阅读说明：本技术 二甲双胍在激活雌性生殖干细胞的应用 (Application of metformin in activation of female reproductive stem cells ) 是由 何瑞 吴际 李广永 马会明 王春红 孙亚群 于 2021-10-27 设计创作，主要内容包括：本申请公开了二甲双胍的新应用,即二甲双胍可以激活雌性生殖干细胞,促进其增殖,进而改善多囊卵巢综合征、卵巢早衰等卵泡发育障碍性疾病；公开了二甲双胍用于制备激活雌性生殖干细胞的药物中的应用和二甲双胍通过激活雌性生殖干细胞而用于制备预防和/或治疗多囊卵巢综合征的药物中的应用。实验结果验证了二甲双胍在激活雌性生殖干细胞的应用。二甲双胍不仅可以激活雌性生殖干细胞,促进细胞增殖,改善卵巢储备功能及卵泡发育过程。还可以有效改善PCOS糖耐量异常、体重增加、排卵异常及卵巢多囊病变,通过激活雌性生殖干细胞,改善多囊卵巢综合征。本申请对于直接利用二甲双胍激活雌性生殖干细胞的发生及增殖提供了证据和基础。(The application discloses a new application of metformin, namely metformin can activate female reproductive stem cells and promote the proliferation of the female reproductive stem cells, so that follicular developmental disorder diseases such as polycystic ovarian syndrome, premature ovarian failure and the like are improved; the application of metformin in preparing a medicament for activating female reproductive stem cells and the application of metformin in preparing a medicament for preventing and/or treating polycystic ovary syndrome by activating female reproductive stem cells are disclosed. The experimental result proves the application of the metformin in activating female reproductive stem cells. The metformin can activate female reproductive stem cells, promote cell proliferation, and improve ovarian reserve function and follicle development process. Can also effectively improve PCOS abnormal sugar tolerance, weight gain, anovulation and ovarian polycystic lesion, and improve polycystic ovarian syndrome by activating female reproductive stem cells. The application provides evidence and basis for directly utilizing metformin to activate the generation and proliferation of female reproductive stem cells.)

1. Use of metformin for activating female reproductive stem cells.

2. Use of metformin for the preparation of a product for activating female reproductive stem cells.

3. The application of metformin in preparing a product for preventing and/or treating polycystic ovarian syndrome by activating female reproductive stem cells.

4. Application of metformin in preparing a product for preventing and/or treating premature ovarian failure by activating female reproductive stem cells.

5. Use of metformin for the preparation of a product for the prevention and/or treatment of infertility by activating female germ stem cells.

6. Use according to any of claims 2-5, wherein the product comprises a pharmaceutical, a food product and a nutraceutical product.

7. The use of claim 6, wherein the medicament is in the form of soft capsules, dripping pills, emulsions, tablets, lotions, liniments, ointments, suppositories, or the like.

Technical Field

The application relates to the technical field of application of metformin, in particular to application of metformin in activating female reproductive stem cells.

Background

Stem cells are ideal seed cells with the potential for self-renewal and multiple differentiation, playing a fundamental role in maintaining body homeostasis, cell regeneration and repair. Female Germ Stem Cells (FGSCs) are present in the ovarian surface epithelium as one of many stem cells, and can differentiate into oocytes and promote follicle renewal. Studies have shown that female germ stem cells can be successfully isolated from postnatal mammalian ovaries, while after transplantation of FGSCs, follicles at various stages can be restored and healthy offspring produced. Therefore, the proliferative capacity of FGSCs helps to maintain follicular development and prolong the reproductive cycle.

Metformin is a classic drug for treating obesity type 2 diabetes, and no report on the action of metformin for activating female reproductive stem cells and promoting the proliferation of the female reproductive stem cells is found so far.

Disclosure of Invention

In view of the above, the present application provides a new use of metformin, that is, the application of metformin in activating female reproductive stem cells, so as to solve the problem that metformin has no activating effect on female reproductive stem cells at present.

In a first aspect, the present application provides the use of metformin for activating female reproductive stem cells.

In a second aspect, the present application provides the use of metformin for the manufacture of a product for activating female reproductive stem cells.

In a third aspect, the application provides the application of metformin in preparing a product for preventing and/or treating polycystic ovarian syndrome by activating female reproductive stem cells.

In a fourth aspect, the application provides the application of metformin in preparing a product for preventing and/or treating premature ovarian failure by activating female reproductive stem cells.

In a fifth aspect, the present application provides the use of metformin for the preparation of a product for the prevention and/or treatment of infertility by activation of female reproductive stem cells.

Further, the products include pharmaceuticals, foods and nutraceuticals.

Furthermore, the dosage form of the medicine comprises soft capsules, dripping pills, emulsions, tablets, lotions, liniments, ointments or suppositories.

The application discovers for the first time that the metformin can activate female reproductive stem cells and promote the proliferation of the female reproductive stem cells. And the action and mechanism of the metformin for activating the female reproductive stem cells are verified through rigorous scientific experiments. The metformin can activate female reproductive stem cells, promote cell proliferation, and improve ovarian reserve function and follicle development process. Meanwhile, the PCOS sugar tolerance abnormality, the weight increase, the ovulation abnormality and the ovarian polycystic lesion can be effectively avoided. The application provides evidence and basis for the occurrence of activating female reproductive stem cells directly with metformin.

Drawings

In order to more clearly explain the technical solution of the present application, the drawings needed to be used in the embodiments will be briefly described below, and it is obvious to those skilled in the art that other drawings can be obtained according to the drawings without creative efforts.

FIG. 1A is a bar graph of body mass index results for various groups of mice;

FIG. 1B is a line graph of oral glucose tolerance before treatment in groups of mice;

figure 1C is a line graph of oral glucose tolerance after treatment in groups of mice;

FIG. 1D is a bar graph of the area under the oral glucose tolerance break before treatment in each group of mice;

FIG. 1E is a bar graph of the area under the oral glucose tolerance break after treatment in groups of mice;

FIG. 2A is a bar graph of testosterone levels in various groups of mice;

FIG. 2B is a bar graph of estrogen levels in groups of mice;

figure 2C is a bar graph of luteinizing hormone levels in various groups of mice;

FIG. 2D is a bar graph of the ratio of luteinizing hormone to follicle stimulating hormone levels in various groups of mice;

FIG. 2E is a bar graph of AMH levels in groups of mice;

FIG. 2F shows the results of immunohistochemical detection of anti-Dolleran hormone in each group of mice;

FIG. 3A shows the results of experiments on the estrus cycle of mice in each group;

FIG. 3B is a bar graph of follicle counts in various groups of mice;

FIG. 3C shows the H & E staining results of the oestrus cycle of the groups of mice;

FIG. 3D shows the H & E staining of ovarian tissues of various groups of mice;

FIG. 4A shows the results of immunohistochemical studies of DDX4/MVH in various groups of mice;

FIG. 4B shows the results of immunofluorescence analysis of DDX4/MVH and PCNA in various groups of mice;

FIG. 4C is a bar graph of the number of FGSCs in each group of mice;

FIG. 4D shows the results of analysis of DDX4, PCNA, cyclin D2 in each group of mice;

FIG. 4E shows the relative expression level of DDX4/MVH protein in each group of mice;

FIG. 4F shows the results of the relative expression levels of PCNA proteins in mice of each group;

FIG. 4G shows the relative expression level of cyclin D2 protein in each group of mice.

Detailed Description

Reference will now be made in detail to embodiments, examples of which are illustrated in the accompanying drawings. When the following description refers to the accompanying drawings, like numbers in different drawings represent the same or similar elements unless otherwise indicated. The embodiments described in the following examples do not represent all embodiments consistent with the present application. But merely as exemplifications of systems and methods consistent with certain aspects of the application, as recited in the claims.

In a first aspect, the present application provides the use of metformin for activating female reproductive stem cells.

In a second aspect, the present application provides the use of metformin for the manufacture of a product for activating female reproductive stem cells.

In a third aspect, the application provides the application of metformin in preparing a product for preventing and/or treating polycystic ovarian syndrome by activating female reproductive stem cells.

In a fourth aspect, the application provides the application of metformin in preparing a product for preventing and/or treating premature ovarian failure by activating female reproductive stem cells.

In a fifth aspect, the present application provides the use of metformin for the preparation of a product for the prevention and/or treatment of infertility by activation of female reproductive stem cells.

Further, the products include pharmaceuticals, foods and nutraceuticals.

Furthermore, the dosage form of the medicine comprises soft capsules, dripping pills, emulsions, tablets, lotions, liniments, ointments or suppositories.

The application discovers for the first time that the metformin can activate female reproductive stem cells and promote the proliferation of the female reproductive stem cells. And the action and mechanism of the metformin for activating the female reproductive stem cells are verified through rigorous scientific experiments. The metformin can activate female reproductive stem cells, promote cell proliferation, and improve ovarian reserve function and follicle development process. Meanwhile, the PCOS sugar tolerance abnormality, the weight increase, the ovulation abnormality and the ovarian polycystic lesion can be effectively avoided. The application provides evidence and basis for the occurrence of activating female reproductive stem cells directly with metformin.

To confirm that metformin has the above-mentioned novel use, the following experiment was performed.

1. The technical scheme is as follows:

polycystic ovarian syndrome (PCOS) is a typical disease of follicular dysgenesis, is a good model for researching female reproductive stem cells, and takes a PCOS mouse model as an example to prove that metformin activates the female reproductive stem cells, improves abnormal glucose tolerance, weight gain and ovulation and protects the ovarian function.

Model establishment and treatment of PCOS mice.

Letrozole is an aromatase inhibitor that inhibits the conversion of ovarian androgens to estrogens, thereby causing hyperandrogenism. The method comprises the following specific implementation steps: high fat feeding with gavage letrozole 1 mg/kg^-1d^-1After 21 days of continuous administration, the PCOS model which causes insulin resistance and hyperandrogenism was randomly divided into 3 groups (n ═ 6), which were a blank normal group, a PCOS model group, and a metformin group. The other two groups were fed with high fat diet continuously, except for the normal group. Simultaneous gavage administration of 1 mg/kg letrozole to the model group^-1d^-1In the metformin group, 200 mg-kg of metformin is administered^-1d^-1Gavage, normal group given equal dose of normal saline. Taking materials after 1 month, and collecting serum for hormone detection; taking a mouse ovary, and embedding one side of the mouse ovary by conventional paraffin for histological examination; the other side is stored in a refrigerator at minus 80 ℃ for molecular biology experimental study.

2. Monitoring experimental results and collecting data:

A. vaginal secretion smears are used for estrous cycle determination; during the specific implementation, a dropper is used for sucking a proper amount of normal saline, vaginal secretion is washed and sucked on a glass slide at the same time point, H & E staining is carried out, the cell morphology is observed under a microscope, and the estrus cycle is judged. The estrus cycle was measured 7 consecutive days before material selection.

B. Oral Glucose Tolerance Test (OGTT); in particular, the OGTT test is carried out 20 days after the model building and 27 days after the treatment. The method comprises the following steps: fasting for 14-16h, measuring body weight before filling sugar, and measuring blood sugar at 0, 15, 30, 60, and 120 min.

Detecting sex hormone by ELISA method; in the specific implementation, the ELISA method is adopted to detect the content of T, E2, LH, FSH and AMH in each serum sample, and the operation is strictly carried out according to the requirements of a kit instruction.

D. Detecting the tissue morphology; in specific implementation, detection includes H & E staining, immunohistochemistry, immunofluorescence. Observing under a mirror, and taking a picture for storage. Wherein the rabbit anti-AMH (1:50), the rabbit anti-DDX 4/MVH (1:1000) in immunohistochemistry; MVH rabbit antibody (1:400), PCNA murine antibody (1:200), corresponding fluorescent secondary antibody (1:300) in immunofluorescence.

Westernblot detects the expression of DDX4/MVH, PCNA, cyclinD2, AMPK, p-AMPK, mTOR and p-mTOR proteins; in the specific implementation, ovarian tissue protein is extracted, a BCA kit is quantified, the sample is loaded, electrophoresis is carried out, the electric conversion is carried out for 200mA and 2h, 5% milk is sealed for 1h, corresponding primary antibodies (except the concentration of beta-actin is 1:2000 and PCNA1:4000, the rest antibodies are 1:1000) are incubated at 4 ℃ overnight, corresponding secondary antibodies are incubated for the next day and room temperature is 1h, autoradiography is carried out, and the gray value of each band is measured by ImageJ image analysis software.

3. The experimental results are as follows:

3.1 high fat feeding in combination with gavage administration of letrozole resulted in the mouse obesity, impaired glucose tolerance phenotype, with significant improvement following metformin administration.

As shown in fig. 1B, the PCOS model was established by combination of high fat feeding and gavage administration of letrozole, and the Body Mass Index (BMI) was calculated by measuring the body mass of mice to prove that the body mass index of mice in the PCOS model group was significantly higher than that in the normal group, and OGTT showed significant impaired glucose tolerance.

Further, as shown in fig. 1A, body weight was significantly reduced after administration of metformin therapy compared to the PCOS model group.

Further, referring to fig. 1D and 1E, OGTT shows the blood glucose values in the metformin-treated group, which were significantly lower than those in the PCOS model group and the normal group at 30min, 60min, 90min, and 120min after administration of glucose, and the area under the curve AUC is also shown.

Further, as shown in fig. 1C, the blood glucose in the metformin group was significantly improved as compared with the normal group.

3.2 high fat feeding in combination with gavage administration of letrozole can cause hyperandrogenism and estrogen disorders in mice, administration of metformin herein ameliorates sex hormone disorders.

Referring to FIGS. 2A, 2C and 2D, by measuring serum sex hormone levels, serum testosterone T, luteinizing hormone LH levels and LH/FSH ratios were significantly increased in PCOS model group mice as compared to normal group mice.

Further, referring to fig. 2B, estradiol levels were also significantly lower than in the normal group, while testosterone, luteinizing hormone LH and the luteinizing hormone LH/follicle stimulating hormone FSH ratio were significantly reduced after metformin drying, and metformin was effective in increasing estrogen levels.

Furthermore, metformin reduces the expression of polycystic ovary AMH, AMH shows the function of ovary reserve, and multiple studies show that a great amount of AMH can be generated in the serum of a PCOS patient, and the AMH level can reflect the severity of PCOS, so that the metformin can be used as an important reference index for clinical diagnosis and prediction of PCOS. As shown in fig. 2F immunohistochemistry, AMH expression (yellow-stained part) was found around follicles and between granulosa cells in the normal group, but the expression level was not high; the model group can obviously see that the expression of AMH is far higher than that of the normal group and other groups; in the metformin group, the area of yellow stain around the follicles and between granulosa cells was significantly smaller than that in the model group, indicating that AMH was highly expressed in the ovaries of PCOS model group mice. Referring to fig. 2E, serum AMH results also demonstrate that metformin herein can reduce the severity of PCOS, preserving ovarian function.

3.3 metformin can improve PCOS mouse ovary morphology and function.

The estrus cycle of the mouse is similar to the menstrual cycle of a human, and the ovarian ovulation function and the endocrine function are reflected. According to the cell types and forms observed by the vaginal secretion smear, the estrus cycle is divided into prophase estrus, anaphase estrus and estrus interval. During specific implementation, the estrus cycle is continuously detected through a mouse vaginal smear, and the rule of the estrus cycle of a normal group of mice is found, so that alternation of the prophase of estrus, the anaphase of estrus and the estrus interval is presented. Wherein, 4-5 days are a cycle and appear regularly.

Referring to FIG. 3C, PCOS model group smears persisted in large numbers of leukocytes, very few spindle epithelial cells, lost normal estrous cycles and at sustained intervals. As can be seen from fig. 3A and 3C, the metformin group was in the same model group before treatment and in the continuous interval, but after treatment, the anteestrus, the estrus and the anaphase of estrus gradually appeared until the normal regular estrus cycle was recovered before material drawing. Therefore, the metformin group can obviously improve the estrus cycle of the mice.

Further, referring to fig. 3D, ovarian morphology experiments were performed and found by H & E staining: the ovary of a normal mouse can see a plurality of follicles and corpus luteum at different developmental stages, and granular cells are complete in shape and are regularly arranged. The ovaries of mice in the PCOS model group showed multiple cavitary follicles, sparse stratum granulosum, and mostly atretic follicles. The number of ovarian atresia follicles of mice in the metformin treatment group is obviously reduced, a plurality of follicles in different development stages can be seen in the ovaries, the follicles close to the mature stage can see oocytes and radial crowns, the granular cell layer is thickened and tightly gathered, and the shape of the granular cell layer is close to that of a normal group.

Further, referring to fig. 3B, follicle count was found to be: compared with the normal group of mice, the number of atretic follicles in the model group is obviously increased, the number of primordial follicles, primary follicles, secondary follicles and mature follicles is obviously reduced, the number of atretic follicles in the metformin group is obviously reduced, and the number of follicles in other stages is increased.

The results show that: the metformin can obviously restore the estrus cycle of the PCOS mouse, improve the pathological polycystic ovary of the PCOS mouse and improve the ovarian function.

3.4 metformin activates female reproductive stem cells and promotes their proliferation.

DDX4/MVH is a specific marker for FGSCs. See FIG. 4A, arrows indicate positive expression of DDX4/MVH by immunohistochemical staining, distributed in the ovarian cortex, demonstrating the presence of FGSCs.

To further quantify and proliferate FGSCs, MVH/DDX4, PCNA co-labeling was performed by immunofluorescence, see fig. 4B. PCNA is proliferating cell nuclear antigen and is an important index reflecting cell proliferation, as shown in the figure, green fluorescence (arrow position) is positive expression of DDX4/MVH, which indicates that the cell is FGSCs, and red fluorescence (arrow position) is positive expression of PCNA, which indicates that the cell has proliferation activity.

Further, referring to fig. 4C, it can be seen that FGSCs were present in each group, especially FGSCs were significantly increased in number in the metformin-treated group, expressed in clusters, and positively stained with PCNA, indicating proliferative activity. Although the PCOS model group has a small amount of FGSCs, most of the cells have no positive expression of PCNA, indicating that the cells have no proliferation.

Further, as shown in FIG. 4D, the results of Westernblot also found that the specific proteins DDX4/MVH, PCNA and cyclinD2 of the metformin group FGSCs are significantly increased compared with the model group, and FIGS. 4E, 4F and 4G are the relative protein quantification results thereof, which all prove that metformin can activate FGSCs and promote the proliferation thereof.

PCOS is associated with a series of metabolic abnormalities such as obesity, insulin resistance, compensatory hyperinsulinemia, glucose intolerance, lipid abnormalities, etc., and disorders such as glycolipid metabolism in turn promote the progress of PCOS.

In the present application, a PCOS model mouse established by letrozole in combination with HFD exhibits obesity, impaired glucose tolerance, hyperandrogenism and estrogen disorders, and develops reproductive disorder phenotypes such as estrus cycle disorders, ovulation disorders, and ovarian dysfunction. After the metformin therapy is given, the characteristics of glycolipid metabolic disorder, hormone level disorder, ovarian dysfunction and the like are obviously improved. Since the main mechanisms of infertility caused by PCOS are follicular developmental disorder and ovulation disorder, high LH/FSH (luteinizing hormone/follicle stimulating hormone) levels are one of the typical features of PCOS. High LH inhibits FSH function, luteinizing granulosa cells, stopping small antral follicle development, and ovarian polycystic. The experimental results also prove that the metformin can regulate the LH/FSH steady state and improve the follicular dysplasia and the granular cell state.

Depletion of FGSCs results in decreased oocyte numbers and ovarian dysfunction. The results of the experiments described above show that PCOS model mice lack proliferation ability although they contain a certain amount of FGSCs. After metformin therapy, FGSCs are significantly increased in number and have proliferative capacity. Meanwhile, Westernblot results show that specific markers DDX4/MVH protein, cell proliferation protein PCNA and cyclinD2 of the female stem cells of mice in a metformin-treated group are obviously up-regulated, and further prove that the metformin can activate FGSCs and promote the proliferation of the FGSCs. The discovery provides a new direction for promoting oocyte regeneration and supplementing the follicle pool so as to increase pregnancy rate.

In summary, the application proves that the metformin improves the effects of PCOS glycolipid metabolic disorder, sex hormone disorder, ovulation disorder and the like through a PCOS animal model. Metformin is found and verified to improve the developmental maturity of follicles by regulating the AMH homeostasis. The application provides new clues for the pathogenesis research and clinical treatment of ovary dysfunction diseases such as PCOS, premature ovarian failure and the like.

The embodiments provided in the present application are only a few examples of the general concept of the present application, and do not limit the scope of the present application. Any other embodiments extended according to the scheme of the present application without inventive efforts will be within the scope of protection of the present application for a person skilled in the art.