New application of ferroptosis inhibitor

Abstract

The invention aims to provide a novel application of a ferroptosis inhibitor, and the ferroptosis inhibitor is Liproxstatin-1. Research is carried out on the ferroptosis phenomenon in the follicle atresia process of egg laying poultry, it is found that the ferroptosis inhibitor Liproxstatin-1 can save follicle particle cell death induced by tamoxifen (TMX), can inhibit the oxidative stress level and intracellular iron ion overload caused by TMX treatment, effectively inhibits the follicle ferroptosis phenomenon induced by TMX, and can be used for preparing the follicle atresia inhibitor Liproxstatin-1 and the follicle atresia inhibitor Liproxstatin-1 in the follicle atresia process of egg laying poultry. And finally, the egg laying function of livestock and poultry is recovered, and the traditional Chinese medicine composition can be developed into a medicine for treating follicular atresia of livestock and poultry for treating follicular atresia.

Description

Technical Field

[0001] The present invention relates to the technical field of poultry breeding, and particularly relates to a new application of ferroptosis inhibitors. Background Art

[0002] The reproductive performance of laying poultry mainly depends on the development status of ovarian follicles. For example, when a chicken is born, the number of primordial follicles is about more than 400,000, but only 500 - 1000 can develop normally to the ovulation stage, indicating that the vast majority of follicles in the chicken ovary cannot reach the pre-ovulation stage, but instead undergo atresia and gradually degenerate and disappear. Follicular atresia is a physiological phenomenon of selective cell death regulated by multiple factors, and its pathological characteristics are that the follicles become smaller, bleeding points appear on the surface of the follicles, and the color becomes turbid. With the in-depth research, researchers have found that animal follicular atresia is regulated by different forms of granulosa cell death. However, there is also a lot of evidence that follicular granulosa cell apoptosis is not the only factor leading to follicular atresia, and other cell death methods such as ferroptosis also play a very crucial role in the process of follicular atresia. The present invention explores the regulatory role of ferroptosis in chicken follicular atresia, and then explores new methods to regulate the process of follicular atresia, in order to improve the reproductive performance of laying poultry led by chickens, and provide new ideas for studying the mechanism of poultry follicular atresia. Summary of the Invention

[0003] The present invention conducts research on the ferroptosis phenomenon during the follicular atresia process of laying poultry, and discovers that by subcutaneously injecting Liproxstatin-1 (Lip-1), a ferroptosis inhibitor, it can rescue the tamoxifen (TMX)-induced granulosa cell death, and then provides a new application of ferroptosis inhibitors, aiming to promote follicular development and maturation through modern molecular breeding technology to reduce the occurrence of follicular atresia, thereby improving the reproductive performance of poultry, increasing the breeding efficiency and economic benefits, and promoting the sustainable development of the poultry breeding industry. The technical solution of the present invention is as follows:

[0004] In the first aspect, the present invention provides an application of a ferroptosis inhibitor in the preparation of a drug for treating follicular atresia and / or restoring egg-laying function, and the ferroptosis inhibitor is selected from Liproxstatin-1.

[0005] Preferably, the drug for treating follicular atresia and / or restoring egg-laying function is used for livestock and poultry, and preferably, the livestock and poultry include chickens, ducks, and geese.

[0006] Further preferably, the dosage of the ferroptosis inhibitor is 5 - 20 mg / kg of livestock and poultry, and more preferably 8 - 12 mg / kg of livestock and poultry.

[0007] In a second aspect, the present invention provides a drug for treating follicular atresia and / or restoring egg-laying function, comprising an iron death inhibitor selected from Liproxstatin-1.

[0008] Preferably, it further comprises: pharmaceutically acceptable excipients.

[0009] Preferably, the drug is used for livestock and poultry.

[0010] More preferably, in the drug, the dosage of the iron death inhibitor is 5-20 mg / kg of livestock and poultry, and more preferably 5-8 mg / kg of livestock and poultry.

[0011] In a third aspect, the present invention provides a method for promoting the proliferation and development of granulosa cells in livestock and poultry for non-diagnostic and non-therapeutic purposes, comprising: using an iron death inhibitor for livestock and poultry, and the iron death inhibitor is selected from Liproxstatin-1.

[0012] Preferably, the way of using the iron death inhibitor for livestock and poultry includes injection.

[0013] More preferably, the injection includes subcutaneous injection or intraperitoneal injection.

[0014] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0015] The present invention constructs a follicular atresia model by TMX induction, and further uses this model to verify that the iron death inhibitor Lip-1 can rescue the death of follicular granulosa cells induced by TMX, and can inhibit the oxidative stress level and intracellular iron ion overload caused by TMX treatment, and effectively inhibit the follicular iron death phenomenon induced by TMX, and thus has the potential to be developed into a drug for treating follicular atresia for the treatment of follicular atresia in livestock and poultry. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] The drawings described herein are used to provide a further understanding of the present invention, and constitute a part of the present invention. The schematic embodiments of the present invention and their descriptions are used to explain the present invention, and do not constitute an improper limitation to the present invention. In the drawings:

[0017] Figure 1In Example 1 of the present invention, the level of ferroptosis increased significantly during the process of TMX-induced follicular atresia. Among them, Figures A-C show the egg production rate, ovarian morphology and ovarian weight of chickens (n = 12) treated with TMX and control (ethanol). Figure D shows the concentrations of follicle-stimulating hormone (FSH) and luteinizing hormone (LH) in the plasma of chickens treated with TMX for 9 days (n = 12). Figures E-G are qPCR analyses showing the expression levels of ACSL4, FTH1 and GPX4 mRNA in chicken follicles during TMX treatment (n = 6). Figure H is a Western blot analysis of the protein levels of ACSL4, FTH1 and GPX4 in chicken follicles during TMX treatment (n = 3), with GAPDH as an internal reference control. Figures I-L are Elisa detections of the relative concentrations of GSH, MDA, iron content and GSSG in chicken follicles during TMX treatment (n = 6). The error is expressed as mean ± S.D., *P<0.05 indicates a significant difference, and **P<0.01 indicates a highly significant difference.

[0018] Figure 2 In Example 2 of the present invention, TMX treatment induced ferroptosis in granulosa cells (GC). Among them, Figure A shows the results of cell viability of GC treated with TMX or Erastin measured by the CCK-8 assay (n = 6). Figure B shows the results of cell viability in GC treated with TMX or Erastin measured by propidium iodide (PI) staining (n = 6). Figures C-F show the relative concentrations of GSH, MDA, iron content and GSSG in GC after treatment with TMX or Erastin (n = 6). Figure G is a flow cytometry analysis of the lipid peroxidation level in GC after treatment with TMX or Erastin (n = 6). Figure H is a Western blot detection analysis of the protein levels of ACSL4, FTH1 and GPX4 in GC treated with Erastin, with GAPDH as a standard control (n = 3).

[0019] Figure 3For the effects of Liproxstatin-1 treatment on ferroptosis and its restoration of chicken follicle function after TMX treatment in Example 3 of the present invention, chickens were subjected to intraperitoneal injection experiments with TMX (6 mg / kg), TMX + liproxstatin-1 (5 mg / kg), or ethanol (control group) for 9 days. Among them, Panel A shows the results of Western blot analysis for detecting the protein levels related to ferroptosis in chicken follicles after injecting TMX or TMX + liproxstatin-1 (n = 3). Panels B-D show the results of qPCR for measuring the mRNA expression levels of genes related to ferroptosis in chicken follicles after treatment with TMX or Lip-1 (n = 9). Panels E-N show the relative values of the concentrations of reactive oxygen species, GSH, MDA, iron ions, and GSSG in chicken follicles after treatment with TMX or Lip-1 (n = 6). Panels O-Q show the determination of the egg production rate, ovarian morphology, and ovarian weight of hens after treatment with TMX or Lip-1 (n = 12). Errors are expressed as mean ± S.D., and the T-test was used. *P < 0.05 indicates a significant difference, and **P < 0.01 indicates a highly significant difference.

[0020] Figure 4 For the effects of Fer-1 treatment on TMX-induced follicular atresia in Example 3 of the present invention, hens at the peak of egg production were subjected to intraperitoneal injection experiments with TMX (6 mg / kg) or TMX + Fer-1 (10 mg / kg) for 13 days. Panel A shows the protein expression levels of ACSL4, FTH1, and GPX4 detected by western blot technology in the two groups (n = 12). Panels B-D show the mRNA expression levels of ACSL4, FTH1, and GPX4 detected by qPCR technology (n = 9). Panel E shows the levels of ROS in follicles detected by flow cytometry technology in the two treatment groups (n = 6). Panels F-I show the concentrations of GSH, MDA, Fe 2+ 、and GSSG detected by using an Elisa kit technology in follicles of different treatment groups (n = 6). Panel J shows the egg production rate of hens in the above different treatment groups (n = 12). Panel K shows the ovarian morphology in the above different treatment groups (n = 12). Panel L shows the ovarian weight in the above different treatment groups (n = 12). Detailed implementation manners

[0021] The animal experiments in the example part of the present invention were carried out in accordance with the research protocol approved by the Animal Ethics Committee of Sichuan Agricultural University (approval number 80153 / 2022). The experimental animals used were commercial Roman brown-shell laying hens from the poultry farm of Sichuan Agricultural University. The experimental chickens were kept in individual cages, with free access to water and food, and the light cycle was 14 hours of light and 10 hours of darkness.

[0022] In a specific embodiment of the present invention, the PI staining steps are as follows: First, aspirate the culture medium from each group of cells, wash the adherent granulosa cells with PBS twice, fix them with 4% paraformaldehyde at room temperature for 15 min, and wash them with PBS three times. Then incubate them with 0.1% Triton X-100 at room temperature for 10 min and wash them with PBS three times. Subsequently, add the PI staining solution (5 μg / mL PI + 100 μg / mL RNase A, prepared with PBS), incubate them in the dark for 15 min, and wash them with PBS three times. Finally, observe them under a fluorescence microscope (Ex / Em 535 / 617 nm). The nuclei of dead cells or late apoptotic cells show red fluorescence, and the number of dead cells is counted using Image J software.

[0023] In a specific embodiment of the present invention, the separation of RNA and real-time quantitative PCR includes: Extract total RNA from follicular cells using a total RNA isolation kit (Foregene, Chengdu, China), and use the TaKaRa PrimeScript TM RT kit (TaKaRa, Tokyo, Japan) for cDNA synthesis. The extraction steps and methods are carried out according to the kit instructions. Use Premix Ex TaqⅡ (TaKaRa) for real-time quantitative PCR (qPCR) analysis. To standardize the cycle threshold, each experiment is repeated three times, and the qPCR results are analyzed using the ΔCt method. The oligonucleotide primers used in the present invention are listed in Table 1.

[0024] Table 1. Primers used in the present invention

[0025]

[0026] The reaction steps of fluorescence quantitative PCR refer to the kit instructions, and the annealing temperature of the reaction system is screened and optimized: Pre-denature at 95°C for 30 s; Denature at 95°C for 5 s; Anneal at X°C for 30 s (X is related to the target gene and is screened through temperature gradient); Extend at 72°C for 10 s; A total of 42 cycles; In the melting curve generation stage, the temperature increases from 65°C to 95°C at a rate of 0.5°C per second.

[0027] The reaction system is shown in Table 2 below:

[0028] Table 2 qPCR system

[0029]

[0030] According to the above reaction system and conditions, using GAPDH as the internal reference gene, fluorescence quantitative PCR analysis is carried out on a fluorescence quantitative instrument, and all samples are made with three technical replicates.

[0031] In a specific embodiment of the present invention, Western blotting includes: adding a phosphatase inhibitor (Sigma-Aldrich) and a protease inhibitor (Promega Madison, Wisconsin, USA) to a cell lysis buffer (cell Signaling Technology) for extracting total protein from follicular cells. Protein concentration was quantified using the Bradford assay (Bio-Rad, Hercules, CA, USA). Approximately 20 μg of protein was electrophoresed on an SDS-PAGE gel and then transferred to a PVDF membrane, and the molecular weight was determined by a protein Marker (Sigma-Aldrich). After blocking the PVDF membrane with 5% milk, it was incubated with a primary antibody overnight at 4 °C. The next day, it was incubated with an HRP-labeled secondary antibody for 1 hour, and then exposed and imaged using an ECL luminescent solution (Millipore, Bedford, MA, USA), and the density of the bands was analyzed using ImageJ.

[0032] In a specific embodiment of the present invention, lipid peroxidation, GSH, and iron assays include: culturing cells in a 12-well plate, and after specific treatment, centrifuging to obtain cell lysates. The levels of lipid peroxidation products in the cell lysates were detected using an MDA (cat.no.ab118970, Abcam) or GSSG (cat.no.ab141393, Abcam) lipid peroxidation detection kit according to the manufacturer's instructions, the concentration level of GSH was quantified using a GSH detection kit (cat.no.CS0260; Sigma-Aldrich), and the ferrous concentration was measured using an iron detection kit (cat.no.ab83366, Abcam).

[0033] In a specific embodiment of the present invention, cell viability assays include: according to the manufacturer's instructions, measuring cell survival rate by staining with propidium iodide (PI) (cat.no.S6874, Selleck) and then observing under a fluorescence microscope. According to the instructions of the cell counting kit CCK-8, after specific treatment, cells were seeded into a 96-well plate, 10 μL of CCK8 was added to each well and incubated for 2 hours, and cell viability was measured. The absorbance value at 570 nm was measured using a Varioskan LUX Elisa (ThermoFisher, Waltham, MA, USA) for live cell counting.

[0034] In the specific embodiments of the present invention, the statistical analysis method adopted is: the results are expressed as mean ± S.D. The comparison of the means of two groups uses the t-test, and the comparison of more than two groups uses one-way analysis of variance (ANOVA). When significant results are obtained from the analysis of variance, the least significant difference test is used for post hoc comparison of the differences between groups. The statistical analysis is performed using GraphPad Prism 6.0 software. A P value less than 0.05 indicates significant differences, a P value less than 0.01 indicates extremely significant differences, and non-significant differences are indicated by the symbol "n.s.".

[0035] Next, the technical solutions in the embodiments of the present invention will be clearly and completely described in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention.

[0036] Example 1

[0037] This example studied whether ferroptosis occurs during the atresia process of chicken follicles, specifically as follows:

[0038] (1) Experimental process:

[0039] The experimental animals were Roman brown-shell laying hens, with a number of 24. They were randomly divided into a control group (n = 12) and an experimental group (n = 12). The experimental group was subcutaneously injected with tamoxifen (TMX) (an estrogen inhibitor) at a dose of 6 mg / kg body weight dissolved in 0.3 ml of ethanol, and the control group was subcutaneously injected with an equal amount of ethanol. Until the hens in the experimental group stopped laying eggs, the number of eggs laid by the hens in the experimental group and the control group was counted every day. Subsequently, all the hens were sacrificed by cervical bleeding, and the hierarchical follicles F3 - F1 (F3 < F2 < F1) were quickly isolated from the ovaries, and the granulosa layer was separated. Since the follicles of the hens in the experimental group were atretic and the granulosa layer and theca layer could not be separated, the follicle wall was collected as the granulosa layer. The collected tissue samples were stored at -80 °C for Western Blot, qPCR, and ELISA detection, and the results are as Figure 1 shown.

[0040] (2) Experimental results:

[0041] In this experiment, a follicular atresia model was established by inducing with TMX. The results showed that the hens treated with TMX completely stopped laying eggs on the 9th day ( Figure 1 Figure A in Figure 1In Figures B and C, Figure B shows the results of one sample in the control group and the experimental group (the same applies to those not specifically described in the embodiments of the present invention). Elisa detection found that the levels of follicle-stimulating hormone and luteinizing hormone in hens treated with TMX were significantly reduced ( Figure 1 in Figure D). Subsequently, it was detected whether ferroptosis occurred during follicular atresia. The qPCR results showed that the injection of TMX increased the mRNA expression level of ACSL4, but decreased the mRNA expression levels of GPX4 and FTH1 ( Figure 1 in Figures E - H). In addition, with the extension of the TMX treatment time, Elisa detection also found that the content of GSH was significantly reduced, while the contents of MDA, GSSG, and iron concentration were significantly increased ( Figure 1 in Figures I - L). These results indicate that ferroptosis occurred during the chicken follicular atresia induced by TMX treatment.

[0042] Example 2

[0043] This example studied the effect of TMX regulating ferroptosis of follicular granulosa cells on follicular atresia process, specifically as follows:

[0044] The method for culturing granulosa cells was as follows: Six Lohmann commercial laying hens at the peak laying period of 300 days old were selected. After the hens were sacrificed by cervical bleeding, the whole ovaries were quickly taken out and placed in physiological saline. The hierarchical follicles F3 - F1 (26 - 35 mm; F3 < F2 < F1) were isolated from the ovaries. The hierarchical follicles were cut with a blade to drain the egg yolk. The follicles were clamped with forceps, and the granulosa cell layer was gently shaken out in PBS, washed clean with PBS, and placed in a small beaker containing PBS. The granulosa cell layer was cut into pieces with ophthalmic scissors, and then the mixture was added to a centrifuge tube and centrifuged (1000 rpm, 8 min), and the supernatant was discarded. 5 - 10 mL of 0.1% type II collagenase was added, and it was placed in a 37°C water bath for digestion for 5 - 10 min. An equal volume of complete medium containing 10% FBS (fetal bovine serum) was added to terminate digestion; centrifuged (1000 rpm, 8 min), the liquid was carefully poured out, and the cells were resuspended with 20 ml of complete medium (DMEM + 10% FBS + 1% double antibody), filtered through a 200 - mesh (70 μm) cell sieve, and the filtrate was collected. Centrifuged (1000 rpm, 8 min), and the cells were resuspended with medium; the cell suspension was inoculated into a 24 - well culture plate at a cell density of 1×10 6 cells per well and cultured in an incubator at 37°C, 5% CO2, 95% air, and saturated humidity.

[0045] Granulosa cells play an important role in maintaining the development and atresia of follicles by secreting estrogen, follicular fluid and nutrients. To investigate whether TMX induces follicular atresia through ferroptosis of chicken follicular granulosa cells, when the granulosa cells were cultured to 80%, different concentrations of TMX (1 μM, 2 μM, 3 μM) were used to treat for 24 h, and at the same time, the classical ferroptosis inducer Erastin (positive control, 5 μM) was used to treat for 24 h. The obtained granulosa cells were detected by CCK8, PI and Western blot. The results are as Figure 2 shown. CCK8 and PI detection found that the cell viability of granulosa cells treated with Erastin or TMX was significantly reduced ( Figure 2 Figures A and B in Figure 2 ). In addition, changes in the relative concentrations of GSH, MDA, iron content and GSSG in GC were also observed, which also indicated that, like Erastin treatment, TMX treatment resulted in GSH depletion, lipid peroxidation accumulation and iron overload ( Figure 2 Figures C - G in

[0046] Example 3

[0047] This example studied reagents that can promote the recovery of follicular function by inhibiting ferroptosis of granulosa cells, specifically as follows:

[0048] In this example, two ferroptosis inhibitors were screened: Liproxstatin-1 (Lip-1) and Ferrostatin-1 (Fer-1), both of which are potent and specific ferroptosis inhibitors.

[0049] (1) Whether Lip-1 (5 mg / kg) can rescue the follicular atresia process by inhibiting TMX-induced ferroptosis of follicular granulosa cells.

[0050] In vivo experiment: Commercial Roman brown-shell laying hens at 300 days old in the peak laying period were selected and divided into a control group, experimental group 1, and experimental group 2 according to a completely randomized grouping design, with 12 hens in each group. In experimental group 1, TMX (dissolved in 0.2 mL of 0.9% normal saline containing 0.05% fetal bovine serum albumin) was injected at a dose of 6 mg / kg, and the injection site was the abdominal area below the sternum; in experimental group 2, Lip-1 (5 mg / kg) was simultaneously injected into the hens injected with TMX; the control group was injected with an equal amount of solvent (normal saline). The number of eggs laid was recorded regularly every day. After the hens in the experimental groups completely stopped laying eggs, ovarian tissues were quickly collected and the levels of lipid peroxidation, glutathione metabolism, iron metabolism, and the expression levels of key ferroptosis genes in the follicles were detected.

[0051] In vitro experiment: Granulosa cells were isolated from large follicles (F1 - F3 grade follicles) of healthy hens. First, the follicles were aseptically removed, washed with PBS, and then the follicles were punctured to release the granulosa cell mass. The cell mass was placed in collagenase (at 37 °C for 20 - 30 minutes), and the cells were collected by centrifugation (1000 rpm, 5 minutes). The cells were washed 2 - 3 times with PBS or culture medium to remove oocytes and residual tissues. The cells were inoculated into a culture plate at a density of 1×10 5 ~5×10 5 cells / mL and cultured in an incubator at 37 °C, 5% CO2, and saturated humidity. When the cell concentration grew to 80%, the granulosa cells were treated with PBS (control group) and TMX (5 μM, experimental group) for 24 hours respectively, and the cell viability, lipid peroxidation level, glutathione metabolism, iron metabolism, and the expression levels of key ferroptosis genes were detected respectively.

[0052] Western blot and qPCR detection found that compared with the TMX treatment group, Lip-1 treatment could significantly promote the expression of FTH1 and GPX4 and inhibit the expression of ACSL4 ( Figure 3 Figures A - D in it). Elisa detection found that Lip-1 treatment could inhibit the reduction of glutathione, increase in reactive oxygen species, elevation of oxidative stress, and iron overload caused by TMX treatment ( Figure 3 Figures E - N in it). The above results showed that Lip-1 treatment significantly inhibited TMX-induced follicular ferroptosis. In addition, on the 9th day after TMX treatment, the hens completely stopped laying eggs, while after Lip-1 treatment, the egg production rate of the hens still remained above 50% ( Figure 3 Figure O in it). Morphological observation found that after Lip-1 treatment, the ovarian weight increased significantly, the distribution of yellow-grade follicles was clear, and there was no follicular atrophy or atresia ( Figure 3 Figures P and Q in it). The above results showed that TMX could induce follicular atresia by promoting ferroptosis of follicular granulosa cells, while inhibiting ferroptosis could promote the functional recovery of follicles.

[0053] (2) The ferroptosis inhibitor Ferrostatin-1 (Fer-1, 10 mg / kg) was used to treat according to the in vivo experiment in (1) and the in vitro experiment in (2): Commercial Lohmann brown laying hens at the peak laying period of 300 days old were selected and divided into a control group and an experimental group with 12 hens in each group by completely random grouping design. The control group was injected with TMX (dissolved in 0.2 mL of 0.9% normal saline containing 0.05% fetal bovine serum albumin) at a dose of 6 mg / kg, and the injection site was the abdominal area below the sternum; the experimental group was to inject Fer-1 (10 mg / kg) into the hens injected with TMX at the same time. The number of eggs laid was recorded regularly every day. After the hens in the experimental group completely stopped laying eggs, ovarian tissues were collected for relevant detection and analysis. The results showed that by simultaneously injecting TMX and Fer-1 subcutaneously into the hens at the peak laying period, Fer-1 treatment could also significantly inhibit TMX-induced follicular ferroptosis ( Figure 4 in Figures A-I). The experimental results showed that although Fer-1 treatment could delay the decline in egg production induced by TMX to a certain extent ( Figure 4 J), on the 13th day of treatment, all the hens in the experimental group still showed the phenomenon of completely stopping egg production. Although morphological observations showed that the follicle morphology and weight parameters were improved ( Figure 4 K, 4L), compared with the Lip-1 treatment group, the rescue effect of Fer-1 on follicular atresia was relatively limited and had no positive effect on egg production, so it did not have further research and development prospects. At present, only the ferroptosis inhibitor Lip-1 has a good effect on follicular atresia and has the potential to be developed into related drugs.

[0054] Although the embodiments of the present invention have been shown and described, those of ordinary skill in the art can understand that various changes, modifications, substitutions and variations can be made to these embodiments without departing from the principles and spirit of the present invention. The scope of the present invention is defined by the appended claims and their equivalents. The above-described embodiments only represent several embodiments of the present invention, and their descriptions are relatively specific and detailed, but they should not be construed as limiting the scope of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the concept of the present invention, several modifications and improvements can still be made, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the present invention should be subject to the appended claims.

Claims

1. Use of a ferroptosis inhibitor in the preparation of a drug for treating follicular atresia and / or restoring egg-laying function, characterized in that: The ferroptosis inhibitor is selected from Liproxstatin-1.

2. The use according to claim 1, characterized in that: The drug for treating follicular atresia and / or restoring egg-laying function is used for livestock and poultry. Preferably, the livestock and poultry include chickens, ducks and geese.

3. The use according to claim 2, characterized in that: The dosage of the ferroptosis inhibitor is 5-20 mg / kg livestock and poultry, preferably 5-8 mg / kg livestock and poultry.

4. A drug for treating follicular atresia and / or restoring egg-laying function, characterized in that: It comprises a ferroptosis inhibitor, wherein the ferroptosis inhibitor is selected from Liproxstatin-1.

5. The drug according to claim 4, characterized in that Also includes: Pharmaceutically acceptable excipients.

6. The drug according to claim 4 or 5, characterized in that The medicine is used for livestock and poultry.

7. The drug according to claim 6, characterized in that In the drug, the dosage of the ferroptosis inhibitor is 5-20 mg / kg livestock and poultry, preferably 8-12 mg / kg livestock and poultry.

8. A method for promoting the proliferation and development of livestock and poultry granulosa cells for non-diagnostic and non-therapeutic purposes, characterized in that: include: A ferroptosis inhibitor is used for livestock and poultry, and the ferroptosis inhibitor is selected from Liproxstatin-1.

9. The method according to claim 8, characterized in that The methods of using ferroptosis inhibitors for livestock and poultry include injection.

10. The method according to claim 9, characterized in that The injection includes subcutaneous injection or intraperitoneal injection.

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