Application of MY-1B in preparation of medicine for improving ovarian aging

Abstract

The invention discloses application of MY-1B in preparation of medicines for improving ovarian aging, belongs to the technical field of biological medicines, and aims to solve the problems of how to effectively delay ovarian aging and improve ovarian functions. Experiments prove that the mRNA m5C modification level of ovarian granular cells in the ovarian aging process is increased, and the expression level of methylated transferase Nsun2 is increased. Ovarian aging caused by mRNA m5C modification level increase can be improved by supplementing the MY-1B, which is embodied in that the MY-1B can reduce the mRNA m5C modification level of aging nest granular cells; the MY-1B can be used for effectively correcting the orbital cycle disorder of the aged ovary and delaying the decline of the ovarian function; the MY-1B plays an important role in reducing the apoptosis level of granulosa cells in senescent ovaries; the MY-1B can improve the storage capacity of ovaries and enhance the quality of oocytes, so that the fertilization rate and the embryonic development potential of the oocytes are improved.

Description

Technical Field

[0001] The present invention belongs to the technical field of molecular biomedicine, and in particular relates to the application of MY-1B in the preparation of drugs for improving ovarian aging. Background Art

[0002] Ovarian aging (OA) is a physiological process that occurs naturally in women as they age. It is mainly manifested by the gradual decline of ovarian function, which directly affects women's fertility and overall health. Ovarian aging is usually accompanied by a decrease in ovarian reserve, a decrease in the number and quality of oocytes, and ovarian endocrine dysfunction. With age, the ovarian function in women gradually weakens, and the fluctuation and instability of hormone levels will trigger a series of physiological changes, leading to irregular menstruation, follicular development disorders and other manifestations, which may eventually lead to menopause. With the decline of ovarian function, the endocrine system in the body becomes dysregulated, which may lead to dysfunction of multiple organs and systems, thereby increasing the risk of diseases such as ovarian cancer, breast cancer, cardiovascular disease and type 2 diabetes. Ovarian aging is also closely related to delayed childbearing. Especially in modern society, more and more women choose to postpone childbearing, but increasing age is accompanied by a higher risk of infertility, miscarriage and birth defects. Therefore, ovarian aging is not only a physiological phenomenon, but has also become a social issue that affects the fertility of the national population.

[0003] Ovarian aging is mainly manifested by a decrease in the number and quality of oocytes and apoptosis of granulosa cells. With age, the reserve of oocytes in the ovaries gradually decreases, and the quality decreases significantly, which is mainly affected by factors such as DNA damage, telomere shortening, oxidative stress, and mitochondrial dysfunction. The accumulation of DNA damage and the gradual shortening of telomeres lead to a decrease in the genomic stability of oocytes, the increase in reactive oxygen species (ROS) further damages the function of cells, and the decline of mitochondrial function affects the energy metabolism of cells, ultimately affecting ovarian function and fertility. With the accumulation of these damages, the endocrine function of the ovaries and the quality of oocytes further deteriorate, making women face fertility difficulties. Although the relevant mechanisms of ovarian aging have been studied to a certain extent, the specific molecular regulatory mechanisms are still not fully understood. How to effectively delay ovarian aging and improve ovarian function is still an urgent problem to be solved.

[0004] mRNA 5 C modification (5-methylcytosine modification) is a post-transcriptional modification of RNA that occurs mainly on cytosine (C) residues in mRNA molecules and is usually catalyzed by methyltransferases (such as proteins of the NSUN family). 5C modification plays an important role in RNA stability, transport, translation regulation, and other processes, and is closely related to a variety of biological processes and diseases. We found that during ovarian aging, mRNA m 5 C modification level increased significantly and m 5 C-modifying methyltransferase Nsun2 The increased expression level indicates that the intervention Nsun2 mRNA 5 Increased levels of C modification may help alleviate ovarian aging. MY-1B is an RNA methyltransferase Nsun2 Covalent inhibitor of MY-1B stereoselective ligand Nsun2 Active site cysteine ​​residue (C271). Studies have shown that MY-1B can significantly reduce m 5 C level, so MY-1B has the potential to be used in the treatment of m 5 Abnormally increased C levels have the potential to cause diseases. However, its role in the ovarian aging process and its use as a target for the treatment of ovarian aging have not been reported. Summary of the invention

[0005] In view of the above problems, the present application provides the use of MY-1B in the preparation of a drug for improving ovarian aging. Supplementation of MY-1B can improve the expression of mRNA mRNA in ovarian granulosa cells. 5 Increased C modification levels lead to ovarian aging, which is reflected in the fact that MY-1B can reduce the mRNA m 5 C modification level, proving that MY-1B can be used as an effective ingredient in the preparation of drugs to improve ovarian aging.

[0006] In a first aspect, the present invention provides the use of MY-1B in the preparation of a drug for preventing and / or delaying ovarian aging, wherein the molecular structure of the MY-1B is: .

[0007] In some preferred examples of this aspect, the MY-1B can reduce the m 5 C modification level.

[0008] In some preferred examples of this aspect, the ovarian aging is manifested as disordered estrous cycle of animals.

[0009] In some preferred examples in this aspect, the ovarian aging is manifested by changes in serum neutral hormones, specifically: a decrease in E2 level and AMH level, and an increase in FSH level.

[0010] In some preferred examples of this aspect, the ovarian aging is manifested by a significant decrease in the number of primordial follicles, primary follicles, secondary follicles, antral follicles and growing follicles of the ovaries, and an increase in the number of atretic follicles.

[0011] In some preferred examples of this aspect, the ovarian aging is manifested by a decrease in the number of ovarian oocytes and a decrease in the quality of ovarian oocytes; wherein the ovarian oocyte quality evaluation indicators are: the proportion of oocytes discharged from the first polar body and the proportion of fragmented oocytes.

[0012] In some preferred examples of this aspect, the ovarian aging is manifested by a decrease in the fertilization rate of ovarian oocytes and a decrease in the embryonic development potential; wherein the evaluation indicators of embryonic development potential include: 2-cell occurrence rate and morula formation rate.

[0013] In a second aspect, the present invention provides a method for providing an ovarian granulosa cell m comprising MY-1B. 5 C modification inhibitor, said inhibitor enables expression of m 5 Ovarian granulosa cells with elevated levels of C modification are contacted with a therapeutically effective amount of MY-1B.

[0014] In a third aspect, the present invention provides a drug for preventing or delaying ovarian aging, comprising MY-1B and a pharmaceutically acceptable carrier or excipient.

[0015] Wherein, the drug is used to reduce or inhibit m 5 C modified activity.

[0016] In some preferred examples in this aspect, the carrier or excipient includes (but is not limited to): diluents such as starch, pregelatinized starch, lactose, dextrin, sucrose, MCC, mannitol, sorbitol, inorganic calcium salts (calcium hydrogen phosphate / calcium carbonate / calcium sulfate); binders such as water, ethanol, starch slurry, hydroxycellulose, PVP, PEG, gelatin; lubricants such as MS, micropowder silica gel, talc, hydrogenated vegetable oil, PEG, sodium / magnesium lauryl sulfate, etc.

[0017] Compared with the prior art, the present invention has the following advantages: The present invention uses drugs on aged mice, and experiments prove that the use of MY-1B can reduce the mRNA m 5 Abnormally increased C modification levels correct estrous cycle disorders, improve hormone levels in aged mice (manifested by increased E2 and AMH), increase ovarian reserve in aged mice and reduce follicular atresia, reduce aging or apoptosis of ovarian granulosa cells in aged mice, improve the quality of aged ovarian oocytes and increase their fertilization rate and embryo development potential. It is highly safe and has great application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 Ovarian m of young and old mice 5 C level test results. Among them, A is the ovarian slice m 5 C Representative images of immunofluorescence staining and statistical results of granulosa cell fluorescence density, scale bar is 100 μm. B is the m 5 Statistical results of C level detection (ELISA). C is m of ovarian granulosa cells 5 Statistical results of C level detection (ELISA).

[0019] Figure 2 The following are the results of ovarian morphology and function test of three groups of mice. A is a representative picture of vaginal smear of mouse estrous cycle and the statistical results of estrous cycle of three groups of mice. B is a representative picture of ovarian morphology of three groups of mice. C is the statistical results of serum AMH hormone levels of three groups of mice. D is the statistical results of serum E2 hormone levels of three groups of mice.

[0020] Figure 3 The results of ovarian follicle counting in three groups of mice. A is a representative picture of HE staining of ovarian sections of three groups of mice, with a scale of 200 μm. B is a representative picture of ovarian follicles at all levels and atretic follicles in mice, with a scale of 20 μm, as well as the statistical results of the three groups of mice.

[0021] Figure 4 The following are the results of immunofluorescence staining related to proliferation and apoptosis of ovarian slices of three groups of mice. A is a representative picture of Ki67 immunofluorescence staining of ovarian slices and the statistical results of granulosa cell fluorescence density, with a scale bar of 100 μm. B is a representative picture of γH2AX immunofluorescence staining of ovarian slices and the statistical results of granulosa cell fluorescence density, with a scale bar of 100 μm. C is a representative picture of TUNEL staining of ovarian slices and the statistical results of granulosa cell fluorescence positivity, with a scale bar of 100 μm. D is Nsun2 , Fbln1 , Cdkn1a , Cdkn1b , IL-6 , IL-8 Statistical results of mRNA expression level detection.

[0022] Figure 5 The superovulation test results of three groups of mice. A is a representative picture of superovulated oocytes of three groups of mice, with a scale of 50 μm. B is the statistical results of superovulated oocyte counts and first polar body extrusion rates of three groups of mice.

[0023] Figure 6The results of oocyte quality staining test of three groups of mice. Among them, A is the representative pictures and statistical results of mitochondrial staining of oocytes of three groups of mice, and the scale bar is 50μm. B is the representative pictures and statistical results of mitochondrial membrane potential staining of oocytes of three groups of mice, and the scale bar is 50μm. C is the representative pictures and statistical results of reactive oxygen species staining of oocytes of three groups of mice, and the scale bar is 50μm. D is the representative pictures and statistical results of apoptosis signal staining of oocytes of three groups of mice, and the scale bar is 50μm. E is the representative picture of spindle and chromosome arrangement of oocytes of three groups of mice, and the scale bar is 50μm. F is the statistical result of the abnormal spindle ratio of oocytes of three groups of mice. G is the statistical result of the disordered chromosome arrangement ratio of oocytes of three groups of mice.

[0024] Figure 7 The figures are the oocyte fertilization rate and embryo development potential of the two groups of mice, as well as the test results of mouse fertility. Among them, A is the microscopic morphological comparison of the fertilized eggs of the two groups of mice at each stage of development and the statistical results of the fertilization rate and morula formation rate. B is the statistical result of the pregnancy rate of the two groups of mice. C is the statistical result of the number of litters per litter of the two groups of mice. DETAILED DESCRIPTION

[0025] In order to make the purpose, technical scheme and advantages of the present invention clearer, the present invention is further described in detail below in conjunction with embodiments and accompanying drawings. It should be understood that the specific embodiments described are only for explaining the specific application of the present invention, and are not intended to limit the scope of the present invention. Through these embodiments, it is intended to help understand the principles, operating methods and advantages of the present invention, but it is not excluded that appropriate adjustments and modifications can be made during the implementation of the present invention.

[0026] The experimental animals used in this study were C57 mice, purchased from Beijing Sibeifu Laboratory Animal Technology Co., Ltd., including female mice aged 8 weeks and 10 months, and male mice aged 3 months. The housing environment of the mice was set at a temperature of 22-25°C, a humidity of no more than 50%, and a 12-hour light-dark cycle. During the experiment, the mice received sufficient drinking water and feed, and the bedding was changed once a week. Unless otherwise specified, the experimental methods in this study were standard techniques in the field or performed under the conditions recommended by the supplier.

[0027] Embodiment 1: Twenty 2-month-old C57 female mice were used as the young group (Young), and 40 10-month-old C57 female mice were randomly divided into two groups: the aged control group (Aged) and the aged group injected with MY-1B (Aged+MY-1B).

[0028] Five mice in the Young group and five mice in the Aged group were randomly selected and sacrificed to collect ovaries and ovarian granulosa cells for ovarian section immunofluorescence staining and ELISA detection. The results are shown in Figure 1 .

[0029] from Figure 1 In A, we can see the ovarian granulosa cells of aged mice. 5 C immunofluorescence density increased significantly. Figure 1 In B and C, we can see the aging ovarian granulosa cells m 5 C modification level was significantly increased.

[0030] Figure 1 The results showed that granulosa cells m 5 C modification level was significantly increased.

[0031] Embodiment 2: The aged group injected with MY-1B was intraperitoneally injected with MY-1B at 20 mg / kg / 3d, and the aged control group was injected with an equal amount of MY-1B dissolving agent every day, which was a mixture of 10% DMSO + 40% PEG300 + 5% Tween-80 + 45% Saline.

[0032] Five weeks after the injection of the old group and the old group injected with MY-1B, 10 mice were randomly selected from each group. Vaginal exfoliated cells were collected every morning for 30 consecutive days to make smears, and hematoxylin and eosin staining was performed to detect their estrous cycle. These 10 mice were still used in other subsequent experiments. Five mice were randomly sampled and killed, and serum and ovaries were collected. The collected serum was tested by ELISA (Elabscience) for AMH and E2. The results are shown in Figure 1 .

[0033] from Figure 2 In A, we can see that the estrous cycle irregularity rate of the old group is significantly higher than that of the young group; and the estrous cycle regularity rate of the old group of mice injected with MY-1B is higher than that of the old group. Figure 2 In B, we can see that the ovaries of the MY-1B-injected aged mice are significantly larger than those of the aged mice. Figure 2 Middle C and Figure 2 As can be seen in D, the serum hormone AMH and E2 levels of the old group mice injected with MY-1B were significantly higher than those of the old group.

[0034] Figure 2 The results showed that injection of MY-1B could correct estrous cycle disorders and increase serum AMH and E2 hormone levels in aged mice.

[0035] Embodiment 3: Five weeks after the intraperitoneal injection of the old group and the old group injected with MY-1B, three mice were randomly selected from each group, together with the young control group that did not undergo the operation. The ovaries of the three groups of mice were fixed in 4% formaldehyde and made into 5μm paraffin sections. After HE staining, the morphology of the primordial follicles, primary follicles, secondary follicles, antral follicles and atretic follicles of the three groups of mice were observed under a microscope and counted. The results are shown in Figure 3 .

[0036] from Figure 3 As can be seen from A and B, during the process of ovarian aging, the number of follicles at each level decreases. The number of primordial follicles, primary follicles, secondary follicles, and antral follicles in the ovaries of MY-1B-injected old mice are more than those in the control old mice, while the number of atretic follicles is less than that in the control old mice.

[0037] Figure 3 The results showed that injection of MY-1B could improve the ovarian follicle reserve capacity of aged mice.

[0038] Embodiment 4: The ovarian sections of the elderly group, the elderly group injected with MY-1B, and the young control group were subjected to immunofluorescence staining to detect Ki67, γH2AX, and TUNEL staining. At the same time, RNA was extracted from the collected ovaries for fluorescence quantitative PCR detection. The results are shown in Figure 4 .

[0039] from Figure 4 In A, we can see that the ovarian proliferation rate (Ki67 + ) Granulocytes decreased significantly, while those in the MY-1B-injected elderly group increased; Figure 4 Figure B shows that the ovarian DNA damage (γH2AX) in the elderly group was higher than that in the young group. + ) Granulocytes increased significantly, while those in the MY-1B-injected elderly group decreased; Figure 4 Middle C shows that the number of apoptotic granulosa cells in the ovaries of the elderly group increased significantly compared with the young group, while it decreased in the elderly group injected with MY-1B; Figure 4 D in the middle shows the elderly group after MY-1B injection Nsun2 The expression of genes related to aging was significantly lower than that in the elderly control group.

[0040] Figure 4 The results showed that injection of MY-1B could correct the m 5 C level, thereby improving the apoptosis of granulosa cells and promoting proliferation, delaying the aging level.

[0041] Embodiment 5: Superovulation experiment was performed on 6 mice randomly selected from each group of the control aged group, the MY-1B injected aged group and the young control group. The specific operation was as follows: 10 IU of pregnant mare serum gonadotropin (PMSG) was injected into the three groups of mice, and 10 IU of human chorionic gonadotropin (hCG) was injected 48 hours later. After 14 hours, the cumulus oocyte complexes (COCs) discharged into the ampulla of the oviduct were removed, and then 0.1% hyaluronidase was used to remove the granulosa cells around the oocytes. The morphology of the oocytes was observed under a microscope and counted.

[0042] from Figure 5 It can be seen that Figure 5 A in the middle shows that the number of ovulations in the control elderly group decreased compared with the young group, while the number of ovulations in the MY-1B-injected elderly group increased compared with the control elderly group; Figure 5 Figures B and C show that the number of oocytes released from the first polar body in the control old group decreased compared with the young group, while the number of oocytes in the old group injected with MY-1B increased.

[0043] Figure 5 The results showed that injection of MY-1B could improve the number and normal ratio of superovulation in aged mice.

[0044] Embodiment 6: The oocytes of three groups of mice were obtained by the same method as in Example 3. The obtained oocytes were subjected to the following operations: 1. Mitotracker staining of oocytes: Use Opti-MEM medium to dilute Mito-Tracker-Red to 200nM, transfer oocytes into the droplet, and incubate at 37℃ in the dark for 30min. Transfer to PBS containing 0.1% BSA and wash three times, then transfer to a glass-bottomed confocal dish and cover with paraffin oil. Observe and collect images under a confocal microscope within 1h.

[0045] 2. Oocyte mitochondrial membrane potential staining: Use the mitochondrial membrane potential detection kit for the experiment. Take an appropriate amount of 1μl JC-10 (200x) probe and dilute it with 160μl ultrapure water, and add 40μl JC-10 staining buffer to mix and make droplets. Transfer the oocytes into the droplets and incubate them at 37℃ in the dark for 20 min. After transferring to the staining buffer (1x) and washing 3 times, transfer to a glass-bottomed confocal dish and cover it with paraffin oil. Observe and collect images under a confocal microscope within 1 hour.

[0046] 3. Oocyte reactive oxygen staining: Dilute 2,7-dichlorofluorescein diacetate (DCFH-DA) to 10 μM using Opti-MEM medium, transfer the oocytes into the droplets, and incubate at 37°C in the dark for 30 minutes. Transfer to PBS containing 0.1% BSA and wash three times, then transfer to a glass-bottomed confocal dish and cover with paraffin oil. Observe and collect images under a confocal microscope within 1 hour.

[0047] 4. Detection of oocyte apoptosis signal: The Annexin V-mCherry apoptosis detection kit was used for the experiment. According to the instructions, 5 μl of Annexin V-mCherry was added to 195 μl of binding solution. The oocytes were transferred into the freshly prepared Annexin V-mCherry binding solution and stained at room temperature in the dark for 20 min. After washing three times in PBS containing 0.1% BSA, the oocytes were transferred into a glass-bottomed confocal dish and covered with paraffin oil. The oocytes were observed and images were collected under a confocal microscope within 1 hour.

[0048] 5. The oocytes were fixed in paraformaldehyde for 30 minutes, permeabilized with 0.5% TritonX-100 for 20 minutes, blocked with 1% BSA for 1 hour, and incubated with 1:200 anti-α-tubulin monoclonal antibody at 4°C overnight. After washing with IVF solution three times, incubated with FITC-conjugated secondary antibody at room temperature for 1 hour, and then stained with DAPI for nuclei, transferred into 10μ antifade droplets, and observed spindle morphology and chromosome arrangement under Zeiss laser confocal LSM880 microscope. Results are shown in Figure 6 .

[0049] from Figure 6 As can be seen in middle A, compared with control aged mice, the abnormal mitochondrial aggregation and distribution in the oocytes of MY-1B-injected aged mice were reduced; Figure 6 Middle B shows that compared with the control aged mice, the mitochondrial membrane potential of the oocytes of the aged mice injected with MY-1B was significantly increased; Figure 6 Middle C shows that compared with the control aged mice, the reactive oxygen level in the oocytes of the aged mice injected with MY-1B was significantly reduced; Figure 6 D in the middle shows that compared with the control aged mice, the apoptosis signal level of the oocytes of the aged mice injected with MY-1B was significantly reduced; Figure 6 Figures E, F, and G show that the incidence of abnormal spindles and chromosome disorder in oocytes of MY-1B-injected aged mice was significantly reduced compared with that of control aged mice.

[0050] Figure 6 The results showed that injection of MY-1B could improve the quality of oocytes in aged mice.

[0051] Embodiment 7: The COCs obtained in Example 3 were fertilized in vitro with sperm of 3-month-old male mice that had been treated with in vitro capacitation in IVF droplets for 6 hours, and then the fertilized eggs were transferred to KSOM culture droplets, washed and transferred to new KSOM droplets, and cultured for another 6 hours. The pronuclear formation was observed, and the fertilization rate (2 cells) and morula rate were counted. The above three groups of mice were subjected to a continuous 8-week cage test and their pregnancy rate and average litter size were counted. The results are shown in Figure 7 .

[0052] from Figure 7 As can be seen in A, B and C, compared with the old group, the fertilization rate and morula rate of oocytes in the old group injected with MY-1B were significantly increased. The pregnancy rate and average litter size of the old group injected with MY-1B were significantly higher than those of the old control group.

[0053] Figure 7 The results showed that injection of MY-1B could improve the fertilization rate of oocytes and embryonic development potential of aged mice, as well as improve the fertility of aged mice.

[0054] The above are only a few embodiments of the present application and do not constitute any form of limitation to the present application. Although the present application is disclosed as above with preferred embodiments, it is not intended to limit the present application. Any technician familiar with the profession, without departing from the scope of the technical solution of the present application, using the technical content disclosed above to make slight changes or modifications are equivalent to equivalent implementation cases and fall within the scope of the technical solution.

Claims

1. Application of MY-1B in the preparation of drugs for preventing and / or delaying ovarian aging.

2. The use according to claim 1, characterized in that: The MY-1B can reduce the m 5 C modification level.

3. The use according to claim 1, characterized in that: The manifestations of ovarian aging are decreased proliferation, increased apoptosis and DNA damage of granulosa cells.

4. The use according to claim 3, characterized in that: The ovarian aging is manifested as disordered estrous cycles in animals.

5. The use according to claim 4, characterized in that: The ovarian aging is manifested by changes in serum neutral hormones, specifically: a decrease in E2 and AMH levels, and an increase in FSH levels.

6. The use according to claim 4, characterized in that: The manifestation of ovarian aging is a significant decrease in the number of primordial follicles, primary follicles, secondary follicles, antral follicles and growing follicles of the ovaries, and an increase in the number of atretic follicles.

7. The use according to claim 4, characterized in that: The ovarian aging is manifested by a decrease in the number of ovarian oocytes and a decrease in the quality of ovarian oocytes; wherein the ovarian oocyte quality evaluation indicators are: the proportion of oocytes discharged from the first polar body and the proportion of fragmented oocytes.

8. The use according to claim 4, characterized in that: The ovarian aging is manifested by a decrease in the fertilization rate of ovarian oocytes and a decrease in the embryonic development potential; wherein the evaluation indicators of the embryonic development potential include: 2-cell occurrence rate and morula formation rate.

9. A MY-1B-containing protein in ovarian granulosa cells 5 C modification inhibitor, characterized in that The inhibitor enables expression of m 5 Ovarian granulosa cells with elevated levels of C modification are contacted with a therapeutically effective amount of MY-1B.

10. A drug for preventing or delaying ovarian aging, characterized in that: It comprises MY-1B and a pharmaceutically acceptable carrier or excipient.

11. The drug according to claim 10, characterized in that The drug is used to reduce or inhibit m 5 C modified activity.

Images