A pharmaceutical composition for preventing and / or treating reproductive damage to testicular spermatogonia caused by combined exposure to toxins, and its application thereto.

Abstract

This invention belongs to the field of pharmaceutical and reproductive toxicology, specifically relating to a pharmaceutical composition for the prevention and / or treatment of reproductive damage to testicular spermatogonia caused by combined exposure to toxins, and its application. This invention is the first to demonstrate that combined exposure to MC-LR and NaNO2 can induce ferroptosis in testicular spermatogonia, leading to testicular structural damage, sex hormone imbalances, and decreased sperm density. The pharmaceutical composition provided by this invention uses Fer-1 as the main active ingredient. Fer-1 alleviates iron overload and oxidative stress in testicular spermatogonia by inhibiting the ferroptosis process, scavenging reactive oxygen species, reducing lipid peroxidation, increasing the activity of GSH, GPX4, and SOD, reversing the abnormal expression of ferroptosis-related proteins, regulating serum reproductive hormone levels, improving testicular structure, and significantly improving sperm quality. This invention provides a novel targeted intervention strategy for male reproductive damage caused by combined environmental pollution, and has significant public health value and social significance.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical and reproductive toxicology technology, and specifically relates to a pharmaceutical composition for the prevention and / or treatment of combined exposure to toxins and its application. Background Technology

[0002] Male reproductive health is fundamental to human reproduction. In recent years, the global prevalence of male infertility has been on the rise, with declining semen quality being a significant contributing factor, and exposure to environmental pollutants being a key risk factor. Statistics show that approximately 17.5% of the global adult population is affected by infertility, with male factors accounting for about half. Furthermore, the number of male infertility cases worldwide increased by 74% between 1990 and 2021, indicating a continuously increasing disease burden. Therefore, exploring the mechanisms by which environmental pollutants cause damage to male reproductive health and developing effective interventions is of significant public health importance.

[0003] With industrialization, urbanization, and the increasing eutrophication of water bodies, microcystins (MCs) and nitrites have become typical compound pollutants in water. MCs, as secondary metabolites of cyanobacteria, are most prevalent and toxic, with microcystin-LR (MC-LR) being the most widely distributed. It can enter the human body through various routes, including drinking water, the food chain, and skin contact, exhibiting multi-organ toxicity. Its reproductive toxicity has been confirmed, damaging testicular tissue, inducing germ cell apoptosis, and interfering with sex hormone secretion, but the specific toxic mechanism is not fully understood. Nitrites are widely present in water and food, originating from both natural reduction and anthropogenic emissions. Long-term exposure can trigger oxidative stress and generate carcinogenic nitrosamines. Research on its toxicity to the male reproductive system is fragmented, and systematic studies on its toxic mechanisms and combined exposure effects are lacking. In natural environments, combined exposure to MCs and nitrites is extremely common. In eutrophic waters, cyanobacterial blooms produce MCs while hypoxic environments lead to increased nitrite concentrations, making combined exposure easy for humans through drinking water and other routes. Existing studies have confirmed that combined exposure to these two substances can synergistically exacerbate damage to the reproductive system in animals, but there are few related studies, and the specific molecular mechanisms by which they cause reproductive damage remain unclear.

[0004] Ferrostatin-1 (Fer-1) is a specific inhibitor of ferroptosis that blocks the ferroptosis process by inhibiting lipid peroxidation and protecting the antioxidant system. Patent CN114903997A discloses the use of the ferroptosis inhibitor Fer-1 in the preparation of drugs for the prevention and treatment of male reproductive dysfunction, specifically targeting reproductive damage caused by type 1 diabetes (STZ-induced), which falls under the category of complications of endogenous metabolic diseases. However, this invention only involves single Fer-1 administration and does not address the scenario of exposure to multiple environmental pollutants. Furthermore, diabetic metabolic damage and in situ damage to spermatogonia directly targeted by multiple aquatic pollutants are fundamentally different in terms of their causes, pathological models, molecular mechanisms, and application scenarios. Existing technology cannot predict the synergistic reproductive toxicity and intervention strategies resulting from combined exposure to two aquatic pollutants based solely on hyperglycemic metabolic damage.

[0005] Given that the mechanism of reproductive damage to testicular spermatogonia caused by combined exposure to MCs and nitrite is unclear and there is a lack of targeted drugs to improve it, it is of great significance to clarify the mechanism of reproductive damage to testicular spermatogonia caused by combined exposure to these two substances and to develop relevant drugs to improve it. Summary of the Invention

[0006] This invention proposes a pharmaceutical composition for preventing and / or treating reproductive damage to testicular spermatogonia caused by combined exposure to toxins and its application. It solves the problems of ferroptosis in spermatogonia, testicular structural damage, and spermatogenesis disorders induced by combined exposure to microcystin LR and nitrite. Based on the specific ferroptosis inhibitor Ferrostatin-1 (Fer-1), it provides a pharmaceutical composition for preventing or alleviating ferroptosis in testicular spermatogonia induced by combined exposure to MC-LR and NaNO2, achieving the goals of blocking ferroptosis, protecting the testes and spermatogenic cells, repairing spermatogenesis, and improving sperm quality. It provides a safe and effective intervention for male reproductive damage caused by environmental compound pollution.

[0007] The technical solution of this invention is implemented as follows:

[0008] This study, using C57BL / 6 female mice and the mouse spermatogonial cell line (GC-1) as models, is the first to demonstrate that combined exposure to MC-LR and NaNO2 resulted in abnormal changes in serum hormone levels, destruction of testicular structure, a significant decrease in sperm density, and a significant reduction in the number of spermatogenic cells in mouse testicular tissue. The study also revealed abnormal changes in ferrous ions (Fe2+) in mouse testicular tissue and GC-1 cells. 2+Increased levels of ferroptosis (FER) and reactive oxygen species (ROS) and malondialdehyde (MDA), and decreased levels of glutathione (GSH) and the activities of glutathione peroxidase 4 (GPX4) and superoxide dismutase (SOD); abnormal expression of ferroptosis-related proteins such as SLC7A11 was observed in the MC-LR and NaNO2 co-exposure group, indicating activation of the ferroptosis pathway. Fer-1 intervention reversed the MC-LR and NaNO2 co-exposure-induced FER in mouse testes and GC-1 cells. 2+ The changes in accumulation and oxidation levels alleviated the microstructural changes of spermatogonia in mouse testicular tissue and significantly alleviated the abnormal expression of ferroptosis-related genes and proteins. Fer-1 intervention can significantly alleviate ferroptosis in GC-1 cells and mouse testicular spermatogonia induced by combined exposure to MC-LR and NaNO2.

[0009] Based on this, the present invention provides a pharmaceutical composition for preventing and / or treating reproductive damage to testicular spermatogonia caused by combined exposure to toxins, wherein the active ingredient of the pharmaceutical composition comprises Fer-1 and a pharmaceutically acceptable carrier.

[0010] Preferably, the above-mentioned active ingredients further include reduced glutathione and L-cysteine; the pharmaceutical composition is an oral or injectable formulation. The pharmaceutical composition comprises a core active component and pharmaceutically acceptable excipients: the active component consists of Fer-1, reduced glutathione (GSH), and L-cysteine, and is available in two preferred formulations depending on the route of administration (intraperitoneal injection, oral administration) and application scenario (treatment, prevention), with all proportions expressed in parts by weight.

[0011] Preferably, the formulation of the above-mentioned injectable preparation contains 1 to 5 parts of Fer-1, 20 to 80 parts of reduced glutathione, 5 to 20 parts of L-cysteine, and is supplemented to 1000 parts with physiological saline or phosphate buffer for injection (this formulation is suitable for in vivo animal intervention and clinical injection preparation, targeting and blocking ferroptosis + supplementing endogenous antioxidants).

[0012] Preferably, the preparation steps of the above-mentioned injectable formulation are as follows:

[0013] (1) Under sterile conditions, take L-cysteine ​​and reduced glutathione according to the formula and add them to physiological saline or PBS buffer for injection. Stir until completely dissolved. Then add Fer-1 dissolved in organic solvent and continue to stir and mix.

[0014] (2) Filter and sterilize, dispense into sterile vials, seal, and store at 2-8℃ away from light to obtain the injectable preparation;

[0015] In step (1), the organic solvent is DMSO, and the final concentration of DMSO is <0.1%.

[0016] The specific steps are as follows:

[0017] (1) Environmental requirements: The operation shall be carried out in a sterile clean bench and all reagents and containers shall be sterilized by high temperature and high pressure.

[0018] (2) Take the injection saline or PBS buffer according to the ratio, stir at room temperature, add L-cysteine ​​and reduced glutathione in sequence, stir until completely dissolved, avoid high temperature, and prevent GSH oxidation and inactivation.

[0019] (3) Add Fer-1 (a small amount of DMSO can be used to aid dissolution first, the final concentration of DMSO is <0.1%, and it is non-cytotoxic), and continue stirring for 10-15 min until completely mixed;

[0020] (4) The mixture is sterilized by filtration through a 0.22 μm sterile filter membrane, dispensed into sterile vials, sealed, and stored at low temperature (2-8℃) away from light to obtain the composition for injection.

[0021] Preferably, the formulation of the above-mentioned oral preparation contains 1 to 4 parts of Fer-1, 30 to 90 parts of reduced glutathione, 6 to 25 parts of L-cysteine ​​and pharmaceutical excipients, totaling 100 to 300 parts; the pharmaceutical excipients are any one or more of starch, lactose, magnesium stearate and sodium carboxymethyl cellulose (this formulation is suitable for oral administration (tablets, suspensions, granules), for environmental exposure protection or chronic exposure intervention in the general population, with the addition of oral stabilizers and excipients).

[0022] Preferably, the preparation steps of the above oral formulation are as follows:

[0023] (1) Fer-1, reduced glutathione and L-cysteine ​​are mixed evenly according to the formula to obtain active powder;

[0024] (2) Mix starch, lactose and active powder, pass through an 80-mesh sieve 2-3 times; then add sodium carboxymethyl cellulose aqueous solution to make soft material, granulate through a 14-mesh sieve, and dry at 35-45℃;

[0025] (3) After drying, the dried granules are mixed with magnesium stearate, granulated and then compressed into oral tablets; or granules are prepared by packaging.

[0026] Preferably, in the above preparation steps, based on a total formula mass of 100-300 parts, starch comprises 40-120 parts, lactose comprises 40-120 parts, magnesium stearate comprises 0.5-5 parts, and the mass concentration of sodium carboxymethyl cellulose aqueous solution is 2%-8%.

[0027] The specific steps are as follows:

[0028] (1) Mixing active ingredients: Mix Fer-1, glutathione and L-cysteine ​​evenly according to the ratio;

[0029] (2) Mixing of excipients: Mix starch, lactose and active ingredients, and pass through an 80-mesh sieve 2 to 3 times to ensure uniformity of materials;

[0030] (3) Granulation and drying: Add sodium carboxymethyl cellulose aqueous solution to make soft material, granulate through a 14-mesh sieve, and dry at a low temperature of 40°C to prevent GSH oxidation;

[0031] (4) Granulation and tableting: Add magnesium stearate to the dried granules, mix well, granulate and then compress into tablets to make oral tablets; or directly package into granules.

[0032] Secondly, this invention claims protection for the use of the above-described pharmaceutical composition in the preparation of a medicament for preventing and / or treating reproductive damage to testicular spermatogonia caused by combined exposure to toxins.

[0033] Preferably, the combined exposure to the above-mentioned toxins is a combination of microcystin and nitrite exposure; reproductive damage includes testicular structural damage, sex hormone disorders, and decreased sperm quality; the pharmaceutical composition prevents and / or treats the above-mentioned reproductive damage by inhibiting the ferroptosis process of testicular spermatogonia.

[0034] Preferably, the above-mentioned drug is used to relieve iron overload (ferrous ions Fe) in testicular spermatogonia. 2+ It can alleviate oxidative stress, reverse abnormal expression of ferroptosis-related proteins, regulate serum reproductive hormone levels, and improve testicular structure and sperm quality.

[0035] Preferably, the above-mentioned methods for alleviating oxidative stress include reducing ROS and MDA accumulation and increasing the activity of GSH, GPX4, and SOD; reversing the abnormal expression of ferroptosis-related proteins includes downregulating the expression of ACSL4, HIF-1α, and HO-1 proteins and upregulating the expression of SLC7A11, GPX4, and FTH1 proteins; regulating serum reproductive hormone levels includes downregulating follicle-stimulating hormone levels and upregulating testosterone levels; and improving sperm quality includes increasing the number of spermatogonia and sperm density.

[0036] The present invention has the following beneficial effects:

[0037] 1. This invention provides a pharmaceutical composition for preventing and / or treating reproductive damage to testicular spermatogonia caused by combined exposure to toxins. The main active ingredients are Fer-1, reduced glutathione, and L-cysteine. This application is the first to demonstrate that combined exposure to microcystin-LR and NaNO2 can induce ferroptosis in testicular spermatogonia, leading to testicular structural damage, sex hormone disorders, and decreased sperm density. It is also the first to effectively block ferroptosis in spermatogonia and testicular tissue induced by the specific ferroptosis inhibitor Fer-1, clarifying that ferroptosis is a key and modifiable target for reproductive damage caused by this combined pollution, filling a gap in the field.

[0038] 2. This invention demonstrates for the first time that the active ingredient Fer-1 can significantly reduce ferrous ion overload, significantly alleviate oxidative stress and lipid peroxidation, reduce ROS and MDA accumulation, restore the function of antioxidant systems such as GSH, GPX4, and SOD, reverse the abnormal expression of ferroptosis-related proteins, significantly reduce the expression of ACSL4, HIF-1α, and HO-1 proteins, and significantly increase the expression of SLC7A11, GPX4, and FTH1 proteins. This indicates that exogenous administration of Fer-1 can significantly reduce the toxic effects of the above two pollutants on cells by targeting and inhibiting the ferroptosis process, achieving precise protection from the source of damage. The mechanism of action is clear and the effect is stable.

[0039] 3. This invention demonstrates for the first time that Fer-1 can significantly improve testicular structural damage and sperm quality decline induced by combined exposure to MC-LR and NaNO2, reverse the decrease in testosterone levels caused by combined exposure, significantly alleviate testicular microstructural damage, restore seminiferous tubule structure, restore spermatogenic epithelial layers, and significantly reduce cell shedding and necrosis. This indicates that combined exposure to MC-LR and NaNO2 can significantly damage the microstructure of mouse testicular tissue, and the ferroptosis inhibitor Fer-1 can effectively alleviate this damage.

[0040] 4. This invention utilizes the proven safety of the specific inhibitor Fer-1, with a clear target and well-defined intervention pathway. It can be directly converted into a drug for preventing and treating male reproductive damage caused by environmental pollutants, possessing significant public health value and clinical application potential. Based on the specific ferroptosis inhibitor Fer-1, this invention provides a pharmaceutical composition and its application for preventing or alleviating ferroptosis in testicular spermatogonia induced by combined exposure to MC-LR and NaNO2. This achieves the goals of blocking ferroptosis, protecting the testes and spermatogenic cells, repairing spermatogenesis, and improving sperm quality, providing a safe and effective intervention for male reproductive damage caused by combined environmental pollution. Attached Figure Description

[0041] 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.

[0042] Figure 1This study illustrates the effects of Fer-1 on various ferroptosis markers in GC-1 cells. Figure A shows the effect of different concentrations of Fer-1 on GC-1 cell viability; Figure B shows the rescue effect of Fer-1 intervention on GC-1 cell viability under combined MC-LR and NaNO2 exposure; Figure C shows that Fer-1 can reverse changes in MDA content induced by MC-LR and NaNO2 exposure; Figure D shows that Fer-1 can reverse changes in GSH content induced by MC-LR and NaNO2 exposure; Figure E shows that Fer-1 can reverse changes in GPX4 activity induced by MC-LR and NaNO2 exposure; Figure F shows that Fer-1 can reverse changes in SOD activity induced by MC-LR and NaNO2 exposure; and Figure G shows the detection of Fe in GC-1 cells using FerroOrange fluorescent probe staining. 2+ Accumulation amount, Figure H shows Fe 2+ The relative fluorescence intensity is shown in Figure IJ, which shows the ROS content in GC-1 cells as determined by flow cytometry. Figure K shows the level of ferroptosis-related proteins in GC-1 cells as detected by Western blot. Figure L shows the quantitative statistics of ferroptosis-related protein expression levels in GC-1 cells.

[0043] Figure 2 The effects of Fer-1 on various indicators of ferroptosis in mouse testicular tissue are shown in Figure A, which shows the rescue effect of Fer-1 intervention on ferrous ion overload in mouse testicular tissue induced by combined exposure to MC-LR and NaNO2. Figures BC show the ROS content in mouse testicular tissue measured by flow cytometry. Figure D shows the reversible effect of Fer-1 on changes in MDA content in mouse testicular tissue induced by MC-LR and NaNO2 exposure. Figure E shows the reversible effect of Fer-1 on changes in GSH content in mouse testicular tissue induced by MC-LR and NaNO2 exposure. Figure F shows the reversible effect of Fer-1 on changes in GPX4 activity in mouse testicular tissue induced by combined exposure to MC-LR and NaNO2. Figure G shows the reversible effect of Fer-1 on changes in SOD activity in mouse testicular tissue induced by MC-LR and NaNO2 exposure. Figure H shows the levels of ferroptosis-related proteins in testicular tissue detected by Western blot. Figure I shows the quantitative statistical analysis of the expression levels of ferroptosis-related proteins in testicular tissue.

[0044] Figure 3 Fer-1 improved testicular structure and sperm quality. Figures A and C show how Fer-1 alleviated the changes in serum hormone levels in mice induced by combined exposure to MC-LR and NaNO2. Figure D shows fluorescently labeled spermatogenic cells, with red fluorescence corresponding to spermatogenic cells and blue fluorescence labeling cell nuclei. The magnification was 200× and the scale bar length was 100 μm. Figure E shows the number of spermatogenic cells. Figure F shows the results of mouse sperm density measurement. Figure G shows that Fer-1 can alleviate the abnormal changes in testicular tissue microstructure in mice induced by combined exposure to MC-LR and NaNO2. Detailed Implementation

[0045] 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.

[0046] 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.

[0047] This study addresses the activation of the ferroptosis pathway and induction of ferroptosis in spermatogonia and GC-1 cells in mouse testes by combined exposure to MC-LR and NaNO2. It provides an intervention composition and its application to prevent or alleviate ferroptosis induced by MC-LR and NaNO2 combined exposure in testicular spermatogonia, thereby blocking ferroptosis, protecting the testes and spermatogenic cells, repairing spermatogenesis, and improving sperm quality. This provides a safe and effective intervention for male reproductive damage caused by environmental pollution. Specifically, this invention uses Fer-1 to specifically inhibit ferroptosis, reduce iron overload, oxidative stress, and lipid peroxidation, restore the antioxidant system, reverse the ferroptosis phenotype, protect spermatogenic cells, and improve sperm quality, thus achieving protection of the male reproductive system.

[0048] Materials and Methods:

[0049] 1. Experimental reagents

[0050] Microcystin-LR (MC-LR, purity >95%) was purchased from Beijing Apruis Technology Development Co., Ltd.

[0051] Sodium nitrite (NaNO2) was purchased from Shanghai Maclean Biochemical Technology Co., Ltd.

[0052] Fer-1 was purchased from MedChemexpress (MCE) Biotechnology Company in the United States;

[0053] The tissue iron assay kit was purchased from Nanjing Jiancheng Bioengineering Institute;

[0054] FerroOrange probes were purchased from MedChemexpress (MCE) Biotechnology Company in the United States;

[0055] The SOD, MDA, GPX4, and GSH detection kits were purchased from Nanjing Jiancheng Bioengineering Institute.

[0056] The ROS detection kit was purchased from Shanghai Beyotime Biotechnology Co., Ltd.

[0057] The CCK-8 kit was purchased from Shanghai Huaxiang Biotechnology Co., Ltd.;

[0058] The ROS detection kit was purchased from Shanghai Beyotime Biotechnology Co., Ltd.;

[0059] Antibodies such as Anti-HO-1, Anti-HIF-1α, Anti-FTH1, Anti-SLC7A11, Anti-GPX4, Anti-ACSL4, and Anti-β-actin were purchased from Wuhan Sanying Biotechnology Co., Ltd.;

[0060] The WB reagents were purchased from Wuhan Sevier Biotechnology Co., Ltd.;

[0061] Other reagents were analytical grade reagents.

[0062] 2. Experimental animals

[0063] The 6-week-old specific pathogen-free (SPF) grade male BALB / c mice used in this invention were ordered from Beijing Vital River Laboratory Animal Technology Co., Ltd. (SCXK (Beijing) 2021-0006) and were housed in the barrier animal house of the School of Public Health, Zhengzhou University (SYXK (Henan) 2023-0013). All animal experiments have obtained the approval of the Experimental Animal Ethics Committee of Zhengzhou University (Ethical number: ZZUIRB2023-023).

[0064] 3. Experimental cells

[0065] The mouse spermatogonial cells GC-1 used in this invention were provided by Beijing Beina Biotechnology Co., Ltd.

[0066] Cell resuscitation and culture: Take out the frozen GC-1 cell cryopreservation tube from the liquid nitrogen tank, quickly place it in a 37°C constant temperature water bath and gently shake to thaw. After the cryopreservation solution is completely melted, transfer it to a centrifuge tube and centrifuge at a speed of 1000 r / min for 5 min. Discard the supernatant, resuspend it with DMEM high-glucose medium containing 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin double antibody, and then transfer it to a 25 cm² cell culture flask containing 4 mL of medium. Gently pipette to mix evenly and place it in a constant temperature incubator at 37°C, 5% CO2, and saturated humidity for culture. Observe the growth status of the cells daily. The CCK8 experiment was used to determine the activity of GC-1 cells.

[0067] Cell passage: When the cell density reaches 80%-90%, first discard the original culture medium in the culture flask, then gently wash the cell surface twice with 2 mL of autoclaved sterile PBS solution, aspirate any residual liquid, and add 1 mL of 0.25% EDTA-containing trypsin digestion solution preheated to 37°C to the culture flask. Observe the cell morphology under a microscope. When some cells have detached, gently tap the culture flask. When most cells have detached, add 1 mL of culture medium to stop the digestion. Gently pipette the culture flask wall to completely detach the cells, transfer the cell suspension to a centrifuge tube, centrifuge at 1000 r / min for 5 min, discard the supernatant, resuspend in culture medium, divide equally into two culture flasks, add 4 mL of culture medium to each flask and mix by pipetting, then place in an incubator for continued culture.

[0068] Cell cryopreservation: GC-1 cells in logarithmic growth phase and in good growth condition were digested and centrifuged according to the passage procedure, and the cell pellet was collected. The cells were resuspended in 1 mL of pre-cooled cell cryopreservation solution, transferred to cryovials, and placed in a cell cryopreservation box for programmed cooling at -80°C. Subsequently, the cells were transferred to liquid nitrogen for long-term storage.

[0069] 4. Mouse exposure and intervention: Mice were exposed to MC-LR (100 μg / L) and NaNO2 (300 mg / L) via free drinking water for 6 months; Fer-1 was administered intraperitoneally at a dose of 1 mg / kg, three times a week. GC-1 cell exposure and intervention: GC-1 cells were exposed to MC-LR (12 μM) and NaNO2 (8 mM NaNO2) either alone or in combination for 24 h; during intervention, cells were incubated with Fer-1 in culture medium along with MC-LR or NaNO2 for 24 h.

[0070] 5. Testing Indicators and Methods

[0071] Hematoxylin-eosin (H&E) staining was used to observe the structure of mouse testes; serum sex hormone levels in mice were detected by ELISA; the number of spermatogenic cells in mouse testicular tissue was assessed by immunofluorescence; and mouse sperm density was detected using a dedicated sperm analysis system. The contents of MDA, GSH, GPX4, and SOD in mouse testicular tissue and GC-1 cells were detected using corresponding kits; ROS content was measured by flow cytometry; and Fe in mouse testicular tissue was detected using a reference kit. 2+ Accumulation levels were measured using FerroOrange fluorescent probe staining in GC-1 cells. 2+ Accumulation levels. Western blot analysis was used to detect the expression levels of ferroptosis-related proteins.

[0072] 6. Statistical Analysis

[0073] 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 27.0 (Armonk, NY, USA, 2012) was used to analyze the experimental data. GraphPad Prism7 (LaJolla, USA) was used for image processing.

[0074] Example 1: Combined exposure to MC-LR and NaNO2 induces ferroptosis in GC-1 cells

[0075] After 24 h of combined exposure to MC-LR and NaNO2, GC-1 cells showed a significant decrease in cell viability compared to the blank control group in all groups treated with MC-LR, NaNO2 alone, and in combination. P <0.05). However, when cells were treated with 0.1 μM Fer-1 in combination with the above-mentioned toxins for 24 h, it was able to effectively reverse the cell viability decline induced by the combined exposure of MC-LR and NaNO2, and the treatment group was significantly different from the toxin-exposed group alone. P <0.05)( Figure 1 AB). After GC-1 cells were exposed to MC-LR + NaNO2 for 24 h, Fe 2+ ROS and MDA were significantly elevated, while GSH, GPX4, and SOD were significantly decreased; after Fer-1 intervention, iron overload, oxidative stress, and lipid peroxidation returned to control levels, and the antioxidant system recovered. Figure 1 CJ). After 24 h of combined exposure to MC-LR and NaNO2, the expression of ferroptosis-related proteins in GC-1 cells changed abnormally. Compared with the control group, the expression of ACSL4, HIF-1α, and HO-1 proteins was significantly increased. P <0.01), while the expression of ferroptosis inhibitors SLC7A11, GPX4, and iron storage protein FTH1 were significantly downregulated, suggesting that combined exposure can significantly induce ferroptosis in GC-1 cells; while Fer-1 intervention can effectively reverse the above protein changes, and the expression of ACSL4, HIF-1α, and HO-1 proteins in the MHNH+Fer-1 group was significantly reduced, basically returning to the control group level ( P <0.05), while the expression of SLC7A11, GPX4, and FTH1 significantly rebounded and returned to the control group level ( Figure 1 (KL), indicating that Fer-1 can effectively alleviate ferroptosis induced by combined exposure.

[0076] The above data fully demonstrate that the ferroptosis pathway is a key molecular mechanism mediating GC-1 cell damage caused by combined exposure to MC-LR and NaNO2; at the same time, exogenous administration of Fer-1 can significantly reduce the toxic effects of the two pollutants on cells by targeting and inhibiting the ferroptosis process.

[0077] Example 2: Combined exposure to MC-LR and NaNO2 induces ferroptosis in mouse testicular spermatogonia.

[0078] After mice were exposed to a combination of MC-LR and NaNO2 for 6 months, the Fe in their testicular tissue was... 2+ The levels of ROS and MDA were significantly increased, while the activities of GSH, GPX4, and SOD were significantly decreased. After Fer-1 intervention, iron overload, oxidative stress, and lipid peroxidation were significantly alleviated and returned to the control group levels. The iron death indices of testicular spermatogonia were completely reversed, and redox homeostasis was restored. Figure 2 AG). After 6 months of combined exposure to MC-LR and NaNO2, mice showed abnormal expression of ferroptosis-related proteins in testicular tissue, with significantly increased expression of ACSL4, HIF-1α, and HO-1 proteins. P <0.05), while the expression of ferroptosis inhibitors SLC7A11, GPX4, and iron storage protein FTH1 were all significantly downregulated ( P <0.01 indicates that combined exposure can significantly induce ferroptosis in mouse testicular tissue; Fer-1 intervention can effectively reverse the above changes, and the expression of ACSL4, HIF-1α and HO-1 proteins in the MHNH+Fer-1 group was significantly reduced, basically returning to the control group level ( P <0.01), while the expression of SLC7A11, GPX4, and FTH1 significantly rebounded and returned to the control group level ( P <0.05, indicating that Fer-1 can effectively alleviate testicular ferroptosis induced by combined exposure ( Figure 2 HI).

[0079] Example 3: Fer-1 improves testicular structure and sperm quality

[0080] Co-exposure to MC-LR and NaNO2 significantly disrupted the balance of serum reproductive hormones in mice, manifested as elevated FSH and decreased LH and T levels. After Fer-1 intervention, compared with the control group, FSH levels in the treated group significantly decreased, essentially returning to normal reference values, while T levels significantly increased, essentially returning to control levels. This significantly reversed the decrease in testosterone levels caused by the combined exposure, indicating that the ferroptosis inhibitor Fer-1 has a significant alleviating effect on the changes in serum reproductive hormone levels in mice induced by MC-LR and NaNO2 exposure. Figure 3AC). Combined exposure to MC-LR and NaNO2 significantly reduced the number of spermatogenic cells, with numerous empty areas visible in the seminiferous tubules; after Fer-1 intervention, the number of spermatogenic cells recovered significantly, and fluorescence intensity increased, especially in the combined exposure + Fer-1 group, where the arrangement and number of spermatogenic cells significantly improved, reaching levels comparable to the control group. Figure 3 DE). Combined exposure to both MC-LR and NaNO2 significantly decreased sperm density; however, with the addition of Fer-1 intervention, the sperm density in the combined exposure + Fer-1 group significantly increased, essentially returning to the control group level. P <0.01), significantly reversing the decrease in sperm density caused by combined exposure ( Figure 3 F). Combined exposure to MC-LR and NaNO2 resulted in severe damage to the testicular microstructure, significant dilation of seminiferous tubules, marked thinning of the seminiferous epithelium, extensive detachment of spermatogenic cells into the lumen, and necrosis of spermatogenic cells in some areas. Numerous detached cell fragments were observed within the lumen, indicating severe impairment of spermatogenic function. After Fer-1 intervention, the testicular microstructural damage was significantly alleviated, the seminiferous tubule structure became more intact, the seminiferous epithelial layers recovered, and cell detachment and necrosis were significantly reduced. This indicates that combined exposure to MC-LR and NaNO2 can significantly disrupt the testicular tissue microstructure in mice, while the ferroptosis inhibitor Fer-1 can effectively alleviate this damage. Figure 3 G).

[0081] In summary, combined exposure to MC-LR and NaNO2 induces ferroptosis in mouse testicular spermatogonia, leading to testicular structural damage, sex hormone imbalance, decreased sperm quality, and ultimately, impaired spermatogenesis. This study demonstrates that the ferroptosis inhibitor Fer-1 can significantly alleviate MC-LR and NaNO2-induced ferroptosis at the cellular and testicular tissue levels, significantly reduce testicular damage induced by combined MC-LR and NaNO2 exposure, and protect testicular structure and function. Therefore, this invention, based on the specific ferroptosis inhibitor Fer-1, provides an intervention composition and its application for preventing or alleviating MC-LR and NaNO2-induced ferroptosis in testicular spermatogonia. This achieves the goals of blocking ferroptosis, protecting the testes and spermatogenic cells, repairing spermatogenesis, and improving sperm quality, providing a safe and effective intervention for male reproductive damage caused by environmental pollution.

[0082] 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. A pharmaceutical composition for preventing and / or treating reproductive damage to testicular spermatogonia caused by combined exposure to toxins, characterized in that: The active ingredient of the pharmaceutical composition comprises Fer-1, and a pharmaceutically acceptable carrier.

2. The pharmaceutical composition according to claim 1, characterized in that: The active ingredients also include reduced glutathione and L-cysteine; the pharmaceutical composition is an oral or injectable formulation.

3. The pharmaceutical composition according to claim 2, characterized in that: The formulation of the injectable preparation, by weight, contains 1 to 5 parts of Fer-1, 20 to 80 parts of reduced glutathione, 5 to 20 parts of L-cysteine, and is supplemented to 1000 parts by physiological saline or phosphate buffer for injection.

4. The pharmaceutical composition according to claim 3, characterized in that, The preparation steps of the injectable formulation are as follows: (1) Under sterile conditions, take L-cysteine ​​and reduced glutathione according to the formula and add them to physiological saline or PBS buffer for injection. Stir until completely dissolved. Then add Fer-1 dissolved in organic solvent and continue to stir and mix. (2) Filter and sterilize, dispense into sterile vials, seal, and store at 2-8℃ away from light to obtain the injectable preparation.

5. The pharmaceutical composition according to claim 4, characterized in that: In step (1), the organic solvent is DMSO, and the final concentration of DMSO is <0.1%.

6. The pharmaceutical composition according to claim 2, characterized in that: The oral formulation comprises, by weight, 1 to 4 parts of Fer-1, 30 to 90 parts of reduced glutathione, 6 to 25 parts of L-cysteine, and pharmaceutical excipients, totaling 100 to 300 parts; the pharmaceutical excipients are any one or more of starch, lactose, magnesium stearate, and sodium carboxymethyl cellulose.

7. The pharmaceutical composition according to claim 6, characterized in that, The preparation steps of the oral formulation are as follows: (1) Fer-1, reduced glutathione and L-cysteine ​​are mixed evenly according to the formula to obtain active powder; (2) Mix starch, lactose and active powder, pass through an 80-mesh sieve 2-3 times; then add sodium carboxymethyl cellulose aqueous solution to make soft material, granulate through a 14-mesh sieve, and dry at 35-45℃; (3) After drying, the dried granules are mixed with magnesium stearate, granulated and then compressed into oral tablets; or granules are prepared by packaging.

8. The pharmaceutical composition according to claim 7, characterized in that: In the preparation steps, based on a total formula mass of 100-300 parts, starch comprises 40-120 parts, lactose comprises 40-120 parts, magnesium stearate comprises 0.5-5 parts, and the mass concentration of sodium carboxymethyl cellulose aqueous solution is 2%-8%.

9. Use of the pharmaceutical composition according to any one of claims 1-8 in the preparation of a medicament for the prevention and / or treatment of reproductive damage to testicular spermatogonia caused by combined exposure to toxins.

10. The application according to claim 9, characterized in that: The combined exposure to toxins is the combined exposure to microcystin and nitrite; reproductive damage includes testicular structural damage, sex hormone disorders and decreased sperm quality, and the pharmaceutical composition prevents and / or treats reproductive damage by inhibiting the ferroptosis process of testicular spermatogonia.

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