Application of adiponectin and receptor stimulant thereof in preparation of medicine for delaying ovarian aging

Abstract

The invention relates to application of adiponectin and a receptor stimulant thereof in preparation of a medicine for delaying ovarian aging. The medicine inhibits premature activation of primordial follicles and optimizes an ovarian microenvironment through multiple action mechanisms: the primordial follicles are regulated and blocked to be converted into primary follicles through an AMPK / mTOR pathway, and meanwhile, the H3K27ac level of a KITL promoter region is reduced through epigenetic regulation; the ovarian microenvironment is adjusted, the NF-kappa B pathway is inhibited, proinflammatory factor secretion is reduced, the angiogenesis structure is optimized, and nutrition supply is improved; the anti-oxidative stress is enhanced, the SIRT1 / PGC-1 alpha pathway is activated, the mitochondrial function is enhanced, and the autophagic flow is induced. Clinical application shows that the medicine can remarkably reduce the infertility rate and the spontaneous abortion rate, and is particularly suitable for prevention and treatment of people with ovarian hypofunction more than 35 years old. Formulations encompass injections, oral formulations, and topical administration formulations, and support combined use with other ovarian protection ingredients. The traditional Chinese medicine composition has the remarkable advantages of delaying follicle depletion, improving oocyte quality and expanding a fertility window.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technology, and in particular to the application of adiponectin and its receptor agonist in the preparation of drugs for delaying ovarian aging. Background Art

[0002] With the rapid development of modern society, women's childbearing age has been gradually postponed. However, with the increase of age, women's fertility has declined significantly, mainly manifested in the continuous increase of infertility rate and spontaneous abortion rate. The core reason for this phenomenon lies in the physiological changes of the ovaries and the reduction of follicle reserves. Unlike men who have continuously renewed spermatogonial stem cells, the number of primordial follicles in women's ovaries no longer increases from birth. With the increase of age, the number of follicles gradually decreases, and most follicles will experience natural atresia. Only a small number of primordial follicles can develop into antral follicles and eventually ovulate. Studies have shown that the decrease in the number of follicles accelerates after the age of 35. By the time of menopause, the reserve of primordial follicles in women's ovaries has dropped from 1 million to 2 million at birth to only about 1,000. In this process, the occurrence of ovarian aging is inevitable, leading to the loss of fertility. Ovarian aging not only affects fertility, but is also closely related to a variety of age-related health problems, such as osteoporosis and cardiovascular disease.

[0003] Therefore, how to delay ovarian aging and protect ovarian function has become one of the hot topics in current medical research. By regulating endocrine factors and molecular pathways related to ovarian aging or using new drug interventions, the ovarian aging process can be effectively delayed, women's reproductive health can be improved, and new solutions can be provided for improving women's fertility. Summary of the invention

[0004] In order to solve the problems of the prior art, the present invention provides the use of adiponectin and its receptor agonist in the preparation of a drug for delaying ovarian aging.

[0005] In order to solve the above technical problems, the present invention is achieved through the following technical solutions: First, the use of adiponectin and its receptor agonists in the preparation of drugs for delaying ovarian aging. The drugs inhibit the premature activation of primordial follicles in the ovaries, delay follicle exhaustion, thereby extending the female reproductive window and improving ovarian function.

[0006] Application of adiponectin and its receptor agonists

[0007] Primordial follicle activation inhibition mechanism

[0008] Regulation through the AMPK / mTOR pathway: Adiponectin binds to AdipoR1 and activates AMPK, which phosphorylates TSC2 and inhibits mTORC1 activity, thereby blocking the molecular switch for the transformation of primordial follicles to primary follicles.

[0009] Epigenetic regulation: Histone modification analysis showed that drug treatment could reduce the H3K27ac level in the KITL promoter region, inhibit Kit ligand expression, and reduce the recruitment of primordial follicles.

[0010] Ovarian microenvironment regulation

[0011] Immunomodulation: AdipoR2-mediated NF-κB pathway inhibition reduces the secretion of pro-inflammatory factors such as TNF-α and IL-6, and improves mitochondrial function of granulosa cells.

[0012] Angiogenesis regulation: Promote VEGF-A expression while inhibiting Ang-2, optimize the capillary network structure around the follicles, and improve the nutrient supply to the oocytes.

[0013] Enhanced resistance to oxidative stress

[0014] Mitochondrial protection: Activates the SIRT1 / PGC-1α pathway, promotes mitochondrial biogenesis, increases SOD2 activity in ovarian tissue, and decreases MDA content.

[0015] Autophagy regulation: Induces the formation of Atg5-Atg12 complex, enhances oocyte autophagic flux, and removes damaged organelles and oxidized proteins.

[0016] In a specific embodiment of the first aspect, the drug regulates the ovarian microenvironment, reduces ovarian inflammatory response and enhances anti-oxidative stress capacity by activating adiponectin receptors AdipoR1 and / or AdipoR2.

[0017] In a specific embodiment of the first aspect, the drug is used to improve female fertility, specifically by reducing the infertility rate and spontaneous abortion rate.

[0018] In a specific embodiment of the first aspect, the drug is suitable for the prevention or treatment of ovarian dysfunction in women over 35 years old.

[0019] In a specific embodiment of the first aspect, the drug preparation comprises an injection, an oral preparation or a topical preparation.

[0020] In one embodiment of the first aspect, the drug can be used alone or in combination with other ovarian protective ingredients.

[0021] In a second aspect, a pharmaceutical composition for delaying ovarian aging comprises a therapeutically effective amount of adiponectin or a receptor agonist thereof, and a pharmaceutically acceptable carrier or excipient.

[0022] In a specific embodiment of the second aspect, the receptor agonist is an agonist of AdipoR1 and / or AdipoR2.

[0023] In a specific embodiment of the second aspect, the drug is used to delay ovarian aging-related fertility decline.

[0024] Figure 1 It is a schematic diagram of the experimental design of AdipoRon treatment of adult mice of the present invention.

[0025] Figure 2 It is a schematic diagram of H&E staining of ovarian tissue and DDX4 / FOXL2 immunofluorescence co-localization of the present invention.

[0026] Figure 3 It is a statistical schematic diagram of the number of ovarian primordial follicles, growing follicles and total follicles of the present invention.

[0027] Figure 4 It is a schematic diagram of Western blot analysis of anti-Mullerian hormone (AMH) protein expression of the present invention.

[0028] Figure 5 It is a schematic diagram of the ELISA test results of the serum adiponectin level of the present invention.

[0029] Figure 6 It is a schematic diagram of Western blot analysis of the adiponectin receptor AdipoR1 protein expression of the present invention.

[0030] Figure 7 It is a schematic diagram of Western blot analysis of adiponectin (APN) protein expression in ovarian tissue of the present invention.

[0031] Figure 8 It is a schematic diagram of Western blot analysis of the expression of p-mTOR and p-rpS6 proteins of the present invention.

[0032] Fig. 9 It is a schematic diagram of the experimental design of the AdipoRon of the present invention for extending the reproductive lifespan of aged female mice.

[0033] Fig.10 It is a schematic diagram of H&E staining of ovarian tissue of aged mice of the present invention.

[0034] Fig.11 It is a statistical schematic diagram of the number of ovarian follicles in aged mice of the present invention.

[0035] Fig.12 It is a schematic diagram of Western blot analysis of p-mTOR and p-rpS6 protein expression in ovaries of aged mice of the present invention.

[0036] Fig.13 It is a schematic diagram of H&E staining of ovarian tissue of 14-month-old mice of the present invention.

[0037] Fig.14 It is a statistical schematic diagram of the number of primordial follicles and total follicles in the ovaries of 14-month-old mice of the present invention.

[0038] Fig.15 It is a schematic diagram of the effect of AdipoRon of the present invention on the cumulative litter size of female mice.

[0039] The beneficial effects of the present invention are:

[0040] 1. The present invention innovatively achieves the systematic delay of the ovarian aging process through the multiple mechanisms of action of adiponectin and its receptor agonists. The drug accurately inhibits the premature activation of primordial follicles through the AMPK / mTOR pathway, effectively prolongs the follicle reserve cycle, and provides a new strategy for expanding the female reproductive window. At the same time, the histone modification level in the KITL promoter region is reduced through epigenetic regulation, blocking the recruitment of primordial follicles at the gene expression level. In addition, the drug regulates the ovarian microenvironment, including inhibiting the NF-κB pathway to reduce the secretion of inflammatory factors, optimizing the angiogenesis structure to improve nutrient supply, and activating the SIRT1 / PGC-1α pathway to enhance mitochondrial function and autophagy flow, forming a multi-dimensional synergistic protective effect and significantly improving ovarian function;

[0041] 2. The drug has shown significant advantages in clinical applications. By improving fertility-related indicators (reducing infertility rates and spontaneous abortion rates), it is particularly suitable for the prevention and treatment of ovarian dysfunction in people over 35 years old. Its preparations are diversified (injections, oral preparations, local administration), which can be flexibly selected according to individual needs, and can be used in combination with other ovarian protective ingredients, expanding clinical application scenarios. The drug has a clear mechanism of action and strong targeting. It can not only slow down the rate of follicle depletion, but also improve the quality of oocytes. It provides an efficient and safe treatment plan for diseases related to ovarian aging, and has important social value and clinical application prospects. DETAILED DESCRIPTION

[0042] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0043] Application of adiponectin and its receptor agonists in the preparation of drugs for delaying ovarian aging.

[0044] Please refer to Figures 9 to 15 Intraperitoneal injection of a receptor agonist reduces primordial follicle loss in young mice.

[0045] AdipoRon treatment extends ovarian and reproductive lifespan in older females.

[0046] Fig. 9 The AdipoRon treatment strategy was used in female mice at the late reproductive stage. After 1 month of AdipoRon or vehicle treatment, 8-month-old female mice were mated to test their fertility.

[0047] Figures 10 to 15 Group experiment: Mice were Figure 8 The mice treated with AdipoRon were sacrificed for analysis.

[0048] Fig.10 , Representative images of H&E staining of ovarian tissues of control mice and AdipoRon-treated mice at 8 months of age. Red arrows: primordial follicles. Yellow arrows: growing follicles. Scale bar: 200 μm.

[0049] Fig.11 , Statistics of the number of primordial follicles, growing follicles and total follicles in the ovaries of 8-month-old mice (n=6).

[0050] Fig.12 , Western blot analysis of p-mTOR and p-rpS6 in ovarian tissues of control mice and AdipoRon-treated mice at 8 months of age (n=3).

[0051] Fig.13 , Representative images of H&E staining of ovarian tissues of control mice and AdipoRon-treated mice at 14 months of age. Scale bar: 200 μm.

[0052] Fig.14 , Statistics of the number of primordial follicles and total follicles in the mouse ovaries at 14 months of age.

[0053] (n=6).

[0054] Fig.15 , Comparison of the cumulative litter size of females in the control group (black) and the AdipoRon-treated group (red). (n=12).

[0055] Comparison of the average litter size per female in the AdipoRon-treated group and the control group during the two reproductive periods of 8-12 months and 12-14 months (n=6).

[0056] Error bars represent mean ± SD. Data were compared between the two groups using two-tailed Student's unpaired t-test. ns, no statistically significant difference. *P<0.05, **P<0.01, ***P<0.001.

[0057] Please refer to Figures 1 to 8Shown: Intraperitoneal injection of a receptor agonist reduces primordial follicle loss and extends reproductive lifespan in middle-aged mice.

[0058] AdipoRon treatment reduces the loss of primordial follicles and increases adiponectin levels in adult mice

[0059] Figure 1 , AdipoRon treatment strategy for female mice. 2-month-old female mice received AdipoRon treatment (5 mg / kg body weight, once every other day) by intraperitoneal injection (ip) for 1 month.

[0060] Figures 2 to 8 Mice were treated with AdipoRon as described in (a) and then sacrificed for analysis.

[0061] Figure 2 , Representative hematoxylin-eosin (H&E) staining results of ovarian tissues of control and AdipoRon-treated mice, and immunofluorescence co-localization staining of DDX4 and FOXL2 in the ovaries. Red arrows: primordial follicles; yellow arrows: growing follicles. Scale bar: 200 μm.

[0062] Figure 3 , Statistics of the number of primordial follicles, growing follicles and total follicles in the ovaries (n=6).

[0063] Figure 4 , Western blot analysis of the expression of anti-Mullerian hormone (AMH) in the ovaries of mice in the control group and AdipoRon treatment group (n=3).

[0064] Figure 5 , ELISA was used to detect the adiponectin protein level in the serum of mice without AdipoRon treatment and after 1 month of treatment (n=6).

[0065] Figure 6 , Western blot analysis of the expression of adiponectin receptor 1 (ADIPOR1) in the ovaries of mice in the control group and the AdipoRon treatment group (n=3).

[0066] Figure 7 , Western blot analysis of the expression of adiponectin (APN) in the ovaries of mice in the control group and AdipoRon treatment group (n=3).

[0067] Figure 8 , Western blot analysis was performed to analyze the expressions of phosphorylated mTOR (p-mTOR) and phosphorylated rpS6 (p-rpS6) in the ovaries of mice in the control group and AdipoRon treatment group (n=3).

[0068] Error bars represent mean ± standard deviation. Two-tailed Student's unpaired t test was used for comparison between the two groups. ns: not significant; *P<0.05, **P<0.01, ***P<0.001.

[0069] Example 1: Preparation of adiponectin receptor agonist composition

[0070] Recipe composition

[0071] Active ingredient: AdipoR1 agonist Compound A (15 mg);

[0072] Auxiliary ingredients: resveratrol (30mg), N-acetylcysteine ​​(100mg);

[0073] Carrier system: DSPC / CHOL liposomes (molar ratio 7:3);

[0074] Excipients: mannitol (50 mg), citrate buffer (pH 6.8);

[0075] Preparation method

[0076] Dissolve Compound A and auxiliary components in a chloroform / methanol mixed solution (3:1);

[0077] The lipid film was formed by rotary evaporation, and PBS was added for hydration to prepare liposomes;

[0078] After ultrasonic treatment for 15 min, the samples were sterilized by passing through a 0.22 μm filter membrane;

[0079] Freeze-drying process: pre-freeze at -50℃ for 2 hours, sublimation drying for 24 hours;

[0080] Quality Control

[0081] Encapsulation efficiency: HPLC test showed 89.2%;

[0082] Particle size distribution: dynamic light scattering measurement 115 ± 12 nm;

[0083] Stability: Drug retention rate >95% after storage at 4°C for 6 months.

[0084] Example 2: Animal experiment on ovarian protection effect;

[0085] Experimental design Model establishment: 35-week-old C57BL / 6 mice (n=30);

[0086] Group processing:

[0087] Control group: normal saline (0.1 ml / day);

[0088] Experimental group: the composition of Example 1 (200 μg / kg, twice a week); Positive control group: DHEA (25 mg / kg, daily gavage);

[0089] Period: Continuous intervention for 4 weeks;

[0090] Detection indicators

[0091] Ovarian histology: HE staining to observe the number of follicles;

[0092] Oocyte quality: spindle morphology assessment, mitochondrial membrane potential detection; oxidative stress: tissue homogenate SOD activity, MDA content determination;

[0093] Reproductive function: After being caged with male mice, the pregnancy rate and number of live fetuses were counted;

[0094] Experimental Results

[0095]

[0096]

[0097] *p<0.05vs control group.

[0098] Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. The use of adiponectin and its receptor agonists in the preparation of drugs for delaying ovarian aging, characterized in that: The drug works by inhibiting the premature activation of primordial follicles in the ovaries and delaying follicle exhaustion, thereby extending the female reproductive window and improving ovarian function.

2. The use of adiponectin and its receptor agonist according to claim 1 in the preparation of a drug for delaying ovarian aging, characterized in that: The drug regulates the ovarian microenvironment, reduces ovarian inflammatory response and enhances the ability to resist oxidative stress by activating adiponectin receptors AdipoR1 and / or AdipoR2.

3. The use of adiponectin and its receptor agonist in the preparation of a drug for delaying ovarian aging according to claim 1, characterized in that: The drug is used to improve female fertility, specifically by reducing infertility and spontaneous abortion rates.

4. The use of adiponectin and its receptor agonist in the preparation of a drug for delaying ovarian aging according to claim 1, characterized in that: The medicine is suitable for preventing or treating ovarian dysfunction in women over 35 years old.

5. The use of adiponectin and its receptor agonist in the preparation of a drug for delaying ovarian aging according to claim 1, characterized in that: The drug preparation forms include injections, oral preparations or topical preparations.

6. The use of adiponectin and its receptor agonist in the preparation of a drug for delaying ovarian aging according to claim 1, characterized in that: The drug can be used alone or in combination with other ovarian protective ingredients.

7. A pharmaceutical composition for delaying ovarian aging, characterized in that: The invention comprises a therapeutically effective amount of adiponectin or a receptor agonist thereof, and a pharmaceutically acceptable carrier or excipient.

8. A pharmaceutical composition for delaying ovarian aging according to claim 7, characterized in that: The receptor agonist is an agonist of AdipoR1 and / or AdipoR2.

9. A pharmaceutical composition for delaying ovarian aging according to claim 7, characterized in that: The drug is used to delay the decline in fertility associated with ovarian aging.

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