Application of MY-1B in the preparation of drugs for improving ovarian senescence

Abstract

The present invention discloses the application of MY-1B in the preparation of drugs for improving ovarian senescence, belonging to the technical field of biomedicine, aiming to solve the problem of how to effectively delay ovarian senescence and improve ovarian function. Experiments have proved that during ovarian senescence, the mRNA m<supgt;5< / supgt;C modification level and the expression level of methyltransferase in ovarian granulosa cells increase. Supplementation with MY-1B can improve the condition caused by mRNA m Nsun2 expression.

Description

Technical Field

[0001] The present invention belongs to the technical field of molecular biomedicine, and particularly 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 naturally occurs 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 reduction in ovarian reserve, a decrease in the quantity and quality of oocytes, and disorders in the endocrine function of the ovary. As women age, the ovarian function in their bodies gradually weakens, and the fluctuations and instability of hormone levels can trigger a series of physiological changes, resulting in manifestations such as irregular menstruation and follicle development disorders, and may ultimately lead to menopause. With the decline of ovarian function, the endocrine system in the body becomes disordered, which may cause dysfunctions in multiple organs and systems, thereby increasing the risk of diseases such as ovarian cancer, breast cancer, cardiovascular diseases, 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 with increasing age, there are higher risks of infertility, miscarriage, and birth defects. Therefore, ovarian aging is not only a physiological phenomenon but also a social problem that affects the national fertility.

[0003] Ovarian aging is mainly manifested by a decrease in the quantity and quality of oocytes and apoptosis of granulosa cells. As women age, the reserve of oocytes in the ovary gradually decreases, and the quality significantly declines, 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 cell functions, and the decline of mitochondrial function affects cell energy metabolism, ultimately affecting ovarian function and fertility. With the accumulation of these damages, the endocrine function of the ovary and the quality of oocytes deteriorate further, thus making it difficult for women to conceive. Although certain studies have been conducted on the related mechanisms of ovarian aging, the specific molecular regulatory mechanisms are still not fully understood. How to effectively delay ovarian aging and improve ovarian function remains an urgent problem to be solved currently.

[0004] mRNA m 5 C modification (5-methylcytosine modification) is a post-transcriptional modification of RNA that mainly occurs on cytosine (C) residues in mRNA molecules and is usually catalyzed by methyltransferases (such as proteins of the NSUN family). m 5N⁶-methyladenosine (m⁶A) modification plays an important role in processes such as RNA stability, transport, and translational regulation, and is closely related to various biological processes and diseases. We found that during ovarian senescence, the m⁶A modification level in ovarian granulosa cells was significantly increased, and the expression level of the m⁶A modification methyltransferase was elevated, suggesting that intervening in the increased m⁶A modification level caused by [methyltransferase] might help alleviate ovarian senescence. MY-1B is a covalent inhibitor of the RNA methyltransferase [methyltransferase]. MY-1B stereoselectively coordinates to the active site cysteine residue (C271). Previous studies have shown that MY-1B can significantly reduce the m⁶A level in human embryonic kidney cells and various cancer cell lines. Therefore, MY-1B has the potential to treat diseases with abnormally elevated m⁶A levels. However, its role in the process of ovarian senescence and its use as a target for treating ovarian senescence have not been reported yet. 5 and the m⁶A 5 modification methyltransferase Nsun2 expression level was increased, indicating that intervening in the increased m⁶A Nsun2 modification level of mRNA caused by [methyltransferase] might help alleviate ovarian senescence. MY-1B is a covalent inhibitor of the RNA methyltransferase 5 m⁶A. MY-1B stereoselectively coordinates to the Nsun2 active site cysteine residue (C271). Previous studies have shown that MY-1B can significantly reduce the m⁶A Nsun2 level in human embryonic kidney cells and various cancer cell lines. Therefore, MY-1B has the potential to treat diseases with abnormally elevated m⁶A levels. However, its role in the process of ovarian senescence and its use as a target for treating ovarian senescence have not been reported yet. 5 m⁶A 5 m⁶A 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 senescence. Supplementing MY-1B can improve ovarian senescence caused by the increased m⁶A modification level of ovarian granulosa cell mRNA, manifested as MY-1B being able to reduce the m⁶A modification level of mRNA in senescent ovarian granulosa cells, proving that MY-1B can be used as an effective ingredient in the preparation of a drug for improving ovarian senescence. 5 m⁶A 5 m⁶A

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

[0007] .

[0008] In some preferred examples of this aspect, the MY-1B can reduce the m⁶A 5 modification level of ovarian granulosa cells.

[0009] In some preferred examples of this aspect, the manifestation of ovarian senescence is the disorder of the estrous cycle in animals.

[0010] In some preferred examples of this aspect, the manifestation of ovarian senescence is the change in serum sex hormones, specifically: the decrease in the levels of E2 and AMH, and the increase in the level of FSH.

[0011] In some preferred examples in this regard, the manifestations of ovarian senescence are a significant decrease in the number of primordial follicles, primary follicles, secondary follicles, antral follicles, and growing follicles in the ovary, as well as an increase in the number of atretic follicles.

[0012] In some preferred examples in this regard, the manifestations of ovarian senescence are a decrease in the number of ovarian oocytes and a decline in the quality of ovarian oocytes; among them, the evaluation indicators for the quality of ovarian oocytes are: the proportion of oocytes that extrude the first polar body and the proportion of fragmented oocytes.

[0013] In some preferred examples in this regard, the manifestations of ovarian senescence are a decline in the fertilization rate of ovarian oocytes and a decline in the embryonic development potential; among them, the evaluation indicators for embryonic development potential are: the incidence of 2-cell stage and the formation rate of morula.

[0014] In a second aspect, the present invention provides an m 5 C modification inhibitor in ovarian granulosa cells containing MY-1B, and the inhibitor enables ovarian granulosa cells with elevated m 5 C modification level to contact with a therapeutically effective amount of MY-1B.

[0015] In a third aspect, the present invention provides a drug for preventing or delaying ovarian senescence, including MY-1B and a pharmaceutically acceptable carrier or excipient.

[0016] Among them, the drug is used to reduce or inhibit the activity of m 5 C modification in ovarian granulosa cells.

[0017] In some preferred examples in this regard, 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 paste, hydroxycellulose, PVP, PEG, gelatin; lubricants such as MS, colloidal silica, talc, hydrogenated vegetable oil, PEG, sodium / magnesium lauryl sulfate, etc.

[0018] Compared with the prior art, the present invention has the following advantages:

[0019] By administering drugs to senescent mice, experiments prove that using MY-1B can reduce the abnormally elevated mRNA m 5 C modification level in ovarian granulosa cells of senescent mice, correct the disorder of estrous cycle, improve the hormone levels of senescent mice (manifested as an increase in E2 and AMH), increase the ovarian reserve of senescent mice and reduce follicular atresia, reduce the senescence or apoptosis of ovarian granulosa cells in senescent mice, improve the quality of oocytes in senescent ovaries and increase their fertilization rate and embryonic development potential. It has relatively high safety and good application value. Description of the Drawings

[0020] Figure 1 Detection results of m 5 C levels in the ovaries of young and old mice. Among them, A is a representative picture of m 5 C immunofluorescence staining of ovarian sections and the statistical results of fluorescence density in granulosa cells. The scale bar is 100 μm. B is the statistical result of the detection of m 5 C levels in the ovaries (ELISA). C is the statistical result of the detection of m 5 C levels in ovarian granulosa cells (ELISA).

[0021] Figure 2 Detection results of ovarian morphology and function in three groups of mice. Among them, A is a representative picture of vaginal smears of the estrous cycle of mice and the statistical results of the estrous cycle in three groups of mice. B is a representative picture of ovarian morphology in three groups of mice. C is the statistical result of the serum AMH hormone level in three groups of mice. D is the statistical result of the serum E2 hormone level in three groups of mice.

[0022] Figure 3 Follicle counting results of the ovaries in three groups of mice. Among them, A is a representative picture of HE staining of ovarian sections in three groups of mice, and the scale bar is 200 μm. B is a representative picture of follicles at all levels and atretic follicles in the mouse ovary, with a scale bar of 20 μm, and the statistical results of three groups of mice.

[0023] Figure 4 Results of immunofluorescence staining related to proliferation and apoptosis of ovarian sections in three groups of mice. Among them, A is a representative picture of Ki67 immunofluorescence staining of ovarian sections and the statistical results of fluorescence density in granulosa cells, with a scale bar of 100 μm. B is a representative picture of γH2AX immunofluorescence staining of ovarian sections and the statistical results of fluorescence density in granulosa cells, with a scale bar of 100 μm. C is a representative picture of TUNEL staining of ovarian sections and the statistical results of positive fluorescence in granulosa cells, with a scale bar of 100 μm. D is Nsun2 、 Fbln1 、 Cdkn1a 、 Cdkn1b 、 IL-6 、 IL-8 Statistical results of the detection of mRNA expression levels of

[0024] Figure 5 Superovulation detection results in three groups of mice. Among them, A is a representative picture of oocytes of superovulation in three groups of mice, and the scale bar is 50 μm. B is the statistical result of the oocyte count and the first polar body extrusion rate of superovulation in three groups of mice.

[0025] Figure 6Results of staining detection of oocyte quality in three groups of mice. Among them, A is a representative picture of mitochondrial staining of oocytes in three groups of mice and the statistical results, with a scale bar of 50 μm. B is a representative picture of mitochondrial membrane potential staining of oocytes in three groups of mice and the statistical results, with a scale bar of 50 μm. C is a representative picture of reactive oxygen species staining of oocytes in three groups of mice and the statistical results, with a scale bar of 50 μm. D is a representative picture of apoptosis signal staining of oocytes in three groups of mice and the statistical results, with a scale bar of 50 μm. E is a representative picture of staining of spindles and chromosome alignment in oocytes in three groups of mice, with a scale bar of 50 μm. F is the statistical result of the abnormal spindle ratio in oocytes of three groups of mice. G is the statistical result of the chromosome misalignment ratio in oocytes of three groups of mice.

[0026] Figure 7 Results of the fertilization rate and embryo development potential of oocytes, as well as the fertility of mice in two groups of mice. Among them, A is a microscopic morphological comparison diagram of each stage of the development of fertilized eggs in two groups of mice and the statistical results of the fertilization rate and morula formation rate. B is the statistical result of the pregnancy rate in two groups of mice. C is the statistical result of the number of offspring per litter in two groups of mice. Detailed implementation manners

[0027] To make the objectives, technical solutions and advantages of the present invention more clear and understandable, the present invention will be further described in detail below with reference to the embodiments and the accompanying drawings. It should be understood that the specific embodiments described are only for explaining the specific applications of the present invention, and are not used to limit the scope of the present invention. Through these embodiments, it is intended to help understand the principles, operation methods and advantages of the present invention, but it does not exclude appropriate adjustments and adaptations during the implementation process of the present invention.

[0028] The experimental animals used in this study were C57 mice, purchased from Beijing Sibefu Experimental Animal Technology Co., Ltd., including female mice at 8 weeks old and 10 months old, and male mice at 3 months old. The breeding environment of the mice was set at a temperature of 22-25 °C, a humidity not exceeding 50%, and a 12-hour light-dark alternating light cycle. During the experiment, the mice were provided with sufficient drinking water and feed, and the bedding was changed once a week. Unless otherwise specified, the test methods in this study were all standard techniques in the art or were carried out according to the conditions recommended by the suppliers.

[0029] Example 1:

[0030] Twenty 2-month-old C57 female mice were used as the young group (Young). Forty 10-month-old C57 female mice were randomly and evenly divided into two groups: the aged control group (Aged) and the aged group injected with MY-1B (Aged+MY-1B).

[0031] Randomly sample 5 mice from the Young group and 5 mice from the Aged group, sacrifice them, collect the ovaries and ovarian granulosa cells, and perform immunofluorescence staining and ELISA detection on ovarian sections. The results are shown in Figure 1 .

[0032] From Figure 1 Figure A, it can be seen that the immunofluorescence density of m 5 C in ovarian granulosa cells of senescent mice is significantly increased. From Figure 1 Figure B in Figure 1 and Figure C in Figure 1, it can be seen that the m 5 C modification level in senescent ovarian granulosa cells is significantly increased.

[0033] Figure 1 The results show that the m 5 C modification level in granulosa cells is significantly increased during ovarian senescence.

[0034] Example 2:

[0035] For the aged group injected with MY-1B, mice were intraperitoneally injected with MY-1B at 20 mg / kg / 3d. The aged control group was injected with an equal amount of the solvent of MY-1B every day. The solvent was prepared by mixing 10% DMSO + 40% PEG300 + 5% Tween-80 + 45% Saline.

[0036] Five weeks after the injection operation in the aged group and the aged group injected with MY-1B, 10 mice were randomly sampled from each of the aged group, the aged group injected with MY-1B, and the young control group without operation. Vaginal exfoliated cells were collected every morning for 30 consecutive days to make smears, and hematoxylin and eosin staining was performed for observation to detect their estrous cycles. These 10 mice were still used in subsequent other experiments. Five mice were randomly sampled for sacrifice and serum and ovaries were collected. ELISA (Elabscience) detection of AMH and E2 was performed on the collected serum. The results are shown in Figure 1 .

[0037] From Figure 2 Figure A, it can be seen that compared with the young group, the irregular estrous cycle rate of mice in the aged group is significantly higher than that in the young group; while the regular estrous cycle rate of mice in the aged group injected with MY-1B is increased compared with the aged group. From Figure 2 Figure B, it can be seen that the ovarian size of mice in the aged group injected with MY-1B is significantly larger than that of mice in the aged group. From Figure 2 Figure C and Figure 2 Figure D, it can be seen that the serum hormone levels of AMH and E2 in mice in the aged group injected with MY-1B are significantly higher than those in the aged group.

[0038] Figure 2 The results show that injecting MY-1B can correct the estrous cycle disorder of senescent mice and increase the serum levels of AMH and E2 hormones.

[0039] Example 3:

[0040] After 5 weeks of intraperitoneal injection in the aged group and the aged group injected with MY-1B, 3 mice were randomly sampled from each group, together with the young control group without any operation. The ovaries of the three groups of mice were fixed in 4% formaldehyde and made into 5-μm paraffin sections. After HE staining, the morphology of primordial follicles, primary follicles, secondary follicles, antral follicles and atretic follicles in the three groups of mice was observed under a microscope and counted. The results are shown in Figure 3 .

[0041] From Figure 3 A and B, it can be seen that during the process of ovarian senescence, the number of follicles at all levels decreases. The number of primordial follicles, primary follicles, secondary follicles and antral follicles in the ovaries of aged mice injected with MY-1B is more than that of control aged mice, while the number of atretic follicles is less than that of control aged mice.

[0042] Figure 3 The results show that injecting MY-1B can improve the ovarian follicle reserve capacity of senescent mice.

[0043] Example 4:

[0044] Immunofluorescence staining was performed on ovarian sections of the aged group, the aged group injected with MY-1B and the young control group 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 .

[0045] From Figure 4 A, it can be seen that compared with the young group, the proliferative (Ki67 + ) granulosa cells in the ovaries of the aged group were significantly reduced, while those in the aged group injected with MY-1B increased; Figure 4 B shows that compared with the young group, the ovarian DNA damage (γH2AX + ) granulosa cells in the aged group were significantly increased, while those in the aged group injected with MY-1B decreased; Figure 4 C shows that compared with the young group, the apoptotic granulosa cells in the ovaries of the aged group were significantly increased, while those in the aged group injected with MY-1B decreased; Figure 4 D shows that after treatment with MY-1B in the aged group Nsun2 and the expression of senescence-related genes were significantly lower than those in the aged control group.

[0046] Figure 4 The results show that after injecting MY-1B, the m 5 C level of ovarian granulosa cells in senescent mice can be corrected, thereby improving the apoptosis of granulosa cells and promoting proliferation, and delaying the level of senescence.

[0047] Example 5:

[0048] Superovulation experiments were performed on six randomly selected mice in each of the control aged group, the aged group injected with MY-1B, and the young control group. The specific procedures were as follows: The three groups of mice were injected with 10 IU of Pregnant Mare Serum Gonadotropin (PMSG). After 48 hours, 10 IU of human Chorionic Gonadotropin (hCG) was injected. Fourteen hours later, the cumulus oocyte complexes (COCs) discharged into the ampulla of the fallopian tube were taken out. Then, the granulosa cells around the oocytes were removed with 0.1% hyaluronidase. The morphology of the oocytes was observed and counted under a microscope.

[0049] From Figure 5 It can be seen that Figure 5 in A, it shows that the number of ovulations in the control aged group is reduced compared with the young group, while the number of ovulations in the aged group injected with MY-1B is increased compared with the control aged group; Figure 5 in B and C, it shows that the number of oocytes with the first polar body discharged in the control aged group is reduced compared with the young group, while the number of oocytes in the aged group injected with MY-1B is increased.

[0050] Figure 5 The results show that injecting MY-1B can improve the superovulation quantity and normal ratio of aging mice.

[0051] Example 6:

[0052] Oocytes of three groups of mice were obtained using the same method as in Example 3. The obtained oocytes were respectively subjected to the following operations:

[0053] 1. Mitotracker staining of oocytes: Dilute Mito-Tracker-Red to 200 nM using Opti-MEM medium. Transfer the oocytes into the droplet and incubate at 37°C in the dark for 30 min. Transfer them into PBS containing 0.1% BSA and wash three times. Then transfer them into a confocal dish with a glass bottom and cover with paraffin oil. Observe and collect images under a confocal microscope within 1 h.

[0054] 2. Mitochondrial membrane potential staining of oocytes: Perform the experiment using a mitochondrial membrane potential detection kit. Take an appropriate amount of 1 μl of JC-10 (200x) probe, dilute it with 160 μl of ultrapure water, and add 40 μl of JC-10 staining buffer and mix well to make a droplet. Transfer the oocytes into the droplet and incubate at 37°C in the dark for 20 min. Transfer them into the staining buffer (1x) and wash three times. Then transfer them into a confocal dish with a glass bottom and cover with paraffin oil. Observe and collect images under a confocal microscope within 1 h.

[0055] 3. Oocyte Reactive Oxygen Species Staining: Dilute 2',7'-Dichlorodihydrofluorescein diacetate (DCFH-DA) to 10 μM using Opti-MEM medium. Transfer the oocytes into the droplet and incubate them in the dark at 37 °C for 30 min. After transferring them into PBS containing 0.1% BSA and washing three times, transfer them into a confocal dish with a glass bottom and cover with paraffin oil. Observe and collect images under a confocal microscope within 1 h.

[0056] 4. Oocyte Apoptosis Signal Detection: Conduct the experiment using the AnnexinV-mCherry Apoptosis Detection Kit. According to the instructions, add 5 μl of AnnexinV-mCherry to 195 μl of binding buffer. Transfer the oocytes into the freshly prepared Annexin V-mCherry binding buffer and stain them in the dark at room temperature for 20 min. After transferring them into PBS containing 0.1% BSA and washing three times, transfer them into a confocal dish with a glass bottom and cover with paraffin oil. Observe and collect images under a confocal microscope within 1 h.

[0057] 5. Fix the obtained oocytes in paraformaldehyde for 30 min, then permeabilize them with 0.5% TritonX-100 for 20 min, and block them with 1% BSA for 1 h. Incubate them overnight at 4 °C with anti-α-tubulin monoclonal antibody at a ratio of 1:200. After washing three times with IVF solution, incubate them with FITC-conjugated secondary antibody at room temperature for 1 h, then stain the nuclei with DAPI, transfer them into a 10 μl anti-quenching agent droplet, and observe the spindle morphology and chromosome arrangement under a Zeiss LSM880 confocal laser microscope. The results are shown in Figure 6 。

[0058] As can be seen from Figure 6 Figure A, compared with the control old mice, the abnormal aggregation and distribution of mitochondria in the oocytes of old mice injected with MY-1B decreased; Figure 6 Figure B shows that compared with the control old mice, the mitochondrial membrane potential of the oocytes of old mice injected with MY-1B increased significantly; Figure 6 Figure C shows that compared with the control old mice, the reactive oxygen species level of the oocytes of old mice injected with MY-1B decreased significantly; Figure 6 Figure D shows that compared with the control old mice, the apoptosis signal level of the oocytes of old mice injected with MY-1B decreased significantly; Figure 6Among E, F, and G, it shows that the incidence of abnormal spindles and chromosome misalignment in the oocytes of aged mice injected with MY-1B is significantly reduced compared to the control aged mice.

[0059] Figure 6 The results indicate that injecting MY-1B can improve the quality of oocytes in aging mice.

[0060] Example 7:

[0061] The COCs obtained in Example 3 were in vitro fertilized with the sperm of 3-month-old male mice that had undergone in vitro capacitation treatment in an IVF droplet for 6 hours. Then, the fertilized eggs were transferred into a KSOM culture droplet, washed and then transferred to a new KSOM droplet, and cultured for another 6 hours. The formation of pronuclei was observed, and the fertilization rate (2-cell stage) and morula rate were counted. The above three groups of mice were subjected to a continuous 8-week cohabitation test, and their pregnancy rates and average litter sizes were counted. The results are shown in Figure 7 。

[0062] From Figure 7 A, B, and C, it can be seen that compared with the aged group, the fertilization rate and morula rate of the oocytes in the aged group injected with MY-1B are both significantly increased. The pregnancy rate and average litter size of the aged group of mice injected with MY-1B are both significantly higher than those of the control aged group.

[0063] Figure 7 The results indicate that injecting MY-1B can improve the fertilization rate of oocytes in aging mice, enhance the embryonic development potential, and improve the fertility of aging mice.

[0064] As described above, these are only several embodiments of the present application and do not impose any form of limitation on the present application. Although the present application is disclosed with preferred embodiments as above, it is not intended to limit the present application. Any person skilled in the art, without departing from the scope of the technical solution of the present application, making some changes or modifications using the disclosed technical content is equivalent to equivalent implementation cases and all fall within the scope of the technical solution.

Claims

1. Use of MY-1B in the preparation of a medicament for preventing and / or delaying ovarian senescence; The ovarian senescence mentioned refers to the physiological process in which a woman's ovarian function gradually declines, ovarian reserve decreases, and the quantity and quality of oocytes decline as she ages.

2. The application according to claim 1, wherein The described MY-1B can reduce the m 5 C modification level in ovarian granulosa cells.

3. The application according to claim 1, characterized in that The manifestations of the ovarian senescence are reduced granulosa cell proliferation, increased apoptosis and DNA damage.

4. The application according to claim 3, wherein The manifestations of the ovarian senescence are disrupted estrous cycles in animals.

5. The application according to claim 4, characterized in that, The manifestations of the ovarian senescence are changes in serum sex hormones, specifically: a decrease in the levels of E2 and AMH, and an increase in the level of FSH.

6. The application according to claim 4, characterized in that, The manifestations of the ovarian senescence are a significant decrease in the number of primordial follicles, primary follicles, secondary follicles, antral follicles and growing follicles in the ovary, as well as an increase in the number of atretic follicles.

7. The application according to claim 4, wherein The evaluation indexes for the quality of ovarian oocytes are: the proportion of oocytes that extrude the first polar body and the proportion of fragmented oocytes.

8. The application according to claim 4, characterized in that The manifestations of the ovarian senescence are a decrease in the fertilization rate of ovarian oocytes and a decrease in the developmental potential of embryos; among them, the evaluation indexes for the developmental potential of embryos are: the incidence of 2-cell stage and the formation rate of morulae.

Images