Use of hdac inhibitor ms-275 in the manufacture of a medicament for ovarian function preservation

Abstract

The application discloses application of an HDAC inhibitor MS-275 in preparation of an ovarian function protection drug and belongs to the technical field of biology and pharmacy. The application finds that MS-275 can safely and effectively improve the endocrine function of ovaries, ovarian reserve, fertility and the like of mice in a drug protection group. Therefore, the application proposes that MS-275 can delay ovarian aging, and plays an important role in delaying ovarian aging and / or repairing ovarian damage and / or improving ovarian reserve function and / or reconstructing ovarian function and the like.

Description

TECHNICAL FIELD

[0001] The application belongs to the fields of biology and pharmacy, and particularly relates to an application of an HDAC inhibitor MS-275 in preparing an ovary function protection drug. BACKGROUND

[0002] Due to the aggravation of population aging, the concepts of "healthy aging" and "aging management" gradually enter the vision of people. In 2015, Nature magazine proposed that aging has entered a state that needs to be treated, and reducing the aging state and promoting healthy aging can help prevent and treat other aging-related diseases. Ovarian aging is a special type of organ aging, and in addition to affecting the fertility of women, changes in hormone levels can also cause physical and psychological discomfort such as hot flashes, sweating, insomnia, and irritability. In addition, ovarian aging can also start and accelerate the aging of brain, heart, bone and other organs, leading to multi-system dysfunction. At present, anti-ovarian aging has become the focus of widespread attention in the biomedical and pharmaceutical industries.

[0003] At present, the exploration of ovarian function protection is increasingly valued, and researchers strive to explore its protection measures in various directions. The reported prevention and treatment measures for ovarian aging mainly include the following aspects: drug intervention, stem cell therapy, mitochondrial transplantation, in vitro ovarian activation, ovarian transplantation, artificial 3D ovary, etc. Looking at the above research directions, there are some problems, such as uncertain efficacy, ethical issues, immature technology, and low patient acceptance. Looking back at drug treatment, it is conservative but safe, and has high public acceptance.

[0004] Histone deacetylase inhibitors (HDAC inhibitors) are considered to be the most promising drugs for treating age-related chronic diseases. HDAC inhibitors are more widely studied in fruit flies, and studies have reported that phenylbutyric acid, sodium butyrate, trichostatin A, and hydroxamic acid can prolong the lifespan of fruit flies, and have very strong anti-aging potential. In mammals, treatment with HDAC inhibitors can produce significant anti-aging and promote healthy lifespan effects. There are many types of HDAC inhibitors, and there are fewer reports about MS-275, especially there is no report about combining it with ovarian aging and ovarian damage. SUMMARY

[0005] In view of the deficiencies in the prior art, the purpose of the present application is to provide an application of an HDAC inhibitor MS-275 in preparing an ovary function protection drug. The present application found that HDAC inhibitor MS-275 can significantly improve the reduction of ovarian reserve, endocrine and reproductive function during ovarian aging, and plays an important role in delaying ovarian aging.

[0006] To achieve the above purpose, the technical scheme of the present application is as follows:

[0007] The application provides application of an HDAC inhibitor MS-275 in preparation of an ovary function protection drug.

[0008] Preferably, the ovary function protection comprises delaying ovary aging and / or repairing ovary damage and / or improving ovary reserve function.

[0009] Further, the ovary aging is ovary aging caused by genetics, environment, behavior, infection or immunity.

[0010] Further, the ovary damage is ovary damage caused by genetics, environment, behavior, infection or immunity.

[0011] Further, the ovary damage and the ovary aging are both caused by oxidative stress and inflammation.

[0012] Preferably, the HDAC inhibitor MS-275 is used for inhibiting follicle number reduction in the ovary aging process, and / or inhibiting estrogen and progesterone secretion reduction, and / or inhibiting fertility function reduction in the ovary function protection.

[0013] Preferably, the drug is in the form of injection.

[0014] Compared with the prior art, the application has the following advantages and beneficial effects:

[0015] The application specifically detects the improvement effect of MS-275 on a D-gal induced chronic ovary aging model. By using an in vitro ovary aging cell model, oxidative stress, inflammation and cell aging level of ovary granulosa cells are detected, and it is found that the ovary cell function can be improved after MS-275 treatment. By using a D-gal induced ovary aging mouse model, the safety of MS-275 and the protection effect of MS-275 on ovary aging are explored, including detection of mouse health status, ovary endocrine function, ovary reserve and fertility, and it is found that MS-275 can obviously improve the reduction of ovary reserve, endocrine and fertility function in the ovary aging process, and plays an important role in delaying ovary aging. BRIEF DESCRIPTION OF DRAWINGS

[0016] Figure 1 Figure 1 is a diagram of the improvement effect of the HDAC inhibitor MS-275 on a D-gal induced ovary aging cell model of the application; wherein, Figure 1 A is the expression change of oxidative stress indexes detected by RT-PCR and WB after MS-275 intervention on COV434, Figure 1 B is the total antioxidant capacity of COV434 cells after MS-275 intervention, Figure 1 C is the MDA content in COV434 cells, Figure 1D is the expression change of inflammation and aging indicators detected by RT-PCR and WB after MS-275 intervention in COV434, Figure 1 E is β-gal staining and statistical analysis.

[0017] Figure 2 Figure is a biological safety evaluation chart of the HDAC inhibitor MS-275 after intervention in mice in the present application, wherein, Figure 2 A is a mouse weight curve, Figure 2 B is a general observation of each organ of mice, Figure 2 C is an index of each organ of mice, wherein, Figure 2 C1 is a heart index, Figure 2 C2 is a liver index, Figure 2 C3 is a spleen index, Figure 2 C4 is a lung index, Figure 2 C5 is a kidney index, and Figure 2 C6 is a uterus index.

[0018] Figure 3 Figure is a chart for detecting endocrine function of mice after intervention of the HDAC inhibitor MS-275 in the present application, wherein, Figure 3 A is a statistical chart of the proportion of regular and irregular estrus cycles, Figure 3 B is the serum FSH level of mice in each group, Figure 3 C is the serum E2 level of mice in each group, Figure 3 D is the serum AMH level of mice in each group.

[0019] Figure 4 Figure is a chart for detecting ovarian reserve of mice after intervention of the HDAC inhibitor MS-275 in the present application, wherein, Figure 4 A is the change of ovarian index in each group, Figure 4 B is the follicle count result in each group; PMF: primordial follicle; PF: primary follicle; SF: secondary follicle; ANF: antral follicle; ATF: atretic follicle; CL: corpus luteum.

[0020] Figure 5 Figure is a chart for detecting fertility of mice after intervention of the HDAC inhibitor MS-275 in the present application, wherein, Figure 5 A is a picture of typical female mice and offspring in each group, Figure 5 B is a pregnancy rate statistics of each group, Figure 5 C is the average number of offspring per litter, Figure 5 D is the average birth weight of mice in each group, Figure 5 E is the sex ratio of offspring of mice in each group. DETAILED DESCRIPTION

[0021] The application will be further described in conjunction with the accompanying drawings, examples and experimental examples. Of course, the protection scope of the application is not limited to the following examples. Those skilled in the art can understand that various changes and modifications can be made to the application without departing from the spirit of the application. The application generally and / or specifically describes the materials used in the experiments and the experimental methods. Although many materials and operation methods used to achieve the purpose of the application are well known in the art, the application is still described as detailed as possible. The following examples are further to illustrate the application, rather than to limit the application. Any formal but not substantial equivalent transformation made according to the concept of the application should be regarded as the scope of the technical solutions of the application.

[0022] The test methods or test methods described in the following examples are all conventional methods unless otherwise specified; the reagents and materials are obtained from conventional commercial channels or prepared by conventional methods unless otherwise specified.

[0023] At present, with the deepening of population aging and the opening of the two-child and three-child policy, it is urgent to solve the problem of ovarian aging and promote female fertility. How to solve the technical problem and protect the function of the ovary is a technical problem that those skilled in the art need to solve.

[0024] Definitions and uses of terms

[0025] Ovarian aging: The application mainly refers to the process that the function of female ovary gradually declines with the increase of age, which is affected by multiple factors such as heredity, environment and lifestyle, based on the decline of follicle number and oocyte quality, and finally shows menopause, and affects multiple organs of the whole body, leading to a pathological process of related diseases.

[0026] Improving or enhancing ovarian reserve function: The ovarian reserve function of the application refers to the ability of primordial follicles in the ovarian cortex to develop into fertilizable oocytes. The current clinical indicators for evaluating ovarian reserve function mainly include age, basal endocrine hormones, cytokines, and basal antral follicle count (AFC).

[0027] D-gal: D-galactose (D-galactose) is a hexosamine with a molecular formula of C6H12O6 and a relative molecular mass of 180.16. It is widely used in biological experiments to cause organ function damage models such as liver damage, neurodegeneration, and ovarian aging.

[0028] MS-275: MS-275 is a HDAC inhibitor with a molecular formula of C21H20N4O3 and a relative molecular mass of 376.41.

[0029] Currently, there are no safe and effective measures to protect ovarian fertility in clinical practice. Moreover, since the factors affecting ovarian aging are very extensive, involving genetic, environmental, and psychological aspects, etiological treatment is crucial. This invention mainly studies how to delay the decline in ovarian function caused by ovarian aging, providing a new measure for drug-induced delay of ovarian aging.

[0030] The specific technical solution is as follows:

[0031] 1. The effect of MS-275 on improving a D-gal-induced in vitro ovarian aging model: Aging is a chronic, gradual process. Studies have shown that D-galactose (D-gal) can induce accelerated aging models both in vitro and in vivo, primarily through increasing intracellular reactive oxygen species (ROS) levels, leading to oxidative stress, inflammation, mitochondrial dysfunction, and apoptosis. Based on literature review, this invention subsequently used 100 mM D-gal to treat for 48 hours to construct ovarian senescent granulosa cells and theca interstitial cells models. Next, this invention treated COV434 cells with MS-275 at concentrations of 0 μM, 0.1 μM, 1 μM, 2 μM, 5 μM, and 10 μM for 48 hours, and then measured CCK8 levels. The results showed that high concentrations of the inhibitor significantly inhibited cell viability. Since the inhibitor's effect on cell viability increased significantly with increasing concentration, 0.25 μM MS-275 was ultimately selected for subsequent experiments. After co-intervention with D-gal in COV434 cells, MS-275 significantly upregulated D-gal, leading to a decrease in the expression of intracellular antioxidant enzymes. Changes in the RNA and protein levels of antioxidant enzymes were detected. Figure 1 A). To further explore the effects of MS-275 on cellular oxidative stress, we examined the total antioxidant capacity of cells and found that the antioxidant capacity of cells in the D-gal group was significantly decreased compared to the control group, while the antioxidant capacity of cells in the MS group was significantly increased compared to the D-gal group. Figure 1 B). Next, we assessed the degree of intracellular oxidative damage by detecting intracellular MDA levels. The results showed that D-gal significantly increased intracellular oxidative damage, while the degree of oxidative damage in the MS group was significantly lower than that in the D-gal group. Figure 1 C). This study further investigated whether MS-275 could alleviate the inflammatory response of granulocytes and mesenchymal cells in vitro. First, the RNA and protein levels of inflammatory factors were detected. The results showed that MS-275 significantly inhibited the increase in inflammatory factors induced by D-gal (C). Figure 1 D). In addition, inflammatory responses can lead to cellular senescence, inducing ovarian aging. We examined cellular senescence-related indicators and found that the senescence indicators in the MS-275 group were significantly lower than those in the D-gal group. β-gal staining revealed that MS-275 could reduce the proportion of senescent cells to varying degrees.Figure 1 E).

[0032] 2. Establishment of a mouse model of ovarian aging and drug treatment: A mouse model of ovarian aging mediated by oxidative stress was induced by subcutaneous injection of D-gal for 42 consecutive days. During D-gal administration, low-concentration (2 mg / kg / d, MS1 group) and high-concentration (10 mg / kg / d, MS2 group) MS-275 were administered intraperitoneally every 2 days until the end of D-gal administration. On the day of administration, D-gal was administered subcutaneously 2 hours after the intraperitoneal injection of MS-275. Body weight was continuously monitored throughout the process.

[0033] 3. General condition and safety assessment of mice

[0034] The results showed that mice experienced a decrease in body weight in the early stages of drug administration, possibly due to maladaptation to the intervention, followed by steady weight gain in the later stages. Compared to the control group, mice in the D-gal group had a significantly lower body weight, while the decrease was not significant in either of the MS groups. Figure 2 A). After the intervention, organs were removed from each group of mice, such as... Figure 2 As shown in B, the mice were weighed. Assessment of organ indices in the mice showed no significant differences between groups. Figure 2 C). The above results indicate that MS-275 has no adverse effects on the health of mice.

[0035] 4. Endocrine function testing in mice

[0036] After confirming the biocompatibility of MS-275, 14-day estrous cycle monitoring revealed that, compared to the control group, the proportion of regular estrous cycles was significantly lower in the D-gal group, while the proportion of regular estrous cycles was significantly higher in the MS1 and MS2 groups compared to the D-gal group. Figure 3 A). Hormone level testing revealed that compared to the control group, the D-gal group showed a significant increase in FSH levels, a significant decrease in AMH levels, and no significant change in E2 levels; while in the MS group, compared to the D-gal group, the MS1 group showed a decreasing trend in FSH levels, but without statistical significance, while E2 levels were significantly decreased and AMH levels were significantly increased; the MS2 group showed a significantly decreased FSH level compared to the D-gal group, while both E2 and AMH levels were significantly increased. Figure 3 (BD). In summary, the experiments show that both low and high concentrations of MS-275 can improve endocrine function in mice.

[0037] 5. Detection of ovarian reserve in mice

[0038] There was no significant difference in ovarian index among the mouse groups. Figure 4A) The follicle count results showed that the primordial follicle number was significantly decreased and the atretic follicle number was significantly increased in D-gal group compared with control group. The primordial follicle number was significantly increased and the atretic follicle number was significantly decreased in MS intervention groups compared with D-gal group Figure 4 B) The results showed that MS-275 could improve the ovarian function decreased by D-gal by inhibiting primordial follicle activation and reducing atretic follicle.

[0039] 6. Evaluation of the fertility of mice

[0040] Five mice in each group were selected to evaluate the fertility of mice. By calculating the pregnancy rate, it was found that the pregnancy rate of MS1 and MS2 groups had a significant upward trend compared with D-gal group Figure 5 A, B) The average number of offspring per litter in D-gal group was significantly decreased compared with control group, while the number of offspring in MS groups was significantly increased compared with D-gal group Figure 5 C) There was no significant statistical difference in the birth weight of offspring mice and the ratio of male and female offspring in each group Figure 5 D, E) In summary, MS-275 had a certain alleviating effect on the decrease of ovarian function and fertility caused by D-gal.

[0041] The above only describes the preferred embodiments of the present application and is not intended to limit the present application. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. Use of the HDAC inhibitor MS-275 for the manufacture of a medicament for delaying ovarian aging caused by oxidative stress and inflammation, wherein delaying ovarian aging is achieved by inhibiting the decrease in the number of follicles during ovarian aging, and / or, inhibiting the decrease in the secretion of estrogens and progestogens, and / or, inhibiting the decrease in the reproductive function.

2. Use according to claim 1, characterized in that: The ovarian aging is genetically, environmentally, behaviorally, infectiously or immunologically caused.

3. Use according to claim 1, characterized in that: The medicament is in the form of an injection.

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