Use of vitamin d in preparation of protective drug for avoiding chemotherapy-induced ovarian injury

Abstract

Use of vitamin D in the preparation of a protective drug for avoiding chemotherapy-induced ovarian injury, pertaining to the field of biotechnology. It has been discovered for the first time that in experiments involving intraperitoneal injection of calcitriol (the active form of vitamin D) and retinoic acid into newborn mice, both calcitriol and retinoic acid, when used individually, can partially reverse the DNA damage caused by the chemotherapeutic drugs cyclophosphamide and doxorubicin, and significantly reduce the number of primordial follicle apoptosis induced by chemotherapeutic drugs (including cyclophosphamide and doxorubicin); the combined drug use (calcitriol + retinoic acid) has a better effect. This discovery expands the application of vitamins in the female reproductive system, provides new ideas for preventing damage to ovarian follicle development caused by chemotherapeutic drugs and for the in vivo protection of fertility, and is also of great significance for safeguarding the quality of life of female patients after chemotherapy.

Description

Application of Vitamin D in the Preparation of Protective Drugs to Prevent Ovarian Damage Caused by Chemotherapy Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to the application of vitamin D in the preparation of protective drugs to prevent ovarian damage caused by chemotherapy. Background Technology

[0002] Cancer is a recognized global public health problem, and chemotherapy is often used to treat it. Chemotherapy drugs can be broadly classified into six categories: alkylating agents, antimetabolites, antitumor antibiotics, herbal anticancer drugs, hormones, and others. 1) Alkylating agents, such as cyclophosphamide and ifosfamide; ifosfamide is an antitumor drug, primarily working by inhibiting DNA synthesis to achieve its antitumor effect. Cyclophosphamide mainly exerts its antitumor effect by inhibiting phosphodiesterase. 2) Antimetabolites: These drugs compete with each other for the binding of nucleic acid metabolites to enzymes, such as gemcitabine, methotrexate, pemetrexed, eufluridine, and hydroxyurea. 3) Antibiotics: Antibiotics with antitumor effects, such as anthracyclines like doxorubicin, actinomycin like bleomycin, and bleomycin like pingyangmycin. 4) Antitumor herbal medicines: Vincristine and paclitaxel act on microtubules and tubulin, interfering with intracellular spindle formation and causing cells to arrest in metaphase of mitosis; camptothecin and podophyllotoxins act on topoisomerases; homoharringtonine and indirubin inhibit tumor cell DNA synthesis, etc. 5) Hormones: These can alter the internal environment, thereby affecting tumor growth, such as tamoxifen, toremifene, flutamide, and letrozole. 6) Others: Such as platinum-based drugs like cisplatin, carboplatin, and oxaliplatin. Cyclophosphamide is the most widely used alkylating agent, and doxorubicin, oxaliplatin, and vincristine are commonly used chemotherapy drugs. However, while treating cancer patients, chemotherapy drugs often cause the death of a large number of follicles in the ovaries of female patients.

[0003] At birth, women establish a fixed and non-renewable pool of primordial follicles as a reserve for female fertility. Chemotherapy drugs cause irreversible follicle apoptosis, leading to a sharp decline in fertility, premature ovarian failure, and infertility in over 90% of female patients. With the increasing prevalence of cancer among younger patients and rising survival rates, especially among young women with hematological malignancies, there is a strong desire to restore ovarian function and fertility after recovery to improve their quality of life, maintain family stability, and contribute to social harmony. Currently, the commonly used method for fertility preservation both domestically and internationally involves cryopreserving ovarian tissue or oocytes before chemotherapy and then transplanting the ovarian tissue in situ after chemotherapy to restore fertility. However, this method requires surgery and carries risks such as low success rates and high tissue preservation costs.

[0004] Vitamin D (VD) is a steroid hormone primarily synthesized by the skin under ultraviolet radiation, with a small portion obtained from diet. VD is converted to 25-hydroxyvitamin D by enzymes secreted by the liver, and then to its active form, 1,25-dihydroxyvitamin D (also known as calcitriol), by enzymes secreted by the kidneys. A key function of VD is maintaining calcium homeostasis and bone formation, but it also regulates energy metabolism, innate and adaptive immunity, and cell growth, differentiation, and apoptosis. VD activates the vitamin D receptor (VDR) and binds tightly to the retinoic acid receptor (RXR) to form the active signal transduction complex VD-VDR-RXR, which enters the cell nucleus and regulates the transcription of approximately 1000 downstream target genes. VD can also exert its effects through rapid non-genomic effects; VDR can respond to VD at the cell membrane. VDR is not only present in calcium-regulating tissues such as the intestine, bone, and parathyroid glands, but also in reproductive organs such as the ovary, uterus, and placenta. However, whether vitamin D can protect primordial follicles from chemotherapy drugs requires further investigation.

[0005] Summary of the Invention

[0006] In order to overcome the shortcomings and deficiencies of the existing technology, the primary objective of this invention is to provide the application of vitamin D in the preparation of protective drugs to avoid ovarian damage caused by chemotherapy.

[0007] Application of vitamin D combined with vitamin A in the preparation of protective drugs to prevent ovarian damage caused by chemotherapy.

[0008] The objective of this invention is achieved through the following technical solution:

[0009] The application of vitamin D is any one of the following applications (1) and (2):

[0010] (1) Application of vitamin D in the preparation of protective drugs to avoid ovarian damage caused by chemotherapy.

[0011] (2) Application of vitamin D combined with vitamin A in the preparation of protective drugs to avoid ovarian damage caused by chemotherapy.

[0012] Furthermore, the chemotherapy drugs include at least one of cyclophosphamide (Cy), doxorubicin (DOX), oxaliplatin (OXA), and vincristine (VIN).

[0013] Furthermore, the ovarian damage includes at least one of DNA damage and primordial follicle apoptosis.

[0014] Furthermore, the vitamin D includes vitamin D and its derivatives; the vitamin A includes vitamin A and its derivatives.

[0015] Furthermore, the vitamin D derivative includes calcitriol, the active form of vitamin D; the vitamin A derivative includes retinoic acid (RA), a ligand for the retinoic acid receptor (RAR) and the retinol X receptor (RXR).

[0016] Furthermore, the ovary is a mammalian ovary; the mammals include humans and mice.

[0017] Furthermore, when the mammal is a human, the effective dose of the drug is calculated based on individual body weight, with calcitriol administered at 0.1–5000 μg / kg, preferably 2.50–50 μg / kg, every other day; and retinoic acid administered at 0.1–2500 mg / kg, preferably 1.50–25 mg / kg, every other day.

[0018] Furthermore, when the mammal is a mouse, the effective dose of the drug is calculated based on the individual's body weight, with calcitriol administered at 0.1–5000 μg / kg, preferably 25–50 μg / kg, every other day; and retinoic acid administered at 0.1–2500 mg / kg, preferably 15–25 mg / kg, every other day.

[0019] Furthermore, the ovarian function-preserving drug also contains a pharmaceutically acceptable carrier.

[0020] Furthermore, the ovarian function-preserving drug is administered orally or via intramuscular injection.

[0021] The inventors of this invention have discovered that chemotherapy drugs cyclophosphamide and doxorubicin can increase DNA damage in oocytes, leading to apoptosis of primordial follicles. In experiments involving intraperitoneal injection of calcitriol and retinoic acid in newborn mice, both calcitriol and retinoic acid partially reversed the DNA damage induced by cyclophosphamide and doxorubicin, significantly reducing the number of primordial follicles apoptotic due to chemotherapy. This discovery expands the application of vitamin D and vitamin A in the female reproductive system, providing new insights into preventing chemotherapy-induced damage to ovarian follicle development and in vivo fertility protection. It also has significant implications for protecting the quality of life of female patients after chemotherapy.

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

[0023] Chemotherapy has improved overall survival and disease-free survival rates for cancer patients. However, chemotherapy drugs can damage follicles, ovarian blood vessels, and stroma, ultimately leading to ovarian reserve loss and severely impairing ovarian function, which has a significant impact on women who wish to have children. Current fertility preservation treatments for female cancer patients may delay cancer treatment or cause further damage to the patient's body. Therefore, it is essential to find an oral medication to protect the ovarian reserve of young women with cancer and prevent ovarian function damage caused by chemotherapy drugs.

[0024] In this invention, we discovered that retinoic acid and calcitriol can partially reverse DNA damage caused by chemotherapy drugs (cyclophosphamide and doxorubicin) and reduce the number of primordial follicle apoptosis induced by chemotherapy drugs. Vitamin D and vitamin A, as essential nutrients, can be taken orally and have promising applications. Attached Figure Description

[0025] Figure 1 shows the ovarian morphology (a) and primordial follicle count (b) of newborn mice injected intraperitoneally with PBS, cyclophosphamide (Cy), or cyclophosphamide (Cy) + different concentrations of calcitriol until 8 days postpartum (scale bar: 50 μm).

[0026] Figure 2 shows the ovarian morphology (a) and primordial follicle count (b) of newborn mice injected intraperitoneally with PBS, doxorubicin (DOX), or doxorubicin (DOX) + different concentrations of calcitriol until 8 days postpartum (scale bar: 50 μm).

[0027] Figure 3 shows the ovarian morphology (a) and primordial follicle count (b) of newborn mice injected intraperitoneally with PBS, cyclophosphamide (Cy), cyclophosphamide (Cy) + calcitriol (50 μg / kg Calcitriol), cyclophosphamide (Cy) + calcitriol (50 μg / kg Calcitriol), and retinoic acid (25 mg / kg RA) until 8 days postpartum (scale bar: 50 μm).

[0028] Figure 4 shows the ovarian morphology (a) and primordial follicle count (b) of newborn mice injected intraperitoneally with PBS, doxorubicin (DOX), doxorubicin (DOX) + calcitriol (50 μg / kg), and retinoic acid (25 mg / kg RA) until 8 days postpartum (scale bar: 50 μm). Detailed Implementation

[0029] The present invention will be further described in detail below with reference to the embodiments and accompanying drawings, but the embodiments of the present invention are not limited thereto.

[0030] Unless otherwise specified, the experimental methods described in the following examples are generally performed under standard experimental conditions or as recommended by the manufacturer. Unless otherwise specified, the materials and reagents used are commercially available.

[0031] Example 1

[0032] I. Preparation of Reagents for In Vitro Culture

[0033] 1.1 Preparation of drug solution

[0034] In the dark, add 1 mg of calcitriol powder to 1 mL of DMSO to prepare a concentrated stock solution; add 30 mg of retinoic acid powder to 1 mL of DMSO to prepare a concentrated stock solution. Add 40 mg of cyclophosphamide powder to 1 mL of PBS to prepare a cyclophosphamide solution; add 10 mg of doxorubicin powder to 1 mL of PBS to prepare a doxorubicin solution; add 0.1 mg of vincristine powder to 1 mL of PBS to prepare a vincristine solution; add 2 mg of oxaliplatin powder to 1 mL of PBS to prepare an oxaliplatin solution. After the solids have completely dissolved, filter through a 0.22 μm filter, seal with sealing film, aliquot, and store at -20°C.

[0035] 1.2 Preparation of DMEM / F12 culture medium (Table 1)

[0036] Table 1. DMEM / F12 culture medium

[0037] After the solid has completely dissolved, filter it through a 0.22μm filter, operate in a clean bench, seal it with sealing film, and store it at 4℃.

[0038] 1.3 Preparation of Phosphate Buffered Solution (PBS) (Table 2)

[0039] Table 2. PBS buffer

[0040] Autoclave and store at 4°C.

[0041] 2. Preparation of immunofluorescence-related reagents

[0042] 2.1 Preparation of 10% Blocked Serum

[0043] Donkey serum and PBS are mixed at a ratio of 1:10 as needed.

[0044] 2.2 Primary Antibody Preparation

[0045] The corresponding primary antibody: 10% blocking serum is prepared at a ratio of 1:200 as needed.

[0046] 2.3 Preparation of secondary antibodies

[0047] Depending on the primary antibody species, the corresponding secondary antibody is prepared in PBS at a ratio of 1:200 as needed.

[0048] 2.4 Preparation of DAPI working solution

[0049] DAPI stock solution: Prepare PBS at a ratio of 1:200 as needed.

[0050] 2.5 Preparation of working solution for citrate buffer (Tables 3, 4, and 5)

[0051] Table 3.0.1M Citric Acid (Solution A)

[0052] Table 4.0.1M Sodium Citrate (Solution B)

[0053] Table 5. Citric Acid Working Solution

[0054] II. Intraperitoneal injection and ovarian isolation in mice

[0055] 1. Intraperitoneal injection in mice

[0056] 1.1 Preparation of intraperitoneal injection solutions: Dilute 1 mg / ml calcitriol stock solution (dissolved in DMSO) with PBS to the corresponding concentrations (25 μg / kg or 50 μg / kg for administration) to prepare calcitriol injection solution for intraperitoneal injection; dilute 30 mg / ml retinoic acid stock solution (dissolved in DMSO) with corn oil to the corresponding concentrations (25 mg / kg for administration) to prepare retinoic acid injection solution for intraperitoneal injection.

[0057] 1.2 Intraperitoneal injection protocol:

[0058] Experimental animals were randomly divided into a control group, a chemotherapy drug group (Cy / DOX), and a chemotherapy drug and treatment drug group (including Calcitriol / Calcitriol+RA combination therapy). Starting with 3-day-ped mice, the injection regimens for each group were as follows:

[0059] Chemotherapy + treatment group: 3 intraperitoneal injections, once every 48 hours; 3-day-pp mice received the first intraperitoneal injection of treatment drug (Calcitriol / Calcitriol + RA combination), 5-day-pp mice received the second intraperitoneal injection of treatment drug (Calcitriol / Calcitriol + RA combination), 1 hour later, chemotherapy drug (Cy 75mg / kg or DOX 10mg / kg) was administered, and 7-day-pp mice received the third injection of treatment drug (Calcitriol / Calcitriol + RA combination);

[0060] Chemotherapy group (Cy / DOX): The above group received intraperitoneal injection of Cy (75 mg / kg) or DOX (10 mg / kg) while receiving chemotherapy; the above group received intraperitoneal injection of an equal volume of PBS while receiving treatment group (including Calcitriol / Calcitriol+RA combination therapy).

[0061] Control group: The above groups received chemotherapy drugs or treatment drugs, and at the same time, an equal amount of PBS or corn oil was injected into their abdominal cavity.

[0062] Ovaries were harvested 24 hours after the last administration of Calcitriol or PBS. Ovaries from mice 8 days post-treatment were used for further experiments.

[0063] 1.3 Intraperitoneal injection method: Weigh the mouse and draw the corresponding volume of drug using a microsyringe. Position the mouse head down, with the index and ring fingers holding the base of the hind limbs, and the thumb and index finger securing the upper limbs and head, gently stretching to taut the abdominal skin. Insert the microsyringe needle, with the tip facing upwards, subcutaneously a short distance, then insert the syringe at a 45° angle into the abdominal cavity. Once a feeling of emptiness is felt, inject the corresponding drug.

[0064] 2. Isolation of mouse ovaries

[0065] 2.1 Surgical instruments (ophthalmic straight scissors, 10cm toothed forceps, pointed forceps, 1mL syringe), 90mm culture dishes, six-well plates, etc. were placed in a clean bench and irradiated with ultraviolet light for 30 minutes, and then irradiated with ultraviolet lamps in the cell compartment for 30 minutes.

[0066] 2.2 The mice were euthanized by cervical dislocation. The abdomen was disinfected with 70% alcohol. A V-shaped incision was made at the umbilicus to open the abdominal cavity. The mice were divided into head and tail parts along the upper edge of both kidneys. The tail part was placed in pre-cooled PBS.

[0067] 2.3 Under a stereomicroscope, the mouse ovary was removed along with the surrounding connective tissue, and the connective tissue around the ovary was cleaned off with a 1mL injection needle.

[0068] III. Hematoxylin staining

[0069] 1. Ovarian paraffin embedding

[0070] 1.1 Sample fixation: The mouse ovaries obtained in step 2 were fixed in 4% paraformaldehyde at 4°C for 12-16 hours;

[0071] 1.2 Dehydration and clearing using alcohol concentration gradients: 70% alcohol, 80% alcohol, 95% alcohol / eosin staining solution, 95% alcohol, anhydrous alcohol, anhydrous ethanol / xylene (volume 1:1), xylene, 5 minutes for each gradient, blot dry with filter paper for the last xylene.

[0072] 1.3 Paraffin Impregnation and Embedding: The tissue was impregnated in a 60℃ water bath for 3 hours, then transferred to an iron paraffin box, covered with an embedding box, and removed to solidify at room temperature.

[0073] 2. Slice

[0074] 2.1 Turn on the microtome, adjust the section thickness to 5μm, fix the wax block and start continuous sectioning.

[0075] 2.2 Display: After the wax ribbon has been fully stretched in a 42℃ water bath, it is lifted out with a glass slide.

[0076] 2.3 Baking slices: overnight at 42℃.

[0077] 3 Hematoxylin staining

[0078] 3.1 Dewaxing and Rehydration: The slides with attached tissue were sequentially passed through xylene, xylene, anhydrous ethanol, anhydrous ethanol, 95% ethanol, 80% ethanol, and 70% ethanol. Each gradient lasted 5 minutes, and finally the slides were rinsed with deionized water.

[0079] 3.2 Staining: Stain with hematoxylin solution for 1 minute and 30 seconds, then rinse with deionized water.

[0080] 3.3 Dehydration and sealing: The slides are sequentially passed through 70% alcohol, 80% alcohol, 95% alcohol, anhydrous alcohol, anhydrous alcohol, xylene, xylene, each gradient for 3 minutes, and then sealed with neutral resin.

[0081] 4. Slide scanning: Count the stained consecutive sections. Take one section out of every five consecutive sections for counting. The final statistical result is the sum of the counts multiplied by 5. Abnormal follicles are characterized by: apoptotic oocytes and / or ≥2 apoptotic granulosa cells; and asymmetrical follicle morphology.

[0082] IV. Immunofluorescence staining

[0083] 1. Ovarian tissue paraffin block sectioning, dewaxing, and rehydration.

[0084] 2. Antigen retrieval: The slides were placed in citrate buffer working solution and microwaved on high for 4 minutes, then on low for 4 minutes for 3 times, and then allowed to cool naturally to room temperature.

[0085] 3. Wash slides: PBS 5 min × 2.

[0086] 4. Blocking: Block with 10% donkey serum at room temperature for 1 hour.

[0087] 5. Incubate primary antibody: overnight at 4°C.

[0088] 6. Wash slides: PBS 5 min × 6.

[0089] 7. Incubate the secondary antibody: 37℃ for 1 hour. From this step onwards, all operations must be carried out in the dark.

[0090] 8. Wash slides: PBS 5 min × 6.

[0091] 9. DAPI: Incubate for 1 minute.

[0092] 10. Wash slides: PBS 5 min × 3.

[0093] 11. Covering: 20 μL of anti-fluorescence quenching agent, cover with a coverslip.

[0094] 12. Laser confocal microscopy imaging. One fluorescence image is taken for every three sections. Five sections from one ovary are counted and statistically analyzed, and the mean of the sections is calculated.

[0095] V. Immunoblotting

[0096] 1. Protein extraction and concentration determination.

[0097] 2. Preparation of polyacrylamide gel, electrophoresis solution, and transfer solution.

[0098] 3. Load 20 μg of protein onto the sample and perform electrophoresis at 60V for 40 minutes, then at 100V for 1 hour.

[0099] 4. Transfer film at 100V for 1 hour.

[0100] Seal with 5.5% skim milk at room temperature for 1 hour.

[0101] 6. Incubate the corresponding primary antibody overnight at 4°C.

[0102] 7. Incubate the corresponding species' secondary antibodies at room temperature for 1 hour.

[0103] 8. Exposure imaging to calculate relative protein expression levels.

[0104] VI. Results

[0105] Figure 1 shows the ovarian morphology (a) and follicle count (b) of newborn mice injected intraperitoneally with PBS, cyclophosphamide (Cy), and cyclophosphamide (Cy) + different concentrations of calcitriol at 8 days postpartum (3 days postpartum) (scale bar: 50 μm). Wherein: a) compares the ovarian morphology among the control group, the cyclophosphamide group, and the groups with different concentrations (25 μg / kg, 50 μg / kg) of calcitriol + cyclophosphamide. Arrows point to surviving primordial oocytes; b) shows the total follicle count of the entire ovary. As shown in the figure, compared with the control group, the number of surviving primordial follicles was significantly reduced in the cyclophosphamide group (4257±156.95 vs 1092.2±91.80, p<0.001). Compared with the cyclophosphamide group, the number of surviving primordial follicles was significantly increased in the 25 μg / kg and 50 μg / kg calcitriol + cyclophosphamide groups (1092.2±91.80 vs 2198±560.79, p<0.01; 1092.2±91.80 vs 2291.2±154.40, p<0.001).

[0106] Figure 2 shows the ovarian morphology (a) and primordial follicle count (b) of newborn mice injected intraperitoneally with PBS, doxorubicin (DOX), and doxorubicin (DOX) + different concentrations of calcitriol up to 8 days postpartum (dpp) (scale bar: 50 μm). Wherein: a is a comparison of ovarian morphology among the control group, the doxorubicin group, and the groups with different concentrations (25 μg / kg, 50 μg / kg) of calcitriol + doxorubicin. Arrows point to surviving primordial oocytes; b is the total ovarian follicle count. As shown in the figure, compared with the control group, the number of surviving primordial follicles was significantly reduced in the doxorubicin group (4257±156.95 vs 209.4±65.18, p<0.001). Compared with the doxorubicin group, the number of surviving primordial follicles was significantly increased in the 25 μg / kg and 50 μg / kg calcitriol + doxorubicin groups (209.4±65.18 vs 420±46.47, p<0.001; 209.4±65.18 vs 967.4±104.83, p<0.001).

[0107] Figure 3 shows the ovarian morphology (a) and primordial follicle count (b) of newborn mice that were intraperitoneally injected with PBS, cyclophosphamide (Cy), cyclophosphamide (Cy) + calcitriol (50 μg / kg Calcitriol), cyclophosphamide (Cy) + calcitriol (50 μg / kg Calcitriol), and retinoic acid (25 mg / kg RA) up to 8 days postpartum (scale bar: 50 μm). Wherein: a is a comparison of ovarian morphology among the control group, Cy group, Cy + Calcitriol group, and combined drug + Cy group. Arrows point to surviving primordial oocytes; b is the total ovarian follicle count. Compared with the Cy group, the combined medication showed a very significant rescue effect on Cy (1092.2±91.80 vs 3717.4±606.6, p<0.001), and the number of rescued primordial follicles was close to that of the control group; compared with the Cy+Calcitriol group, the effect was even better (2291.2±154.40 vs 3717.4±606.6, p<0.001).

[0108] Figure 4 shows the ovarian morphology (a) and primordial follicle count (b) of newborn mice that were intraperitoneally injected with PBS, doxorubicin (DOX), doxorubicin (DOX) + calcitriol (50 μg / kg), and retinoic acid (25 mg / kg RA) at 8 days postpartum (scale bar: 50 μm). Wherein: a is a comparison of ovarian morphology among the control group, DOX group, DOX + Calcitriol group, and combined drug + DOX group. Arrows point to surviving primordial oocytes; b is the total ovarian follicle count. Compared with the DOX group, the combination therapy showed a very significant rescue effect on DOX (209.4±65.18 vs 2012.5±410.87, p<0.001); compared with the DOX+Calcitriol group, the combination therapy showed an even better rescue effect on DOX (967.4±104.83 vs 2012.5±410.87, p<0.001).

[0109] The above results indicate that chemotherapy reduces the number of primordial follicles in the ovary. Both calcitriol and retinoic acid, used alone, can significantly increase the number of surviving primordial follicles after chemotherapy. The combined use of calcitriol and retinoic acid is more effective than that used alone. This suggests that retinoic acid and calcitriol can avoid ovarian damage caused by chemotherapy drugs (including cyclophosphamide and doxorubicin) and play a protective role in the primordial follicles of mouse ovaries.

[0110] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. The application of vitamin D, characterized by: For any of the following applications (1) and (2): (1) Application of vitamin D in the preparation of protective drugs to avoid ovarian damage caused by chemotherapy. (2) Application of vitamin D combined with vitamin A in the preparation of protective drugs to avoid ovarian damage caused by chemotherapy.

2. The application according to claim 1, characterized in that: The vitamin D mentioned includes vitamin D and its derivatives; the vitamin D derivatives include calcitriol; the vitamin A mentioned includes vitamin A and its derivatives; the vitamin A derivatives include retinoic acid.

3. The application according to claim 1 or 2, characterized in that: The chemotherapy drugs mentioned include at least one of cyclophosphamide, doxorubicin, vincristine, and oxaliplatin.

4. The application according to claim 1 or 2, characterized in that: The ovarian damage mentioned includes at least one of DNA damage and primordial follicle apoptosis.

5. The application according to claim 1 or 2, characterized in that: The ovary mentioned is a mammalian ovary.

6. The application according to claim 5, characterized in that: The mammals mentioned include humans and mice.

7. The application according to claim 6, characterized in that: When the mammals are humans, the effective dose of the drug is calculated based on individual body weight, with calcitriol administered at 0.1–5000 μg / kg and retinoic acid at 0.1–2500 mg / kg, administered every other day. When the mammal is a mouse, the effective dose of the drug is calculated based on the individual's body weight, with calcitriol administered at 0.1–5000 μg / kg and retinoic acid at 0.1–2500 mg / kg, administered every other day.

8. The application according to claim 7, characterized in that: When the mammals are humans, the effective dose of the drug is calculated based on individual body weight, with calcitriol administered at 2.5–50 μg / kg and retinoic acid at 1.5–25 mg / kg, administered every other day. When the mammal is a mouse, the effective dose of the drug is calculated based on the individual's body weight, with calcitriol administered at 25-50 μg / kg and retinoic acid at 15-25 mg / kg, administered every other day.

9. The application according to claim 1 or 2, characterized in that: The ovarian function protection drug also contains a pharmaceutically acceptable carrier.

10. The application according to claim 1 or 2, characterized in that: The ovarian function protection drug is administered orally or via intramuscular injection.

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