Use of retinoic acid in preparation of drug for protecting ovarian function

Abstract

The use of retinoic acid in the preparation of a drug for protecting an ovarian function. Retinoic acid can partially reverse excessive PI3K / Akt activation and DNA damage caused by the chemotherapy drug doxorubicin and can reduce the number of apoptotic follicles caused by doxorubicin. The intraperitoneal injection of retinoic acid in mice can also partially reverse the follicular apoptosis caused by doxorubicin. Retinoic acid is used as a nutrient element that is necessary for the body, can be used for oral administration, and has good application prospects.

Description

Application of retinoic acid in the preparation of ovarian function protection drugs Technical Field

[0001] The invention belongs to the field of biotechnology, and particularly relates to the application of retinoic acid in the preparation of ovarian function protection drugs. Background Art

[0002] Cancer is a recognized global public health problem. Advances in early diagnosis and treatment methods have led to improved overall and disease-free survival rates for cancer patients with adjuvant chemotherapy. However, chemotherapy can cause both short-term and long-term side effects, particularly premature ovarian insufficiency. Chemotherapeutic agents can be broadly categorized into six major groups: alkylating agents, antimetabolites, antitumor antibiotics, botanical anticancer drugs, hormones, and others. Alkylating agents include cyclophosphamide and busulfan. These drugs produce highly reactive alkylating groups, inhibiting DNA synthesis and interfering with RNA function. Antimetabolites include antifolates like methotrexate, antipurines like mercaptopurine, and antipyrimidines like gemcitabine. These drugs compete with enzymes for the binding of nucleic acid metabolites, inhibiting DNA synthesis. Antibiotics include anthracyclines like doxorubicin (also known as adriamycin), actinomycins like dactinomycin, and bleomycins like bleomycin. These drugs can directly intercalate into DNA molecules, inhibiting both DNA and RNA synthesis. Anti-tumor botanicals include vincristine and paclitaxel, which act on microtubules and tubulin, disrupting spindle formation and arresting cells in metaphase. Camptothecin specifically inhibits topoisomerase activity, exerting its anti-cancer effects. Hormonal drugs include tamoxifen, dexamethasone, and letrozole, which exert their anti-cancer effects by specifically binding to hormone receptors. Other anti-cancer drugs include platinum-based drugs such as cisplatin, carboplatin, and oxaliplatin. Cyclophosphamide is the most widely used alkylating agent, while doxorubicin, oxaliplatin, and vincristine are all commonly used chemotherapy drugs.

[0003] The oocytes in female ovarian follicles are very sensitive to chemotherapy. Since the oocytes in the ovaries cannot be regenerated, more than 90% of female patients will experience varying degrees of ovarian failure after chemotherapy. Currently, the number of such patients has increased sharply and they are getting younger, with a high survival rate after treatment. Taking non-malignant blood system diseases such as severe aplastic anemia (SAA) and thalassemia as examples, the cure rate exceeds 85%. After being cured, such patients are eager to restore ovarian function and fertility, improve their quality of life, and maintain family stability. Currently, fertility preservation for such young female cancer patients requires cryopreservation of ovarian tissue, which is then transplanted back into the body at an optional date after the end of chemotherapy. This approach requires surgery and increases the risk of DNA damage. Therefore, it is becoming increasingly urgent to screen oral and injectable drugs that can protect patients' ovarian function in vivo while undergoing chemotherapy.

[0004] Women establish a non-renewable reservoir of primordial follicles at birth, which serve as the ovarian reserve that determines their future reproductive potential. Numerous growth factors, cytokines, and protein kinases regulate the activation of primordial follicles. Activation of the mTOR (mTOR) signaling in pregranulosa cells increases the secretion of KIT ligand (KITL). KITL binds to KIT on the oocyte surface, activating phosphatidylinositol 3-kinase (PI3K)-protein kinase B (Akt) signaling in the oocyte. KITL then phosphorylates forkhead box O3a (FOXO3a), causing its translocation from the nucleus and relieving its inhibitory effect on oocyte growth, leading to the initiation of follicle growth. Chemotherapeutic drugs can cause follicular atresia by directly damaging oocyte and granulosa cell DNA, or indirectly accelerate primordial follicle depletion by upregulating Akt phosphorylation, ultimately leading to loss of ovarian reserve and decreased ovarian function. Animal studies have shown that intraperitoneal administration of quercetin can reduce cyclophosphamide-induced primordial follicle loss by inhibiting the PI3K / Akt / FOXO3a pathway. Intraperitoneal administration of resveratrol can restore the number of primordial follicles after doxorubicin treatment and reduce DNA damage and apoptosis in preantral and early antral follicles, thereby protecting the ovaries. This suggests that inhibiting Akt phosphorylation and reducing ovarian DNA damage can help mitigate the effects of chemotherapy on ovarian reserve function. However, there is currently a lack of effective oral medications to prevent chemotherapy-induced ovarian damage.

[0005] Retinoic acid (RA) is a biologically active derivative of the fat-soluble vitamin A (retinol). The distribution and levels of RA in tissues are strictly controlled. Its synthesis is regulated by specific retinol and retinal dehydrogenases, while its degradation is controlled by the cytochrome P450 26 subfamily (CYP26s), thereby maintaining RA homeostasis. Previous studies have shown that RA plays important roles in the female reproductive system, such as initiating embryonic germ cell meiosis, regulating follicular growth and development, regulating embryonic Müllerian duct development, and regulating endometrial receptivity during pregnancy. Furthermore, one study found that RA, through binding to the Toll-like receptor 9 ligand, could prevent spontaneous, radiation-induced, and doxorubicin-mediated apoptosis in human B cells. These studies suggest the potential application of retinoic acid in female reproductive system diseases and as an anti-apoptotic agent, but there are no reports on its use in preventing ovarian damage from chemotherapy drugs.

[0006] Summary of the Invention

[0007] In order to overcome the shortcomings and deficiencies in the prior art, the primary purpose of the present invention is to provide the use of retinoic acid in the preparation of ovarian function protection drugs.

[0008] The purpose of the present invention is achieved through the following technical solutions:

[0009] Application of retinoic acid in the preparation of ovarian function protection drugs.

[0010] Furthermore, the ovarian function protection drug is a drug for preventing and / or treating ovarian function damage.

[0011] Furthermore, the ovarian damage includes iatrogenic ovarian function damage.

[0012] Furthermore, the iatrogenic ovarian damage includes ovarian function damage caused by chemotherapy.

[0013] Furthermore, the chemotherapy drug includes doxorubicin.

[0014] Furthermore, the ovarian function protection drug has the effects of reversing or partially reversing PI3K / Akt overactivation, DNA damage and reducing follicle apoptosis.

[0015] Furthermore, the retinoic acid includes retinoic acid and pharmaceutically acceptable salts of retinoic acid.

[0016] Furthermore, the ovarian function protection drug also contains a pharmaceutically acceptable carrier.

[0017] Furthermore, the effective dose of the ovarian function protection drug is calculated based on the retinoic acid agent and is administered at 0.1-1 mg / kg / day.

[0018] Furthermore, the ovarian function protection drug is administered orally or by intramuscular injection.

[0019] The inventors of the present invention have discovered that the chemotherapy drug doxorubicin can overactivate the PI3K / Akt signaling pathway in oocytes, increase oocyte DNA damage, and lead to apoptosis of primordial and growing follicles. In an in vitro neonatal mouse ovarian culture experiment, retinoic acid was able to partially reverse the PI3K / Akt overactivation and DNA damage caused by the chemotherapy drug doxorubicin and reduce the number of follicular apoptosis caused by doxorubicin. Intraperitoneal injection of retinoic acid in mice was also able to partially reverse the follicular apoptosis caused by doxorubicin. This discovery expands the application of retinoic acid in the female reproductive system, provides new ideas for preventing chemotherapy drugs from damaging ovarian follicle development and protecting fertility in vivo, and is also of great significance for protecting the quality of life of female patients after chemotherapy.

[0020] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0021] Chemotherapy has improved overall survival and disease-free survival in cancer patients. However, chemotherapeutic agents can damage follicles, ovarian vasculature, and stroma, ultimately leading to loss of ovarian reserve and severely impairing ovarian function, significantly impacting women seeking fertility. Current fertility preservation therapies for cancer patients can delay cancer treatment or cause further damage to the patient's body. Therefore, it is crucial to develop an oral medication that can protect ovarian reserve and prevent chemotherapy-induced ovarian damage in young women with cancer. Different chemotherapeutic agents exhibit similar cell damage patterns. In this study, using doxorubicin as an example of cell damage, we found that retinoic acid can partially reverse the PI3K / Akt overactivation and DNA damage induced by doxorubicin, reducing the number of follicular apoptosis caused by doxorubicin. Intraperitoneal administration of retinoic acid in mice also partially reversed doxorubicin-induced follicular apoptosis. As an essential nutrient, retinoic acid can be taken orally and holds great promise for its application. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 shows the morphological observation (a) and follicle statistics (b) of the ovaries of newborn mice at 3 days postpartum (3 dpp) after addition of doxorubicin and doxorubicin + retinoic acid of the present invention for 4 days in vitro; among them, in the retinoic acid + doxorubicin group, doxorubicin was added after 5 hours of pre-culture with retinoic acid (scale bar: 50 μm).

[0023] Figure 2 shows immunofluorescence staining of the apoptosis marker γ-H2AX (a) and statistical graph of the mean number of cells positive for apoptosis signals in a single section of 3 dpp newborn mouse ovaries cultured in vitro for one day with the addition of doxorubicin and doxorubicin + retinoic acid of the present invention (b); in the retinoic acid + doxorubicin group, doxorubicin was added after a 5-hour pre-culture with retinoic acid (scale bar: 50 μm).

[0024] Figure 3 shows the protein immunoblotting (a) and relative protein expression statistics (b) of 3dpp newborn mouse ovaries after addition of doxorubicin and doxorubicin + retinoic acid of the present invention for one day in vitro culture; in the retinoic acid + doxorubicin group, doxorubicin was added after 5 hours of pre-culture with retinoic acid.

[0025] Figure 4 shows the ovarian morphology (a) and follicle count statistics (b) of newborn mice injected intraperitoneally with doxorubicin and doxorubicin + retinoic acid until 6 days old. In the retinoic acid + doxorubicin group, retinoic acid was pre-injected 1 day before doxorubicin was co-injected (scale bar: 50 μm). DETAILED DESCRIPTION

[0026] The present invention will be described in further detail below with reference to the embodiments and drawings, but the embodiments of the present invention are not limited thereto.

[0027] In the following examples, experimental methods without specific experimental conditions were generally performed under conventional experimental conditions or those recommended by the manufacturer. Materials and reagents used were commercially available unless otherwise specified.

[0028] Example 1

[0029] 1. Preparation of Reagents Related to In Vitro Culture

[0030] 1.1 Preparation of drug solution

[0031] Prepare a retinoic acid solution by adding 50 mg of retinoic acid powder to 1 mL of DMSO in the dark. Prepare a doxorubicin solution by adding 1 mg of doxorubicin powder to 10 mL of purified water. Once the solids are fully dissolved, filter through a 0.22 μm filter, seal with parafilm, and aliquot. Store at -20°C.

[0032] 1.2 DMEM / F12 medium preparation (Table 1)

[0033] Table 1. DMEM / F12 culture medium

[0034] After all the solids are dissolved, filter with a 0.22 μm filter, operate in a clean bench, seal with sealing film, and store at 4°C.

[0035] 1.3 Preparation of phosphate buffered solution (PBS) (Table 2)

[0036] Table 2. PBS buffer

[0037] Autoclave and store at 4°C.

[0038] 2. Preparation of immunofluorescence-related reagents

[0039] 2.1 Preparation of 10% blocking serum

[0040] Donkey serum and PBS were prepared at a ratio of 1:10 as needed.

[0041] 2.2 Primary Antibody Preparation

[0042] Corresponding primary antibody: 10% blocking serum at a ratio of 1:200, prepared as needed.

[0043] 2.3 Secondary Antibody Preparation

[0044] Prepare the corresponding secondary antibody in PBS at a ratio of 1:200 according to the species of the primary antibody.

[0045] 2.4 Preparation of DAPI working solution

[0046] DAPI storage solution: PBS at a ratio of 1:200, prepared as needed.

[0047] 2.5 Preparation of citric acid buffer working solution (Table 3, Table 4, Table 5)

[0048] Table 3. 0.1M citric acid (Solution A)

[0049] Table 4. 0.1M Sodium Citrate (Solution B)

[0050] Table 5. Citric acid working solution

[0051] 2. Isolation and in vitro culture of mouse ovaries, intraperitoneal injection of mice

[0052] 1. Isolation and Culture of Mouse Ovaries

[0053] 1.1 Surgical instruments (ophthalmic straight scissors, 10 cm toothed forceps, pointed forceps, 1 mL syringe), 90 mm culture dishes, six-well plates, etc. were placed in a clean bench and irradiated with UV light for 30 minutes. Intercellular UV light was then used for 30 minutes.

[0054] 1.2 In this experiment, 3-day-old ICR female mice (3 dpp) were sacrificed by cervical dislocation. The abdomen was disinfected with 70% alcohol, and a V-shaped incision was made at the umbilicus to open the abdominal cavity. The mice were divided into the head and tail along the upper edge of the kidneys, and the tail was placed in pre-cooled PBS.

[0055] 1.3 Under a stereomicroscope, remove the mouse ovary along with the surrounding connective tissue and use a 1 mL injection needle to remove the surrounding connective tissue.

[0056] 2. In vitro culture of mouse ovaries

[0057] 2.1 Add 3 mL of DMEM / F12 medium to the wells of a six-well plate. Add 1.8 μL of DMSO to the control group; add 1.5 μL of doxorubicin solution to the doxorubicin group to a final concentration of 0.05 μg / mL; add 1.8 μL of retinoic acid and 1.5 μL of doxorubicin solution to the retinoic acid + doxorubicin group to achieve a final concentration of 1 μM retinoic acid and 0.05 μg / mL doxorubicin. Mix thoroughly by pipetting, place a 0.44 μm culture membrane in the wells, and incubate in a 37°C incubator for 30 min.

[0058] 2.2 Place the isolated ovaries on a culture membrane and culture in a 37°C, 5% CO2 incubator;

[0059] 2.3 Cultivate for 4 days: Change the medium every two days.

[0060] 3. Intraperitoneal Injection of Mice

[0061] 1.1 Preparation of intraperitoneal injection: Dissolve 50 mg of retinoic acid in 1 ml of sesame oil to prepare retinoic acid injection; dissolve 1 mg of doxorubicin in 10 mL of 0.9% saline to prepare doxorubicin injection.

[0062] 1.2 Intraperitoneal Injection Protocol: Control group: Newborn mice were injected with the appropriate volume of sesame oil twice daily for 3 days, starting at 3 days postpartum. Doxorubicin group: Newborn mice were injected with doxorubicin at a single dose of 0.05 mg / kg twice daily for 2 days, starting at 4 days postpartum. Retinoic acid + doxorubicin group: Newborn mice were injected with retinoic acid at a single dose of 0.3 mg / kg twice daily for 3 days, starting at 3 days postpartum. Doxorubicin was co-injected with 0.05 mg / kg twice daily for 2 days, starting at 4 days postpartum. Ovaries were harvested from mice at 6 days postpartum for the next experiment.

[0063] 1.3 Intraperitoneal Injection: Weigh the mouse and draw up the appropriate volume of drug using a microsyringe. Position the mouse head downward. Grasp the base of the lower limbs with your index and ring fingers. Secure the upper limbs and head with your thumb and index finger, gently stretching the abdominal skin to tighten it. With the microsyringe needle facing upward, insert the needle parallel to the skin and slightly penetrate the skin. Then, insert the syringe at a 45° angle into the abdominal cavity. Inject the drug when you feel a drop in the air.

[0064] 3. Hematoxylin staining

[0065] 1. Paraffin Embedding of Ovaries

[0066] 1.1 Sample fixation: Mouse ovaries cultured for 4 days were fixed in 4% paraformaldehyde at 4°C for 12-16 hours;

[0067] 1.2 Dehydration and transparency in alcohol concentration gradient: 70% alcohol, 80% alcohol, 95% alcohol / eosin staining solution, 95% alcohol, anhydrous alcohol, anhydrous ethanol / xylene (volume 1:1), xylene, each gradient for 5 minutes, and the last xylene was absorbed with filter paper.

[0068] 1.3 Wax immersion and embedding: Wax the tissue in a 60℃ water bath for 3 hours, transfer the tissue to an iron paraffin box, cover the box, take it out and let it solidify at room temperature.

[0069] 2. Slicing

[0070] 2.1 Turn on the microtome, adjust the slice thickness to 5 μm, and start continuous sectioning after fixing the wax block.

[0071] 2.2 Spreading: After the wax strip is fully stretched in a 42℃ water bath, pick it up with a glass slide.

[0072] 2.3 Bake the slices at 42℃ overnight.

[0073] 3. Hematoxylin staining

[0074] 3.1 Dewaxing and Rehydration: Place the slides with tissue attached in a series of xylene, xylene, anhydrous alcohol, anhydrous alcohol, 95% alcohol, 80% alcohol, and 70% alcohol steps. Each step lasts 5 minutes, and the slides are rinsed with deionized water.

[0075] 3.2 Staining: Stain with hematoxylin solution for 1 minute and 30 seconds, then rinse with deionized water.

[0076] 3.3 Dehydration and sealing: The slides were sequentially dehydrated with 70% alcohol, 80% alcohol, 95% alcohol, anhydrous alcohol, anhydrous alcohol, xylene, and xylene, each gradient for 3 minutes, and then sealed with neutral resin.

[0077] 4. Scan the slides and count the stained serial sections. Count one of every five consecutive sections. The final statistical result is the sum of the counts multiplied by 5. Abnormal follicles are manifested by apoptotic oocytes and / or ≥2 apoptotic granulosa cells, and asymmetric follicles.

[0078] IV. Immunofluorescence staining

[0079] 1. Section, dewax, and rehydrate the ovarian tissue wax blocks.

[0080] 2. Antigen retrieval: Place the slices in citric acid buffer working solution and microwave on high heat for 4 minutes, low heat for 4 minutes × 3, and cool naturally to room temperature.

[0081] 3. Wash the slides: PBS 5min×2.

[0082] 4. Blocking: Block with 10% donkey serum at room temperature for 1 hour.

[0083] 5. Incubate with primary antibody: overnight at 4°C.

[0084] 6. Wash slides: PBS 5min×6.

[0085] 7. Incubate with secondary antibody at 37°C for 1 hour. From this step onwards, operate in the dark.

[0086] 8. Wash slides: PBS 5min×6.

[0087] 9.DAPI: Incubate for 1 minute.

[0088] 10. Wash slides: PBS 5 min × 3.

[0089] 11. Sealing: Add 20 μL of anti-fluorescence quencher and cover with a coverslip.

[0090] 12. Photograph using a laser confocal microscope. Take a fluorescence photograph of every three sections. Count five sections for each ovary and calculate the mean value.

[0091] 5. Western Blot

[0092] 1. Protein extraction and concentration determination.

[0093] 2. Prepare polyacrylamide gel, electrophoresis fluid, and transfer fluid.

[0094] 3. Load 20 μg of protein and run electrophoresis at 60V for 40 minutes and 100V for 1 hour.

[0095] 4. Transfer the membrane at 100V for 1 hour.

[0096] 5. Block with 5% skim milk at room temperature for 1 hour.

[0097] 6. Incubate with corresponding primary antibody at 4°C overnight.

[0098] 7. Incubate with corresponding species secondary antibody at room temperature for 1 hour.

[0099] 8. Expose and image, and calculate relative protein expression.

[0100] 6. Results

[0101] 1. Ovarian follicle count results

[0102] Figure 1 shows the results of 4-day in vitro culture of 3-day-old neonatal mouse ovaries. (a) Comparison of ovarian morphology between the control group, doxorubicin group, and retinoic acid + doxorubicin group. Yellow arrows point to apoptotic granulosa cells, and red arrows point to apoptotic oocytes. Scale bar: 50 μm. (b) Abnormal follicle counts. Compared with the control group, the number of abnormal follicles in the doxorubicin group was significantly increased (75 ± 15 vs. 936.67 ± 196.36, p < 0.01). The number of abnormal follicles in the retinoic acid + doxorubicin group was significantly increased (75 ± 15 vs. 556.67 ± 129.65, p < 0.01). Compared with the doxorubicin group, the number of abnormal follicles in the retinoic acid + doxorubicin group was significantly decreased (936.67 ± 196.36 vs. 556.67 ± 129.65, p < 0.05).

[0103] 1.2 Results of intraperitoneal injection of 3 dpp mice are shown in Figure 4. (a) Comparison of ovarian morphology between the control group, doxorubicin group, and retinoic acid + doxorubicin group. Arrows point to abnormal follicles. Scale bar: 50 μm. (b) Abnormal follicle count. Compared with the control group, the number of abnormal follicles in the doxorubicin group was significantly increased (38.33±17.56 vs. 260±20, p<0.001). The number of abnormal follicles in the retinoic acid + doxorubicin group was significantly increased (38.33±17.56 vs. 166.67±30.55, p<0.01). Compared with the doxorubicin group, the number of abnormal follicles in the retinoic acid + doxorubicin group was significantly decreased (260±20 vs. 166.67±30.55, p<0.05).

[0104] 2. Immunofluorescence staining and counting results

[0105] Figure 2 shows the results of 1-day in vitro culture of 3-day-old neonatal mouse ovaries. Figure 2 shows immunofluorescence staining of γ-H2AX in the control, doxorubicin, and retinoic acid + doxorubicin groups. White arrows point to cells positive for apoptosis signals. Scale bar: 50 μm. Figure 2 shows the average number of cells positive for apoptosis signals per section. Compared with the control group, the number of apoptotic signals in the doxorubicin group was significantly increased (2.63±1.40 vs 8.87±0.50, p<0.01). Compared with the doxorubicin group, the number of apoptotic signals in the retinoic acid + doxorubicin group was significantly decreased (8.87±0.50 vs 4.75±0.25, p<0.001).

[0106] 3. Grayscale value analysis of protein bands in immunoblotting

[0107] The results of one-day in vitro culture of 3-day-old neonatal mouse ovaries are shown in Figure 3, where (a) is a Western blotting image. (b) is a statistical graph of relative protein expression. Compared with the control group, the relative expression of phosphorylated Akt (p-Akt) protein in the doxorubicin group was significantly increased (p < 0.05), and the relative expression of γ-H2AX protein was significantly increased (p < 0.05). The relative expression of phosphorylated Akt protein in the retinoic acid + doxorubicin group was significantly decreased (p < 0.05). Compared with the doxorubicin group, the relative expression of phosphorylated Akt protein in the retinoic acid + doxorubicin group was significantly decreased (p < 0.001), and the relative expression of γ-H2AX protein was significantly decreased (p < 0.05).

[0108] The present invention adds doxorubicin, retinoic acid + doxorubicin, or no drug to the ovaries of 3dpp newborn mice and cultures them for 1 day or 4 days, with retinoic acid added 5 hours or 1 day earlier than doxorubicin. Statistical analysis of serial sections of cultured ovarian tissue shows that retinoic acid can significantly reduce the increase in abnormal follicles caused by doxorubicin. Immunofluorescence and protein immunoblotting results show that retinoic acid can inhibit the overactivation of PI3K / Akt caused by doxorubicin and reduce the expression of γ-H2AX protein. Doxorubicin, retinoic acid + doxorubicin, or normal saline are injected intraperitoneally into newborn mice at 4dpp, and statistical analysis of serial sections of ovarian tissue shows that retinoic acid can significantly reduce the increase in abnormal follicles caused by doxorubicin. The above results indicate that retinoic acid can protect the ovaries of mice treated with doxorubicin.

[0109] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be considered as equivalent replacement methods and are included in the scope of protection of the present invention.

Claims

1. Application of retinoic acid in the preparation of drugs for protecting ovarian function.

2. The use of retinoic acid according to claim 1 in the preparation of a drug for protecting ovarian function, characterized in that: The ovarian function protection drug is a drug for preventing and / or treating ovarian function damage.

3. The use of retinoic acid in the preparation of a drug for protecting ovarian function according to claim 2, characterized in that: The ovarian damage includes iatrogenic ovarian function damage.

4. The use of retinoic acid in the preparation of a drug for protecting ovarian function according to claim 3, characterized in that: The iatrogenic ovarian damage includes ovarian function damage caused by chemotherapy.

5. The use of retinoic acid in the preparation of a drug for protecting ovarian function according to claim 4, characterized in that: The chemotherapy drug includes doxorubicin.

6. Use of retinoic acid according to any one of claims 1 to 5 in the preparation of a drug for protecting ovarian function, characterized in that: The ovarian function protection drug has the effects of reversing or partially reversing PI3K / Akt overactivation, DNA damage and reducing follicle apoptosis.

7. Use of retinoic acid according to any one of claims 1 to 5 in the preparation of a drug for protecting ovarian function, characterized in that: The retinoic acid includes retinoic acid and pharmaceutically acceptable salts of retinoic acid.

8. Use of retinoic acid according to any one of claims 1 to 5 in the preparation of a drug for protecting ovarian function, characterized in that: The ovarian function protection drug further comprises a pharmaceutically acceptable carrier.

9. Use of retinoic acid according to any one of claims 1 to 5 in the preparation of a drug for protecting ovarian function, characterized in that: The effective dose of the ovarian function protection drug is calculated based on the retinoic acid agent and is administered at 0.1-1 mg / kg / day.

10. Use of retinoic acid according to any one of claims 1 to 5 in the preparation of a drug for protecting ovarian function, characterized in that: The ovarian function protection drug is administered orally or by intramuscular injection.

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