A method for evaluating the effect of a compound on the hypothalamic-pituitary reproductive endocrine axis

Abstract

The application discloses a method for evaluating the hypothalamus-pituitary reproductive endocrine axis interference effect of a compound, and belongs to the technical field of chemical environmental health risk evaluation. The hypothalamic neuron cell line and the pituitary gonadotropin cell line are selected as in-vitro evaluation models, the exposure concentration gradient of the compound affecting cell activity is obtained through cell culture and cytotoxicity experiment, the cell exposure experiment of the hypothalamic neuron cell line and the pituitary gonadotropin cell line is carried out at the exposure concentration gradient, the expression amount of a target gene in the cells is determined after the exposure is finished, the dose-effect curve of the compound interfering with the expression of the target gene is obtained according to the exposure concentration gradient and the expression amount of the target gene, and the interference effect of the compound on the hypothalamus-pituitary reproductive endocrine axis is evaluated by using a toxicology priority index ToxPi.

Description

Technical Field

[0001] This invention belongs to the field of chemical environmental health risk assessment technology, specifically relating to a method for evaluating the hypothalamic-pituitary reproductive endocrine axis interference effect of compounds. Background Technology

[0002] Various compounds are widely present in the environment and can enter the human body and accumulate through ingestion, skin contact, and inhalation. Currently, many types of compounds have been detected in various human tissues and organs, including blood, fat, liver, and brain. Many of these compounds have attracted widespread attention due to their endocrine disrupting effects. The hypothalamus-pituitary-reproductive axis is the core of the mammalian reproductive endocrine system, composed of the hypothalamus and pituitary gland, and regulates the hormone cascades required for key physiological functions such as reproduction, growth and development, and metabolism. Many studies have analyzed the endocrine disrupting effects of various chemicals on the hypothalamus-pituitary-reproductive axis from epidemiological and toxicological perspectives. However, due to differences in research subjects and experimental designs, the results are difficult to compare, and issues such as long experimental cycles, high costs, and ethical controversies surrounding animal experiments exist. Currently, a high-throughput assessment method for the disruption of the hypothalamus-pituitary-reproductive axis by compounds is still lacking.

[0003] Chinese patent document CN109815532A discloses a method for high-throughput screening of endocrine disruptors. This invention targets the primary, secondary, and tertiary warning structures of compounds extracted from nuclear receptors, then uses these structures to form a nuclear receptor high-throughput screening model. The model is then used to match the target compounds to these warning structures, and ligand-receptor binding patterns are analyzed, along with semi-quantitative predictions of binding and disruptive activities. Chinese patent document CN109827934A discloses a rapid detection kit and method for the endocrine disrupting activity of chemicals. The kit contains a recombinant human nuclear receptor ligand-binding domain hNR-LBD protein solution, a recombinant human nuclear receptor co-activator-EcoRI fusion protein solution, a specific TaqMan probe, A-DTT buffer, and B buffer. The kit enables rapid detection of the endocrine disrupting activity of chemicals on microplates. The aforementioned high-throughput screening methods primarily rely on the binding affinity of compounds to nuclear receptors. However, the binding affinity of most compounds to nuclear receptors is far lower than that of natural steroid hormones. This low binding affinity makes it difficult to accurately interpret the endocrine-disrupting effects of compounds, especially at low concentrations, where the endocrine-disrupting effects may be underestimated or ignored. Furthermore, these methods mainly focus on interactions at the nuclear receptor level, neglecting other key aspects of the endocrine system. High-throughput screening methods that solely rely on nuclear receptor structure cannot comprehensively reflect the disruptive effects of compounds on the entire endocrine system.

[0004] In vitro cell evaluation is also an important method for toxicity assessment, providing a more efficient means for high-throughput screening of high-risk compounds. It has advantages such as short cycle time, low cost, minimal interference, and ease of analyzing toxicity mechanisms. However, toxicity assessment based on in vitro single-cell levels is difficult to predict the true performance of compounds in biological systems. How to use cell models to achieve a comprehensive assessment of the interference effects of the hypothalamus-pituitary reproductive endocrine axis remains a key breakthrough. Summary of the Invention

[0005] This invention provides a method for evaluating the hypothalamic-pituitary reproductive endocrine axis interference effect of compounds, which has broad application prospects in screening high-risk compounds.

[0006] The specific technical solution adopted is as follows:

[0007] A method for evaluating the hypothalamic-pituitary reproductive endocrine axis interference effect of compounds includes the following steps:

[0008] (1) Hypothalamic neuronal cell lines and pituitary gonadotropin cell lines were selected as in vitro evaluation models. Hypothalamic neuronal cell lines and pituitary gonadotropin cell lines were cultured, and compound concentration gradients were set. The effects of the compounds at gradient concentrations on the cell activity of hypothalamic neuronal cell lines and pituitary gonadotropin cell lines were tested respectively, and the exposure concentration gradients affecting the activity of each cell type were obtained (each cell type was counted separately).

[0009] (2) Based on the exposure concentration gradient obtained in step (1), select non-cytotoxic concentrations to conduct cell exposure experiments on hypothalamic neuronal cell lines and pituitary gonadotropin cell lines respectively. After exposure, collect cell RNA, reverse the RNA to cDNA, and measure the expression level of the target gene in the cells. Based on the exposure concentration gradient and the expression level of the target gene, obtain the dose-response curve of the compound interfering with the expression of the target gene, and calculate the concentration ECx when the change in the expression of the target gene reaches x%.

[0010] (3) The ToxPi value was used to evaluate the endocrine disruption effect of the compound on the hypothalamic-pituitary axis. The ToxPi value was calculated as follows:

[0011] Where n is the number of target genes, w i r represents the weight of the i-th target gene. i Let r be the sector radius corresponding to the i-th target gene. i =y i / y max , where y i =-log 10 EC i x, y max For yi Maximum value, EC i x represents the ECx of the compound interfering with the expression of the i-th target gene;

[0012] The ToxPi value was further normalized to the 0-1 range. The closer it is to 1, the stronger the interference effect of the compound on the hypothalamic-pituitary reproductive endocrine axis.

[0013] This invention first conducts cell culture and toxicity experiments to determine the cytotoxicity of the compounds. By analyzing the interference effect of compound exposure on the expression of key target genes related to reproductive endocrine function in hypothalamic neuronal cell lines and pituitary gonadotropic cell lines, the invention screens hypothalamic-pituitary reproductive endocrine axis disruptors based on the ToxPi method, thus achieving high-throughput evaluation of the compounds' interference effect on the hypothalamic-pituitary reproductive endocrine axis.

[0014] Optionally, the hypothalamic neuron cell line is the mouse hypothalamic GnRH neuron cell line, specifically GT1-7 cells, and the pituitary gonadotropin cell line is the mouse pituitary gonadotropin cell line, specifically LβT2 cells. The target genes of the hypothalamic neuron cell line include the GnRH gene (Gene ID 14714) and / or the GPR54 gene (Gene ID 114229), and the target genes of the pituitary gonadotropin cell line include at least one of the following: CGα gene (Gene ID 12640), LHβ gene (Gene ID 16866), FSHβ gene (Gene ID 14308), and GnRHR gene (Gene ID 14715).

[0015] GT1-7 cells are a mouse hypothalamic GnRH neuronal cell line that expresses the hypothalamic gonadotropin-releasing hormone gene GnRH and the kiss1 hormone receptor gene GPR54, making them suitable for evaluating the interference effects of compounds on hypothalamic GnRH hormone synthesis. LβT2 cells are a mouse pituitary gonadotropin cell line that expresses the pituitary gonadotropin subunit genes CGα, LHβ, and FSHβ, as well as the GnRH hormone receptor gene GnRHR, making them suitable for evaluating the interference effects of compounds on pituitary gonadotropin synthesis.

[0016] Preferably, before conducting cell exposure experiments, cells are placed in a serum-free culture medium for serum starvation treatment for 4–25 hours. The starvation treatment time can be selected based on factors such as cell type, cell growth state, signal molecule change time, and experimental objectives.

[0017] Specifically, Livak analysis of quantitative PCR was performed using real-time quantitative PCR, with GAPDH (Gene ID14433) as the reference gene. The relative expression level of the target gene mRNA in cells was measured, and the dose-response curve of the compound interfering with the expression of the target gene was obtained based on the exposure concentration gradient and the relative expression level of the target gene mRNA.

[0018] Furthermore, the primer nucleotide sequences for GnRH are shown in SEQ ID NO.1 and SEQ ID NO.2, and / or the primer nucleotide sequences for GPR54 are shown in SEQ ID NO.3 and SEQ ID NO.4, and / or the primer nucleotide sequences for CGα are shown in SEQ ID NO.5 and SEQ ID NO.6, and / or the primer nucleotide sequences for LHβ are shown in SEQ ID NO.7 and SEQ ID NO.8, and / or the primer nucleotide sequences for FSHβ are shown in SEQ ID NO.9 and SEQ ID NO.10, and / or the primer nucleotide sequences for GnRHR are shown in SEQ ID NO.11 and SEQ ID NO.12, and / or the primer nucleotide sequences for GAPDH are shown in SEQ ID NO.13 and SEQ ID NO.14.

[0019] Optionally, the concentrations EC10, EC20, or EC50 at which the induced change in target gene expression reaches 10%, 20%, or 50% can be calculated, and EC10, EC20, or EC50 can be used as parameters for calculating the ToxPi value.

[0020] When calculating the ToxPi value, if EC i If x exceeds the maximum exposure concentration gradient or has no significant effect on the expression of the target gene, then y i =0, r i =0.

[0021] Target gene weight w i You can choose integers or fractions. For example, the weights of 6 genes can be {1, 1, 1, 1, 1, 1} or {1 / 6, 1 / 6, 1 / 6, 1 / 6, 1 / 6, 1 / 6}, and the weights of 3 genes can be {4, 1, 1} or {2 / 3, 1 / 6, 1 / 6}.

[0022] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0023] (1) This invention establishes a comprehensive evaluation system based on the hypothalamus-pituitary two-cell model, overcoming the limitations of existing technologies that only focus on a single target or a single link. By integrating the hypothalamic GnRH neuron cell line (GT1-7) and the pituitary gonadotropin cell line (LβT2), it simulates the functional linkage between upstream and downstream cells of the hypothalamus-pituitary axis, directly capturing the interference effects of compounds on gene expression (such as abnormal expression of GnRH and FSH / LH genes) and their cascade effects. It can comprehensively evaluate the interference effects of compounds on key links of the reproductive endocrine axis, including the synthesis and release of hypothalamic GnRH hormones and the synthesis and secretion of pituitary gonadotropins. This systematic evaluation method is more consistent with the actual regulatory mechanism of the reproductive endocrine axis in vivo, significantly improving the reliability and predictive value of the evaluation results.

[0024] (2) This invention enables the quantitative evaluation of the endocrine-disrupting effects of compounds by establishing dose-response curves and calculating ECx values. This invention innovatively applies the ToxPi analysis method to the evaluation of reproductive endocrine disruption effects, and by establishing a standardized evaluation index system, it achieves integrated analysis of changes in the expression of multiple target genes. This method not only simultaneously assesses the interference effects of compounds on multiple key genes but also reduces the interference of single gene expression fluctuations on the results, improving the stability and reliability of the evaluation. The ECx and ToxPi values ​​obtained by this method can provide important references for the safe use concentration of compounds and have significant practical application value. This method is not only applicable to the evaluation of single compounds but can also be used for risk assessment of complex environmental mixtures, providing a new technical means for the environmental health risk assessment of chemicals.

[0025] (3) The method of this invention is highly operable and reproducible. By establishing a standardized evaluation process, different laboratories can obtain consistent evaluation results, which is conducive to the promotion and application of the method. At the same time, this invention allows for flexible adjustment of evaluation indicators and weight settings according to specific research needs, and has good adaptability.

[0026] (4) Compared with traditional animal experiments, the in vitro cell model used in this invention avoids the ethical controversies brought about by animal experiments, reduces experimental costs, and has significant social benefits. Attached Figure Description

[0027] Figure 1 The dose-response curves for BPA interference with the expression of GnRH(a), GPR54(b), CGα(c), LHβ(d), FSHβ(e), and GnRHR(f) genes.

[0028] Figure 2 ToxPi diagram showing the interference effects of various compounds on the hypothalamic-pituitary reproductive endocrine axis. Detailed Implementation

[0029] To make the objectives, features, and advantages of this invention more apparent and understandable, a detailed description is provided below through specific embodiments. Many specific details are set forth in the following description to provide a thorough understanding of the invention. However, the invention can be practiced in many other ways different from those described herein, and those skilled in the art can make similar modifications without departing from the spirit of the invention. Therefore, the invention is not limited to the specific embodiments disclosed below. Technical features in various embodiments of the invention can be combined appropriately without mutual conflict.

[0030] Unless otherwise specified, the operating methods in the following examples are generally performed under conventional conditions or as recommended by the manufacturer. Contents not described in detail in this specification are prior art known to those skilled in the art. Unless otherwise specified, the experimental materials used in the examples below can be purchased from conventional biochemical reagent companies.

[0031] Example 1

[0032] 1. Cell culture experiment

[0033] Cells were cultured in DMEM high-glucose medium supplemented with 10% fetal bovine serum and 1% antibiotic solution, and placed in a constant temperature incubator at 37°C, saturated humidity, and 5% CO2. Cell culture dishes / flasks were coated with poly-L-lysine solution for at least 5 minutes to promote cell adhesion.

[0034] Remove the cryovials containing GT1-7 and LβT2 cells from the liquid nitrogen container and quickly transfer them to a 37°C water bath, gently agitating horizontally until the cryopreservation solution is completely thawed (approximately 1-2 minutes). Then, transfer the cells to a 10mL centrifuge tube containing 4mL of complete culture medium, centrifuge at 1000rpm for 5 minutes, and discard the supernatant. Add 2mL of complete culture medium and mix well by pipetting. Transfer the cell suspension to a 25cm agar tube containing 3mL of complete culture medium (DMEM high-glucose medium supplemented with 10% fetal bovine serum and 1% antibiotic solution). 2 In a culture flask, shake well and place in an incubator. Cell passage is performed when confluence reaches 80%–90%, with a passage ratio of 1:2–1:3. Aspirate the culture medium and wash twice with PBS buffer. Dilute 0.25% trypsin 5-fold and add to the culture dish. Digest at 37°C for 2–3 minutes, add culture medium to stop digestion, gently pipette to detach cells, collect, and centrifuge at 1000 rpm for 5 minutes. Discard the supernatant, add fresh culture medium, gently pipette to resuspend the cells, seed in culture dishes according to the specified ratio, and return to the incubator.

[0035] 2. Cytotoxicity assay

[0036] GT1-7 cells and LβT2 cells in the logarithmic growth phase were selected for experiments. Cells were cultured at 1×10⁻⁶ cells per cell line. 4 Cells were seeded at a density of 100 μL of complete culture medium per well in 96-well plates, and cell status was observed the next day. Subsequent experiments could be performed when there were no significant differences between wells and the degree of polymerization reached 60–80%. Based on the compound solubility and exposure concentration, the test chemical was dissolved in dimethyl sulfoxide (DMSO) to prepare a stock solution at a concentration 1000 times the highest exposure concentration and stored at -20°C. The compound stock solution was diluted with phenol red-free medium to create a gradient concentration, such as a 10:10 concentration gradient for bisphenol A (BPA). -4 10 -5 10 -6 10 -7 10 -8 10 -9 M, maintain DMSO content ≤0.1%, discard the original culture medium in the wells, add 100 μL of a gradient concentration of compound solution to each well, and add 100 μL of phenol red-free culture medium containing 0.1% DMSO to each well of the control group. Both experimental and control groups were set up in 4–6 replicates. After incubation in a constant temperature incubator for 24 h, cell viability was tested using an MTS kit. Add 20 μL of MTS reagent to each well, gently shake the 96-well plate, and incubate in a 37°C CO2 incubator for 1–4 h. After removal, measure the optical density (OD) at 490 nm using a microplate reader, and calculate the relative cell viability = (OD of experimental wells - OD of blank wells) / (OD of control wells - OD of blank wells) × 100%. Use SPSS 9.0 to analyze whether there is a significant difference in cell viability between the experimental and control groups. Select non-cytotoxic concentrations for subsequent exposure experiments, such as BPA at 10... -4 At concentration M, both GT1-7 and LβT2 cells showed significant toxicity; the concentration used in subsequent exposure experiments was 10%. -5 10 -6 10 -7 10 -8 10 -9 M.

[0037] 3. Dose-response relationship of target gene expression

[0038] Cells were seeded in 12-well plates, and their growth status was observed. Cell exposure experiments were performed when there were no significant differences between wells and the degree of polymerization reached 70-80%. Before cell exposure, the medium was replaced with serum-free, phenol red-free, high-glucose DMEM for starvation treatment: GT1-7 cells were starved for 4-6 hours, and LβT2 cells for 12-14 hours. Then, the cells were exposed to the concentration of the compound contained in step 2, along with serum-free, phenol red-free, high-glucose medium. In the control group, 1 mL of phenol red-free medium containing 0.1% DMSO was added to each well. Both the experimental and control groups were set up in quadruplicates.

[0039] After 24 hours of incubation in a constant temperature incubator, the exposure period ended, and RNA was extracted from the cells. 1 mL of Trizol was added to each well, and the cells were thoroughly detached by pipetting. The liquid was transferred to 1.5 mL sterile RNase-free centrifuge tubes. 200 μL of trichloroethane was added to each tube, and the mixture was vigorously vortexed until fully combined. The mixture was allowed to stand for 3–10 minutes. Centrifuged at 13000 rpm for 15 minutes at 4°C. 400 μL of the supernatant was transferred to a new centrifuge tube, and 400 μL of isopropanol was added. The mixture was gently mixed and allowed to stand for 10 minutes. Centrifuged at 13000 rpm for 10 minutes at 4°C until a white precipitate formed at the bottom of the tube. The liquid was poured off, and the RNA was washed with 75% ethanol (prepared with DEPC water). The mixture was centrifuged at 7600 g for 5 minutes at 4°C. After removing the liquid, the RNA was air-dried and dissolved in 20 μL of DEPC water.

[0040] RNA quality and concentration were determined using an ELISA reader. Reverse transcription experiments could only be performed when the absorption wavelength ratio of 260nm / 280nm was between 1.8 and 2.0. The RNA was diluted to the required concentration for reverse transcription, and residual genomic DNA was removed using a reverse transcription kit before reverse transcription of the RNA into cDNA.

[0041] Real-time quantitative PCR was performed using the SYBR Green dye method. The relative expression levels of GnRH and GPR54 genes in GT1-7 cells and the relative expression levels of CGα, LHβ, FSHβ, and GnRHR genes in LβT2 cells were measured. The relative expression levels of the target gene mRNA were analyzed using 2... -△△CT The Livak method was normalized to the internal reference gene GAPDH. The primer sequences used for quantitative PCR are shown in Table 1.

[0042] Table 1: Gene and primer sequences determined by real-time quantitative PCR

[0043]

[0044] After obtaining the relative expression data of the target gene, GraphPad Prism 9.5 was used to plot the dose-response scatter plot of the compound interfering with the expression of the target gene and to perform curve fitting to obtain the dose-response curve. The concentration EC50 at which the target gene expression change reached 50% was calculated.

[0045] BPA is a typical endocrine disruptor. The dose-response curves of BPA interfering with the expression of GnRH, GPR54, CGα, LHβ, FSHβ, and GnRHR genes are shown below. Figure 1 As shown in (a)-(f) in the figure; organophosphates are a class of widely used flame retardants and plasticizers. The organophosphate flame retardants tested in this example include triphenyl phosphate (TPHP), tricresyl phosphate (TMPP), 2-ethylhexyl diphenyl phosphate (EHDPHP), trioctyl phosphate (TEHP), tributyl phosphate (TNBP), tri(butoxyethyl) phosphate (TBEP), tri(2-chloroethyl) phosphate (TCEP), tri(1-chloro-2-propyl) phosphate (TCIPP), tri(1,3-dichloroisopropyl) phosphate (TDCIPP), and bisphenol A-bis(diphenyl phosphate) (BDP). The EC50 values ​​of BPA and the 10 organophosphates on the interference of GnRH, GPR54, CGα, LHβ, FSHβ, and GnRHR gene expression are shown in Table 2.

[0046] Table 2: EC50 of the effects of the tested compounds on target gene expression

[0047]

[0048] 4. Evaluation of comprehensive endocrine disruption effects

[0049] The ToxPi value, a toxicological priority index, was used to evaluate the endocrine disruption effects of compounds on the hypothalamic-pituitary axis. The ToxPi value was calculated as follows: Where n is the number of target genes, w i r represents the weight of the i-th target gene. i Let r be the sector radius corresponding to the i-th target gene (the size of the sector radius represents the strength of the interference effect of the test compound on the expression of the target gene). i =y i / y max , where y i =-log 10 EC i x, y max For y i Maximum value, EC i x represents ECx, which represents the compound's interference with the expression of the i-th target gene; if ECx iIf x exceeds the experimental maximum concentration or has no significant effect on the expression of the target gene, then y i =0, r i =0.

[0050] The ToxPi values ​​were further normalized to the 0-1 range. The magnitude of the ToxPi value indicates the strength of the hypothalamic-pituitary axis endocrine interference effect of the tested compound; the closer to 1, the stronger the interference effect. If the ToxPi value is 0, it means that the tested compound has no significant hypothalamic-pituitary endocrine interference effect. The ToxPi values ​​of each tested compound are shown in Table 3, and the ToxPi visualization results are shown in […]. Figure 2 The experimental results showed that BPA had a ToxPi value of 0.533, ranking first among the tested compounds. BDP had a ToxPi value of 0.494, the highest among organophosphate compounds, which may indicate a strong endocrine disruption effect on the hypothalamus-pituitary axis.

[0051] Table 3: ToxPi values ​​for testing the hypothalamic-pituitary endocrine interference effect of compounds

[0052]

[0053] The embodiments described above provide a detailed explanation of the technical solutions of the present invention. It should be understood that the above descriptions are merely specific embodiments of the present invention and are not intended to limit the present invention. Any modifications, additions, or similar substitutions made within the scope of the principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for evaluating the hypothalamic-pituitary reproductive endocrine axis interference effect of compounds, characterized in that, Includes the following steps: (1) Hypothalamic neuronal cell lines and pituitary gonadotropin cell lines were selected as in vitro evaluation models. Hypothalamic neuronal cell lines and pituitary gonadotropin cell lines were cultured, and compound concentration gradients were set. The effects of the compounds at gradient concentrations on the cell activity of hypothalamic neuronal cell lines and pituitary gonadotropin cell lines were tested respectively, and the exposure concentration gradients affecting the activity of each cell were obtained. (2) Based on the exposure concentration gradient obtained in step (1), select non-cytotoxic concentrations to conduct cell exposure experiments on hypothalamic neuronal cell lines and pituitary gonadotropin cell lines respectively. After exposure, collect cell RNA, reverse the RNA to cDNA, and measure the expression level of the target gene in the cells. Based on the exposure concentration gradient and the expression level of the target gene, obtain the dose-response curve of the compound interfering with the expression of the target gene, and calculate the concentration ECx when the change in the expression of the target gene reaches x%. (3) The ToxPi value was used to evaluate the endocrine disruption effect of the compound on the hypothalamic-pituitary axis. The ToxPi value was calculated as follows: Where n is the number of target genes, w i r represents the weight of the i-th target gene. i Let r be the sector radius corresponding to the i-th target gene. i = y i / y max , where y i = -log 10 EC i x, y max For y i Maximum value, EC i x represents the ECx of the compound interfering with the expression of the i-th target gene; If EC i If x exceeds the maximum exposure concentration gradient or has no significant effect on the expression of the target gene, then y i = 0, r i = 0; Further normalizing the ToxPi value to the 0-1 range, the closer it is to 1, the stronger the compound’s interference effect on the hypothalamic-pituitary reproductive endocrine axis; The hypothalamic neuronal cell line is the mouse hypothalamic GnRH neuronal cell line, and the pituitary gonadotropin cell line is the mouse pituitary gonadotropin cell line. The target genes of the hypothalamic neuronal cell line include... GnRH Genes and GPR54 Genes, target genes in pituitary gonadotropic cell lines include CGα Gene, LHβ Gene, FSHβ Genes and GnRHR Gene.

2. The method for evaluating the hypothalamic-pituitary-reproductive endocrine axis interference effect of a compound according to claim 1, characterized in that, GnRH The gene's Gene ID is 14714. GPR54 The gene's Gene ID is 114229. CGα The gene's Gene ID is 12640. LHβ The gene's Gene ID is 16866. FSHβ The gene's Gene ID is 14308. GnRHR The gene's Gene ID is 14715.

3. The method for evaluating the hypothalamic-pituitary-reproductive endocrine axis interference effect of a compound according to claim 1, characterized in that, Before conducting cell exposure experiments, cells were placed in a serum-free culture medium for serum starvation treatment for 4–25 hours.

4. The method for evaluating the hypothalamic-pituitary-reproductive endocrine axis interference effect of a compound according to claim 1, characterized in that, Livak analysis was performed using real-time quantitative PCR to... GAPDH Using a reference gene, the relative expression level of the target gene mRNA in cells was measured, and the dose-response curve of the compound interfering with the expression of the target gene was obtained based on the exposure concentration gradient and the relative expression level of the target gene mRNA.

5. The method for evaluating the hypothalamic-pituitary-reproductive endocrine axis interference effect of a compound according to claim 4, characterized in that, against GnRH The primer nucleotide sequences are shown in SEQ ID NO.1 and SEQ ID NO.2, targeting... GPR54 The primer nucleotide sequences are shown in SEQ ID NO.3 and SEQ ID NO.4, targeting... CGα The primer nucleotide sequences are shown in SEQ ID NO.5 and SEQ ID NO.6, targeting... LHβ The primer nucleotide sequences are shown in SEQ ID NO.7 and SEQ ID NO.8, targeting... FSHβ The primer nucleotide sequences are shown in SEQ ID NO. 9 and SEQ ID NO. 10, and are for... GnRHR The primer nucleotide sequences are shown in SEQ ID NO.11 and SEQ ID NO.

12.

6. The method for evaluating the hypothalamic-pituitary-reproductive endocrine axis interference effect of a compound according to claim 1, characterized in that, Calculate the concentrations EC10, EC20, or EC50 at which the induced change in target gene expression reaches 10%, 20%, or 50%, and use EC10, EC20, or EC50 as parameters for calculating the ToxPi value.

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