatant is taken as a crude enzyme solution and is stored in the dark, DCFH-DA (2,7-dichlorofluorescein diacetate) probes are used to detect the active oxygen of the crude enzyme solution of the control group and the experimental group, and the inhibition rate of the experimental group is calculated. ​ 8. The method for evaluating the immunological effects induced by plasticizers using C. elegans according to claim 1, wherein the C. elegans is C. elegans strain CL3035. In the "respectively determining the median life span of nematodes after being contaminated, intestinal damage, in vivo bacterial content, active oxygen content and antioxidant enzyme activity", the determination of antioxidant enzyme activity of nematodes after being contaminated specifically includes: the total protein (TP) content, total superoxide dismutase (SOD) activity, catalase (CAT) activity and glutathione peroxidase (GSH-PX) activity of the crude enzyme solution are measured by using a kit, and the inhibition rate of the experimental group is calculated. ​

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