Application of abscisic acid in preparation of medicine for improving or treating oocyte maturation disorder and improving ovarian reserve function

Abstract

The invention discloses an application of abscisic acid in preparation of a medicine for promoting oocyte maturation and improving an ovarian reserve function. The invention further discloses a medicine which comprises abscisic acid and a pharmaceutically acceptable carrier or auxiliary material, and the medicine can improve or treat oocyte maturation disorder and improve the ovarian reserve function. The invention further discloses a health care product which comprises abscisic acid and a pharmaceutically acceptable carrier or auxiliary material, and the health care product can improve or treat oocyte maturation disorder and improve the ovarian reserve function. The invention finds that the abscisic acid can promote the maturation of oocytes and improve the ovarian reserve function.

Description

Technical Field

[0001] The present invention relates to the technical field of ovarian aging, and in particular to an application of abscisic acid in the preparation of a medicine for improving or treating oocyte maturation disorders and enhancing ovarian reserve function. Background Art

[0002] Aging is an intrinsic, multifactorial, and gradual process. It is an inevitable process that everyone must go through. Its essential feature is organ aging. Aging, especially ovarian aging, has become a world problem and a frontier hotspot. Elucidating the pathogenesis of ovarian aging and exploring therapeutic strategies and drugs to delay ovarian aging have become urgent needs of an aging society, which is not only of great theoretical significance, but also of great practical significance. The ovaries gradually age with the increase of female age. Studies have shown that the rate of functional decline is more obvious after the age of 35. Therefore, ovarian aging occurs earlier than other organs in the body, and its aging rate is much faster than other organs. At present, the average age of menopause in women is about 51 years old. With the extension of overall life expectancy, the life process of women after menopause will occupy 1 / 3 of their lives. Ovarian aging is mainly manifested in a decrease in the number and quality of eggs, resulting in a decrease in female fertility and reproductive quality. Studies have found that transplanting the ovaries of young mice into old mice can prolong the life of old mice. Therefore, ovarian aging is also called the pacemaker of female body aging, and anti-ovarian aging has become a focus of widespread attention in the biomedical and pharmaceutical industries.

[0003] Abscisic acid (ABA) is an important dorsal terpene plant hormone that regulates the plant's response to environmental stresses, such as drought, cold, high salt, and nutrient deficiency. Therefore, it is also called a stress hormone. Generally speaking, abscisic acid is synthesized in plants such as figs, apricots, bananas, and potatoes. However, humans and other animals also often obtain abscisic acid from nutritional sources such as fruits and vegetables. It is currently believed that it has important physiological functions in plant embryonic development, seed dormancy, fruit ripening, and adverse stress. Adversities such as drought, cold, salinity, and waterlogging can rapidly increase the amount of ABA in plants, while enhancing the plant's ability to resist adverse environmental factors and inducing many gene expressions and protein synthesis. Studies have found that abscisic acid is not only present in plants, but also in human tissues including the brain, liver, lungs, and other tissues and cells including macrophages and granulocytes. In many physiological and pathological conditions, abscisic acid can be produced and released in a variety of animal and human cells (immune cells, cardiovascular cells, hepatocytes, and mesenchymal stem cells). It is reported that as an endogenously produced anti-inflammatory secondary metabolite, even very high doses of ABA are well tolerated in mice without causing adverse reactions. These special properties make ABA a potential safer drug or nutritional supplement. However, the role and molecular mechanism of ABA in the ovarian aging process have not been reported. Summary of the invention

[0004] Based on the technical problems existing in the background technology, the present invention proposes an application of abscisic acid in the preparation of a drug for improving or treating oocyte maturation disorders and improving ovarian reserve function. The present invention has found through research that abscisic acid can improve or treat oocyte maturation disorders and improve ovarian reserve function.

[0005] The invention also proposes the use of abscisic acid in preparing a medicine for promoting oocyte maturation.

[0006] Preferably, oocyte maturation disorders are improved or treated by improving oocyte DNA damage.

[0007] Preferably, the oocyte is an oocyte in an aged ovary.

[0008] The invention also proposes the use of abscisic acid in preparing a medicine for improving ovarian reserve function.

[0009] Preferably, the ovary is an aged ovary.

[0010] Oocytes are oogonia that undergo meiosis during oogenesis, and their maturation process mainly includes the following stages: Oocytes undergo meiosis during oogenesis and are divided into primary oocytes, secondary oocytes, and mature oocytes. In this process, primary oocytes undergo the first meiotic division to form secondary oocytes and the first polar body; secondary oocytes undergo the second meiotic division to form mature eggs and the second polar body. Primary oocytes, secondary oocytes, and mature oocytes are the products of oogonia differentiation and DNA replication, the first meiotic division, and the second meiotic division, respectively. The quality of oocytes largely reflects the health of the ovaries and has an important impact on reproductive capacity.

[0011] The maturation of oocytes is a natural developmental process, which is jointly affected by many aspects of the internal and external environment, such as DNA damage, mitochondrial functional state, oxidative stress, apoptosis and autophagy. Although inhibiting oxidative stress and apoptosis can indirectly promote the maturation of oocytes to a certain extent, these factors are not decisive and can only play an auxiliary effect. The present invention innovatively discovered that abscisic acid can efficiently repair DNA damage in oocytes, thereby directly improving oocyte maturation disorders, and provides a new solution for improving oocyte quality.

[0012] In human ovaries, there are about 1 million oocytes at birth, but only about 500 oocytes will mature and ovulate in a lifetime, and most of the others are gradually consumed or die. With age, the ovarian reserve function continues to decline until it is basically exhausted at about 51 years old. Ovarian reserve function and oocyte quality have important research and application potential in repairing ovarian damage and delaying ovarian aging.

[0013] The present invention also provides a medicine, comprising: abscisic acid and a pharmaceutically acceptable carrier or excipient;

[0014] The medicine can improve or treat oocyte maturation disorders and enhance ovarian reserve function.

[0015] Preferably, the dosage form of the drug is an oral preparation and an injection preparation.

[0016] The above-mentioned oral preparations can be tablets, capsules, granules, powders, oral liquids, etc.

[0017] The above-mentioned liquid injection can be injection solution, injection powder, injection tablet and the like.

[0018] The present invention also provides a health product, comprising: abscisic acid and a pharmaceutically acceptable carrier or excipient;

[0019] The health product can improve or treat oocyte maturation disorders and enhance ovarian reserve function.

[0020] The above-mentioned pharmaceutically acceptable carrier can be a microcapsule, a microsphere, a nanoparticle, a liposome, etc., and the above-mentioned pharmaceutically acceptable excipient can be an adhesive, a disintegrant, a filler, a lubricant, a wetting agent, an osmotic pressure regulator, a stabilizer, a glidant, a flavoring agent, a preservative, a suspending agent, a coating material, a fragrance, an anti-adhesive agent, an integrator, a penetration enhancer, a pH regulator, a buffer, a plasticizer, a surfactant, a foaming agent, a defoaming agent, a thickener, a inclusion agent, a humectant, an absorbent, a diluent, a flocculant and a deflocculating agent, a filter aid, a release retardant, a solubilizing agent, a solubilizing agent, an emulsifier, a colorant, etc.

[0021] Although CN 113398110A discloses the use of abscisic acid in preparing products for treating and / or improving polycystic ovary syndrome, it mainly discloses the use of abscisic acid in preparing drugs for treating and / or improving hyperandrogenemia, insulin resistance and inflammation in polycystic ovary syndrome, and it mainly improves the metabolic problems of hyperandrogenemia and insulin resistance in polycystic ovary syndrome by regulating hormone levels.

[0022] Polycystic ovary syndrome is a reproductive endocrine disease characterized by hyperandrogenism, ovulatory disorders, and polycystic ovarian changes, with a higher incidence in adolescents and women of childbearing age. In contrast, ovarian aging is a natural physiological process with complex and diverse mechanisms, including a decrease in the number and quality of oocytes, accumulation of DNA damage, apoptosis of granulosa cells, abnormal genetic and epigenetic regulation, and the effects of environmental toxins. It can be seen that there are significant differences in the mechanisms of polycystic ovary syndrome and ovarian aging.

[0023] The present invention discovered for the first time that abscisic acid can upregulate the AMH level in aged ovarian tissue and significantly improve the ovarian reserve function of aged mice; and abscisic acid can also improve oocyte DNA damage, thereby improving or treating oocyte maturation disorders. Studies have found that the mitochondrial membrane potential in oocytes of aged mice is significantly decreased, while the expression level of Ki-67, a proliferation marker, in ovarian granulosa cells is downregulated, and proteins related to mitochondrial homeostasis (such as DRP1, OPA1, SDHA) show an imbalanced expression state; and abscisic acid can reverse these abnormal phenomena.

[0024] These results indicate that abscisic acid and its related drug preparations can improve or treat oocyte maturation disorders, enhance ovarian reserve function, have important potential in repairing ovarian damage or delaying ovarian aging, and have good clinical application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 The results of observation and detection of abscisic acid in improving oocyte maturation disorder in aged mice by phase contrast microscopy in Example 2, wherein A represents the GV stage oocytes from young mice, aged mice, and aged mice + abscisic acid treatment group cultured in M16 medium for 14 h to observe PB emission, scale = 100 μm; BD represents the total number of oocytes, the ratio of GVBD and the ratio of PB emission from young mice, aged mice, and aged mice + abscisic acid treatment group.

[0026] Figure 2 This is the result of using immunofluorescence staining to detect the effect of abscisic acid on improving the ovarian reserve function of aged mice in Example 3, wherein AB used immunofluorescence staining to detect the expression level and quantitative analysis of AMH in the ovaries of young mice, aged mice, and aged mice + abscisic acid treatment group, scale = 20 μm.

[0027] Figure 3 In Example 4, immunofluorescence staining was used to detect the role of abscisic acid in improving DNA damage in oocytes of aged mice; wherein, AB used immunofluorescence staining to detect the expression level and quantitative analysis of γH2AX in the ovaries of young mice, aged mice, and aged mice + abscisic acid treatment group, scale = 20 μm.

[0028] Figure 4 These are the results of using TMRE staining to detect the effect of abscisic acid in improving the mitochondrial membrane potential level of oocytes in aged mice in Example 5, wherein A is a confocal microscope image of the mitochondrial membrane potential level of oocytes in young mice, aged mice, and aged mice + abscisic acid treated mice detected by TMRE live cell dye, and B is the result of quantification and comparison of the mitochondrial membrane potential level of oocytes in each group of mice, and scale bar = 20 μm.

[0029] Figure 5 This is the result of using immunofluorescence staining to detect the effect of abscisic acid in improving the expression of Ki-67, an indicator related to the proliferation of ovarian granulosa cells in aged mice, in Example 6. Among them, AB used immunofluorescence staining to detect the expression level and quantitative analysis of Ki-67 in the ovaries of young mice, aged mice, and aged mice + abscisic acid treatment group, and the scale bar = 20μm.

[0030] Figure 6 In Example 7, Western Blot was used to analyze the effect of abscisic acid on improving the expression of mitochondrial dynamics-related proteins (DRP1 and OPA1) and nuclear gene-encoded proteins (Mitofilin and SDHA) in ovarian granulosa cells of aged mice; AC was used to detect and quantitatively analyze the expression levels of DRP1 and OPA1 in the ovaries of young mice, aged mice, and aged mice + abscisic acid treatment group using Western Blot; DF was used to detect and quantitatively analyze the expression levels of Mitofilin and SDHA in the ovaries of young mice, aged mice, and aged mice + abscisic acid treatment group using Western Blot. DETAILED DESCRIPTION

[0031] The technical solution of the present invention is described in detail below through specific embodiments.

[0032] Example 1 Construction of aging group mice + abscisic acid model

[0033] Six healthy female mice aged 2 months were selected as the young group mice, denoted as Young.

[0034] Six healthy female mice aged 10 months were selected as the aging group mice, denoted as Aged.

[0035] Six healthy female mice aged 10 months were given intraperitoneal injection of abscisic acid aqueous solution (once a day, 30 mg / kg each time) for 14 days as the aging group + abscisic acid treatment group, recorded as Aged+ABA.

[0036] The young mice, the aged mice, and the aged mice + abscisic acid treatment group were raised in the same way. In the young mice and the aged mice, the intraperitoneal injection of abscisic acid aqueous solution was replaced by an equal amount of normal saline.

[0037] Example 2

[0038] Phase contrast microscopy observation and detection of the role of abscisic acid in improving oocyte maturation disorders in aged mice

[0039] 1. Take each group of mice in Example 1 and kill them quickly by cervical dislocation, and then spray the abdomen of the mice with 75% disinfectant alcohol for disinfection. Pull up the mouse abdominal skin with tweezers, cut the mouse skin and peritoneum with scissors, and find the ovarian tissue on both sides along the uterus; after taking out the ovaries, place them in F12 culture medium for thorough washing, chop the ovarian tissue, and collect the oocytes of the young group mice, the aged group mice, and the aged group mice + abscisic acid treatment group under a stereomicroscope;

[0040] 2. Under the stereomicroscope, use a homemade mouth pipette to select oocytes in the GV stage with good morphology, transfer the selected GV stage oocytes into F12 culture medium, rinse them repeatedly several times to remove excess impurities;

[0041] 3. Then transfer to pre-warmed M16 culture droplets and place at 37°C, 5% CO 2 , cultured in an incubator with saturated humidity, and the oocytes were cultured for 2h, 8h, 9.5h, 10h, and 14h, which corresponded to the GVBD stage, MI, the late first meiotic division (anaphase I, AI), the late first meiotic division (telophase I, TI), and MII stages of the oocyte meiotic maturation process, respectively. According to the experimental needs, oocytes at different stages were collected, observed under a phase contrast microscope, and corresponding tests and statistics were performed. Statistical analysis was performed using AVONA analysis, and graphPad was used for plotting (data are mean ± SEM, *p<0.05, **p<0.01, ***p<0.001). The results are shown in Figure 1 shown.

[0042] Figure 1 The results of observation and detection of abscisic acid in improving oocyte maturation disorder in aged mice by phase contrast microscopy in Example 2, wherein A represents the GV stage oocytes from young mice, aged mice, and aged mice + abscisic acid treatment group cultured in M16 medium for 14 h to observe PB emission, scale = 100 μm; BD represents the total number of oocytes, the ratio of GVBD and the ratio of PB emission from young mice, aged mice, and aged mice + abscisic acid treatment group.

[0043] Depend on Figure 1It can be seen that abscisic acid significantly improved the oocyte acquisition rate, GVBD rate and first polar body extrusion rate of the aged mice, and can improve the oocyte maturation disorder of aged mice.

[0044] Example 3 Immunofluorescence staining to detect the role of abscisic acid in improving the level of AMH, a marker of ovarian reserve function in aged mice

[0045] The ovarian tissues of the mice in each group of Example 1 were taken and made into paraffin sections, which were sequentially placed in xylene I for 30 min, xylene II for 30 min, 100% anhydrous ethanol I for 10 min, 100% anhydrous ethanol II for 10 min, 90% ethanol aqueous solution for 5 min, 80% ethanol aqueous solution for 5 min, 70% ethanol aqueous solution for 3 min, and then washed with PBS on a shaker for 3 min, and repeated 3 times for sufficient washing;

[0046] First, add 200 ml of PBS solution to a 2L glass cup, heat it in a microwave oven for 2 minutes, then add 600 μl of Triton and stir thoroughly, cool to room temperature, then put the above slices in the permeabilization solution, keep it away from light for 45 minutes, and then wash it with PBS 3 times, 3 minutes each time;

[0047] Then put the slices into a wet box, add appropriate amount of water in advance to prevent the slices from drying, then use a brush to circle the tissues one by one, then drop appropriate amount of 10% donkey serum on each tissue, and finally put them into a 37℃ oven for sealing for 2 hours;

[0048] Then, the required primary antibodies were added to the tissue sections of the young group mice, the aged group mice, and the aged group + abscisic acid treatment group mice, respectively, and the sections were placed in a humidified box and incubated overnight in a 4°C refrigerator. The next day, the sections were washed 3 times with PBS for 3 minutes each time to fully remove the residual primary antibodies in the tissues. Subsequently, the corresponding secondary antibodies were added under light-proof conditions and incubated at 37°C for 2 hours. Finally, the sections were washed again with PBS 3 times for 3 minutes each time;

[0049] Then add anti-fluorescence quenching agent to each tissue, add a small amount of colorless nail polish to the four corners of the slide, and then slowly cover the slide with a cover slip. The entire sealing process should be kept away from light, and finally stored in a -20℃ refrigerator away from light. Immunofluorescence staining was used for detection, and Image J was used to quantitatively analyze the fluorescence intensity bands shown. AVONA analysis was used for statistical analysis, and graphPad was used for plotting (data are mean ± SEM, *p<0.05, **p<0.01). The results are shown in Figure 2 shown.

[0050] Figure 2This is the result of using immunofluorescence staining to detect the effect of abscisic acid on improving the ovarian reserve function of aged mice in Example 3, wherein AB used immunofluorescence staining to detect the expression level and quantitative analysis of AMH in the ovaries of young mice, aged mice, and aged mice + abscisic acid treatment group, scale = 20 μm.

[0051] Depend on Figure 2 It can be seen that the AMH expression level in the Aged+ABA group was significantly higher than that in the Aged group, indicating that abscisic acid significantly improved the ovarian reserve function of the aged mice and could delay ovarian aging.

[0052] Example 4 Immunofluorescence staining to detect the role of abscisic acid in improving oocyte DNA damage in aging mice

[0053] The oocytes of the young group mice, the aged group mice, and the aged group mice + abscisic acid treatment group were collected (the steps were the same as steps 1-2 of Example 2); then, after being quickly washed once in the cleaning solution, the oocytes were immediately transferred to the fixing and permeabilization solution for 50 minutes; then, the oocytes were washed in the cleaning solution for 5 minutes, transferred to the blocking solution containing 2% BSA, and incubated at room temperature for 1 hour;

[0054] Then the oocytes were transferred to the primary antibody diluent, incubated in a humidified chamber at 4°C overnight or at 37°C for 1 h, and then transferred to the secondary antibody diluent after washing, and incubated in a humidified chamber at 37°C for 1 h; after washing, the oocytes were transferred to the DAPI (10 μg / mL) solution, stained at room temperature for 10 min, and washed clean;

[0055] Select a suitable slide, add 5-10 μL of DABCO anti-fluorescence quencher to the corresponding central position of the slide, and add a small amount of support agent around it; then transfer the oocyte or early embryo to the DABCO liquid, cover it with a coverslip, and slowly press the coverslip with the tip of a gun under a stereoscope to press the oocyte or early embryo to the appropriate size, seal the slide, and observe and collect images using a fluorescence microscope or laser confocal microscope. Use Image J software to calculate the fluorescence value, use Image J to quantitatively analyze the fluorescence intensity shown, use AVONA analysis for statistical analysis, and use graphPad to draw graphs (data are mean ± SEM, ***p<0.001, ****p<0.0001). The results are shown in Figure 3 shown.

[0056] Figure 3 In Example 4, immunofluorescence staining was used to detect the role of abscisic acid in improving DNA damage in oocytes of aged mice; wherein, AB used immunofluorescence staining to detect the expression level and quantitative analysis of γH2AX in the ovaries of young mice, aged mice, and aged mice + abscisic acid treatment group, scale = 20 μm.

[0057] Depend on Figure 3 It can be seen that abscisic acid significantly reduced the degree of DNA damage in the oocytes of the aging group of mice, and the improvement of DNA damage can directly alleviate the maturation disorder of the oocytes.

[0058] Example 5 TMRE staining to detect the role of abscisic acid in improving the mitochondrial membrane potential level of aged mouse oocytes

[0059] To detect mitochondrial membrane potential, MII stage oocytes were placed in IVF medium, 5 mM TMRE reagent (prepared according to the instructions of TMRE mitochondrial membrane potential indicator) was added, and incubated at 37°C and 5% CO 2 Incubate in the incubator for 20 min; then wash three times with IVF culture medium, and collect oocyte images under a laser scanning confocal microscope. Fluorescence values ​​were calculated using Image J software, and the fluorescence intensity shown was quantitatively analyzed using Image J. Statistical analysis was performed using AVONA analysis, and graphPad was used for plotting (data are mean ± SEM, *p < 0.05, **p < 0.01). The results are shown in Figure 4 shown.

[0060] Figure 4 These are the results of using TMRE staining to detect the effect of abscisic acid in improving the mitochondrial membrane potential level of oocytes in aged mice in Example 5, wherein A is a confocal microscope image of the mitochondrial membrane potential level of oocytes in young mice, aged mice, and aged mice + abscisic acid treated mice detected by TMRE live cell dye, and B is the result of quantification and comparison of the mitochondrial membrane potential level of oocytes in each group of mice, and scale bar = 20 μm.

[0061] Depend on Figure 4 It can be seen that abscisic acid significantly improved the mitochondrial membrane potential level of oocytes in the aging group of mice, enhanced mitochondrial function, and further improved the quality and maturity of oocytes.

[0062] Example 6 Immunofluorescence staining to detect the role of abscisic acid in improving the expression of Ki-67, a proliferation-related indicator of ovarian granulosa cells in aged mice

[0063] The ovarian tissues of the mice in each group of Example 1 were taken and made into paraffin sections, which were sequentially placed in xylene I for 30 min, xylene II for 30 min, 100% anhydrous ethanol I for 10 min, 100% anhydrous ethanol II for 10 min, 90% ethanol aqueous solution for 5 min, 80% ethanol aqueous solution for 5 min, 70% ethanol aqueous solution for 3 min, and then washed with PBS on a shaker for 3 min, and repeated 3 times for sufficient washing;

[0064] First, add 200 ml of PBS solution to a 2L glass cup, heat it in a microwave for 2 minutes, then add 600 μl of Triton and stir thoroughly, cool to room temperature, then place the slices in the permeabilization solution, keep it away from light for 45 minutes, and then wash it with PBS three times, 3 minutes each time;

[0065] Then put the slices into a wet box, add appropriate amount of water in advance to prevent the slices from drying, then use a brush to circle the tissues one by one, then drop appropriate amount of 10% donkey serum on each tissue, and finally put them into a 37℃ oven for sealing for 2 hours;

[0066] Then, the primary antibody rabbit anti-Ki67 was added to the tissue sections of the young mice, the aged mice, and the aged mice + abscisic acid treatment group, respectively, and then placed in a wet box and kept in a refrigerator at 4°C overnight. The next day, the sections were washed with PBS three times, each time for 3 minutes, to remove the primary antibody on the tissue, and then the corresponding secondary antibody was added in the dark, and then incubated at 37°C for 2 hours. Finally, the sections were washed with PBS three times, each time for 3 minutes;

[0067] Then add anti-fluorescence quenching agent to each tissue, add a small amount of colorless nail polish to the four corners of the slide, and then slowly cover the slide with a cover slip. The entire sealing process should be kept away from light, and finally stored in a -20℃ refrigerator away from light. Immunofluorescence staining was used for detection, and Image J was used to quantify the fluorescence intensity bands shown. AVONA analysis was used for statistical analysis, and graphPad was used for plotting (data are mean ± SEM, *p < 0.05). The results are shown in Figure 5 shown.

[0068] Figure 5 This is the result of using immunofluorescence staining to detect the effect of abscisic acid in improving the expression of Ki-67, an indicator related to the proliferation of ovarian granulosa cells in aged mice, in Example 6. Among them, AB used immunofluorescence staining to detect the expression level and quantitative analysis of Ki-67 in the ovaries of young mice, aged mice, and aged mice + abscisic acid treatment group, and the scale bar = 20μm.

[0069] Depend on Figure 5 It can be seen that abscisic acid significantly improved the Ki-67 expression level of aging group mice and significantly improved the proliferation ability of ovarian granulosa cells.

[0070] Example 7 Using Western Blot to detect the role of abscisic acid in improving the expression of mitochondrial homeostasis-related proteins in ovarian granulosa cells of aged mice

[0071] Take the ovarian tissue of each group of mice in Example 1, add pre-cooled protein extraction reagent containing inhibitors (including a mixture of protease inhibitors, PMSF and phosphatase inhibitors), and lyse on ice for 20 minutes; place the lysate in a pre-cooled centrifuge, centrifuge at 12,000 rpm for 15 minutes, discard the precipitate, and immediately transfer the supernatant (i.e., the protein of each group) to a new centrifuge tube, heat it at 100° C. for 10 minutes, and then store it at -80° C. for later use;

[0072] Protein samples from each group were separated on 12% SDS-polyacrylamide gel electrophoresis, then transferred to nitrocellulose membranes and detected with Mitofilin, SDHA, DRP1, and OPA1 antibodies; the results are shown in Figure 6 shown.

[0073] Figure 6 In Example 7, Western Blot was used to analyze the effect of abscisic acid on improving the expression of mitochondrial power-related proteins (DRP1 and OPA1) and nuclear gene-encoded proteins (Mitofilin and SDHA) in ovarian granulosa cells of aged mice; wherein AC was detected and quantitatively analyzed by Western Blot for the expression levels of DRP1 and OPA1 in the ovaries of young mice, aged mice, and aged mice + abscisic acid-treated groups; DF was detected and quantitatively analyzed by Western Blot for the expression levels of Mitofilin and SDHA in the ovaries of young mice, aged mice, and aged mice + abscisic acid-treated groups. The bands shown were quantitatively analyzed using Image J, statistical analysis was performed using AVONA analysis, and graphPad was used for plotting (data are mean ± SEM, *p < 0.05).

[0074] Depend on Figure 6 It can be seen that compared with the young group, the expression of Mitofilin in the aging group did not change, but the expression of DRP1 decreased, while the expression of OPA1 and SDHA increased significantly; this phenomenon can be reversed after abscisic acid treatment; abscisic acid significantly improved the expression levels of DRP1, OPA1 and SDHA in the aging group mice, effectively promoted the recovery of mitochondrial dynamic balance (fission and fusion process), and maintained mitochondrial homeostasis. At the same time, this effect further optimized the function of ovarian granulosa cells.

[0075] In summary, the present invention discovered for the first time that abscisic acid can upregulate the AMH level in aged ovarian tissue and significantly improve the ovarian reserve function of aged mice; and abscisic acid can also improve oocyte DNA damage, thereby improving or treating oocyte maturation disorders. Studies have found that the mitochondrial membrane potential in the oocytes of aged mice is significantly reduced, while the expression of Ki-67 in ovarian granulosa cells is downregulated, and the dynamic balance of mitochondria in ovarian granulosa cells is also disturbed; and abscisic acid can reverse these abnormal phenomena. The above results show that abscisic acid and its related pharmaceutical preparations have significant effects in improving oocyte maturation disorders and improving ovarian reserve function, and show great potential in repairing ovarian damage and delaying ovarian aging, and have broad prospects for clinical application.

[0076] The above description is only a preferred specific implementation manner of the present invention, but the protection scope of the present invention is not limited thereto. Any technician familiar with the technical field can make equivalent replacements or changes according to the technical scheme and inventive concept of the present invention within the technical scope disclosed by the present invention, which should be covered by the protection scope of the present invention.

Claims

1. A use of abscisic acid in the preparation of a drug for improving or treating oocyte maturation disorders.

2. The use of abscisic acid according to claim 1 in the preparation of a medicament for improving or treating oocyte maturation disorders, characterized in that: Improve or treat oocyte maturation disorders by improving oocyte DNA damage.

3. The use of abscisic acid in the preparation of a medicament for improving or treating oocyte maturation disorders according to claim 1 or 2, characterized in that: The oocyte is an oocyte in an aged ovary.

4. Use of abscisic acid in the preparation of a drug for improving ovarian reserve function.

5. The use of abscisic acid in the preparation of a drug for improving ovarian reserve function according to claim 4, characterized in that: The ovary is an aged ovary.

6. A drug, characterized in that include: Abscisic acid and a pharmaceutically acceptable carrier or excipient; The medicine can improve or treat oocyte maturation disorders and enhance ovarian reserve function.

7. The drug according to claim 6, characterized in that: The dosage form of the drug is oral preparation and injection preparation.

8. A health product, characterized in that: include: Abscisic acid and a pharmaceutically acceptable carrier or excipient; The health product can improve or treat oocyte maturation disorders and enhance ovarian reserve function.

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