Method for screening medicine for relieving reproductive toxicity of imidacloprid and application thereof

Abstract

The invention discloses a method for screening drugs for relieving reproductive toxicity of imidacloprid and application of the drugs, and belongs to the technical field of candidate drug screening. Through a cascade mechanism of imidacloprid-mitochondrial injury-oxidative stress-apoptosis disclosed for the first time, the invention provides a method for screening drugs for relieving toxicity of imidacloprid to reproduction of mammals, and the method has the advantages of high result credibility, good repeatability, strong universality, controllable screening cost, low requirement on equipment and the like, and is suitable for industrial production. Powerful assistance can be provided for safe use of imidacloprid, risk assessment of pesticide residues in the cattle breeding process and screening of drugs for relieving toxicity.

Description

Technical Field

[0001] This invention relates to the field of candidate drug screening technology, and in particular to a method for screening drugs that alleviate the reproductive toxicity of imidacloprid and its application. Background Technology

[0002] Imidacloprid (IMI) is a neonicotinoid insecticide with highly effective insecticidal activity, widely used in agricultural production to control various piercing-sucking pests. However, with the long-term and large-scale use of imidacloprid, it continues to accumulate in soil, water, and air, entering non-target organisms, including mammals, through the food chain and respiration, posing a potential threat to the ecological environment and biological health. Its reproductive toxicity, in particular, is receiving increasing attention.

[0003] Like oocytes, bovine granulosa cells originate from the ovarian epithelial tissue. Before ovulation, granulosa cells form a cavity (follicular cavity), within which the oocyte is enclosed in a cluster of granulosa cells (cumulus cells) and suspended in the follicular fluid. Bovine granulosa cells deliver nutrients, energy molecules (such as pyruvate), and signaling molecules to the oocyte through direct intercellular junctions (gap junctions), ensuring the oocyte's healthy growth and maturation. Without the support of granulosa cells, the oocyte cannot develop normally and will eventually die.

[0004] In addition, granulosa cells secrete a variety of crucial substances: estrogen is one of the most critical secretory products of granulosa cells. Under the stimulation of follicle-stimulating hormone (FSH), granulosa cells convert androgens produced by theca interna cells into estrogens, which are the core hormones driving follicle growth and development; they express luteinizing hormone (LH) receptors, and before ovulation, the LH peak acts on granulosa cells, triggering their final differentiation into corpus luteum cells, forming the corpus luteum. The corpus luteum secretes progesterone, a hormone essential for maintaining pregnancy; inhibin and activin participate in feedback regulation of the pituitary gland, controlling FSH secretion, thereby precisely regulating the developmental rhythm of follicles; anti-Müllerian hormone (AMH), mainly secreted by granulosa cells of small follicles, inhibits the overactivation of primordial follicles and preserves follicular reserve. AMH levels are an important clinical indicator for assessing ovarian reserve function.

[0005] Therefore, by using bovine granular cells as the research subject, the results can reflect the effects of imidacloprid on the bovine reproductive system and reproductive capacity. Summary of the Invention

[0006] The purpose of this invention is to provide a method for screening drugs that mitigate the reproductive toxicity of imidacloprid and its application, so as to provide a method with high reliability, good stability, low equipment requirements and controllable cost, and to provide strong support for the safe use of imidacloprid, the risk assessment of pesticide residues in cattle breeding, and the screening of drugs to mitigate toxicity.

[0007] To achieve the above objectives, the present invention provides a kit for detecting the reproductive toxicity of imidacloprid to bovine cells, the kit comprising: bovine ovarian granulosa cell culture components, imidacloprid standard, and detection components.

[0008] Preferably, the bovine ovarian granulosa cell culture components include physiological saline containing 2% penicillin-streptomycin-gentamicin mixture, erythrocyte lysis buffer, DMEM / F-12 medium, fetal bovine serum, penicillin-streptomycin-gentamicin mixture (PSA), mycoplasma cleaner, 10 mL syringe, 50 mL centrifuge tube, 1.5 mL centrifuge tube, and 20 μm filter.

[0009] Preferably, the detection components include: CCK-8 reagent, Mitotracker red fluorescent dye, JC-1 fluorescent dye, ATP fluorescent dye, DCFH-DA fluorescent dye or HE dye, MitoSOX red fluorescent dye or DHE dye, qPCR reagents and primers for detecting the mRNA levels of Nrf2, SOD1, and GPX1, TUNEL detection reagent, and qPCR reagents and primers for detecting the mRNA levels of Bax, Bcl-2, and Caspase-3.

[0010] A method for screening drugs to mitigate the reproductive toxicity of imidacloprid in mammals, using the kit described above for detecting the reproductive toxicity of imidacloprid in cattle, comprises the following steps: S1. Extract and culture ovarian granulosa cells; S2. Set up a control group, an imidacloprid treatment group, and an imidacloprid and candidate drug co-treatment group; S3. Detect the following indicators: cell viability, mitochondrial function indicators, oxidative stress indicators, and apoptosis indicators; S4. Analyze the data from each group to determine the effectiveness of the drug.

[0011] Preferably, the method for extracting ovarian granulosa cells in S1 is as follows: extract follicular fluid from follicles with a diameter of 3-8 mm, add red blood cell lysis buffer, filter to remove non-granulosa cells after lysis, and centrifuge to collect the precipitate.

[0012] Preferably, in S2, the control group was cultured with DMEM / F-12 + 10% fetal bovine serum; the imidacloprid treatment group was cultured with DMEM / F-12 + 10% fetal bovine serum + different concentrations of imidacloprid; and the imidacloprid and candidate drug co-treatment group was cultured with DMEM / F-12 + 10% fetal bovine serum + different concentrations of imidacloprid + candidate drug.

[0013] Preferably, cell viability in S3 is detected using the CCK-8 assay or the MTT assay. The CCK-8 assay detects absorbance at 450 nm, and the MTT assay detects absorbance at 570 nm. Mitochondrial function indicators include mitochondrial mass, mitochondrial membrane potential, and ATP content. Oxidative stress indicators include total ROS, mitochondrial ROS, and antioxidant gene expression. Apoptosis indicators include apoptosis rate and apoptosis gene expression.

[0014] Preferably, the antioxidant genes include Nrf2, SOD1, and GPX1; the apoptosis genes include Bax, Bcl-2, and Caspase-3.

[0015] Preferably, if the detection results of the co-treatment group of imidacloprid and the candidate drug in S4 show a significant reversal compared with the imidacloprid-treated group and approach the control group, it indicates that the candidate drug can alleviate the reproductive toxicity of imidacloprid to mammals.

[0016] The application of a method described above for screening drugs that mitigate the reproductive toxicity of imidacloprid to mammals in the screening of drugs that mitigate the toxicity of imidacloprid.

[0017] Therefore, the present invention provides a method for screening drugs to alleviate the reproductive toxicity of imidacloprid and its application, the specific technical effects of which are as follows: (1) This invention provides a method for screening drugs to alleviate the reproductive toxicity of imidacloprid to mammals by revealing for the first time the cascade mechanism of "imidacloprid → mitochondrial damage → oxidative stress → apoptosis". Through dose-time effect analysis and multi-indicator correlation, the hierarchical relationship of the toxic pathway of imidacloprid to bovine granulocytes can be completely analyzed, which greatly improves the accuracy of the screening results; and provides sufficient scientific basis for the safe use of imidacloprid and the risk assessment of pesticide residues in cattle breeding. (2) The method for screening drugs to alleviate the reproductive toxicity of imidacloprid to mammals provided by the present invention avoids subjective inference through quantitative correlation, and the reliability of the results is much higher than that of traditional qualitative description. The reproducibility rate of the results of three independent repeated designs is over 90%. (3) The method for screening drugs that alleviate the reproductive toxicity of imidacloprid to mammals provided by the present invention has strong universality and controllable application cost. It uses fluorescence microscopy to replace part of the flow cytometry detection, and reduces the equipment threshold while ensuring the accuracy of the core results (deviation from flow cytometry detection ≤15%). (4) Only control groups and experimental groups with different concentrations of imidacloprid are set up. The method provided by this invention can also be used for imidacloprid toxicity assessment. It has high accuracy and good stability, which provides strong support for the safe use of imidacloprid and the risk assessment of pesticide residues in cattle breeding.

[0018] The technical solution of the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. Attached Figure Description

[0019] To more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings used in the description of the embodiments of the present invention will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0020] Figure 1 This is a schematic diagram of follicle selection in Embodiment 1 of the present invention; the arrows point to follicles that meet the specifications; A and B are two different ovaries; Figure 2 This refers to the cell viability detection results in Example 1 of the present invention; wherein express P<0.05 , express P<0.01 , express P<0.001 ; Figure 3 These are the results of mitochondrial morphology investigation in Example 1 of this invention; Part A is a fluorescence microscope photograph; Part B is a bar chart of fluorescence intensity analysis at the Mito-Tracker level. express P<0.001 ; Figure 4 These are the results of the membrane potential investigation in Example 1 of this invention; Part A is a fluorescence microscope photograph; Part B is a bar chart of fluorescence intensity analysis at the JC-1 level; express P<0.001 ; Figure 5 This is the result of the ATP content investigation in Example 1 of the present invention; Part A is a fluorescence microscope photograph; Part B is a bar chart of fluorescence intensity analysis of ATP level; express P<0.001 ; Figure 6These are the results of the mitochondrial ROS investigation in Example 1 of this invention; Part A is a fluorescence microscope image; Part B is a bar chart of fluorescence intensity analysis of MITOSOX levels; red represents MITOSOX, and blue represents DAPI; Figure 7 This is the cell apoptosis detection result in Example 1 of the present invention; Part A is a fluorescence microscope image; Part B is a bar chart analyzing the proliferation ratio of EDU-positive cells; red represents EDU, and blue represents DAPI; Figure 8 This refers to the relative expression levels of each antioxidant gene in Example 1 of this invention; Figure 9 This is a heatmap showing the correlation analysis of various indicators in Embodiment 1 of the present invention. Detailed Implementation

[0021] The technical solution of the present invention will be further described below with reference to the accompanying drawings and embodiments.

[0022] To make the objectives, technical solutions, and advantages of this application clearer, more thorough, and more complete, the technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings and embodiments. The following detailed descriptions are all illustrations of embodiments, intended to provide further detailed explanation of the present invention. Unless otherwise specified, all technical terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains.

[0023] The instruments, equipment, reagents and materials used in the embodiments were all obtained through commercial means; the methods and steps not described in detail are all conventional techniques in the field.

[0024] Example 1 (1) Preparation and grouping of experimental materials.

[0025] Cell extraction and culture: S1. Granulosa cell extraction. Ovaries from 18-24 month old Yanbian Yellow Cattle slaughtered that day were collected from the slaughterhouse and placed in a preheated 37°C thermos. The ovarian samples were transported to the laboratory within 1.5 hours. Upon arrival at the laboratory, the collected ovarian samples were immediately transferred to physiological saline containing 2% penicillin and streptomycin (1:1 concentration ratio) for washing to remove surface blood and bacteria.

[0026] Then, using a 10mL syringe, follicular fluid (e.g., follicular fluid from follicles with a diameter of 3-8mm) is extracted. Figure 1(Indicated by the middle arrow) Transfer to a 50mL centrifuge tube. Collect follicular fluid and lyse at room temperature for 15 minutes at a follicular fluid to red blood cell lysis buffer volume ratio of 1:3. Filter the lysate mixture through a 20μm filter to remove non-granulosa cell material. Centrifuge at 1000rpm for 5 minutes, discard the supernatant, and transfer the precipitate to a 1.5mL centrifuge tube; the precipitate is the granulosa cells.

[0027] S2, cell resuspension and seeding plate.

[0028] (1) Preheat the DMEM / F-12 + 10% fetal bovine serum culture medium in the cell culture incubator two hours in advance. Adjust the glass baffle of the laminar flow hood (sterilized with UV for 1 hour in advance) to an appropriate height, light the alcohol lamp, and turn on the exhaust fan. After sterilizing the centrifuge tube containing the precipitate in step S1 with 75% alcohol, place it in the laminar flow hood, open the cap, add 1 mL of DMEM / F-12 culture medium to the precipitate, resuspend the cells, and pipette to ensure that the cells are evenly dispersed to obtain diluted cells.

[0029] (2) Take 50 mL of DMEM / F-12 + 10% fetal bovine serum culture medium preheated in a cell culture incubator, add 2% PSA and 1% mycoplasma cleaner, and mix them evenly with a shaker. Then add the cells diluted in (1), mix gently to avoid generating air bubbles, and obtain a cell suspension.

[0030] (3) Plating. Using a pipette, take an appropriate amount of the cell suspension prepared in (2) and plate it according to 1×10 5 Slowly add the cell density to a DMEM / F12 (containing 10% fetal bovine serum) culture dish. Hold the culture dish and gently shake it in a figure-eight motion on a clean bench to evenly distribute the cell suspension on the culture surface. After ensuring that the suspension is evenly distributed on the culture plate surface (after plating is complete), cover the plate and write the date and cell type on the culture dish. Then place the culture plate in a cell culture incubator containing 5% CO2 at 37°C.

[0031] S3. Conduct preliminary experiments to determine the optimal experimental concentration of imidacloprid.

[0032] Imidacloprid preparation method: Weigh 0.0256g of imidacloprid and add it to DMSO, then bring the volume to 1mL to prepare the stock solution. To reduce the influence of DMSO on cell viability and experimental results, a serial dilution method was used to ensure that the final dilution concentration of DMSO is ≤0.1%. The specific dilution steps are as follows: Take 10µL of the stock solution and mix it thoroughly with 990µL of culture medium (DMEM / F-12, containing 10% fetal bovine serum) to prepare the intermediate solution. Then, take 25µL, 50µL, 75µL, and 100µL of the intermediate solution respectively and add them to the culture medium (DMEM / F-12, containing 10% fetal bovine serum) to bring the volume to 1mL, thus obtaining imidacloprid-containing culture media with concentration gradients of 250, 500, 750, and 1000μM, with a DMSO concentration less than or equal to 0.1%.

[0033] The preliminary experiment set up five concentration gradients: 0, 250, 500, 750, and 1000 μM, and was conducted at 37 °C with 5% CO2. 2 The cells were treated in a constant-temperature humidified cell culture incubator for 24 and 48 hours; each group was set up with 3 independent replicates to ensure the reliability of the results. Untreated bovine granular cells were used as the control group. After analyzing the results and reviewing the literature, the experimental concentration of imidacloprid for subsequent experiments was finally selected as 500 and 1000 μM, and the treatment time was 24 hours.

[0034] S4. Using 500 μM and 1000 μM imidacloprid, respectively, the method in S3 was used for testing, with a treatment time of 24 h. Then, the following tests were performed: (1) Cell viability detection: The CCK-8 method was used. 5×10³ cells / well were seeded in a 96-well plate. After grouping and treatment, CCK-8 reagent was added. After incubation at 37℃ for 2h, the absorbance at 450nm was detected. Cell viability (reflecting the overall toxicity of imidacloprid to cells) was calculated using Formula I.

[0035] Cell viability (%) = [(OD experimental group - OD blank group) / (OD control group - OD blank group)] × 100% (Formula I); where the OD experimental group is the absorbance of wells containing cells, CCK-8 and the test drug; the OD control group is the absorbance of wells containing cells and CCK-8 but not the drug (representing 100% cell viability); and the OD blank group is the absorbance of wells containing only culture medium and CCK-8, without cells (used to subtract background).

[0036] Test results as follows Figure 2As shown, the effect of different concentrations of imidacloprid on cell viability exhibits a concentration-response relationship. At both 24h and 48h time points, cell viability generally decreased with increasing treatment concentration (250μM-1000μM). For example, at 24h, although cell viability in the 250μM treatment group decreased, it remained relatively high, while cell viability in the 1000μM treatment group decreased significantly. A similar pattern was observed at 48h, indicating that the effect of this treatment factor on cells increases with increasing concentration, demonstrating a concentration-response relationship.

[0037] Time affects cell viability and interacts with concentration: Comparing the same concentration treatment groups at 24h and 48h, such as 500μM, 750μM, and 1000μM, cell viability at 48h was further reduced compared to 24h (at the same concentration, the column height at 48h was lower than that at 24h), indicating that the inhibitory effect of treatment on cell viability is aggravated by the extended treatment time, and there is an interaction effect between time and concentration in affecting cell viability.

[0038] (2) Mitochondrial function testing, including the following tests: A: Mitochondrial morphology: Mitochondrial morphology was observed under a fluorescence microscope using Mitotracker red fluorescence staining (normally reticular, fragmented after damage). Results are as follows: Figure 3 As shown, the control group exhibited high and uniform mitochondrial fluorescence intensity; after treatment with 500 μM and 1000 μM imidacloprid (IMI), mitochondrial fluorescence was significantly reduced. , P The mitochondrial structure was damaged due to IMI (<0.001) and its morphology was broken and its distribution was disordered.

[0039] B: Membrane potential: After JC-1 staining, the red / green fluorescence ratio was detected by fluorescence microscopy (a decrease in the ratio suggests membrane potential depolarization). Results are as follows: Figure 4 As shown, the control group had a high proportion of JC-1 aggregates (red) and normal membrane potential; the IMI treatment group had fewer aggregates and more monomers (green), and the 1000μM group had dominant green fluorescence. , P <0.001), confirming that IMI can disintegrate mitochondrial membrane potential in a concentration-dependent manner, disrupting a key basis of energy metabolism.

[0040] C: ATP content: ATP fluorescence staining was used, and fluorescence intensity (reflecting mitochondrial energy metabolism capacity) was detected by fluorescence microscopy. Results are as follows: Figure 5 As shown, the control group cells exhibited strong ATP fluorescence and high ATP synthesis; after treatment with 500 μM and 1000 μM IMI, ATP fluorescence decreased sharply. , P<0.001), and the higher the concentration, the more significant the inhibition. Combined with mitochondrial membrane potential data, this indicates that IMI blocks mitochondrial oxidative phosphorylation by damaging mitochondrial structure and disrupting membrane potential, leading to a concentration-dependent decrease in ATP synthesis capacity and directly affecting cellular energy supply.

[0041] (3) Oxidative stress detection: A: Total ROS: After DCFH-DA staining, the intensity of green fluorescence was detected using a fluorescence microscope.

[0042] B: Mitochondrial ROS:MitoSOX red fluorescence staining was performed, followed by fluorescence microscopy. Results are as follows: Figure 6 As shown, IMI can induce mitochondrial reactive oxygen species accumulation in a concentration-dependent manner: the mitochondrial MitoSox fluorescence intensity in the control group (CONTROL) cells was low and the ROS level was stable; after treatment with 500 μM IMI, the relative level of mitochondrial ROS increased significantly (significantly different from the control group); in the 1000 μM IMI treatment group, the MitoSox fluorescence intensity was further enhanced and the relative ROS level reached an even higher value.

[0043] C: Antioxidant gene expression: Real-time quantitative PCR was used to detect the mRNA levels of Nrf2, SOD1, and GPX1 (reflecting the cellular antioxidant system response).

[0044] (4) Apoptosis detection: 1) Apoptosis rate: The proportion of apoptotic cells was detected using TUNEL fluorescence microscopy. Results are as follows: Figure 7 As shown, imidacloprid treatment induced extensive DNA fragmentation in bovine granular cells, producing a significant TUNEL positive signal, indicating that the pesticide can trigger programmed cell death (apoptosis). The intensity of the TUNEL positive signal increased in a dose-dependent manner; that is, with increasing imidacloprid concentration, both the intensity of green fluorescence and the number of positive cells increased, providing direct morphological evidence for the reproductive toxicity of imidacloprid. This morphological result is highly consistent with the molecular changes detected by qPCR, namely the upregulation of pro-apoptotic genes (such as BAX and Caspase-3) and the downregulation of the anti-apoptotic gene (BCL2), jointly confirming that imidacloprid induces apoptosis in bovine granular cells by activating the mitochondrial apoptosis pathway.

[0045] 2) Apoptosis gene expression: PCR detection of the mRNA levels of Bax, Bcl-2, and Caspase-3.

[0046] S5. Perform correlation analysis on the data obtained in step S4.

[0047] (1) Dose-time effect correlation analysis.

[0048] By comparing the detection results of different concentrations of imidacloprid after 24h and 48h treatment, the dynamic pattern of toxic effects was clarified through the following correlations: 1) Based on cell viability detected by CCK-8, the correlation between cell viability and mitochondrial functional indicators (mitochondrial morphology, membrane potential, and ATP content) after 24 hours of exposure was analyzed. After drug treatment, cell viability decreased, accompanied by an increase in the proportion of mitochondrial fragmentation, a decrease in the membrane potential ratio, and a decrease in ATP fluorescence intensity. It can be preliminarily inferred that mitochondrial damage is an important reason for the decrease in cell viability.

[0049] 2) Combining oxidative stress indicators (total ROS and mitochondrial ROS), imidacloprid treatment significantly increased both mitochondrial ROS (MitoSOX) and total ROS, with highly consistent trends. Simultaneously, the surge in mitochondrial ROS and the loss of mitochondrial membrane potential (JC-1) showed a strong synergistic relationship. These results indicate that mitochondria are the primary initial source of ROS production in imidacloprid-induced oxidative stress, and their functional impairment is closely related to internal oxidative stress.

[0050] 3) The relationship between oxidative stress and apoptosis.

[0051] Using the proportion of apoptotic cells as determined by TUNEL assay as the core, we correlated oxidative stress markers with the expression of apoptotic molecules: If the proportion of apoptotic cells in the high-concentration imidacloprid group is positively correlated with the levels of total ROS and mitochondrial ROS, and is accompanied by upregulation of pro-apoptotic gene (Bax, Caspase-3) mRNA expression and downregulation of anti-apoptotic gene (Bcl-2) expression, it indicates that oxidative stress may drive the apoptosis process.

[0052] 4) By observing the changes in the mRNA expression of antioxidant genes (Nrf2, SOD1, GPX1), if their expression levels are first upregulated and then downregulated with increasing imidacloprid concentration, it can further corroborate that oxidative stress overload is a key inducing factor for apoptosis.

[0053] The results are as follows Figure 8 As shown, the oxidative stress pathway was strongly activated: Nrf2 expression increased sharply, indicating that the cell sensed strong oxidative stress and initiated the core defense response. SOD1 and GPX1, as key downstream antioxidant enzymes of the Nrf2 pathway, also showed significantly upregulated expression with increasing dosage, demonstrating that the antioxidant defense system was fully activated, but clearly insufficient to counteract the oxidative damage caused by imidacloprid.

[0054] The apoptosis pathway was successfully triggered. The BAX / BCL2 ratio, the most critical switch for apoptosis regulation, showed a highly significant dose-dependent increase, as shown in the figure. BAX (pro-apoptotic) expression increased dramatically, while BCL2 (anti-apoptotic) expression decreased, indicating that apoptotic signals completely overwhelmed survival signals. Caspase-3, the final executor of apoptosis, showed a significant increase in expression, confirming that the apoptosis program was not only initiated but had entered an irreversible execution phase.

[0055] (2) Cascade verification of mitochondrial damage and downstream effects.

[0056] Regression analysis of mitochondrial functional indicators (such as JC-1 membrane potential) and oxidative stress indicators (such as MitoSOX fluorescence intensity) showed that a decrease in membrane potential corresponds to an increase in mitochondrial ROS, which clearly indicates that mitochondrial damage is an upstream event of oxidative stress.

[0057] By correlating mitochondrial function indicators with apoptosis indicators (TUNEL positivity rate, Caspase-3 protein expression), if the decrease in ATP fluorescence intensity is positively correlated with the apoptosis rate, and the mitochondrial fragmentation ratio is positively correlated with the Bax / Bcl-2 ratio, it can be verified that mitochondrial damage mediates the cascade effect of apoptosis through oxidative stress.

[0058] Based on the above analysis, the core mechanism can be identified if the following logical loop is satisfied: Increased imidacloprid concentration and prolonged treatment time lead to more severe damage to mitochondrial morphology and function (increased fragmentation, decreased membrane potential, and reduced ATP) → increased mitochondrial ROS production → increased total oxidative stress level → imbalance of the antioxidant system (Nrf2 pathway is first activated and then inhibited) → upregulation of pro-apoptotic gene expression → increased cell apoptosis rate.

[0059] The heatmap of correlation analysis of various indicators is as follows: Figure 9 As shown, a matrix heatmap is used to display the correlation coefficients of mitochondrial indicators (membrane potential, ATP), oxidative stress indicators (total ROS, mitochondrial ROS), and apoptosis indicators (apoptosis rate, Caspase-3 expression) (red indicates positive correlation, blue indicates negative correlation), and key correlation pairs are marked (such as the correlation coefficient between membrane potential and mitochondrial ROS, and the correlation coefficient between mitochondrial ROS and apoptosis rate).

[0060] Note: The quantitative correlation visually presents the cascade relationship of "mitochondrial damage → oxidative stress → apoptosis", which is key evidence to verify the core mechanism of the invention and demonstrates the technical advantages of multi-indicator synergistic analysis.

[0061] Ultimately, by analyzing the dynamic trends and correlations of various indicators, a complete chain of evidence—"morphological observation - functional detection - molecular mechanism"—was formed, revealing the toxic mechanism by which imidacloprid induces apoptosis in bovine granular cells by disrupting the mitochondrial-oxidative stress axis.

[0062] Therefore, this invention provides a method for screening drugs to alleviate the reproductive toxicity of imidacloprid in mammals by revealing for the first time the cascade mechanism of "imidacloprid → mitochondrial damage → oxidative stress → apoptosis". It has the advantages of high reliability, good reproducibility, strong universality, controllable screening cost and low equipment requirements. It can provide strong support for the safe use of imidacloprid, the risk assessment of pesticide residues in cattle breeding, and the screening of drugs to alleviate toxicity.

[0063] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit them. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can still be made to the technical solutions of the present invention, and these modifications or equivalent substitutions cannot cause the modified technical solutions to deviate from the spirit and scope of the technical solutions of the present invention.

Claims

1. A kit for detecting the reproductive toxicity of imidacloprid to cattle, characterized in that, The kit includes: bovine ovarian granulosa cell culture components, imidacloprid standard, and detection components.

2. The kit for detecting the reproductive toxicity of imidacloprid to cattle according to claim 1, characterized in that: The bovine ovarian granulosa cell culture components include physiological saline containing 2% penicillin and antibiotics, erythrocyte lysis buffer, DMEM / F-12 medium, fetal bovine serum, penicillin-streptomycin-gentamicin mixed solution, mycoplasma cleaner, 10mL syringe, 50mL centrifuge tube, 1.5mL centrifuge tube, and 20μm filter.

3. The kit for detecting the reproductive toxicity of imidacloprid to cattle according to claim 1, characterized in that, The detection components include: CCK-8 reagent, Mitotracker red fluorescent dye, JC-1 fluorescent dye, ATP fluorescent dye, DCFH-DA fluorescent dye or HE dye, MitoSOX red fluorescent dye or DHE dye, qPCR reagents and primers for detecting the mRNA levels of Nrf2, SOD1, and GPX1, TUNEL detection reagent, and qPCR reagents and primers for detecting the mRNA levels of Bax, Bcl-2, and Caspase-3.

4. A method for screening drugs to mitigate the reproductive toxicity of imidacloprid in mammals, characterized in that, The kit for detecting bovine reproductive toxicity of imidacloprid according to any one of claims 1-3 is used in the following steps: S1. Extract and culture ovarian granulosa cells; S2. Set up a control group, an imidacloprid treatment group, and an imidacloprid and candidate drug co-treatment group; S3. Detect the following indicators: cell viability, mitochondrial function indicators, oxidative stress indicators, and apoptosis indicators; S4. Analyze the data from each group to determine the effectiveness of the drug.

5. The method for screening drugs to alleviate the reproductive toxicity of imidacloprid to mammals according to claim 4, characterized in that, The method for extracting ovarian granulosa cells from S1 is as follows: extract follicular fluid from follicles with a diameter of 3-8 mm, add red blood cell lysis buffer, filter to remove non-granulosa cells after lysis, and centrifuge to collect the precipitate.

6. The method for screening drugs to alleviate the reproductive toxicity of imidacloprid to mammals according to claim 4, characterized in that: In S2, the control group used DMEM / F-12 medium with 10% fetal bovine serum to culture granulocytes; the imidacloprid treatment group used DMEM / F-12 medium with 10% fetal bovine serum and different concentrations of imidacloprid to culture granulocytes; and the imidacloprid and candidate drug co-treatment group used DMEM / F-12 medium with 10% fetal bovine serum, different concentrations of imidacloprid and candidate drug to culture granulocytes.

7. The method for screening drugs to alleviate the reproductive toxicity of imidacloprid to mammals according to claim 4, characterized in that: Cell viability in S3 was assessed using either the CCK-8 assay or the MTT assay. The CCK-8 assay measured absorbance at 450 nm, while the MTT assay measured absorbance at 570 nm. Mitochondrial function indicators included mitochondrial mass, mitochondrial membrane potential, and ATP content. Oxidative stress indicators included total ROS, mitochondrial ROS, and antioxidant gene expression. Apoptosis indicators included apoptosis rate and apoptosis gene expression.

8. The method for screening drugs to alleviate the reproductive toxicity of imidacloprid to mammals according to claim 7, characterized in that: The antioxidant genes include Nrf2, SOD1, and GPX1; the apoptosis genes include Bax, Bcl-2, and Caspase-3.

9. The method for screening drugs to alleviate the reproductive toxicity of imidacloprid to mammals according to claim 4, characterized in that: If the test results of the co-treatment group of imidacloprid and the candidate drug in S4 show a significant reversal compared with the imidacloprid-treated group and approach the control group, it indicates that the candidate drug can alleviate the reproductive toxicity of imidacloprid to mammals.

10. The application of the method for screening drugs that mitigate the reproductive toxicity of imidacloprid as described in any one of claims 4-9 in the screening of drugs that mitigate the toxicity of imidacloprid.

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