Application of FeTMPyP in the preparation of drugs to alleviate ovarian toxicity

Abstract

This invention belongs to the field of biomedicine, and specifically relates to the application of FeTMPyP in the preparation of drugs to alleviate ovarian toxicity. This application addresses the issue that combined exposure to MC-LR and NaNO₂ significantly induces nitrification stress in mouse ovaries, leading to follicular barrier disruption and ovarian reproductive endocrine dysfunction. FeTMPyP was selected as an intervention agent, which effectively alleviated nitrification stress induced by combined exposure to MC-LR and NaNO₂ in mouse ovaries, and improved sex hormone disorders, estrous cycle irregularities, and follicular atresia caused by the combined exposure to these two toxins. Simultaneously, it restored the fertility of mice, indicating that FeTMPyP intervention can alleviate ovarian toxicity induced by combined exposure to MC-LR and NaNO₂ in mice. This invention can be used to prepare drugs to prevent and treat diseases related to ovarian toxicity caused by environmental toxin exposure in mice, providing a new approach for the early identification and targeted intervention of ovarian-related diseases.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine, and specifically relates to a drug for alleviating ovarian toxicity. Background Technology

[0002] Microcystins (MCs) are a class of cyclic heptapeptide endotoxins widely found in water bodies. Produced by abundant cyanobacteria in eutrophic lakes and rivers, these endotoxins can enter organisms through the food chain or direct contact, causing multi-organ damage. With increasing water pollution, the health problems caused by environmental exposure to microcystins are becoming increasingly serious. Among the more than 200 MC isomers discovered, microcystin-LR (MC-LR) is the most abundant and toxic isomer. Numerous studies have shown that MC-LR has multi-organ toxicity, accumulating in multiple tissues in humans and animals, including the liver, brain, ovaries, intestines, and kidneys, causing dysfunction in these systems. The gonads are its primary target organ. Eutrophic water bodies also contain nitrite, the most toxic inorganic nitrogen pollutant in water. Studies have shown that acute exposure to nitrite poses a toxic threat to the female reproductive system. Nitrite in water bodies mainly originates from domestic and industrial wastewater and the conversion of ammonia nitrogen in water bodies. It is worth noting that ammonia nitrogen is also the most abundant nutrient in water bodies, and it can cause eutrophication and algal proliferation, promoting the release of algal toxins. In eutrophic water bodies, nitrite concentration is usually positively correlated with microcystin concentration. Microcystins (MCs) and nitrite often coexist in water bodies, and the two can significantly enhance toxic effects through synergistic effects, especially posing a serious threat to endocrine-sensitive organs (such as the ovaries).

[0003] In recent years, the female reproductive toxicity caused by exposure to microcystins (MCs) and nitrites has received increasing attention. Epidemiological studies have found that nitrite intake is positively correlated with the risk of ovarian cancer in women. Microcystin exposure can cause programmed cell death in ovarian cells, reduce the number and quality of oocytes, and thus affect fertility. Existing studies mostly focus on the toxicity of single pollutants and lack a systematic analysis of the mechanisms of combined exposure. For example, traditional antioxidants (such as vitamin C and N-acetylcysteine) have a certain mitigating effect on single microcystin exposure (Xue Lijian, Li Jinhui, Yang Mingfeng, et al. Effects of combined exposure to N-acetylcysteine ​​and microcystin-LR on apoptosis of ovarian cells in Chinese hamsters [J]. Journal of Environment and Health, 2013, 30(10):879-881.), but it is difficult to block the combined toxicity cascade of MCs and nitrites.

[0004] Therefore, finding effective interventions to alleviate the female reproductive toxicity caused by MCs and nitrite exposure is an important prevention and treatment strategy for alleviating ovarian-related diseases and protecting reproductive health. Summary of the Invention

[0005] To address the issue of ovarian toxicity caused by combined exposure to MCs and NaNO2, this invention proposes the application of FeTMPyP in the preparation of drugs that alleviate ovarian toxicity. Iron(III)tetra(4-methylpyridyl)porphyrin (FeTMPyP) is selected as an intervention agent to target nitrification stress, providing a basis for new drug screening.

[0006] The technical solution of this invention is implemented as follows:

[0007] Co-exposure to MC-LR and NaNO2 significantly induced nitrification stress in mouse ovaries. Nitrification stress alters protein structure and expression, leading to impaired cellular function, and is a core mechanism by which co-exposure to these two toxins disrupts the follicular barrier and impairs ovarian reproductive endocrine function in mice. Therefore, nitrification stress can serve as a potential molecular biomarker for ovarian toxicity caused by co-exposure to microcystins and nitrites. Targeting nitrification stress and finding effective interventions are important strategies for alleviating ovarian-related diseases and protecting women's reproductive health.

[0008] FeTMPyP is an ONOO-decomposition catalyst with a unique mechanism of action, exhibiting the ability to precisely regulate nitrification stress levels in the intracellular environment. This application used BALB / c mice as research subjects, employing the nitrification stress inhibitor FeTMPyP for intervention. It was found that in vivo, FeTMPyP effectively alleviated nitrification stress induced by combined exposure to MC-LR and NaNO2 in mouse ovaries, and improved sex hormone disorders, estrous cycle dysregulation, and follicular atresia caused by this combined exposure, while restoring fertility in mice. This further elucidates that nitrification stress is the core mechanism by which combined exposure to these two toxins leads to follicular barrier disruption and ovarian reproductive endocrine dysfunction in mice. FeTMPyP can serve as a therapeutic agent for ovarian toxicity caused by MCs and nitrite exposure, and can also be used to prepare drugs for the prevention and treatment of ovarian toxicity and related diseases caused by environmental toxin exposure in mice. Targeting nitrification stress is an important preventive and therapeutic approach for alleviating ovarian-related diseases and protecting women's reproductive health.

[0009] Based on this, this application provides the use of FeTMPyP in the preparation of drugs that alleviate ovarian toxicity.

[0010] Preferably, the aforementioned ovarian toxicity is caused by exposure to environmental toxins.

[0011] Preferably, the above-mentioned environmental toxin exposure is a combined exposure to MCs and NaNO2.

[0012] On the other hand, this application also proposes a drug for alleviating ovarian toxicity, comprising FeTMPyP and a pharmaceutically acceptable carrier.

[0013] Preferably, the dosage of FeTMPyP in the above-mentioned drug is 10 mg / kg BW, and the duration of drug action is 3 months.

[0014] Preferably, the above-mentioned drug is administered via intraperitoneal injection once a week.

[0015] The alleviating effect of FeTMPyP on ovarian nitrification stress induced by combined exposure to MC-LR and NaNO2 in BALB / c mice;

[0016] The alleviating effect of FeTMPyP on ovarian toxicity induced by combined exposure to MC-LR and NaNO2;

[0017] Preferably, the aforementioned ovarian toxicity is caused by exposure to environmental toxins.

[0018] Preferably, the above-mentioned environmental toxin exposure is a combined exposure to MCs and NaNO2.

[0019] Furthermore, the diseases caused by the aforementioned ovarian toxicity include damage to tight junctions of granulosa cells, apoptosis, and disruption of the follicular barrier;

[0020] Furthermore, the diseases caused by the aforementioned ovarian toxicity also include sex hormone disorders, estrous cycle irregularities, follicular atresia, and adverse birth outcomes.

[0021] The present invention has the following beneficial effects:

[0022] 1. This application is the first to target the disruption of the follicular barrier and ovarian reproductive endocrine dysfunction in mice caused by the combined exposure of MCs and NaNO2. FeTMPyP was selected as an intervention agent, providing the alleviating effect of FeTMPyP on ovarian toxicity in mice caused by the combined exposure of MCs and nitrite and its application, providing a basis for new drug screening. Unlike traditional antioxidants or anti-inflammatory drugs, FeTMPyP specifically scavenges peroxynitrite (ONOO-) as the intervention target, clearly identifying nitrification stress as the core mechanism of ovarian toxicity caused by the combined exposure of MC-LR and NaNO2. It has a clear molecular mechanism targeting and has the potential to serve as a disease intervention target, providing a new approach for the early identification and targeted intervention of ovarian-related diseases.

[0023] 2. This application also conducted a drug safety assessment of FeTMPyP, showing that FeTMPyP at a dose of 10 mg / kg BW and a treatment cycle of 3 months had no significant systemic toxicity to mice and had good in vivo safety; FeTMPyP can effectively alleviate nitrification stress induced by the combined exposure of MC-LR and NaNO2 in mouse ovaries, and improve sex hormone imbalance, estrous cycle disorder and follicular atresia caused by the combined exposure of these two toxins, reduce cell apoptosis and follicular barrier damage, and restore the fertility of mice.

[0024] 3. This application expands the application field of FeTMPyP. FeTMPyP can be used as a therapeutic agent for ovarian toxicity caused by exposure to MCs and nitrites, and can also be used to prepare drugs for the prevention and treatment of ovarian toxicity and other related diseases caused by exposure to environmental toxins in mice. It provides a new prevention and control strategy for ovarian-related diseases caused by combined exposure to MCs and nitrites, and enriches the theory and technology for preventing and controlling the toxic effects of environmental toxins on human health. Attached Figure Description

[0025] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art 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.

[0026] Figure 1 The general toxicity assessment of FeTMPyP in mice is shown in Figure A, where A represents the change in mouse body weight, B represents the liver index, C represents the kidney index, D represents the heart index, E represents the spleen index, and F represents the ovarian index.

[0027] Figure 2 FeTMPyP was used to alleviate the sex hormone secretion disorder induced by combined exposure to MC-LR and NaNO2 in mice; where A represents estradiol (E2) content, B represents follicle-stimulating hormone (FSH) content, C represents luteinizing hormone (LH) content, and D represents anti-Müllerian hormone (AMH) content.

[0028] Figure 3 FeTMPyP was used to alleviate MC-LR and NaNO2-induced apoptosis in mouse ovarian cells. In this study, A represents the expression level of apoptosis-related proteins in mouse ovarian tissue detected by Western blotting, B represents the quantitative analysis of the expression level of apoptosis-related proteins in mouse ovarian tissue, and C represents the TUNEL fluorescence staining analysis of apoptosis in mouse ovarian cells. Blue fluorescence represents cell nuclei, and green fluorescence represents TUNEL-labeled apoptotic cells.

[0029] Figure 4 FeTMPyP was used to alleviate estrous cycle disorder in mice induced by combined exposure to MC-LR and NaNO2; where A is a trend graph of estrous cycle changes in experimental mice over 21 days, and B is the statistical analysis results of estrous cycle in mice.

[0030] Figure 5 FeTMPyP was used to alleviate follicular atresia induced by combined exposure to MC-LR and NaNO2 in mice; A shows the histopathological changes and follicular development of mouse ovaries observed by H&E staining, and B shows the statistical analysis results of mouse follicle count. Detailed Implementation

[0031] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0032] Unless otherwise specified, the experimental methods used in the following experimental examples are conventional methods; the materials and reagents used are commercially available unless otherwise specified.

[0033] This application is the first to target the disruption of the follicular barrier and ovarian reproductive endocrine dysfunction in mice caused by combined exposure to MCs and NaNO2. FeTMPyP was selected as an intervention agent, providing information on the alleviating effect of FeTMPyP on ovarian toxicity induced by combined exposure to MCs and nitrite in mice and its application, thus providing a foundation for new drug screening. Unlike traditional antioxidants or anti-inflammatory drugs, FeTMPyP specifically targets the scavenging of peroxynitrite (ONOO-), clearly identifying nitrification stress as the core mechanism of ovarian toxicity induced by combined exposure to MC-LR and NaNO2. It has a clear molecular mechanism targeting and the potential to serve as a disease intervention target, providing a new approach for the early identification and targeted intervention of ovarian-related diseases, and also offering a new direction for the intervention of reproductive damage related to environmental toxin exposure.

[0034] Co-exposure to MC-LR and NaNO2 significantly induced nitrification stress in mouse ovaries. Nitrification stress alters protein structure and expression, leading to impaired cellular function, and is a core mechanism by which co-exposure to these two toxins disrupts the follicular barrier and impairs ovarian reproductive endocrine function in mice. Therefore, nitrification stress can serve as a potential molecular biomarker for ovarian toxicity caused by co-exposure to microcystins and nitrites. Targeting nitrification stress and finding effective interventions are important strategies for alleviating ovarian-related diseases and protecting women's reproductive health.

[0035] FeTMPyP is an ONOO-decomposition catalyst with a unique mechanism of action, exhibiting the ability to precisely regulate nitrification stress levels in the intracellular environment. This application used BALB / c mice as research subjects, employing the nitrification stress inhibitor FeTMPyP for intervention. It was found that in vivo FeTMPyP intervention effectively alleviated nitrification stress induced by combined exposure to MC-LR and NaNO2 in mouse ovaries, and improved sex hormone disorders, estrous cycle dysregulation, and follicular atresia caused by this combined exposure, while restoring fertility in mice. This further elucidates that nitrification stress is the core mechanism by which combined exposure to these two toxins leads to follicular barrier disruption and ovarian reproductive endocrine dysfunction in mice. FeTMPyP can serve as a therapeutic agent for ovarian toxicity caused by MCs and nitrite exposure, and can also be used to prepare drugs for the prevention and treatment of ovarian toxicity and related diseases caused by environmental toxin exposure in mice. Targeting nitrification stress is an important preventive and therapeutic approach for alleviating ovarian-related diseases and protecting women's reproductive health.

[0036] Materials and Methods:

[0037] 1. Experimental reagents

[0038] Microcystin-LR (MC-LR, purity >95%) was purchased from Beijing Apruis Technology Development Co., Ltd.

[0039] Sodium nitrite (NaNO2) was purchased from Shanghai Maclean Biochemical Technology Co., Ltd.

[0040] FeTMPyP was purchased from APExBIO Technologies, Inc., USA.

[0041] The apoptosis detection kit was purchased from Beijing Senbo Biotechnology Co., Ltd.

[0042] The nitrite content test kit, estradiol test kit, follicle-stimulating hormone test kit, luteinizing hormone test kit, and anti-Müllerian hormone test kit were purchased from Wuhan Yilairuit Biotechnology Co., Ltd.

[0043] Sodium pentobarbital was purchased from Merck, Inc., USA.

[0044] WB reagent was purchased from Jiangsu Kangwei Century Biotechnology Co., Ltd., and antibodies were purchased from Wuhan Sanying Biotechnology Co., Ltd.

[0045] Other reagents are analytical grade reagents.

[0046] 2. Animal handling

[0047] The 3-week-old specific pathogen-free (SPF) grade BALB / c female mice used in this experiment were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. and housed in the barrier animal facility of the School of Public Health, Zhengzhou University (SYXK(YU)2018-0005). All animal experiments have been approved by the Laboratory Animal Ethics Committee of Zhengzhou University (ethics number: ZZUIRB2023-023).

[0048] 3. Statistical Analysis

[0049] Experimental data are expressed as mean ± standard deviation (SD). One-way ANOVA (Birmingham, UK) was used to analyze the significance of differences between groups, followed by the Student-Newman-Keuls test. P < 0.05 was considered statistically significant. SPSS 21.0 (Armonk, NY, USA, 2012) was used to analyze the experimental data. GraphPad Prism 7 (La Jolla, USA) was used for image processing.

[0050] Example 1: General toxicity assessment of FeTMPyP in mice

[0051] After one week of acclimatization, mice were randomly divided into two groups: a control group and a FeP group. Mice in the control group had free access to distilled water; mice in the FeP group received weekly intraperitoneal injections of 10 mg / kg bw FeTMPyP. The intervention period for the mice was 3 months.

[0052] The general toxicity of FeTMPyP in mice was assessed by changes in body weight and organ index. The results showed that, Figure 1 As shown, after treating mice with 10 mg / kg BW FeTMPyP for 3 months, there was no significant change in the mice's body weight. Further analysis of the mice's organ indices revealed no significant changes in the indices of the liver, kidneys, heart, spleen, and ovaries, indicating that the treatment with 10 mg / kg BW FeTMPyP did not have a toxic effect on the health of the mice.

[0053] Example 2: Study on the alleviating effect of FeTMPyP on MC-LR and NaNO2 combined exposure induced ovarian toxicity in mice

[0054] After one week of acclimatization, mice were randomly divided into four groups (see Table 1): a control group, an M2N2 group, a FeP group, and an M2N2+FeP group. Control group mice had free access to distilled water; M2N2 group mice were treated with a combination of MC-LR (100 μg / L) and NaNO2 (300 mg / L); FeP group mice were treated with weekly intraperitoneal injections of the nitrification stress inhibitor FeTMPyP (10 mg / kg bw); and M2N2+FeP group mice received 10 mg / kg bw FeTMPyP during the MC-LR and NaNO2 combination treatment. The treatment and intervention period for the mice was 3 months.

[0055] Table 1. FeTMPyP intervention regimens in MC-LR and NaNO2-exposed mice.

[0056]

[0057] After the initial exposure to the drug, 15 female mice from each group were paired with unexposed male mice at a 1:1 ratio to assess fertility. During sacrifice, the mice were anesthetized with 1% sodium pentobarbital, followed by arterial blood collection. After sacrifice, the mice were rapidly dissected, ovarian tissue was separated and weighed, flash-frozen in liquid nitrogen, and stored at -80°C. The collected whole blood was allowed to stand at room temperature for 2 hours, then centrifuged (3000 rpm, 4°C, 20 min), and the supernatant serum was collected and stored at -80°C.

[0058] 1. Detection of serum sex hormone changes in mice using ELISA method

[0059] The concentrations of FSH, LH, and AMH in mouse serum were determined using a double-antibody sandwich ELISA method. First, standard wells, blank wells, and sample wells were set up. 100 μL of serially diluted standards were added to the standard wells, and 100 μL of the serum sample to be tested was added to the remaining wells. The OD value of each well was measured at 450 nm using a microplate reader. The concentrations of FSH, LH, and AMH in mouse serum were calculated by fitting the OD values ​​of the samples to a standard curve and combining this with the known concentrations of the standards. Five mice were used in each group for the analysis.

[0060] E2 levels in mouse serum were detected using a competitive ELISA method. The experiment first set up standard wells, blank wells, and sample wells. 50 μL of serially diluted standards were added to the standard wells, and 50 μL of the serum sample to be tested was added to the remaining wells. The OD value of each well was measured at 450 nm using a microplate reader. The concentration of E2 in mouse serum was calculated by fitting the OD values ​​of the samples to a standard curve and combining this with the known concentration of the standards. Five mice were used in each group for the analysis.

[0061] Depend on Figure 2Analysis of serum sex hormone levels in mice showed that, compared with the control group, combined exposure to 100 μg / L MC-LR and 300 mg / L NaNO2 significantly reduced E2 (approximately 35.7%), LH (approximately 41.9%), and AMH (approximately 47.6%) levels, and increased FSH (approximately 188%) levels, with statistically significant differences (P<0.05). Furthermore, intervention with 10 mg / kg BWFeTMPyP significantly improved the decrease in E2 and AMH and the increase in FSH caused by combined exposure to the two toxins (P<0.05; * indicates P<0.05 compared with the control group; # indicates P<0.05 compared with the M2N2 group). Compared with the M2N2 group, the levels of E2, AMH, and FSH recovered by 46.9%, 96.2%, and 43%, respectively, indicating that FeTMPyP intervention can significantly alleviate the sex hormone imbalance in mice caused by combined exposure to MC-LR and NaNO2.

[0062] 2. Analysis of Estrogenic Cycle Changes in Mice Using Vaginal Smear Method

[0063] Using a pipette, 100 μL of physiological saline was drawn and gently inserted into the mouse vagina. The saline was slowly injected, and vaginal secretions were aspirated again using the pipette. The collected secretions were smeared onto an adhesive slide and allowed to air dry at room temperature. The slide was then stained with Wright-Gymsza stain for 2 minutes. The slide was gently rinsed with distilled water to remove excess stain. After the slide was completely dry, it was mounted with neutral resin, and cell morphology was observed under a microscope. The estrous cycle stage of the mouse was determined based on the different cell types observed in the slide. The mice were observed continuously for 14 days, and changes in the estrous cycle were recorded and analyzed. Three mice were used in each group for analysis.

[0064] like Figure 4 As shown, compared with the control group, combined exposure to 100 μg / L MC-LR and 300 mg / L NaNO2 significantly shortened the duration of estrus in mice (by approximately 50%), increased the duration of interestrus (by approximately 85.1%), and led to a prolonged estrous cycle (by approximately 85.1%) (P<0.05). FeTMPyP intervention restored 33.4%, 28.1%, and 34.5% of the shortened estrus, increased interestrus, and prolonged estrous cycle caused by the combined exposure to the two toxins, respectively (P<0.05). This indicates that FeTMPyP can effectively alleviate the estrous cycle disorder in mice caused by combined exposure to MC-LR and NaNO2.

[0065] 3. H&E staining to observe histological changes in mouse ovaries

[0066] Mouse ovaries were rapidly collected, washed with cold PBS, fixed with 4% paraformaldehyde for 24 h, equilibrated with 30% phosphate-buffered sucrose solution for 2 h, embedded in paraffin, and cut into 6 μm sections. The sections were dehydrated with xylene and 100% ethanol, stained with hematoxylin and eosin. Finally, images were observed and acquired under a microscope to analyze follicular development and pathological changes in the ovarian tissue. Ovaries from three mice in each group were used for analysis.

[0067] Figure 5 A shows the histopathological changes and follicular development of mouse ovaries observed by H&E staining (CL: corpus luteum, PF: primary follicle, SF: secondary follicle, GF: mature follicle, and AF: atretic follicle; blue arrows indicate oocyte necrosis, fragmentation, and lysis). Compared with the control group, in the MC-LR or NaNO2 alone and in combination, oocyte necrosis, fragmentation, and lysis were observed after combined exposure to 100 μg / L MC-LR and 300 mg / L NaNO2, accompanied by a significant increase in the number of atretic follicles (1.8-fold increase) (P<0.05). Intervention with 10 mg / kg BW FeTMPyP reduced the number of atretic follicles increased by 35.6% after combined exposure to the two toxins (P<0.05). Figure 5 B) suggests that FeTMPyP treatment may be a potential intervention to alleviate ovarian dysfunction caused by combined exposure to MC-LR and NaNO2.

[0068] 4. Effects of FeTMPyP intervention on MC-LR and NaNO2 combined exposure-induced apoptosis in mouse ovarian cells

[0069] (1) TUNEL test

[0070] Apoptosis was detected using the terminal deoxynucleotidyl transferase (dUTP) nick-end labeling assay (TUNEL) (Roche, Switzerland). In short, testes were fixed in 4% paraformaldehyde for 24 hours, permeated with 0.1% Triton X-100, and washed twice. Then, a mixture of TdT-labeled nucleotides was added to each slide and incubated at 37°C for 1 hour. Observation was performed using a fluorescence microscope (Olympus, Tokyo, Japan) at 488 nm excitation and 530 nm emission. Labeled green fluorescent nuclei were selected using Image-Pro Plus 6.0 (Media Cybernetics, Inc., Rockville, MD, USA) as a unified standard for identifying positive cells in all images. DAPI-labeled blue nuclei were selected as the total cells. The apoptosis rate (%) was calculated as the number of positive cells / total cells × 100.

[0071] (2) Western Blot detection of the expression level of the target protein

[0072] Total protein extraction and concentration determination: An appropriate amount of mouse ovarian tissue was transferred to a 2 mL grinding tube, and 1 mL of pre-chilled RIPA lysis buffer (containing 1% protease inhibitor and 1% phosphatase inhibitor) was added. The mixture was ground on ice for 30 min, with sonication performed every 10 min. After lysis, the sample was centrifuged for 15 min (12000 rf, 4℃), and the supernatant was collected and transferred to a new centrifuge tube. Protein concentration was determined using a BCA kit combined with a multi-mode microplate reader. Based on the measured protein concentration, all samples were adjusted to the same concentration using lysis buffer, and SDS-PAGE loading buffer was added. The samples were then boiled at 100℃ for 10 min to denature the protein.

[0073] Western Blot Experiment: Gels were prepared using a PAGE gel preparation kit according to standard procedures. 20 μg of protein was loaded onto each well. Electrophoresis was performed at a constant voltage of 60 V until bromophenol blue entered the separating gel. The voltage was then increased to 120 V until bromophenol blue reached the bottom of the gel. After electrophoresis, the protein on the gel was transferred to an ethanol-activated PVDF membrane using a wet transfer method at 60 V for 120 min. After transfer, the PVDF membrane was immersed in TBST containing 5% skim milk powder and blocked with shaking at room temperature for 60 min. After blocking, the primary antibody was diluted according to the antibody manufacturer's instructions and incubated overnight at 4°C. The next day, the membrane was washed 5 times with TBST buffer for 6 min each time to remove unbound primary antibody. Subsequently, HRP-labeled secondary antibody matching the primary antibody was added and incubated at room temperature for 1.5 h. The membrane was washed again with TBST 5 times for 6 min each time. Finally, ECL chemiluminescence reagent was used for color development to capture specific bands. The grayscale values ​​of the target protein and the internal control protein GAPDH were analyzed using ImageJ software to calculate the relative expression level of the target protein. Each experiment was performed in triplicate for statistical analysis.

[0074] Depend on Figure 3 It was found that combined exposure to MC-LR and NaNO2 significantly increased the Bax / Bcl-2 ratio, as well as the levels of Cleaved Caspase-3 and Cleaved PARP in mouse ovaries (P<0.05), while FeTMPyP intervention significantly downregulated the levels of these apoptosis markers (P<0.05). Furthermore, immunofluorescence staining results showed that combined exposure to the two toxins increased the proportion of apoptotic cells in the ovaries by 1.7-fold (P<0.05); compared with the M2N2 group, FeTMPyP intervention reduced the proportion of apoptotic cells by approximately 65% ​​(P<0.05). Figure 3(C and D). These results suggest that FeTMPyP intervention can alleviate MC-LR and NaNO2 combined exposure-induced apoptosis in mouse ovarian cells.

[0075] In summary, nitrification stress is the core mechanism of ovarian toxicity in mice induced by the combined exposure to MC-LR and NaNO2, and it has the potential to serve as a target for disease intervention. FeTMPyP intervention can effectively alleviate nitrification stress induced by the combined exposure to MC-LR and NaNO2 in mouse ovaries, and improve hormonal imbalances, estrous cycle disorders, and follicular atresia caused by this combined exposure, while restoring fertility in mice. FeTMPyP can serve as a therapeutic agent for ovarian toxicity induced by MCs and nitrite exposure, and can also be used to prepare drugs for the prevention and treatment of ovarian toxicity and related diseases caused by environmental toxin exposure in mice.

[0076] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. Application of FeTMPyP in the preparation of drugs to alleviate ovarian toxicity induced by combined exposure to MCs and NaNO2.

2. The application according to claim 1, characterized in that: The drug comprises FeTMPyP and a pharmaceutically acceptable carrier.

3. The application according to claim 2, characterized in that: The drug is administered via intraperitoneal injection.

4. The application according to claim 3, characterized in that: The diseases caused by ovarian toxicity include sex hormone disorders, estrous cycle disorders, follicular atresia, and adverse birth outcomes.

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