Application of Ferrostatin-1 in relieving zearalenone-induced ovarian toxicity in mammals

Abstract

The application discloses an application of a ferrostatin-1 in relieving zearalenone-induced ovary toxicity of mammals, and belongs to the technical field of animal breeding. The application reduces the damage of zearalenone to ovary granulosa cells from multiple angles of ferroptosis and cell apoptosis regulation, improves the reproductive health level of mammals, and provides a new intervention strategy for preventing and controlling mycotoxin-induced reproductive disorders of mammals.

Description

Technical Field

[0001] This invention relates to the application of Ferrostatin-1, a ferroptosis inhibitor, in alleviating zearalenone-induced ovarian toxicity in mammals, and belongs to the field of animal husbandry technology. Background Technology

[0002] Zearalenone (ZEA) is a fungal toxin produced by Fusarium fungi. It is widely found in moldy corn, wheat, and feed ingredients, and is one of the most common and harmful mycotoxins in livestock production. ZEA has strong estrogen-like activity and is highly damaging to the reproductive systems of both female and male animals, especially pigs. When sows ingest ZEA-contaminated feed, they often exhibit abnormal follicle development, ultimately leading to follicular atresia and reduced reproductive performance, causing serious economic losses to the pig industry. Therefore, effectively mitigating or preventing ZEA-induced ovarian toxicity in sows has always been an important research direction in the field of animal nutrition and reproductive toxicology.

[0003] Recent studies have shown that ZEA can cause ovarian cell damage through multiple pathways, including inducing oxidative stress, mitochondrial dysfunction, and lipid peroxidation. Apoptosis and ferroptosis are the main forms of cell damage.

[0004] To address the reproductive toxicity caused by Z-anethole (ZEA), some technical solutions have proposed interventions using natural active substances or functional compounds. For example, Chinese patent CN202411637223.9 reports a method for alleviating ZEA-induced damage to the animal reproductive system using glycyrrhetinic acid; Chinese patent CN202411837304.3 also discloses a technical solution for reducing ZEA toxicity using trans-anisole. These technical solutions can alleviate ZEA-induced tissue damage to some extent, but they mainly focus on regulating inflammatory responses and other pathways, and they also exhibit some cytotoxicity. Currently, research on the regulatory mechanisms of ZEA-induced ferroptosis remains relatively limited.

[0005] Ferrostatin-1 (Fer-1), a classic small-molecule lipid peroxidation inhibitor, effectively blocks ferroptosis by inhibiting lipid peroxidation and reducing the accumulation of phospholipid peroxides in cell membranes. Fer-1 has been shown to have significant protective effects in various oxidative stress-related disease models. However, its application in alleviating ZEA-induced ovarian toxicity in female animals requires further investigation. Taurine and melatonin are two of the most studied natural bioactive molecules in antioxidant and cell protection research, exhibiting significant anti-apoptotic effects in the nervous, reproductive, cardiovascular, and tumor treatment systems. Both not only inhibit ROS accumulation but also regulate mitochondrial homeostasis, endoplasmic reticulum stress, autophagy, and inflammatory signaling, thereby exerting cytoprotective effects. Therefore, exploring the application of ferroptosis inhibitors Fer-1, taurine, and melatonin in alleviating ZEA-induced ovarian toxicity in female animals from the perspective of inhibiting apoptosis and regulating ferroptosis is of great significance for revealing the molecular mechanisms of ZEA-induced ovarian damage and developing new prevention and control strategies. Summary of the Invention

[0006] Objectives of this invention: The first objective is to provide the application of the ferroptosis inhibitor Ferrostatin-1 in alleviating zearalenone-induced ovarian toxicity in mammals; the second objective is to provide the application of the ferroptosis inhibitor Ferrostatin-1 in the preparation of feed products that alleviate zearalenone-induced ovarian toxicity in mammals. This invention aims to reduce damage to ovarian granulosa cells from multiple angles, including regulating ferroptosis and apoptosis, thereby improving reproductive health in mammals.

[0007] Technical solution: The present invention provides the application of the ferroptosis inhibitor Ferrostatin-1 in alleviating zearalenone-induced ovarian toxicity in mammals.

[0008] The application of the ferroptosis inhibitor Ferrostatin-1 described in this invention in the preparation of feed products that alleviate zearalenone-induced ovarian toxicity in mammals.

[0009] Furthermore, the feed products for mitigating zearalenone-induced ovarian toxicity in mammals include pharmaceutical preparations or feed additives for mitigating zearalenone-induced ovarian toxicity in mammals.

[0010] Furthermore, the pharmaceutical formulation comprises the ferroptosis inhibitor Ferrostatin-1, an auxiliary active ingredient with antioxidant activity, and a pharmaceutically acceptable carrier; the feed additive comprises the ferroptosis inhibitor Ferrostatin-1, an auxiliary active ingredient with antioxidant activity, and a feed additive carrier.

[0011] Furthermore, the auxiliary active ingredient is selected from one or more of melatonin, taurine and their derivatives.

[0012] Furthermore, the weight ratio of the ferroptosis inhibitor Ferrostatin-1 to the co-active ingredient is 1:10-150.

[0013] Preferably, when the auxiliary active ingredient is melatonin, the mass ratio of Ferrostatin-1 to melatonin is 1:12.5.

[0014] Preferably, when the auxiliary active ingredient is taurine, the mass ratio of Ferrostatin-1 to taurine is 1:125.

[0015] Furthermore, the pharmaceutically acceptable carrier is selected from one or more of the following: microcrystalline cellulose, lactose, starch, pregelatinized starch, dicalcium phosphate, mannitol, povidone, hydroxypropyl methylcellulose, croscarmellose sodium, microcrystalline silica, magnesium stearate, talc, ethanol, propylene glycol, polyethylene glycol, Tween 80, water for injection, and vegetable oil.

[0016] Furthermore, the feed additive carrier is selected from one or more of the following: zeolite powder, bentonite, montmorillonite, silica, corn cob powder, rice husk powder, maifanite, calcium carbonate, dicalcium phosphate, maltodextrin, starch, wheat bran, and rice bran.

[0017] Furthermore, the pharmaceutical preparation is a tablet, capsule, granule or powder, injection, suspension or emulsion.

[0018] Furthermore, the feed additive is a premix, concentrated feed, or complete feed.

[0019] Based on ZEA-induced oxidative stress, iron metabolism disorder, and mitochondrial damage, this study reveals the molecular mechanism of ZEA-induced ovarian damage from the perspectives of ferroptosis and apoptosis.

[0020] Ferrostatin-1 can significantly reduce lipid peroxidation levels and restore antioxidant system function; Ferrostatin-1 combined with melatonin can significantly reduce apoptosis in follicular granulosa cells; Ferrostatin-1 combined with melatonin can significantly restore the level of estrogen synthase CYP19A1 in follicular granulosa cells.

[0021] Ferrostatin-1 combined with melatonin: Building upon the effectiveness of Ferrostatin-1 alone, the therapeutic effect was further enhanced by combining it with melatonin. Melatonin, as a highly effective free radical scavenger and mitochondrial protectant, may synergize with Ferrostatin-1 in the antioxidant defense mechanism—Fer-1 primarily inhibits downstream processes of lipid peroxidation, while melatonin reduces oxidative stress upstream by scavenging reactive oxygen species (ROS), stabilizing mitochondrial membrane potential, and upregulating the expression of endogenous antioxidant enzymes (such as SOD and GSH-Px). The combination of the two can complement each other at different nodes of ferroptosis regulation, thereby more comprehensively alleviating ZEA toxicity and improving the repair efficiency of ovarian tissue. Ferrostatin-1 combined with taurine: This regimen also showed some detoxification effect on ZEA, but its efficacy was not as good as the Ferrostatin-1 combined with melatonin regimen. Although taurine has certain antioxidant and osmotic regulation functions, its target and efficacy in inhibiting the core pathway of ferroptosis and comprehensively repairing ovarian damage may be relatively limited, and its synergistic effect with Ferrostatin-1 is weak.

[0022] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages:

[0023] (1) This invention is the first to apply the ferroptosis inhibitor Ferrostatin-1 to alleviate the toxic damage to mammalian ovaries caused by ZEA. Starting from inhibiting ferroptosis, a key form of cell damage, Ferrostatin-1 effectively inhibits lipid peroxidation and reduces the accumulation of phospholipid peroxides in the cell membrane, thereby blocking the process of ferroptosis. This provides a new target and intervention method for alleviating ZEA-induced ovarian granulosa cell damage and follicular atresia.

[0024] (2) This invention is the first to propose the application of the ferroptosis inhibitor Ferrostatin-1 in combination with taurine or melatonin to alleviate ZEA-induced ovarian toxicity. By combining the ferroptosis inhibition mechanism of Ferrostatin-1 with the anti-apoptotic and antioxidant mechanisms of melatonin, the dual pathway of ferroptosis-apoptosis in ZEA-induced ovarian damage is synergistically regulated, overcoming the limitations of existing single intervention strategies and the cytotoxicity of some compounds, and significantly improving the protective effect on ovarian function.

[0025] (3) This invention provides a new intervention strategy for preventing and controlling reproductive disorders in mammals caused by mycotoxins. By clarifying the protective role of Ferrostatin-1 and its combined use with taurine or melatonin in alleviating abnormal follicle development and reduced reproductive performance in mammals caused by ZEA, this invention not only deepens the understanding of the molecular mechanism of ZEA ovarian toxicity (oxidative stress-mitochondrial damage-ferroptosis / apoptosis cascade), but also provides experimental evidence and candidate schemes for developing safe and efficient mycotoxin antidotes, which has important application value for reducing economic losses caused by mycotoxin pollution in the pig industry. Attached Figure Description

[0026] Figure 1 ZEA interferes with ovarian development and estrogen synthesis in pigs;

[0027] Figure 2 ZEA-induced Fe in pig ovaries 2+ Increased malondialdehyde (MDA) content;

[0028] Figure 3 ZEA causes Fe in pig ovaries 2+ Imbalance between homeostasis and antioxidant systems;

[0029] Figure 4 ZEA induces Fe in porcine ovarian granulosa cells 2+ Imbalance between homeostasis and antioxidant systems;

[0030] Figure 5 Ferrostatin-1 can alleviate ZEA-induced dysfunction of porcine ovarian granulosa cells;

[0031] Figure 6 The combined use of Ferrostatin-1 and melatonin alleviated ovarian damage in mice;

[0032] Figure 7 Ferrostatin-1 and melatonin, when used in combination, inhibited apoptosis in mouse ovarian cells and restored ovarian hormone synthesis function. Detailed Implementation

[0033] The technical solution of the present invention will be further described below with reference to the accompanying drawings.

[0034] Unless otherwise specified, the conditions in the examples were performed under standard conditions or as recommended by the manufacturer. All reagents and instruments used, unless otherwise stated, are commercially available products.

[0035] Zearalenone (ZEA) was purchased from Aladdin, catalog number Z347362; penicillin (500 U / mL) was purchased from Aladdin, catalog number P105489; streptomycin (0.5... (mg / mL) was purchased from Aladdin, catalog number S432672; Ferrostatin-1, a ferroptosis inhibitor, was purchased from MedChemExpress (MCE), catalog number HY-100579; Melatonin was purchased from MCE, catalog number HY-B0075; Taurine was purchased from Maclean's, catalog number T818825; Fluorescence assay kit was purchased from Elabscience, catalog number E-BC-F101; Malondialdehyde (MDA) assay kit was purchased from Nanjing Jiancheng Biotechnology Institute, catalog number A003-1-2; Reduced glutathione (GSH) assay kit was purchased from Nanjing Jiancheng Biotechnology Institute, catalog number A006-2-1; Total glutathione (T-GSH) / oxidized glutathione (GSSG) assay kit was purchased from Nanjing Jiancheng Biotechnology Institute, catalog number A061-1-2; RNA extraction reagent RNAiso Plus was purchased from Takara (Baori Biotechnology (Beijing) Co., Ltd.), catalog number 9109; the RNA reverse transcription kit HiScript II Q RT SuperMix was purchased from Novizan, catalog number R223; the quantitative PCR kit (ChamQSYBR qPCR Master Mix) was purchased from Novizan, catalog number Q311; the ECL chemiluminescence solution was purchased from Novizan, catalog number E432; protein extraction reagents RIPA, PMSF, and QuickBlock™ were also purchased. Western blot primary antibody diluents were purchased from Beyotime Biotechnology Co., Ltd. (catalog numbers P0013B, ST506, and P0256); BCA kits were purchased from Kangwei Century Biotechnology Co., Ltd. (catalog number CW0014S); DEPC-treated water and hematoxylin-eosin (HE) staining kits were purchased from Sangon Biotech (Shanghai) Co., Ltd. (catalog numbers B501005 and E607318); Nrf2 and GPX4 antibodies were purchased from Wuhan Sanying Biotechnology Co., Ltd. (catalog numbers 16396 and 67763, respectively); GAPDH and α-tubulin antibodies were purchased from Beijing Solarbio Science & Technology Co., Ltd. The product numbers are K200057M and K200064M, respectively; GAR-HRP and GAM-HRP were purchased from Beyotime Biotechnology Co., Ltd., product numbers A0208 and A0216, respectively; 4% tissue cell fixative, neutral resin, PBS buffer, TBST buffer, erythrocyte lysis buffer, and 5× protein loading buffer were purchased from Beijing Solarbio Science & Technology Co., Ltd., product numbers P1110, G8590, P1020, T1085, R1013, and P1040, respectively; DMEM / F12 medium was purchased from Sigma-Aldrich, product number D2906; fetal bovine serum was purchased from Biovistech Pty Ltd.The product number is SE100-011.

[0036] The paraffin microtome was purchased from Leica, model RM2235; the inverted microscope was purchased from Leica, model [model missing]; the micro spectrophotometer was purchased from Beijing Kai'ao Technology Development Co., Ltd., model K5600; the real-time fluorescence quantitative PCR instrument was purchased from Applied Biosystems, model StepOne Plus; the microplate reader was purchased from Tecan (Shanghai) Trading Co., Ltd., model Infinite®200 PRO; and the luminescence image analysis system was purchased from Shanghai Tianneng, model Tanon 5200.

[0037] Three-month-old Bama sows were purchased from the Institute of Zoology, Chinese Academy of Sciences, and housed in the general facilities of the Experimental Animal Center of Yangzhou University. The experimental procedures complied with the requirements of the Experimental Animal Ethics Review Committee of Yangzhou University (Batch No.: 202103074).

[0038] Eight-week-old healthy female mice were purchased from the Experimental Animal Center of Yangzhou University and housed in a standard environment. The experimental protocol was approved by the Experimental Animal Ethics Review Committee of Yangzhou University (approval number: 202103168).

[0039] Example 1: Animal Experiment with Sows

[0040] 1. Laboratory animals and grouping:

[0041] Twelve healthy 3-month-old sows were randomly divided into two groups of six each:

[0042] Blank control group: fed a basal diet and treated with the same amount of corn oil carrier as the experimental group. The corn oil was evenly sprayed on the surface of the basal diet, mixed thoroughly, and then fed.

[0043] Zearalenone experimental group: fed with a basal diet and treated with zearalenone (ZEA) at a dose of 3 mg / kg body weight for 7 consecutive days;

[0044] Administration instructions: Calculate the required dosage based on the daily body weight of each sow. Accurately weigh zearalenone (ZEA) and dissolve it thoroughly in corn oil. Then, spray the solution evenly onto the feed surface, stir well, and feed. The control group should be treated with corn oil in the same way. Feed twice daily (08:00 and 17:00). Feed the basal diet normally in the morning, and administer zearalenone (ZEA) and the control diet in the evening. Ensure each sow finishes eating within 30 minutes before supplementing with the remaining basal diet. Provide free access to water. Continue feeding for one week. Four hours after the last feeding, administer a sleep aid. After anesthesia with Heshutai, the sows were euthanized by exsanguination through the jugular vein. Blood was collected from the sows, and the upper serum was obtained by centrifugation after 1 hour. The serum was then placed on dry ice and sent to Beijing Northern Biotechnology Co., Ltd. for estrogen concentration determination. After the sows were euthanized, they were dissected, and ovarian tissue samples were collected. Some samples were cryopreserved for molecular biological testing, while others were fixed for histological testing.

[0045] 2. Test index detection:

[0046] (1) Fixation of porcine ovarian tissue: After fixing porcine ovaries with 10 times the volume of 4% tissue cell fixative at room temperature for 2 hours, they were divided into 4 equal parts from the transverse and sagittal planes and fixed overnight in fresh fixative at 4°C; washed with PBS for 30 minutes and stored in 70% ethanol.

[0047] (2) Preparation of paraffin sections and hematoxylin-eosin staining: Take a fixed ovarian tissue sample and dehydrate it by passing it through 85%, 95%, and 100% alcohol for 30 minutes each time, twice. Clear it with xylene for about 2-3 minutes until the tissue is transparent. Immerse it in molten paraffin at 65℃ for 3 hours, changing the paraffin every hour. Place the fully paraffin-impregnated ovarian tissue in an embedding cassette for paraffin embedding. After solidification, prepare 5 μm thick sections using a paraffin microtome and dry them in an oven at 37℃ for 6-8 hours. Dewax it with xylene for 10 minutes twice, then hydrate it in a gradient of 100%→95%→85%→75%→50% alcohol and distilled water. Stain with hematoxylin for 30 seconds and let it stand in tap water for 1 minute to return to blue. Dehydrate it in a gradient of 50%-100% alcohol, during which time stain it with eosin in 95% alcohol for about 20 seconds. Continue dehydration, xylene impregnation, and mounting with neutral resin. Take images with an inverted microscope.

[0048] The serum estrogen levels were measured by Beijing Northern Biotechnology Co., Ltd. using radioimmunoassay.

[0049] (4) To determine whether the toxic effects of ZEA on sow ovaries are related to ferroptosis, the ovarian tissue samples frozen in step 1 were first thoroughly ground into a homogeneous powder in liquid nitrogen, and then strictly processed according to the cellular ferrous ion (Fe) ion distribution method. 2+ The following kits were used to detect Fe in ovarian tissue: a fluorescence assay kit, a malondialdehyde (MDA) assay kit, a reduced glutathione (GSH) assay kit, and a total glutathione (T-GSH) / oxidized glutathione (GSSG) assay kit. 2+ The changes in lipid peroxidation (marker malondialdehyde, MDA) and the antioxidant system glutathione (GSH) levels were studied. The remaining powder was aliquoted into multiple RNase-free 1.5 mL EP tubes and stored at -80°C.

[0050] (5) Take about 50 mg of the ovarian tissue powder ground in step (4) to extract RNA: Add 200 μL RNAiso Plus, homogenize with a grinding stick suitable for a 1.5 mL EP tube, add 800 μL RNAiso Plus, and mix repeatedly by pipetting; add 200 μL chloroform, vortex to mix, and let stand at room temperature for 10 min, then centrifuge at 4℃ and 12,000 r / min for 15 min; collect the supernatant into a new 1.5 mL EP tube, add 500 μL pre-chilled isopropanol, incubate on ice for 15 min, and then centrifuge at 4℃ and 12,000 r / min for 15 min; discard the supernatant, add 1 mL 75% ethanol to wash, invert 8 times, and then centrifuge at 4℃ and 12,000 r / min for 10 min; discard the supernatant, air dry, and add 20 μL The total RNA solution was obtained by dissolving the RNA in water using DEPC treatment; the RNA concentration and purity (A260 / A280=1.9~2.1; A260 / A230=2.0~2.2) were detected using a KAIAO micro spectrophotometer.

[0051] (6) Reverse transcription: Take 1 μg of RNA solution obtained in step (5), and follow the operating steps of the 20 μL system described in the HiScript II Reverse Transcriptase RNA reverse transcription kit. First, add 4× gDNA wiper Mix and incubate at 42℃ for 2 minutes to remove residual genomic DNA. After incubating on ice for 2 minutes, add 5× SuperMix and incubate at 50℃ for 15 minutes and 85℃ for 5 seconds to obtain cDNA. Store at -20℃.

[0052] (7) Quantitative analysis: The cDNA obtained from reverse transcription in step (6) was diluted 10 times (1:10) with sterile water and used as a qPCR template. Quantitative analysis of CYP19A1, Nrf2, GPX4, FTH1, FTL1, SLC7A11, SLC3A2, and TFR1 genes was performed using a real-time fluorescence kit (ChamQ SYBR qPCR Master Mix). The qPCR reaction system was 10 μL: containing 4.4 μL of diluted cDNA template, 0.2 μL each of gene-specific upstream and downstream primers (10 μM), 5 μL of 5× ChamQ SYBR qPCR Master Mix, and Dye. 0.2 μL of dye. Reaction program: 95℃, 20 s pre-denaturation; 95℃, 1 s, 62℃, 20 s, for a total of 40 cycles. GAPDH was used as an internal reference gene, according to... The formula calculates the relative expression level (RQ) of the target gene. Primers for gene amplification were synthesized by Qingke Biotechnology, and their sequences are shown in Table 1.

[0053] Table 1 Primer sequences for gene amplification

[0054]

[0055] (8) Protein extraction: Process the ovarian tissue powder ground in step (4) according to the RIPA lysis buffer instructions. Before use, add PMSF to the RIPA lysis buffer to a final concentration of 1 mM (i.e., add 10 μL of 100 mM PMSF to each 1 mL of RIPA lysis buffer). Take 200 mg of tissue powder, add 200 μL of the above RIPA lysis buffer, mix repeatedly by pipetting, and repeatedly aspirate 30 times with a 1 mL syringe to break the cells. Incubate on ice for 30 minutes. Centrifuge at 12000 rpm and 4℃ for 15 minutes, and collect the supernatant as the tissue protein solution. According to the BCA kit instructions, use an ELISA reader to determine the protein concentration, and adjust the protein concentration to 1 μg / μL with RIPA lysis buffer. Take 80 μL of protein solution, add 20 μL of 5× protein loading buffer, denature at 99℃ for 5 minutes, aliquot into 11 μL / tube, and store at -80℃.

[0056] (9) Western Blot detection of protein abundance: Take the tissue protein solution prepared in step (8), load 10 μg per group, and perform electrophoresis separation in a 12% polyacrylamide gel. First, perform electrophoresis at 80 V for 30 minutes to concentrate the proteins to the same level, then perform electrophoresis at 100 V for about 60-70 minutes until the bromophenol blue indicator band migrates to the bottom of the gel; transfer the protein to a 0.22 μm PVDF membrane using the 200 mA wet transfer method for 80 minutes; according to the molecular weight of the target protein (Nrf2: 66 kDa; GPX4: 20 kDa; GAPDH: 36 kDa), the protein is separated by electrophoresis. Cut a PVDF membrane (kDa); block with 5% skim milk powder (prepared with TBST) at room temperature for 30 minutes; dilute the primary antibody with Western blotting buffer at 1:2000 and incubate overnight at 4°C; wash the membrane with TBST for 5 minutes × 3 times the next day; dilute the secondary antibody with TBST at 1:10000 (incubate Nrf2 and GPX4 bands with GAR-HRP, and GAPDH bands with GAM-HRP) and incubate at room temperature for 1 hour; wash the membrane with TBST for 5 minutes × 3 times; develop the membrane using a chemiluminescence imaging system with ECL chemiluminescence solution; analyze the gray values ​​using ImageJ software, and express the relative abundance of the target protein as the ratio of the gray values ​​of the target protein to the internal reference protein GAPDH.

[0057] (10) Data processing and statistical analysis: The data obtained from the above experimental indicators were statistically analyzed and plotted using GraphPad Prism 10. The results are expressed as mean ± standard error. The t-test was used for statistical analysis between the two groups.

[0058] (11) Results Analysis:

[0059] Appendix Figure 1 As shown, zearalenone (ZEA) causes a large number of atretic follicles to appear in the ovaries of sows (see attached image). Figure 1 A), and inhibits estrogen synthesis in the ovaries (see appendix). Figure 1 B) and the expression of CYP19A1, a key enzyme in estrogen synthesis (see appendix). Figure 1 C).

[0060] Appendix Figure 2 As shown, ZEA leads to Fe in the ovary 2+ The significantly increased levels of MDA and ZEA indicate that ZEA induced Fe in the ovaries of sows. 2+ Accumulation (with appendix) Figure 2 A) Increased lipid peroxidation (see appendix) Figure 2 B); Although the ratio of reduced glutathione to oxidized glutathione (GSH / GSSH) did not change significantly (see appendix) Figure 2 C), but ZEA significantly reduced the total glutathione (T-GSH) content in the ovaries (see appendix). Figure 2 D). The above results demonstrate that ZEA induces ferrodeogenesis in the porcine ovary.

[0061] Appendix Figure 3 As shown, at the molecular level, ZEA inhibits the expression of antioxidant-related factors Nrf2, GPX4, SLC7A11, and SLC3A2 (see attached diagram). Figure 3 (A, B, E, F), while ZEA promotes the expression of transferrin receptor TFR1 (see appendix). Figure 3 G), Inhibit the expression of ferritin heavy chain FTH1 (see appendix) Figure 3 (C and D), which is also a possible molecular mechanism by which ZEA causes ferrodegeneration in pig ovaries.

[0062] Example 2 Cell Experiment

[0063] 1. Preparation of 30 μM ZEA: Weigh 19 mg ZEA and dissolve it in 0.1 mL DMSO to prepare a 600 mL stock solution. Take 10 μL of this solution and dilute it with 90 μL DMSO to prepare a 60 mL stock solution. For cell treatment, take 60 mL of the ZEA stock solution and dissolve it in 10% FBS DMEM / F12 culture medium at a ratio of 1:2000. For the control group, add DMSO to 10% FBS DMEM / F12 culture medium at a ratio of 1:2000.

[0064] 2. Preparation of 2 μM Ferrostatin-1: Dissolve 5 mg Ferrostatin-1 in 0.195 mL DMSO to prepare a 100 mL stock solution. Take 4 μL of this solution and dilute it with 96 μL DMSO to prepare a 4 mL stock solution. For cell treatment, take 4 mL of ZEA stock solution and dissolve it in 10% FBS DMEM / F12 culture medium at a ratio of 1:2000. For the control group, add DMSO to 10% FBS DMEM / F12 culture medium at a ratio of 1:2000.

[0065] 3. Preparation of 30 μM ZEA + 2 μM Ferrostatin-1: Dissolve 60 mL ZEA stock solution and 4 mL ZEA in the same 10% FBS DMEM / F12 culture medium at a ratio of 1:2000; the control group was prepared by adding DMSO to 10% FBS DMEM / F12 culture medium at a ratio of 1:1000.

[0066] 4. Cell Culture and Processing: Granulosa cells are a type of cell in pig ovaries that synthesize estrogen. Pig ovaries were collected from the slaughterhouse, disinfected by soaking in 75% ethanol for 30 seconds, washed three times with sterile PBS buffer, and finally transferred into PBS buffer containing 5 times the concentration of penicillin and streptomycin (500 U / mL penicillin and 0.5 mg / mL streptomycin), placed in a sterile container, and brought back to the laboratory. In the cell culture room, cells were washed three times with PBS containing 5-fold concentration of penicillin and erythromycin. Follicular fluid from follicles with a diameter of 5–6 mm was aspirated using a 20 mL sterile syringe and collected in a 15 mL sterile centrifuge tube. The cells were centrifuged at 2200 r / min for 5 min to collect the cell pellet. After treatment with a cell volume: erythrocyte lysis buffer ratio of 1:3 for 5 min, the cells were centrifuged at 2200 r / min for 5 min to collect the cell pellet. The cells were resuspended in PBS buffer and washed once, then centrifuged at 2200 r / min for 5 min to collect the cells. The cells were resuspended in DMEM / F12 medium containing 10% FBS and seeded evenly in 24-well cell culture plates. The cells were cultured in a 5% CO2, 37℃ cell culture incubator for 24 h to further detect the toxic effect of ZEA on ovarian granulosa cells. To synchronize cell cycle and metabolic levels, cells were starved using FBS-free DMEM / F12 medium. After 7 h, cells were grouped according to the dilution method described in steps 1-3: the control group was treated with complete medium; the ZEA treatment group was treated with 30 μM ZEA; and the ZEA+Fer-1 treatment group was treated with both 30 μM ZEA and 2 μM Ferrostatin-1. From the start of drug addition until 24 h later, cells were collected using a cell scraper, centrifuged at 2000 r / min to collect granulocytes, and stored at -80℃ for later use.

[0067] 5. Test index detection:

[0068] (1) Add 500 μL of RNAiso Plus to each well of a 24-well cell culture plate and repeatedly pipette until the granule cells collected in step 4 are fully lysed. The RNA extraction is consistent with step (2) of the test index detection in step 2 of Example 1. The difference is that the amount of chloroform, isopropanol and DEPC-treated water added is 100 μL, 250 μL and 8 μL, respectively. The reverse transcription and quantitative analysis are consistent with steps (3) and (4) in Example 1.

[0069] (2) Protein extraction and detection: Add RIPA lysis buffer to each well of a 24-well cell culture plate at a rate of 80 μL. Use a 1 mL syringe to repeatedly aspirate to fully lyse the cells. After incubating on ice for 30 min, centrifuge at 4℃ and 12,000 r / min for 15 min. Collect the supernatant to obtain the total cell protein. Take 60 μL of protein solution and add 15 μL of 5× protein loading buffer. Denature at 99℃ for 5 min to obtain the denatured protein sample. Aliquot the sample into 16 μL tubes and store at -80℃.

[0070] (3) Western Blot method for detecting protein abundance: Take 15 μL of each denatured protein sample from step (2), and perform the remaining operations according to the test index detection method described in Example 1 - Western Blot method for detecting protein abundance. The internal control used is α-tubulin (55 kDa), and the corresponding secondary antibody is GAM-HRP.

[0071] (4) Data processing and statistical analysis: The experimental data were statistically analyzed and plotted using GraphPad Prism 10. The results are expressed as mean ± standard error. The t-test was used to perform statistical analysis between two groups of data, and one-way ANOVA was used to perform statistical comparison between multiple groups of data.

[0072] (5) Results Analysis:

[0073] 30 μM ZEA significantly increased the Fe²⁺ content in cells (see attached image). Figure 4 A), while simultaneously inhibiting the protein expression levels of Nrf2 and GPX4 in granulocytes (see appendix). Figure 4 B). To further verify whether ZEA-induced granulosa cell damage is related to ferroptosis, cultured granulosa cells were co-treated with 2 μM Ferrostatin-1 and 30 μM ZEA for 24 h. The results showed that, compared with the ZEA-only treatment group, the levels of Nrf2 and GPX4 in granulosa cells were significantly increased after co-treatment with Fer-1 and ZEA (see appendix). Figure 5(A–F). This indicates that ZEA can induce ferroptosis in cultured porcine ovarian granulosa cells, and that inhibiting ferroptosis with Fer-1 can significantly improve ZEA-induced granulosa cell toxicity.

[0074] Example 3: In vivo rescue experiment in mice

[0075] 1. Laboratory animals and grouping:

[0076] Fifty-six healthy female mice aged 8 weeks were randomly divided into seven groups (n=8 / group): control group (Vehicle), ZEA group, ZEA + taurine (Tau) group, ZEA + melatonin (MT) group, ZEA + Ferrostatin-1 (Fer-1) group, ZEA + Fer-1 + Tau group, and ZEA + Fer-1 + MT group.

[0077] Oral administration dosage and administration: ZEA 3 mg / kg, Fer-1 0.8 mg / kg, MT 10 mg / kg, Tau 100 mg / kg;

[0078] Control group (Vehicle group): 21 μL of DMSO stock solution was placed in 1 mL of corn oil and vortexed to mix. 200 μL was administered to each mouse by gavage. 200 μL of physiological saline was injected into each mouse intraperitoneally.

[0079] ZEA group: 2 μL of 600 mM ZEA stock solution from Example 2 was placed in 1 mL of corn oil and vortexed to mix. 200 μL was administered to each mouse by gavage. 200 μL of physiological saline was injected into each mouse intraperitoneally.

[0080] ZEA + Taurine (Tau) group: 2 μL of 600 mM ZEA stock solution from Example 2 was placed in 1 mL of corn oil and vortexed to mix. 200 μL was administered to each mouse by gavage. 15 mg of taurine was weighed and dissolved in 1 mL of physiological saline. 200 μL was injected into each mouse intraperitoneally.

[0081] ZEA + melatonin (MT) group: 9.3 mg MT was weighed and dissolved in 100 μL DMSO; 16 μL of the above MT stock solution and 2 μL of 600 mM ZEA stock solution were placed in 1 mL of corn oil and vortexed to mix well. 200 μL was administered to each mouse by gavage; 200 μL of physiological saline was injected into each mouse intraperitoneally.

[0082] ZEA+Ferrostatin-1 (Fer-1) group: 2 μL of 600 mM ZEA stock solution and 3.2 μL of 100 mM Ferrostatin-1 stock solution from Example 2 were placed in 1 mL of corn oil and vortexed to mix. 200 μL was administered to each mouse by gavage. 200 μL of physiological saline was injected into each mouse intraperitoneally.

[0083] ZEA+Fer-1+Tau group: 2 μL of 600 mM ZEA stock solution and 3.2 μL of 100 mM Ferrostatin-1 stock solution from Example 2 were placed in 1 mL of corn oil and vortexed to mix. 200 μL was administered to each mouse by gavage. 15 mg of taurine was weighed and dissolved in 1 mL of physiological saline. 200 μL was injected into each mouse intraperitoneally.

[0084] ZEA+Fer-1+MT group: 9.3 mg MT was weighed and dissolved in 100 μL DMSO; 16 μL of the above MT stock solution, 2 μL of 600 mM ZEA stock solution, and 3.2 μL of 100 mM Ferrostatin-1 stock solution were placed in 1 mL of corn oil and vortexed to mix. 200 μL was administered to each mouse by gavage; 200 μL of physiological saline was injected into each mouse intraperitoneally.

[0085] Administer once every 2 days for 1 week.

[0086] Sample collection: Four hours after the last gavage, the patient was euthanized by cervical dislocation. Both ovaries were rapidly dissected and collected: one ovary was flash-frozen in liquid nitrogen and then transferred to -80°C for storage for RNA extraction, while the other ovary was placed in 4% tissue cell fixative.

[0087] 2. Test index detection:

[0088] Mouse ovarian tissue fixation: Mouse ovaries were fixed overnight at 4°C using 10 times the volume of 4% tissue cell fixative at 4°C; washed with PBS for 30 minutes and stored in 70% ethanol.

[0089] Paraffin section preparation and hematoxylin-eosin staining: The above mouse ovarian tissue was taken and dehydrated by 85%, 95%, and 100% alcohol, respectively, for 15 minutes each time × 2 times; xylene was used for clearing for about 1 minute until the tissue was clear; the remaining paraffin embedding, sectioning, and hematoxylin-eosin staining steps were the same as in Example 1 - Test index detection - Paraffin section preparation and hematoxylin-eosin staining.

[0090] RNA extraction from ovarian tissue: 200 μL of RNAiso Plus was added to one ovary and thoroughly ground using a grinding stick adapted to a 1.5 mL EP tube. Then, 300 μL of RNAiso Plus was added to fully lyse the tissue. The RNA extraction method in the test index detection of Example 1 was followed, and the ratio of chloroform, isopropanol and DEPC-treated water was adjusted to extract RNA from mouse ovarian tissue. The RNA concentration and purity were detected. cDNA was obtained by reverse transcription and the Cyp19a1 gene was quantitatively detected. The primers are shown in Table 1.

[0091] 3. Data processing and statistical analysis: The experimental data were statistically analyzed and plotted using GraphPad Prism 10. The results are expressed as mean ± standard error. One-way ANOVA was used to compare multiple groups of data.

[0092] 4. Results Analysis: The protective effect of Ferrostatin-1 and its combination therapy against ZEA-induced ovarian injury in mice.

[0093] To systematically evaluate the intervention effects of Ferrostatin-1 (Fer-1) and its combination with melatonin (MT) and taurine on zearalenone (ZEA)-induced ovarian damage, this study conducted comparative verification through histological staining and detection of expression of key enzymes in steroid hormone synthesis.

[0094] (1) Histological morphological observation

[0095] Histological staining analysis showed that a large number of follicles in the ovaries of mice in the ZEA group suffered varying degrees of damage, mainly manifested as granulosa cell shedding and follicular atresia, suggesting that ZEA exposure can severely damage the normal structure and function of ovarian tissue. After intervention with different drugs, the ovarian morphology of mice in each treatment group showed varying degrees of recovery: ovarian damage in the ZEA+Taurine group, ZEA+MT group, ZEA+Fer-1 group, and ZEA+Fer-1+Taurine group was reduced compared with the ZEA group, and the integrity of follicular structure was improved to some extent. It is worth noting that the treatment effect of the ZEA+Fer-1+MT combined treatment group was the most significant, and the ovarian tissue morphology of mice in this group was the most completely restored, with no significant difference compared with the control group (see appendix). Figure 6 The above results indicate that Ferrostatin-1 itself has a good rescue effect on ZEA-induced ovarian damage in mammals, and the combination of Ferrostatin-1 and MT can further amplify this protective effect, achieving a repair level close to normal.

[0096] (2) Detection of expression of key enzymes in steroid hormone synthesis

[0097] At the molecular level, this study further examined the mRNA expression level of Cyp19a1 (aromatase), a key enzyme in steroid hormone synthesis in mitochondria. The results showed that ZEA treatment significantly reduced the level of Cyp19a1 mRNA in mouse ovaries, indicating that ZEA exposure can inhibit the ovarian estrogen synthesis pathway, thereby interfering with normal follicle development and maturation. After drug intervention, the Cyp19a1 mRNA level in the ZEA+Fer-1+MT co-treatment group recovered to a level not significantly different from the control group (see appendix). Figure 7 This further confirms, from a functional perspective, the comprehensive alleviating effect of the combined application of Ferrostatin-1 and MT on ZEA ovarian toxicity.

[0098] Through the above comparisons and verifications, this study draws the following conclusions: Ferrostatin-1 itself has a good salvage effect on ZEA-induced ovarian damage in mammals; when used in combination with melatonin, the therapeutic effect is further enhanced, manifested in the most complete restoration of ovarian morphology and the normalization of expression of key enzymes in steroid hormone synthesis; in comparison, the detoxification effect of Ferrostatin-1 combined with taurine on ZEA is better than that of the ZEA-only treatment group, but not as good as the Ferrostatin-1 combined with melatonin regimen. These results suggest that the combined application of Ferrostatin-1 and MT has a synergistic advantage in antagonizing ZEA ovarian toxicity, providing experimental evidence for developing clinical intervention strategies for ZEA poisoning.

[0099] In conclusion, Ferrostatin-1 is an effective intervention to alleviate ZEA ovarian toxicity. Furthermore, the combined use of Ferrostatin-1 and melatonin showed the best therapeutic effect, suggesting that this combination regimen has potential application value in the clinical prevention and treatment of ZEN poisoning or in reproductive health protection strategies.

[0100] This invention is the first to apply the ferroptosis inhibitor Ferrostatin-1 (Fer-1) to alleviate ZEA-induced ovarian toxicity in mammals. By effectively inhibiting lipid peroxidation and reducing the accumulation of phospholipid peroxides in cell membranes, it blocks the ferroptosis process, providing a new target and intervention for alleviating ZEA-induced ovarian granulosa cell damage and follicular atresia. Building on this, this invention further proposes a novel strategy of combining Fer-1 with either taurine or melatonin, integrating the ferroptosis-inhibiting mechanism of Fer-1 with the anti-apoptotic and antioxidant mechanisms of melatonin, thus achieving a control over the ferroptosis-apoptosis process in ZEA-induced ovarian damage. "The synergistic regulation of the dual pathways overcomes the limitations of existing single intervention strategies and the cytotoxicity of some compounds, significantly improving the protective effect on sow ovarian function. This study provides a new intervention strategy for controlling mycotoxin-induced reproductive disorders in mammals. By clarifying the protective effect of Fer-1 and its combined application, it not only deepens the understanding of the molecular mechanism of ZEA ovarian toxicity (oxidative stress-mitochondrial damage-ferroptosis / apoptosis cascade), but also provides experimental evidence and candidate solutions for developing safe and efficient mycotoxin antidotes. It has important application value for reducing the economic losses caused by mycotoxin pollution in the pig industry."

Claims

1. Application of Ferrostatin-1, an inhibitor of ferroptosis, in alleviating zearalenone-induced ovarian toxicity in mammals.

2. Application of Ferrostatin-1, an inhibitor of ferroptosis, in the preparation of feed products that alleviate zearalenone-induced ovarian toxicity in mammals.

3. The application according to claim 2, characterized in that, The feed products that alleviate the ovarian toxicity of zearalenone in mammals include pharmaceutical preparations or feed additives that alleviate the ovarian toxicity of zearalenone in mammals.

4. The application according to claim 3, characterized in that, The pharmaceutical formulation comprises the ferroptosis inhibitor Ferrostatin-1, an auxiliary active ingredient with antioxidant activity, and a pharmaceutically acceptable carrier; the feed additive comprises the ferroptosis inhibitor Ferrostatin-1, an auxiliary active ingredient with antioxidant activity, and a feed additive carrier.

5. The application according to claim 4, characterized in that, The auxiliary active ingredient is selected from one or more of melatonin, taurine and its derivatives.

6. The application according to claim 4, characterized in that, The mass ratio of the ferroptosis inhibitor Ferrostatin-1 to the co-active ingredient is 1:10-150.

7. The application according to claim 4, characterized in that, The pharmaceutically acceptable carrier is selected from one or more of the following: microcrystalline cellulose, lactose, starch, pregelatinized starch, dicalcium phosphate, mannitol, povidone, hydroxypropyl methylcellulose, croscarmellose sodium, microcrystalline silica, magnesium stearate, talc, ethanol, propylene glycol, polyethylene glycol, Tween 80, water for injection, and vegetable oil.

8. The application according to claim 4, characterized in that, The feed additive carrier is selected from one or more of the following: zeolite powder, bentonite, montmorillonite, silica, corn cob powder, rice husk powder, maifanite, calcium carbonate, dicalcium phosphate, maltodextrin, starch, wheat bran, and rice bran.

9. The application according to claim 4, characterized in that, The pharmaceutical preparation is a tablet, capsule, granule or powder, injection, suspension or emulsion.

10. The application according to claim 4, characterized in that, The feed additive is a premix, concentrated feed, or complete feed.

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