Use of fos / ap-1 inhibitors in the preparation of a medicament for ameliorating vitrification cryodamage to ovarian tissue

Abstract

The application relates to application of FOS / AP-1 inhibitors in preparation of drugs for improving vitrification freezing damage of ovary tissues. The application provides a new concept and intervention target for female fertility preservation based on ovary vitrification freezing, and has a good application prospect.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, and specifically relates to the application of FOS / AP-1 inhibitors in the preparation of drugs to improve vitrification cryotrauma of ovarian tissue. Background Technology

[0002] Over the past two decades, advances in cancer treatment have significantly improved survival rates. The fertility needs of young female cancer patients are receiving increasing attention. However, treatments such as chemotherapy (especially alkylating agents), radiation therapy, and surgery can severely impair ovarian function, leading to primary ovarian insufficiency (POI). Therefore, fertility preservation should be considered before undertaking any treatment that may damage the gonads, especially for adolescents and women of reproductive age. Embryo and oocyte cryopreservation are recognized fertility preservation techniques, while ovarian tissue cryopreservation (OTC) is an emerging and promising approach.

[0003] Ovarian tissue cryopreservation has been used for fertility preservation for over 20 years. Since the first report of a successful live birth from autologous transplantation after ovarian cortex thawing in 2004, this technique has been recognized and widely used globally. Currently, OTC and subsequent transplantation are the only clinical strategies that can simultaneously restore fertility and ovarian endocrine function while avoiding POI-related diseases. Cohort meta-analysis shows that over 90% of women regain ovarian function after OTC-transplantation. However, the transplantation outcomes of frozen ovaries are significantly worse than those of fresh ovaries, indicating adverse influencing factors during the OTC process. Furthermore, the live birth rate for ovarian tissue cryopreservation followed by in vitro fertilization (IVF) is only 21%, while the live birth rate for embryo cryopreservation is 41%. Therefore, to improve the quality of follicular development after transplantation, it is necessary to elucidate the cellular analysis mechanisms of damage to ovarian cells caused by temperature stress and cryoprotectant toxicity. Identifying key targets and drugs to mitigate ovarian tissue cryopreservation damage caused by toxic substances such as dimethyl sulfoxide (DMSO) in cryoprotectants and rapid temperature changes is a crucial technological breakthrough at present. Summary of the Invention

[0004] This invention provides the application of FOS / AP-1 inhibitors in the preparation of drugs to improve vitrification cryotherapy damage to ovarian tissue, solving the technical problem that the key genes regulating ovarian tissue damage and their specific mechanisms of action are unclear in the prior art.

[0005] This invention provides the application of FOS / AP-1 inhibitors in the preparation of drugs to improve vitrification cryotrauma of ovarian tissue.

[0006] Preferably, the FOS / AP-1 pathway serves as an intervention target for reversing vitrification and freeze-thaw damage in ovarian tissue.

[0007] Preferably, the FOS / AP-1 inhibitor is T-5224. Specifically, T-5224 inhibits FOS / AP-1, targets and regulates its downstream molecule EGR1, inhibits the excessive accumulation of inflammatory factors such as ILs and MMPs in frozen-thawed ovaries, and improves the in vitro culture status of frozen-thawed ovaries, mainly manifested in angiogenesis maintenance and a reduction in apoptotic cells.

[0008] Preferably, the drug further comprises a pharmaceutically acceptable carrier and / or excipients.

[0009] Preferably, the pharmaceutically acceptable carrier and / or excipient includes at least one of diluents, binders, surfactants, humectants, adsorbents, lubricants, fillers, and disintegrants.

[0010] This invention also provides the application of FOS / AP-1 inhibitors in the preparation of ovarian tissue cryoprotectant or ovarian 3D culture medium.

[0011] Preferably, the final concentration of T-5224 added to the culture medium is 80 μM.

[0012] This invention also provides a method for observing the improvement of ovarian tissue vitrification-freeze-thaw injury-related phenotypes by FOS / AP-1 inhibitors, including:

[0013] (1) In vitro 3D culture and phenotypic observation of mouse ovaries after freeze-thaw;

[0014] (2) Total RNA and protein were extracted from mouse ovarian tissue for real-time quantitative PCR and protein immunoblotting analysis.

[0015] (3) Immunofluorescence staining of whole tissue of mouse ovarian tissue to label blood vessels and extracellular matrix components.

[0016] Beneficial effects

[0017] This invention first performed transcriptome (scRNA) and spatial transcriptome sequencing (Stereo-seq) analysis at the single-cell level to compare in detail and systematically the changes in cellular expression profiles of ovarian tissue after freeze-thaw injury. It was found that FOS / AP-1 is a sensitive and rapidly activated signaling molecule during the complex process of ovarian vitrification and thawing, and it amplifies the accumulation of inflammatory factors through a cascade reaction. Furthermore, a mouse ovarian tissue vitrification and 3D culture model was constructed to verify the regulatory role of FOS / AP-1 in ovarian cryoprotectant injury. By targeting FOS / AP-1 as a mechanism to improve ovarian injury, the addition of T-5224, a FOS / AP-1 inhibitor, to the ovarian cryoprotectant and 3D culture medium can inhibit FOS / AP-1 overactivation, thereby targeting and inhibiting the overexpression of EGR1, ultimately reversing the excessive accumulation of inflammatory factors such as ILs and MMPs. This is manifested in a slowed vascular network degradation and a reduced number of apoptotic cells in the cultured frozen-thawed ovaries. Overall, this approach provides a new concept and intervention target for female fertility preservation based on ovarian vitrification. Attached Figure Description

[0018] Figure 1 AE studies, using scRNA and Stereo-seq sequencing analysis, revealed that FOS / AP-1 is rapidly activated after ovarian cryo-thaw and targets EGR1, promoting the accumulation of inflammatory factors such as ILs and MMPs, thereby accelerating post-cryo-thaw ovarian damage.

[0019] Figure 2 AC demonstrated the construction of a mouse ovary cryo-thaw model and the verification that the FOS / AP-1 pathway is activated during the ovary cryo-thaw recovery process.

[0020] Figure 3 Flowchart for the vitrification-freezing-thawing + T-5224 culture group treatment.

[0021] Figure 4 AB showed that in a mouse ovarian freeze-thaw 3D culture model, the addition of T-5224 inhibited AP-1, thereby improving the ovarian freeze-thaw damage phenotype. Detailed Implementation

[0022] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Furthermore, it should be understood that after reading the teachings of this invention, those skilled in the art can make various alterations or modifications to the invention, and these equivalent forms also fall within the scope defined by the appended claims.

[0023] This invention provides FOS / AP-1 as an important target for improving frozen-thawed ovarian tissue damage. FOS / AP-1 is a dimer formed by the binding of transcription factors c-fos and c-jun protein families, which can bind to the promoter region and participate in the transcriptional regulation of downstream genes.

[0024] This invention also provides T-5224 as an inhibitor of FOS / AP-1, which specifically inhibits the binding of FOS to DNA and inhibits the overexpression of its downstream gene EGR1, thereby reducing inflammation accumulation, improving the microenvironment of the frozen-thawed ovarian cortex, and thus mitigating the negative effects of vitrification and cryoprotectant toxicity on ovarian tissue.

[0025] The molecular formula of T-5224, an inhibitor of FOS / AP-1, is as follows:

[0026]

[0027] This invention also provides the concentration ratio and usage strategy for adding T-5224 to ovarian tissue cryoprotectant and ovarian 3D culture medium.

[0028] This invention also provides a method for observing how T-5524 improves phenotypes related to freeze-thaw damage in ovarian tissue, as detailed below:

[0029] (1) In vitro 3D culture and phenotypic observation of mouse ovaries after freeze-thaw;

[0030] (2) Total RNA and protein were extracted from mouse ovarian tissue for real-time quantitative PCR and protein immunoblotting analysis.

[0031] (3) Immunofluorescence staining of whole tissue of mouse ovarian tissue to label blood vessels and extracellular matrix components.

[0032] FOS (also known as c-Fos) belongs to the activator protein 1 (AP-1) transcription factor superfamily. To function, FOS needs to form a heterodimeric complex with another transcription factor from the Jun family (a basic leucine zipper domain), thus forming the active AP-1 complex. AP-1 is a dimeric protein complex composed of members of the Fos (Fos, FosB, Fosl1, Fosl2), Jun (Jun, Junb, Jund), ATF, and JDP families. FOS / AP-1 has been shown to be widely involved in processes such as cell proliferation, differentiation, hypoxia, angiogenesis, steroid production, and prostaglandin (PG) production. These processes are particularly crucial for follicular development, ovulation, and corpus luteum development. The regulation of FOS / AP-1 activity depends on the concentration and activation level of AP-1 protein after extracellular stimulation. Under physiological conditions, FOS / AP-1 concentration and activity are extremely low; when cells are stimulated, the level of the FOS / AP-1 protein complex increases transiently and rapidly. The FOS / AP-1 transcription factor can be activated by various factors, including chemokines, growth factors, cytokines, hormones, and environmental stress. Activated FOS / AP-1, after nuclear translocation, induces the expression of inflammatory cytokines such as ILs, IFN-γ, and TNF-α, participating in the inflammatory response. Therefore, inhibiting FOS / AP-1 activation can reduce the inflammatory response, thereby contributing to the maintenance of homeostasis during the in vitro culture of frozen-thawed ovaries.

[0033] Therefore, this invention aims to elucidate the cascade signaling pathway by which FOS / AP-1 is rapidly activated and initiates the accumulation of ovarian inflammation. By constructing a 3D culture model of mouse ovaries after vitrification and thawing, T-5224 was used to inhibit FOS / AP-1 activation and suppress EGR1 overexpression, thereby reducing the accumulation of inflammation in the ovaries after vitrification and thawing, and exploring methods to improve ovarian damage after vitrification and thawing.

[0034] The present application will be described below with reference to specific embodiments:

[0035] Example 1

[0036] This embodiment provides a method using scRNA and Stereo-seq sequencing analysis to reveal that FOS / AP-1 is rapidly activated after ovarian cryo-thaw, targeting EGR1 and promoting the accumulation of inflammatory factors such as ILs and MMPs, thereby accelerating post-cryo-thaw ovarian damage. Specifically, it includes the following steps:

[0037] I. Single-cell preparation and library construction (for scRNA-seq):

[0038] First, the obtained ovarian tissue was dissected to obtain approximately 1 mm thick ovarian cortex tissue, which was then divided into a fresh group and a vitrification group. The Cryo group underwent normal ovarian vitrification and was then cryopreserved in liquid nitrogen for 2 weeks. The Fresh group underwent collagenase digestion (2 mg / ml collagenase IV dissolved in DPBS, 3 mL total, preheated to 37°C in a water bath). After obtaining a single-cell suspension, DAPI staining was performed, followed by flow cytometry sorting to obtain DAPI. - Live cells were used. The cell concentration was set to 1000 cells / µL before passing the cells through the 10X Genomics microfluidic instrument, with a final loading volume of 10,000 cells. GEMs were then created and collected, barcoded for reverse transcription, and the first-strand cDNA was purified using magnetic beads. After quality control and quantification of the cDNA, a library was constructed according to the standard protocol (10X Genomics). Library sequencing was performed using the Illumina NovaSeq platform.

[0039] II. Library construction and sequencing of single-cell spatial transcriptomes (for Stereo-seq):

[0040] Fresh ovarian cortical tissue was rinsed twice in pre-chilled PBS to remove surface impurities and gently dried to remove any residual fluid or blood. Next, one portion was processed according to standard human ovarian cortical vitrification freezing and thawing procedures. The other portion was embedded in pre-chilled OCT, and the ovary was positioned using a blunt metal needle to ensure correct placement. The entire OCT block was then rapidly frozen in pre-chilled liquid nitrogen with isopentane, and then transferred to a -80°C freezer for storage before cryosectioning. Two weeks later, the vitrified group underwent the same processing and OCT embedding methods as the fresh group.

[0041] To minimize RNA degradation, both fresh and frozen samples were processed within 30 minutes, with the entire process conducted at -20°C. OCT blocks were transversely sliced ​​to 10 μm thickness using a Leica CM1950 cryostat (Leica). Total RNA was then extracted from the slices using the RNeasy Mini Kit (Qiagen, USA). Samples with an RNA integrity number (RIN) of 7–10, measured using a 2100 Bioanalyzer (Agilent, USA), were subsequently subjected to Stereo-seq sequencing analysis. Target slices were directly adhered to the surface of a sequencing chip (BGI, Qingdao, China) equipped with capture probes containing a 25 bp coordinate identifier barcode (CID), a 10 bp molecular identifier (MID), and a 22 bp polyT for in situ mRNA hybridization. Adjacent slices were stained with H&E for later histological examination. The slices on the chip were incubated at 37°C for 3 minutes, fixed in pre-cooled methanol at -20°C for 30 minutes, and then stained with nucleic acid dyes (Thermo Fisher Scientific) for ssDNA visualization. ssDNA and histological imaging were performed using a Ti-7 Nikon Eclipse microscope (Nikon, Japan). Finally, library construction and sequencing were completed with the assistance of Shanghai Ouyi Biotechnology Co., Ltd.

[0042] Third, by integrating scRNA-seq and Stereo-seq data, we systematically analyzed the changes in the expression profiles of FOS / AP-1 and its downstream gene EGR1 during ovarian vitrification and thawing. Simultaneously, we used the "SCENIC" algorithm to clarify that FOS targets and regulates EGR1.

[0043] IV. Immunofluorescence staining

[0044] 1) Remove the tissue sections cut using Leica CM1950 from -20°C and air dry. Circle the operating area with a hydrophobic pen and wash with PBS for 20 min. 2) Shake off the water and fix with 4% PFA for 30 min to 1 h, then wash with PBS for 10 min each time. 3) Block with 4% BSA containing 0.1% Triton X-100 for 1 h. 4) Incubate with primary antibody at 4°C overnight, then wash with blocking buffer for 5 min each time. 5) Incubate with secondary antibody at room temperature for 1 h, then wash with blocking buffer for 5 min each time. 6) Stain with DAPI for 10 min, then wash with PBS for 5 min each time. 7) Mount with immunofluorescence mounting medium and examine under a microscope.

[0045] See overall results Figure 1 The FOS gene family was significantly upregulated in ovarian tissue cells after vitrification and thawing. Figure 1Immunofluorescence staining also largely confirmed that after ovarian vitrification and thawing, the protein expression level of FOS, especially in perivascular cells, was significantly higher than that in the fresh control group. Figure 1 C). Furthermore, using bioinformatics analysis and the "SCENIC" algorithm, it was found that both FOS and JUND directly target and regulate EGR1 (C). Figure 1 D). Furthermore, immunolabeling results showed that EGR1 protein expression was also upregulated after vitrification and freeze-thaw. Figure 1 E).

[0046] Example 2

[0047] A mouse ovary cryopreservation and thawing 3D culture model was constructed, and the activation of the FOS / AP-1 pathway during the ovary cryopreservation and thawing process was verified.

[0048] Specifically, it includes the following steps:

[0049] I. Preparation of a 3D in vitro culture model of mouse ovaries after freeze-thaw.

[0050] 1. In vitro culture medium: DMEM / F12 1:1 + 1% P / S + 5g / ml ITS + 5% FBS + 100mlU / ml rhFSH.

[0051] 2. Dissection culture medium: L-15 + 10% FBS + 0.5% P / S.

[0052] 3. The experimental animals were 2-3 week old female BALB / c mice. Male and female mice were housed at a 2:1 ratio. The birth date of the pups was counted as 0.5 days post-birth (dpp). Ovaries from female mice at 16 dpp were used for in vitro culture. 0.4 ml of culture medium was added to each 24-well Transwell plate, ensuring the membrane of the intercalated chamber (0.4 μm) was covered with a thin layer of culture medium. The plate was then placed in an incubator for preheating.

[0053] 4. The procedure for freezing and culturing ovarian tissue is as follows:

[0054] Vitrification procedure: 1) Under a stereomicroscope, remove the ovarian tissue, dissect and remove surrounding tissue and the cyst, being careful not to squeeze or poke the ovary. Then, use sharp ophthalmic scissors to divide the ovary into 4 equal parts; 2) Transfer the ovary using a pipette, wash it twice in the thawed culture medium, then wash it once in a dish with cryoprotectant ①, and place it in a new dish with cryoprotectant ①. Gently shake at room temperature for 10 minutes; 3) Aspirate the ovarian tissue from cryoprotectant ①, rinse it once in cryoprotectant ②, and transfer it to a new dish with cryoprotectant ②. Rotate at room temperature for 10 minutes; 4) Place it on a 1ml needle and quickly transfer it into liquid nitrogen. Let it stand for 30 minutes (to prepare the drug preparation culture medium);

[0055] Recovery process: such as Figure 3As shown, 1) Preheat the resuscitation solution ① culture medium at 37℃; prepare resuscitation solutions ②, ③, and ④; 2) Remove the tissue from liquid nitrogen and immediately place it in resuscitation solution ① for 1 min, then transfer it to resuscitation solutions ②, ③, and ④ for 10 min each; 3) Transfer it to a small chamber for culture; add other drugs to the culture medium as needed; 4) Change the medium once every 2 days, and harvest the ovaries after 4 days.

[0056] 5. Fresh ovarian culture:

[0057] 1) Sacrifice the mice and remove the ovaries of 3-day-prep mice under a dissecting microscope, placing them in preheated dissecting culture medium; 2) Transfer and clean the ovaries with a 20ul pipette with a slit, and gently place them on the nested chambers of a 12-well plate with forceps, and incubate them in a 37°C, 5% CO2 incubator; 3) Change the medium every 2 days, and harvest the ovaries after 4 days.

[0058] II. After freezing and thawing mouse ovaries, Western blot was used to detect the expression of key proteins in the FS / AP-1 signaling pathway, including FOS / FOSB / JUN / EGR1 / AKT / p-AKT.

[0059] 1) After thorough grinding, add lysis buffer and lyse on ice for 2 hours (RIPA:Cocktail proteinase inhibitor = 100:1). Then centrifuge at 12000 rpm for 10 minutes at 4°C. Transfer the supernatant to a new EP tube, add 5x concentrated SDS loading buffer, mix well, and incubate in boiling water for 5 minutes to denature the protein. Finally, aliquot the processed sample and store at -80°C for later use. 2) Perform protein quantification using BCA for subsequent standardized sample loading. 3) Prepare the gel and assemble the electrophoresis apparatus, adding electrophoresis buffer. Inject 30 μg of protein marker and protein sample sequentially into the wells, set the voltage, and run the gel until bromophenol blue approaches the bottom of the gel. Stop electrophoresis and transfer the sample to a membrane. Cut the filter paper and PVDF membrane to the same size as the gel, and soak the PVDF membrane in methanol for 15 seconds. Stack the sponge pad, filter paper, gel, PVDF membrane, filter paper, and sponge pad together in sequence, remove air bubbles, and place in the transfer tank. Add transfer buffer. Block with 5% skim milk powder at room temperature for 2 hours. Dilute the primary antibody (FOS, FOSB, JUN, EGR1, AKT, and p-AKT) with the primary antibody dilution buffer according to the antibody instructions, and incubate overnight at 4°C. After warming, recover the primary antibody and wash the membrane three times for 5 minutes with TBST. Incubate with HRP-labeled secondary antibody (1:3000) at room temperature for 1 hour, and wash the membrane three times for 5 minutes with TBST. 4) Finally, add an appropriate amount of ECL luminescence solution to the membrane and take pictures using an integrated chemiluminescence analyzer.

[0060] Please see Figure 2In vitrified and thawed mouse ovaries, the number and quality of mature follicles were significantly lower than in the fresh group. Figure 2 A), and simultaneously, throughout the entire ovarian cryo-thawing process, FOS / AP-1-related proteins (FOS, FOSB, JUN) and their target gene (EGR1) were activated and highly expressed in the early stages and continued to be expressed throughout the entire process. Figure 2 (B and C).

[0061] Example 3

[0062] In a mouse ovarian freeze-thaw 3D culture model, the addition of T-5224 inhibited AP-1, thereby improving the ovarian freeze-thaw damage phenotype.

[0063] I. Whole-body fixation and immunofluorescence staining analysis of mouse ovarian tissue

[0064] Mice were randomly divided into three groups of 12 mice each. (A) Fresh Group: Fresh ovaries were used. After dissection of the surrounding tissues, the ovaries were rinsed with culture medium and directly cultured in a microlab. (B) Cryo Group: After cryopreservation and thawing, the ovaries were rinsed with culture medium and cultured in a microlab. (C) Cryo+T-5224 Group: The cryopreservation and thawing process was completed in cryosol and thawing medium containing 80 μM T-5224. The ovaries were rinsed with culture medium and cultured in medium containing 80 μM T-5224. After 48 hours, the medium was changed to medium without T-5224. The specific procedure is as follows: Figure 3 .

[0065] II. Whole-body fixation and immunofluorescence staining analysis of mouse ovarian tissue

[0066] 1) Fix the in vitro cultured ovaries in cold 4% paraformaldehyde (PFA) overnight at 4°C, while placing the culture dish on a shaker to ensure the embryos are completely immersed in the PFA; 2) Wash the ovaries in PBS for 5 minutes, 3 times, then transfer to cold 100% methanol and incubate overnight at -20°C; 3) Subsequently, rehydrate the ovarian tissue in a series of gradients of methanol (MeOH) / PBST (PBS + 0.2% Triton X-100). Use 75%, 50%, and 25% MeOH / PBST; 5 minutes each time. Finally, wash the ovarian tissue in PBST twice, 5 minutes each time; 4) Block the ovarian tissue in blocking buffer (5% BSA in PBST) on a shaker at room temperature for 2 hours; 5) If necessary, incubate the ovarian tissue overnight in an appropriate primary antibody at 4°C; 6) After incubating the tissue in the antibody overnight, wash the tissue four times with PBST on a shaker at room temperature (10 minutes each time); 7) Incubate the tissue with secondary antibody and / or fluorescently conjugated secondary antibody for 2 hours at room temperature; 8) Wash the tissue four times with PBST (10 minutes each time), avoiding light during the washing process, and finally take pictures and collect data using a laser confocal microscope.

[0067] III. Statistical Analysis of TUNEL Staining in Mouse Ovarian Tissue Sections

[0068] Ovarian paraffin tissue sections were processed using the Yeasen Biotechnology (Shanghai) Co., Ltd. TUNEL Apoptosis Detection Kit (FITC). The procedure was simple: 1) Immerse the paraffin tissue sections in xylene at room temperature for 5 min, repeating once to completely remove the paraffin; then immerse the sections in 100% ethanol at room temperature for 5 min, repeating once; wash once with a gradient of ethanol (90%, 80%, 70%), 3 min each time. 2) Add 100 μL of Proteinase K working solution to each sample, ensuring complete coverage, and incubate at room temperature for 20 min. 3) Add 100 μL of 1×Equilibration Buffer to each sample for equilibration. 4) After equilibration, wash away most of the 100 μL of 1×Equilibration Buffer around the equilibrated area with absorbent paper, then add 50 μL of TdT incubation buffer to a 5 cm x 2 area of ​​cells. Afterward, protect the slides from light and incubate at 37°C for 60 min. 5) After washing with PBS, stain with DAPI for 20 min, then wash the sample, immerse the slide in deionized water, and incubate at room temperature for 5 min. Repeat twice, for a total of 3 washes. 6) Immediately afterwards, analyze the sample under a fluorescence microscope, observing green fluorescence at 520±20 nm using a standard fluorescence filter.

[0069] See Figure 4 In vitrified and thawed mouse ovaries, treatment with T-5224 resulted in improved maintenance of overall vascular density. Figure 4 A), and at the same time, analysis of cell apoptosis showed that in the vitrified cryopreservation group with added T-5224, the overall percentage of apoptosis was improved, but slightly lower than that in the fresh group ( Figure 4 B).

[0070] The vitrified ovarian cortical tissue used in this invention is discarded ovarian cortical tissue from the marginal corners of female patients with ovarian cysts and tumors who underwent surgery at the International Peace Maternity and Child Health Hospital affiliated with Shanghai Jiao Tong University. The procedure was approved by the Ethics Committee of the International Peace Maternity and Child Health Hospital affiliated with Shanghai Jiao Tong University (Review No. B2022269P).

[0071] The experimental animals used in the mouse ovarian vitrification cryopreservation and resuscitation model were obtained from Jiangsu Jicui Yaokang Biotechnology Co., Ltd., and all animal experiments were approved by the Animal Committee of the International Peace Maternity and Child Health Hospital affiliated to Shanghai Jiao Tong University School of Medicine (Approval No. 202201403).

[0072] This invention is based on the molecular and cellular responses of human ovarian cortical tissue to freeze-thaw stress and the toxicity of protective agents. Through 10× single-cell transcriptome sequencing, Stereo-seq spatial transcriptome sequencing technology and immunofluorescence staining localization, it was found that FOS / AP-1 was overactivated in ovarian cells after vitrification and freeze-thaw.

[0073] Furthermore, this invention constructs a mouse ovary cryo-thaw model to investigate the effects of the FOS / AP-1 inhibitor (T-5224) on physiological metabolism and potential signaling pathways after ovarian cryo-thaw recovery. Ovarian vascular density and the expression of AP-1 core components c-Jun and c-Fos were measured. The results showed that pharmacological inhibition of FOS / AP-1 significantly reversed ovarian cryo-thaw damage, demonstrating significant theoretical and clinical value.

Claims

1. The application of FOS / AP-1 inhibitors in the preparation of drugs to improve vitrification cryotrauma of ovarian tissue, characterized in that: The FOS / AP-1 inhibitor is T-5224.

2. The application according to claim 1, characterized in that: The FOS / AP-1 pathway serves as an intervention target for reversing vitrification and freeze-thaw damage in ovarian tissue.

3. The application according to claim 1, characterized in that: The drug also contains pharmaceutically acceptable excipients.

4. The application according to claim 3, characterized in that: The pharmaceutically acceptable excipients include at least one of the following: diluents, binders, surfactants, humectants, adsorbents, lubricants, fillers, and disintegrants.

5. The application of FOS / AP-1 inhibitors in the preparation of ovarian tissue cryoprotective solutions or ovarian 3D culture media, characterized in that: The FOS / AP-1 inhibitor is T-5224.

6. The application according to claim 5, characterized in that: The final concentration of T-5224 added to the culture medium was 80 μM.

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