Application of N-acetyl-L-tryptophan in the preparation of drugs to improve or treat oocyte maturation disorders and enhance ovarian reserve function

Abstract

This invention discloses the application of N-acetyl-L-tryptophan, including the following applications: a. application in the preparation of drugs or health products that improve ovarian reserve function; b. application in the preparation of drugs or health products that improve or treat oocyte maturation disorders. This invention also discloses a drug comprising: N-acetyl-L-tryptophan or a pharmaceutically acceptable salt thereof, a pharmaceutically acceptable carrier, or an excipient; said drug can improve or treat oocyte maturation disorders and improve ovarian reserve function. This invention also discloses a health product comprising: N-acetyl-L-tryptophan or a pharmaceutically acceptable salt thereof, a pharmaceutically acceptable carrier, or an excipient; said health product can improve or treat oocyte maturation disorders and improve ovarian reserve function. This invention is the first to discover that N-acetyl-L-tryptophan has the efficacy of improving or treating oocyte maturation disorders and improving ovarian reserve function.

Description

Technical Field

[0001] This invention relates to the field of ovarian aging technology, and in particular to the application of N-acetyl-L-tryptophan in the preparation of drugs that improve or treat oocyte maturation disorders and enhance ovarian reserve function. Background Technology

[0002] Aging is a progressive life process driven by multiple intrinsic factors, its core characteristic being the gradual decline in the function of various organs. Ovarian function decline in women, as a key link in declining fertility and changes in health status, is regulated by multiple factors including genetics, metabolism, and environment, exhibiting high complexity and irreversibility. Ovarian reserve declines continuously from puberty, accelerating after age 35, manifested as a decrease in the number and quality of oocytes, entering the aging process significantly earlier than other organs. The average age of menopause is approximately 51 years, and the increased life expectancy means that the postmenopausal stage occupies a larger proportion of life, leading to increased long-term health risks. Therefore, developing intervention methods and treatment strategies based on the mechanisms of ovarian aging not only has significant scientific research value but also responds to the real need to delay the degeneration of the reproductive system in the context of an aging population.

[0003] The oocyte is the female reproductive cell. During oogenesis, it gradually develops into a mature ovum through meiosis. This process is divided into three stages: primary oocyte, secondary oocyte, and mature ovum. After the primary oocyte completes the first meiotic division, it produces a secondary oocyte and the first polar body. Subsequently, the secondary oocyte undergoes the second meiotic division to produce a mature ovum and the second polar body. These stages reflect its continuous developmental state in DNA replication and orderly division.

[0004] Oocyte maturation is a natural developmental process influenced by a variety of internal and external environmental factors, including DNA damage, mitochondrial functional status, oxidative stress, apoptosis, and autophagy. While inhibiting oxidative stress and apoptosis can indirectly promote oocyte maturation to some extent, it plays only a supporting role and is not a decisive factor. Currently, effective treatments and drugs for oocyte maturation disorders and decreased ovarian reserve are still relatively scarce.

[0005] N-acetyl-L-tryptophan (L-NAT) is an important aromatic derivative formed by acetylation of the essential amino acid L-tryptophan. It is an endogenous metabolite with multiple regulatory functions. Studies have shown that L-NAT not only participates in neurotransmitter synthesis and protein metabolism, but also possesses antioxidant, anti-inflammatory, and neuroprotective activities, playing a crucial role in maintaining metabolic homeostasis and stress adaptation.

[0006] L-NAT can be synthesized through the tryptophan metabolism pathway in the body, and can also be obtained from natural foods such as milk, eggs, and beans, or through dietary supplements. Studies have confirmed that this molecule exhibits potential physiological effects in regulating sleep rhythms, improving mood, mitigating oxidative stress, stabilizing immune function, and inhibiting low-level chronic inflammation.

[0007] Recent studies have shown that L-NAT, in addition to its role in the central nervous system, can also be expressed in various tissues such as the liver, intestines, and blood. Its synthesis and release in vivo involve multiple cell types, including neurons, immune cells, and epithelial cells. Animal experiments have demonstrated that this molecule possesses good biocompatibility, providing a solid foundation for its development as a neuroprotective agent, an adjuvant anti-inflammatory drug, or a nutritional intervention component. However, systematic research on the mechanism of action and regulatory pathways of L-NAT in ovarian dysfunction is currently lacking. This field remains largely unexplored, and its functional value and application potential require further investigation. Summary of the Invention

[0008] Based on the technical problems existing in the background art, this invention proposes the application of N-acetyl-L-tryptophan in the preparation of drugs to improve or treat oocyte maturation disorders and enhance ovarian reserve function. This invention is the first to discover that N-acetyl-L-tryptophan has the efficacy of improving or treating oocyte maturation disorders and enhancing ovarian reserve function. It can alleviate oocyte maturation disorders by improving the mitochondrial dynamics balance of oocytes, providing a novel approach to improving oocyte quality; simultaneously, it can upregulate AMH levels in aging ovarian tissue, significantly improving the reserve function of aging ovaries.

[0009] This invention proposes an application of N-acetyl-L-tryptophan, including the following applications: a. Application in the preparation of drugs or health products that enhance ovarian reserve function; b. Use in the preparation of drugs or health products that improve or treat oocyte maturation disorders.

[0010] Ovarian aging is mainly characterized by a decrease in the number and quality of oocytes. Its core mechanism is closely related to oxidative stress caused by mitochondrial dysfunction, including the accumulation of reactive oxygen species, loss of mitochondrial membrane potential, DNA damage, and abnormal meiosis, ultimately leading to decreased fertility. This process differs from polycystic ovary syndrome (PCOS), which is characterized by metabolic disorders such as hyperandrogenism and insulin resistance; the two have fundamentally different pathological mechanisms.

[0011] At birth, human females have approximately one million immature oocytes in both ovaries, but only about 500 will fully develop and be released during their lifetime; the rest will degenerate or undergo apoptosis at different stages. With age, especially after middle age, ovarian reserve declines continuously until it is essentially depleted by menopause. Therefore, improving oocyte quality and promoting their normal maturation is not only crucial for delaying the aging process of the female reproductive system, but also provides a theoretical basis and technical approach for maintaining natural fertility and preventing related diseases.

[0012] Oocyte quality is a key factor determining female reproductive potential and an important indicator for assessing ovarian function. Its maturation process is regulated by various endogenous and exogenous factors, including biological events such as DNA damage accumulation, mitochondrial dysfunction, increased oxidative stress, autophagy dysregulation, and programmed apoptosis. While some studies have attempted to optimize the developmental microenvironment through antioxidant or apoptosis-inhibiting methods, most approaches only achieve limited effects and cannot fundamentally reverse the maturation barriers caused by decreased genome stability.

[0013] This invention is the first to discover that N-acetyl-L-tryptophan can upregulate the expression level of anti-Müllerian hormone (AMH) in aging ovarian tissue. As a key marker of antral follicle secretion, the level of AMH directly reflects the strength of ovarian reserve function, suggesting that L-NAT has a significant effect on improving ovarian reserve.

[0014] Furthermore, this invention is the first to discover that N-acetyl-L-tryptophan can effectively improve oocyte maturation disorders in aging mice. Further research in this invention revealed typical mitochondrial dysfunction in oocytes of aging mice: a significant decrease in mitochondrial membrane potential, downregulation of the ovarian granulosa cell proliferation marker Ki-67, and an imbalance in the expression of mitochondrial dynamics-related proteins (such as DRP1, OPA1, MFF, Fis1, MFN1, MFN2, etc.). N-acetyl-L-tryptophan intervention can effectively reverse these abnormalities, restore mitochondrial dynamics balance, maintain normal mitochondrial function, enhance ovarian granulosa cell proliferation, and reduce oocyte DNA damage, thereby optimizing oocyte quality and maturation rate and improving oocyte maturation disorders. These results indicate that N-acetyl-L-tryptophan and its related drug preparations can improve oocyte quality and enhance ovarian reserve function, demonstrating significant potential in delaying ovarian aging and repairing ovarian damage, and providing a new direction for clinical intervention in female reproductive aging.

[0015] Preferably, the ovary is an aging ovary.

[0016] Preferably, N-acetyl-L-tryptophan improves or treats oocyte maturation disorders by enhancing the kinetic balance of oocyte mitochondria.

[0017] Preferably, the oocyte is an oocyte from a senescent ovary.

[0018] The present invention also provides a drug comprising: N-acetyl-L-tryptophan or a pharmaceutically acceptable salt thereof, a pharmaceutically acceptable carrier or excipient; The drug can improve or treat oocyte maturation disorders and enhance ovarian reserve.

[0019] Preferably, the dosage form of the drug is a liquid dosage form, a gas dosage form, a solid dosage form, or a semi-solid dosage form.

[0020] The above-mentioned liquid dosage forms can be injectable preparations, infusion preparations, etc., and injectable preparations can be intraperitoneal injections, intramuscular injections, etc.

[0021] The above-mentioned solid dosage forms can be oral preparations, etc.

[0022] The present invention also proposes a health product comprising: N-acetyl-L-tryptophan or a pharmaceutically acceptable salt thereof, a pharmaceutically acceptable carrier or excipient; The health products mentioned can improve or treat oocyte maturation disorders and enhance ovarian reserve function.

[0023] The pharmaceutically acceptable carriers mentioned above can be: microcapsules, microspheres, nanoparticles, liposomes, etc.

[0024] The pharmaceutically acceptable excipients mentioned above can be: binders, disintegrants, fillers, lubricants, wetting agents, osmotic pressure regulators, stabilizers, flow aids, flavoring agents, preservatives, suspending agents, coating materials, fragrances, anti-adhesion agents, chelating agents, penetration enhancers, pH adjusters, buffers, plasticizers, surfactants, foaming agents, defoamers, thickeners, encapsulating agents, humectants, absorbents, diluents, flocculants and anti-flocculation agents, filter aids, release inhibitors, solubilizers, cosolvents, emulsifiers, colorants, etc.

[0025] This invention is the first to discover that N-acetyl-L-tryptophan has the effect of improving or treating oocyte maturation disorders and enhancing ovarian reserve function. It can alleviate oocyte maturation disorders by improving the mitochondrial dynamics balance of oocytes, providing a novel approach to improving oocyte quality; simultaneously, it can upregulate AMH levels in aging ovarian tissue, significantly improving the reserve function of aging ovaries.

[0026] This invention has promising applications, as it can be used to improve the retrieval rate of oocytes from aging ovaries and provide technical support for the adjuvant treatment of related reproductive disorders. Furthermore, N-acetyl-L-tryptophan exhibits good biocompatibility and safety, making it a potentially safer ingredient for pharmaceuticals or nutritional supplements. It holds significant potential and broad clinical application prospects in repairing ovarian damage, delaying ovarian aging, alleviating oocyte maturation disorders, and improving oocyte quality. Attached Figure Description

[0027] Figure 1 To observe and detect the effects of N-acetyl-L-tryptophan on improving oocyte maturation disorders in aging mice using phase contrast microscopy; where A represents the PBE emission of GV stage oocytes after 14 h of culture in young mice, aging mice, and aging mice + N-acetyl-L-tryptophan treatment group, scale bar = 100 μm; BD represents the total number of oocytes, the proportion of GVBD, and the proportion of PBE emission in the three groups.

[0028] Figure 2 To analyze the effect of N-acetyl-L-tryptophan on improving ovarian reserve function in aging mice by Western blotting; where A represents the detection and quantitative analysis results of AMH expression level in the ovary; B represents the quantitative analysis results of A; and GAPDH is the internal reference protein.

[0029] Figure 3 The results of using immunofluorescence staining in Example 3 to detect the effect of N-acetyl-L-tryptophan on improving DNA damage in oocytes of aging mice are shown in Figure 3. In Figure 3, A represents the expression level of γ.H2AX in oocytes of young mice, aging mice, and aging mice + N-acetyl-L-tryptophan treatment group detected by immunofluorescence staining. B represents the quantitative analysis of γ.H2AX in oocytes in Figure 3. Scale bar = 20 μm.

[0030] Figure 4 The results of TMRE staining to detect the effect of N-acetyl-L-tryptophan on improving the mitochondrial membrane potential level of oocytes in aging mice are shown. Among them, A is a confocal microscopy image of the mitochondrial membrane potential level of three groups of mouse oocytes detected by TMRE live cell dye, and B is the quantitative analysis result of A. Scale bar = 20 μm.

[0031] Figure 5 This is the result of using immunofluorescence staining in Example 6 to detect the effect of N-acetyl-L-tryptophan on improving the proliferation of ovarian granulosa cells in aging mice. In this example, A is the expression level of Ki-67 in ovarian granulosa cells of young mice, aging mice, and aging mice + N-acetyl-L-tryptophan treatment group detected by immunofluorescence staining, and B is the quantitative analysis of A. Scale bar = 40 μm.

[0032] Figure 6 To analyze the effect of N-acetyl-L-tryptophan on improving mitochondrial homeostasis imbalance in the ovaries of aging mice using Western blotting; AD represents the detection and quantitative analysis of the expression levels of OPA1, MFN2, and MFN1 in the ovaries of the three groups, and EH represents the detection and quantitative analysis of the expression levels of DRP1, MFF, and Fis1 in the ovaries of the three groups. Detailed Implementation

[0033] The technical solution of the present invention will be described in detail below through specific embodiments. However, it should be clearly stated that these embodiments are for illustrative purposes only and are not intended to limit the scope of the present invention.

[0034] Example 1: Constructing Each Group of Models Ten healthy 2-month-old female mice were selected as the young group, denoted as Young.

[0035] Ten healthy 10-month-old female mice were selected as the aged group mice and denoted as Aged.

[0036] Ten healthy 10-month-old female mice were given intraperitoneal injections of N-acetyl-L-tryptophan aqueous solution (30 mg / kg once a day) for 14 days. This group was designated as the aged group + N-acetyl-L-tryptophan treatment group, denoted as Aged+L-NAT.

[0037] The three groups were fed the same way, with the young mice and the aging mice receiving an equal amount of physiological saline instead of N-acetyl-L-tryptophan solution via intraperitoneal injection.

[0038] Example 2: Phase Contrast Microscopy Observation and Detection of the Role of N-acetyl-L-tryptophan in Improving Oocyte Maturation Impairment in Aged Mice 1. After the mice in each group of Example 1 were quickly euthanized by cervical dislocation, their abdomens were disinfected by spraying with 75% disinfectant alcohol. The abdominal skin was pulled up with forceps, and the skin and peritoneum were cut open with scissors. The ovarian tissues on both sides were located along the uterus, removed and thoroughly washed in F12 culture medium. The ovarian tissues were chopped up and oocytes from each group were collected under a stereomicroscope.

[0039] Under a stereomicroscope, oocytes in the GV stage with good morphology were selected using a homemade pipette and transferred to F12 culture medium for repeated rinsing to remove excess impurities.

[0040] 2. Oocytes were transferred to preheated M16 medium and cultured in an incubator at 37°C, 5% CO2, and saturated humidity. Cultured for 2 hours, 8 hours, 9.5 hours, 10 hours, and 14 hours corresponded to the GVBD, MI, anaphase I (AI), telophase I (TI), and MII stages of oocyte maturation, respectively. Oocytes at different stages were collected as needed for the experiment, observed under a phase-contrast microscope, and statistically analyzed. Statistical analysis was performed using AVONA, and graphs were plotted using Graphpad (data are mean ± SEM). , The result is as follows: Figure 1 As shown.

[0041] Figure 1 To observe and detect the effects of N-acetyl-L-tryptophan on improving oocyte maturation disorders in aging mice using phase contrast microscopy; where A represents the PBE emission of GV stage oocytes after 14 h of culture in young mice, aging mice, and aging mice + N-acetyl-L-tryptophan treatment group, scale bar = 100 μm; BD represents the total number of oocytes, the proportion of GVBD, and the proportion of PBE emission in the three groups.

[0042] Depend on Figure 1 It can be seen that N-acetyl-L-tryptophan significantly improved the oocyte retrieval rate, GVBD rate and first polar body expulsion rate in aging mice, indicating that it can effectively improve oocyte maturation disorders in aging mice.

[0043] Example 3: Western Blot analysis of the role of N-acetyl-L-tryptophan in improving AMH levels, a marker of ovarian reserve function in aging mice.

[0044] Ovarian tissues from each group of mice in Example 1 were taken and added to a pre-cooled protein extraction reagent containing inhibitors (a mixture of protease inhibitors, RIPA, and phosphatase inhibitors). The mixture was lysed on ice for 60 min. The lysate was placed in a pre-cooled centrifuge and centrifuged at 12,000 rpm for 10 min. The supernatant (i.e., the protein of each group) was transferred to a new centrifuge tube, heated at 100°C for 10 min, and then stored at -80°C for later use to obtain the protein samples of each group.

[0045] Protein samples from each group were separated on a 10% SDS-polyacrylamide gel electrophoresis, transferred to a nitrocellulose membrane, and detected using AMH antibody. Image J was used for quantification of the bands, and ANOVA was used for statistical analysis. Graphpad software was used for plotting (data are expressed as mean ± SEM). , The result is as follows: Figure 2 As shown.

[0046] Figure 2 To analyze the effect of N-acetyl-L-tryptophan on improving ovarian reserve function in aging mice by Western blotting; where A represents the detection and quantitative analysis results of AMH expression level in the ovary; B represents the quantitative analysis results of A; and GAPDH is the internal reference protein.

[0047] Depend on Figure 2 It can be seen that AMH expression was reduced in the aging group compared with the young group; this phenomenon was reversed after treatment with N-acetyl-L-tryptophan; the AMH expression level in the Aged+L-NAT group was significantly higher than that in the Aged group, indicating that N-acetyl-L-tryptophan can significantly improve ovarian reserve function and delay ovarian aging in aging mice.

[0048] Example 4: Immunofluorescence staining to detect the role of N-acetyl-L-tryptophan in improving DNA damage in oocytes of aging mice.

[0049] Oocytes were collected from young mice, aged mice, and aged mice treated with N-acetyl-L-tryptophan (the steps were the same as step 1 in Example 2); after being quickly washed once in the washing solution, they were immediately transferred to the fixation permeabilization solution for 20 min; then the oocytes were washed three times in the washing solution for 5 min each time, and transferred to the blocking solution containing 3% BSA for 1 h at room temperature. Then, the oocytes were transferred to the primary antibody dilution buffer and incubated overnight at 4°C or 1 h at 37°C in a humidified chamber. After washing, they were transferred to the secondary antibody dilution buffer and incubated at 37°C for 1 h in a humidified chamber. After washing, the oocytes were transferred to DAPI (10 µg / mL) solution and stained at room temperature for 20 min. After washing, the oocytes were cleaned. Select a suitable slide and add 5-10 µL of DABCO anti-fluorescence quencher to the corresponding center position, with a small amount of support agent added around the edges. Then, transfer the oocytes or early embryos into the DABCO solution, cover with a coverslip, and gently press the coverslip with a pipette tip under a stereomicroscope to compress the oocytes or early embryos to the appropriate size. Mount the slide and observe and image using a fluorescence microscope or laser confocal microscope. Calculate the fluorescence value using ImageJ software, perform quantitative analysis of the fluorescence intensity using ImageJ, perform statistical analysis using AVONA, and plot the data using Graphpad (data are mean ± SEM). The result is as follows: Figure 3 As shown.

[0050] Figure 3The results of using immunofluorescence staining in Example 3 to detect the effect of N-acetyl-L-tryptophan on improving DNA damage in oocytes of aging mice are shown in Figure 3. In Figure 3, A represents the expression level of γ.H2AX in oocytes of young mice, aging mice, and aging mice + N-acetyl-L-tryptophan treatment group detected by immunofluorescence staining. B represents the quantitative analysis of γ.H2AX in oocytes in Figure 3. Scale bar = 20 μm.

[0051] Depend on Figure 3 It can be seen that the expression level of γ.H2AX in the Aged+L-NAT group was significantly lower than that in the Aged group, indicating that N-acetyl-L-tryptophan can reduce the degree of DNA damage in oocytes of aged mice, and the improvement of DNA damage can directly alleviate the maturation barrier of oocytes.

[0052] Example 5: Detection of the role of N-acetyl-L-tryptophan in improving mitochondrial membrane potential levels in aging mouse oocytes using TMRE staining. To detect mitochondrial membrane potential, MII stage oocytes were placed in IVF medium, and 5 mM TMRE reagent (prepared according to the instructions) was added. The cells were incubated at 37°C and 5% CO2 for 30 min. After washing three times with IVF medium, oocyte images were acquired under a laser scanning confocal microscope. Fluorescence values ​​were calculated using ImageJ software, fluorescence intensity was quantitatively analyzed, and statistical analysis was performed using AVONA. Graphpad was used for plotting (data are mean ± SEM). , The result is as follows: Figure 4 As shown.

[0053] Figure 4 The results of TMRE staining to detect the effect of N-acetyl-L-tryptophan on improving the mitochondrial membrane potential level of oocytes in aging mice are shown. Among them, A is a confocal microscopy image of the mitochondrial membrane potential level of three groups of mouse oocytes detected by TMRE live cell dye, and B is the quantitative analysis result of A. Scale bar = 20 μm.

[0054] Depend on Figure 4 It can be seen that N-acetyl-L-tryptophan significantly improved the mitochondrial membrane potential level of oocytes in the aging group mice, enhanced mitochondrial function, and thus improved the quality and maturation rate of oocytes.

[0055] Example 6: Detection of the role of N-acetyl-L-tryptophan in improving the expression of Ki-67, a proliferation-related marker, in ovarian granulosa cells of aging mice, using immunofluorescence staining. Ovarian tissue from each group of mice in Example 1 was prepared into paraffin sections and then sequentially immersed 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, and 70% ethanol aqueous solution for 3 min. Then, the sections were washed with PBS on a shaker for 5 min, and this process was repeated 3 times to ensure thorough washing.

[0056] Add 200 ml of PBS solution to a 2 L glass and heat in a microwave oven for 2 min. Then add 600 μl of Triton and stir thoroughly. Cool to room temperature and then place the slices in permeabilization solution and keep them in the dark for 60 min. After that, wash with PBS 3 times for 7 min each time. Next, place the slides into a humidified chamber, adding an appropriate amount of water to the chamber beforehand to prevent the slides from drying out. Then, use a pen to circle the tissues one by one, and then drop an appropriate amount of 10% donkey serum onto each tissue. Finally, place the slides in a 37°C incubator for 2 hours. Next, the required primary antibody was added to tissue sections from young mice, aged mice, and aged mice treated with N-acetyl-L-tryptophan, respectively. The sections were then placed in a humidified chamber and incubated overnight at 4°C. The following day, the sections were washed three times with PBS for 7 minutes each time to thoroughly remove any residual primary antibody. Subsequently, the corresponding secondary antibody was added under light-protected conditions, and the sections were incubated at 37°C for 2 hours. Finally, the sections were washed three more times with PBS for 7 minutes each time. Then, an anti-fluorescence quencher was applied to each tissue sample, and a small amount of colorless nail polish was applied to each of the four corners of the slide. The slides were then mounted (protected from light throughout the process) and stored at -20°C in the dark. Immunofluorescence staining was used for detection, and ImageJ was used for quantitative analysis of fluorescence intensity bands. AVONA was used for statistical analysis, and Graphpad was used for plotting (data are mean ± SEM). , The result is as follows: Figure 5 As shown.

[0057] Figure 5 This is the result of using immunofluorescence staining in Example 6 to detect the effect of N-acetyl-L-tryptophan on improving the proliferation of ovarian granulosa cells in aging mice. In this example, A is the expression level of Ki-67 in ovarian granulosa cells of young mice, aging mice, and aging mice + N-acetyl-L-tryptophan treatment group detected by immunofluorescence staining, and B is the quantitative analysis of A. Scale bar = 40 μm.

[0058] Depend on Figure 5 It can be seen that N-acetyl-L-tryptophan can improve the Ki-67 expression level in ovarian granulosa cells of aging mice and effectively enhance the proliferation capacity of ovarian granulosa cells.

[0059] Example 7: Detection of the role of N-acetyl-L-tryptophan in improving mitochondrial homeostasis imbalance in the ovaries of aging mice using Western blotting. Take mouse ovarian tissue from each group in Example 1, add pre-chilled protein extraction reagent containing inhibitors (a mixture of protease inhibitor, RIPA and phosphatase inhibitor), and lyse on ice for 60 min; place the lysate in a pre-chilled centrifuge, centrifuge at 12,000 rpm for 10 min, take the supernatant (i.e., the protein of each group) and transfer it to a new centrifuge tube, heat at 100℃ for 10 min, and store at -80℃ for later use to obtain the protein samples of each group.

[0060] Protein samples from each group were separated on a 10% SDS-polyacrylamide gel electrophoresis, transferred to a nitrocellulose membrane, and detected using antibodies against DRP1, OPA1, MFF, Fis1, MFN1, and MFN2. Image J was used for quantification of the bands, and ANOVA was used for statistical analysis. Graphpad software was used for plotting (data are expressed as mean ± SEM). , , ).

[0061] Figure 6 To analyze the effect of N-acetyl-L-tryptophan on improving mitochondrial homeostasis imbalance in the ovaries of aging mice using Western blotting; AD represents the detection and quantitative analysis of the expression levels of OPA1, MFN2, and MFN1 in the ovaries of the three groups, and EH represents the detection and quantitative analysis of the expression levels of DRP1, MFF, and Fis1 in the ovaries of the three groups.

[0062] Depend on Figure 6 It can be seen that, compared with the young group, the expression of mitochondrial homeostasis-related proteins DRP1, MFF, Fis1, OPA1, MFN1, and MFN2 was imbalanced in the aging group; this phenomenon was reversed after treatment with N-acetyl-L-tryptophan. N-acetyl-L-tryptophan significantly improved the expression levels of DRP1, OPA1, and MFF in aging mice, effectively promoted the restoration of mitochondrial homeostasis (the process of division and fusion), maintained mitochondrial function, and further optimized ovarian function.

[0063] In summary, this invention is the first to discover that N-acetyl-L-tryptophan can upregulate AMH levels in aging ovarian tissue, significantly improving ovarian reserve function in aging mice; simultaneously, it can improve mitochondrial dynamics balance in oocytes, thereby improving or treating oocyte maturation disorders. The mitochondrial membrane potential of oocytes in aging mice was significantly reduced, and Ki-67 expression in ovarian granulosa cells was downregulated, while N-acetyl-L-tryptophan could reverse these abnormalities. These results indicate that N-acetyl-L-tryptophan and its related drug formulations are highly effective in improving oocyte maturation disorders and enhancing ovarian reserve function, possessing great potential and broad clinical application prospects in repairing ovarian damage and delaying ovarian aging.

[0064] The above description is only a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any equivalent substitutions or modifications made by those skilled in the art within the scope of the technology disclosed in the present invention, based on the technical solution and inventive concept of the present invention, should be covered within the scope of protection of the present invention.

Claims

1. An application of N-acetyl-L-tryptophan, characterized in that, Including the following applications: a. Application in the preparation of drugs or health products that enhance ovarian reserve function; b. Use in the preparation of drugs or health products that improve or treat oocyte maturation disorders.

2. The application of N-acetyl-L-tryptophan according to claim 1, characterized in that, The ovary is an aging ovary.

3. The application of N-acetyl-L-tryptophan according to claim 1 or 2, characterized in that, N-acetyl-L-tryptophan improves or treats oocyte maturation disorders by enhancing the dynamic balance of oocyte mitochondria.

4. The application of N-acetyl-L-tryptophan according to any one of claims 1-3, characterized in that, The oocytes are oocytes from aging ovaries.

5. A drug, characterized in that, include: N-acetyl-L-tryptophan or a pharmaceutically acceptable salt thereof, a pharmaceutically acceptable carrier or excipient; The drug can improve or treat oocyte maturation disorders and enhance ovarian reserve.

6. The drug according to claim 5, characterized in that, The dosage form of the drug is liquid, gas, solid, or semi-solid.

7. A health product, characterized in that, include: N-acetyl-L-tryptophan or a pharmaceutically acceptable salt thereof, a pharmaceutically acceptable carrier or excipient; The health products mentioned can improve or treat oocyte maturation disorders and enhance ovarian reserve function.

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