Application of MAS receptor agonist in the preparation of medicine for preventing or treating polycystic ovary syndrome

Abstract

The present invention relates to the field of medical technology, and discloses the use of MAS receptor agonists in the preparation of drugs for preventing or treating polycystic ovary syndrome. Through experiments, it is proved for the first time that MAS receptors affect the occurrence and development of polycystic ovary syndrome. The MAS receptor agonist AVE0991 is used to intervene in PCOS group mice, so that the estrous cycle of mice tends to be normal; fasting blood sugar is reduced, and body weight is significantly reduced; the expression of ovarian sex hormone-related genes is reduced; in addition, the expression of genes related to liver, brown fat and subcutaneous fat metabolism in PCOS group mice intervened by AVE0991 is significantly improved; and AVE0991 significantly improves the fertility of PCOS group mice.

Description

Technical Field

[0001] The present invention relates to the field of medical technology, and in particular to application of a MAS receptor agonist in preparing a medicine for preventing or treating polycystic ovary syndrome. Background Art

[0002] Polycystic ovary syndrome (PCOS) is a female endocrine and metabolic disorder with highly heterogeneous multi-system dysfunction. The main clinical manifestations are reproductive complications (chronic anovulation, irregular menstruation, polycystic ovaries and pregnancy complications), endocrine and metabolic disorders (hyperandrogenism, IR, obesity, non-alcoholic fatty liver disease, cardiovascular disease) and psychological factors (anxiety and depression). Its incidence rate is increasing year by year, seriously affecting the physical and mental health of 5%-20% of women of childbearing age worldwide. The etiology of PCOS is complex and is related to factors such as genetics, neuroendocrine function, inflammation, environment and lifestyle, but its specific pathophysiological mechanism has not been fully elucidated. At present, the treatment methods and methods for PCOS still rely on clinical experience and are not effective.

[0003] Studies have shown that the use of lisinopril (ACE inhibitor) can reduce the levels of testosterone, androstenedione and 17α-hydroxyprogesterone in women with PCOS and hypertension, and increase the expression of sex hormone binding globulin (SHBG). Empagliflozin (SGLT-2 inhibitor) can reduce the blood pressure of PCOS rats by reducing the expression and activity of angiotensin converting enzyme (ACE) induced by androgen, but has no significant effect on serum androgen and lipid metabolism levels. At present, there is a lack of research on drugs related to the renin-angiotensin system (RAS) in the treatment of PCOS. Summary of the invention

[0004] In view of this, the present invention provides an application of a MAS receptor agonist in the preparation of a drug for preventing or treating polycystic ovary syndrome, so as to solve the problem that there is a lack of research on the application of renin-angiotensin system (RAS) related drugs in the treatment of PCOS in the prior art.

[0005] In a first aspect, the present invention provides a use of a MAS receptor agonist in the preparation of a medicament for preventing or treating polycystic ovary syndrome.

[0006] In an optional embodiment, the MAS receptor agonist includes AVE0991 or a preparation thereof, and / or Ang-(1-7) or a preparation thereof.

[0007] In an optional embodiment, the symptoms of polycystic ovary syndrome include multiple cystic dilated follicles visible in the ovaries;

[0008] Optionally, the symptoms of PCOS also include at least one of enlarged ovarian volume, pale ovarian surface, increased serum testosterone level, increased follicle-stimulating hormone level, increased luteinizing hormone level, increased total cholesterol level, and estrous cycle disorder.

[0009] In an optional embodiment, the MAS receptor agonist is used in the preparation of a drug for preventing or treating polycystic ovary syndrome combined with hypertension.

[0010] In an optional embodiment, the symptoms of polycystic ovary syndrome combined with hypertension include multiple cystic dilated follicles visible in the ovaries;

[0011] Optionally, the symptoms of polycystic ovary syndrome combined with hypertension also include at least one of enlarged ovarian volume, pale ovarian surface, increased serum testosterone level, increased follicle-stimulating hormone level, increased luteinizing hormone level, increased total cholesterol level, and estrous cycle disorder.

[0012] In an optional embodiment, the use of the MAS receptor agonist in the preparation of a drug having at least one of the following functions (1)-(7):

[0013] (1) reducing the expression of ovarian sex hormone-related genes, wherein the ovarian sex hormone-related genes include Cyp19a1 and / or Hsd17b2;

[0014] (2) improving the expression of genes related to liver metabolism, wherein the improved liver metabolism-related genes include one or a combination of two or more of AOX, HSL and Fabpl;

[0015] (3) improving the expression of genes related to brown fat metabolism, including COX4 and / or UQCRC2;

[0016] (4) improving the expression of genes related to subcutaneous fat metabolism, wherein the genes related to subcutaneous fat metabolism include one or a combination of two or more of PPARγ, ATGL, PGC1α, AGPAT and SDHB;

[0017] (5) Regulate the estrous cycle;

[0018] (6) Lowering blood sugar;

[0019] (7) Improve fertility.

[0020] In an optional embodiment, the preparation further comprises a pharmaceutically acceptable excipient.

[0021] In an optional embodiment, the preparation includes any pharmaceutically acceptable preparation;

[0022] Optionally, the preparation is in the form of tablets, capsules, oral liquids, granules, pills or injections.

[0023] Compared with the prior art, the present invention has the following beneficial effects:

[0024] The present invention provides an application of a MAS receptor agonist in the preparation of a drug for preventing or treating polycystic ovary syndrome. The experiment proves for the first time that the MAS receptor affects the occurrence and development of polycystic ovary syndrome, which provides an important potential target for the treatment of polycystic ovary syndrome.

[0025] The present invention uses DHEA-induced Mas knockout female mice to form a PCOS group mouse model. Experiments have shown that using MAS receptor agonist AVE0991 to intervene in PCOS group mice can make the estrous cycle of mice tend to normal; fasting blood sugar is reduced, and body weight is significantly reduced; the expression of ovarian sex hormone-related genes (Cyp19a1, Hsd17b2) is reduced; in addition, the expression of metabolism-related genes in the liver (AOX, HSL, Fabpl), brown fat (COX4, UQCRC2) and subcutaneous fat (PPARγ, ATGL, PGC1α, AGPAT and SDHB) of PCOS group mice intervened by AVE0991 is significantly improved; most notably, AVE0991 significantly improves the fertility of PCOS group mice, and the number of mice born in the WT-PCOS+AVE0991 group of mice is significantly increased compared with the WT-PCOS control group. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] In order to more clearly illustrate the specific implementation methods of the present invention or the technical solutions in the prior art, the drawings required for use in the specific implementation methods or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some implementation methods of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0027] Figure 1 The WT-Con group and the Mas - / - -Con group, WT-PCOS group and Mas - / - - Detection results of ovarian morphology of PCOS group mice;

[0028] Figure 2 The WT-Con group and the Mas - / - -Con group, WT-PCOS group and Mas - / - -The test results of hormone levels of PCOS group mice; A is the test result of serum testosterone content; B is the test result of serum follicle-stimulating hormone content; C is the test result of serum corpus luteum content;

[0029] Figure 3 The WT-Con group and the Mas - / - -Con group, WT-PCOS group and Mas - / - -The test results of total cholesterol and serum triglyceride levels in mice in the PCOS group; A is the test result of total cholesterol content; B is the test result of serum triglyceride content;

[0030] Figure 4 The WT-Con group and the Mas - / - -Con group, WT-PCOS group and Mas - / - -The test results of body weight and fasting blood glucose of mice in the PCOS group; C is the body weight test result; D is the fasting blood glucose test result;

[0031] Figure 5 The WT-Con group and the Mas - / - -Con group, WT-PCOS group and Mas - / - -PCOS group mice estrous cycle detection results; A is the estrous cycle monitoring results of WT-Con group mice, B is the Mas - / - -Con group mice estrous cycle monitoring results; C is WT-PCOS group mice estrous cycle monitoring results; D is Mas - / - -Estrous cycle monitoring results of PCOS group mice;

[0032] Figure 6 are the detection results of the estrous cycle of the mice in the Control group, the mice in the DHEA group, and the mice in the DHEA+AVE0991 group in Experimental Example 2 of the present invention; wherein A is the estrous cycle monitoring result of the mice in the Control group, B is the estrous cycle monitoring result of the mice in the DHEA group; and C is the estrous cycle monitoring result of the mice in the DHEA+AVE0991 group;

[0033] Figure 7 are the test results of body weight and fasting blood glucose of mice in the Control group, DHEA group and DHEA+AVE0991 group in Experimental Example 2 of the present invention; wherein C is the body weight test result of the three groups of mice; D is the fasting blood glucose test result of the three groups of mice;

[0034] Figure 8 The expression of ovarian sex hormone-related genes (Lhegr, P450scc, StAR, Cyp19a1, Fshr, Hsd17b2) in the Control group mice, the DHEA group mice and the DHEA+AVE0991 group mice in Experimental Example 2 of the present invention;

[0035] Fig. 9 The expression of liver (AOX, HSL, Fabpl) metabolism-related genes in the Control group mice, DHEA group mice and DHEA+AVE0991 group mice in Experimental Example 2 of the present invention;

[0036] Fig.10 is the expression of genes related to brown fat (COX4, UQCRC2) metabolism in the Control group mice, the DHEA group mice and the DHEA+AVE0991 group mice in Experimental Example 2 of the present invention;

[0037] Fig.11 is the expression of genes related to subcutaneous fat metabolism (PPARγ, ATGL, PGC1α, AGPAT and SDHB) of the Control group mice, the DHEA group mice and the DHEA+AVE0991 group mice in Experimental Example 2 of the present invention;

[0038] In the above figures, * indicates P < 0.05, and ** indicates P < 0.01. DETAILED DESCRIPTION

[0039] The following examples are provided for a better understanding of the present invention, but are not intended to limit the best mode of implementation, nor to limit the content and protection scope of the present invention. Any drug identical or similar to the present invention obtained by anyone under the guidance of the present invention or by combining the features of the present invention with other prior arts shall fall within the protection scope of the present invention.

[0040] Unless otherwise specified or incompatible herewith, the terms and phrases used herein shall have the following meanings:

[0041] In the present application, the selection scope involving "and / or", "or / and", and "and / or" includes any one of two or more related listed items, and also includes any and all combinations of the related listed items, and the arbitrary and all combinations include any two related listed items, any more related listed items, or a combination of all related listed items. It should be noted that when at least three items are connected by at least two conjunctions selected from "and / or", ", it should be understood that the technical solution undoubtedly includes technical solutions that are all connected by "logical and", and undoubtedly includes technical solutions that are all connected by "logical or". For example, "A and / or B" includes three parallel solutions of A, B and A+B.

[0042] In this application, "optionally" refers to optional, that is, it refers to any one of the two parallel solutions of "yes" or "no". If there are multiple "options" in a technical solution, unless otherwise specified and there is no contradiction or mutual restriction, each "option" is independent.

[0043] In this application, in the "first aspect", "second aspect", "third aspect", "fourth aspect", etc., the terms "first", "second", "third", "fourth", etc. are used only for descriptive purposes and cannot be understood as indicating or implying relative importance or quantity, nor can they be understood as implicitly indicating the importance or quantity of the indicated technical features. Moreover, "first", "second", "third", "fourth", etc. only serve the purpose of non-exhaustive enumeration and description, and it should be understood that they do not constitute a closed limitation on quantity.

[0044] In the present application, the technical features described in an open manner include closed technical solutions composed of the listed features, and also include open technical solutions containing the listed features.

[0045] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art to which this application belongs. The terms used herein in the specification of this application are only for the purpose of describing specific embodiments and are not intended to limit this application.

[0046] At present, the treatment methods and approaches for PCOS still rely on clinical experience and are not very effective. There is still a lack of research on RAS-related drugs in the treatment of PCOS. Therefore, further clarifying the etiology, inducing factors and specific pathogenesis of PCOS can provide potential drug targets and new ideas for the clinical treatment of PCOS.

[0047] In order to further study the therapeutic mechanism of RAS-related drugs in PCOS, according to the first aspect of the present invention, the present invention provides a use of a MAS receptor agonist in the preparation of a drug for preventing or treating polycystic ovary syndrome.

[0048] A receptor agonist refers to a compound or drug that can activate a receptor, that is, can cause a receptor response. A MAS receptor agonist refers to a compound or drug that can activate a MAS receptor, that is, can cause a MAS receptor response.

[0049] In some embodiments, the MAS receptor agonist includes AVE0991 and its preparations, and / or Ang-(1-7) and its preparations.

[0050] In some embodiments, the symptoms of PCOS include multiple cystic dilated follicles visible in the ovaries;

[0051] Optionally, the symptoms of PCOS also include at least one of enlarged ovarian volume, pale ovarian surface, increased serum testosterone level, increased follicle-stimulating hormone level, increased luteinizing hormone level, increased total cholesterol level, and estrous cycle disorder.

[0052] In some embodiments, the MAS receptor agonist is used in the preparation of a drug for preventing or treating polycystic ovary syndrome combined with hypertension.

[0053] In some embodiments, the symptoms of polycystic ovary syndrome combined with hypertension include multiple cystic dilated follicles visible in the ovaries;

[0054] Optionally, the symptoms of polycystic ovary syndrome combined with hypertension also include at least one of enlarged ovarian volume, pale ovarian surface, increased serum testosterone level, increased follicle-stimulating hormone level, increased luteinizing hormone level, increased total cholesterol level, and estrous cycle disorder.

[0055] ACE2 (also known as ACEH, angiotensin converting enzyme 2) has a strong affinity for Ang II type 1 and type 2 receptors, regulating blood pressure, fluid balance, inflammation, cell proliferation, hypertrophy and fibrosis. At the same time, the organ and cell-specific expression of this gene suggests that it may play a role in regulating cardiovascular and renal function and fertility. Multiple cystic dilated follicles can be seen in the ovaries. Multiple refers to more than two cystic dilated follicles.

[0056] The MAS receptor agonist of the present invention has a better effect in preventing or treating polycystic ovary syndrome combined with hypertension.

[0057] In some embodiments, the MAS receptor agonist is used in the preparation of a drug having at least one of the following functions (1)-(7):

[0058] (1) reducing the expression of ovarian sex hormone-related genes, wherein the ovarian sex hormone-related genes include Cyp19a1 and / or Hsd17b2;

[0059] (2) improving the expression of genes related to liver metabolism, wherein the improved liver metabolism-related genes include one or a combination of two or more of AOX, HSL and Fabpl;

[0060] (3) improving the expression of genes related to brown fat metabolism, including COX4 and / or UQCRC2;

[0061] (4) improving the expression of genes related to subcutaneous fat metabolism, wherein the genes related to subcutaneous fat metabolism include one or a combination of two or more of PPARγ, ATGL, PGC1α, AGPAT and SDHB;

[0062] (5) Regulate the estrous cycle;

[0063] (6) Lowering blood sugar;

[0064] (7) Improve fertility.

[0065] In some embodiments, the formulation further comprises a pharmaceutically acceptable excipient.

[0066] In some embodiments, the formulation includes any pharmaceutically acceptable formulation;

[0067] Optionally, the preparation is in the form of tablets, capsules, oral liquids, granules, pills or injections.

[0068] The present invention is further described in detail below in conjunction with specific embodiments. These embodiments should not be construed as limiting the scope of protection claimed by the present invention.

[0069] If no specific experimental steps or conditions are specified in the examples, the conventional experimental steps or conditions described in the literature in the field can be used. If no manufacturer is specified for the reagents or instruments used, they are all conventional reagents and drugs that can be purchased commercially.

[0070] In the following experiments, normal feeding specifically means: raising in an SPF animal room, with a room temperature of 22±2°C, a humidity of 60±10%, normal daylight lighting from 7 am to 7 pm, and free access to food and water. The 60% high-fat diet is a rodent diet with 60% fat energy.

[0071] Test Example 1

[0072] 1. Establishment of PCOS mouse model

[0073] 1.1 Experimental method: 3-week-old wild-type C57BL / 6 female mice were randomly divided into 2 groups, 20 mice in each group; 3-week-old Mas gene knockout female mice were randomly divided into 2 groups, 20 mice in each group; namely, the following 4 groups:

[0074] WT-Con group: WT mice high fat + sesame oil;

[0075] Mas - / - -Con Group:Mas - / - Mouse high fat + sesame oil;

[0076] WT-PCOS group: WT mice high fat + DHEA;

[0077] Mas - / - -PCOS group: Mas - / - Mouse high fat + DHEA;

[0078] Among them, the WT-Con group was wild-type mice fed with a 60% high-fat diet and injected subcutaneously with sesame oil at the back of the neck every day (the daily injection volume of mice was 0.1 ml / 100 g mouse body weight);

[0079] Mas - / --Con group is Mas knockout mice fed with 60% high-fat diet and treated with daily subcutaneous injection of sesame oil in the back of the neck (the daily injection volume of mice is 0.1 ml / 100 g mouse body weight);

[0080] The WT-PCOS group consisted of wild-type mice fed a 60% high-fat diet and treated with a dehydroepiandrosterone mixture subcutaneously at the back of the neck every day (the daily injection volume for mice was 6 μL / g mouse body weight);

[0081] Mas - / - -PCOS group: Mas knockout mice were fed a 60% high-fat diet and treated with a dehydroepiandrosterone mixture subcutaneously at the back of the neck every day (the daily injection volume of mice was 6 μL / g mouse body weight);

[0082] Dehydroepiandrosterone mixed solution: 60 mg of dehydroepiandrosterone (brand MCE, model HY-14650, abbreviated as DHEA) is dissolved in 6 ml of sesame oil to form a dehydroepiandrosterone mixed solution.

[0083] WT-Con group, Mas - / - -Con group, WT-PCOS group and Mas - / - The -PCOS group was treated according to the above method until the end of the experiment, and the mice were killed for samples.

[0084] 1.2 Ovarian morphology detection methods and results

[0085] Detection method: On the 22nd day after mice were enrolled in the experiment, paraffin sections were prepared and HE staining was performed to detect the WT-Con group and Mas - / - -Con group, WT-PCOS group and Mas - / - -Ovarian morphology of mice in the PCOS group was observed (including analysis of follicles at different developmental stages, arrangement of granulosa cells in follicles, theca cell layer, surface of the tunica albuginea, follicular atresia, etc.).

[0086] (1) Preparation of paraffin sections

[0087] Sampling → Fixation (10% paraformaldehyde, more than 24h) → Trim blocks (prefer large rather than thick), wash → Dehydration (70% alcohol for 2h, 85% alcohol for 2h, 90% alcohol for 1h, 95% alcohol for 0.5h, 100% alcohol for 0.5h → 100% alcohol for 0.5h) → Transparentization (xylene I for 0.5h → xylene II for 0.5h) → Wax impregnation (soft wax for 0.5h, hard wax for 1h) → Embedding (hard wax) → Sectioning (5μm) → Baking (37℃ overnight or 60℃ for 2-3 hours).

[0088] (2) HE staining

[0089] Dewax the sections (xylene I for 10 min → xylene II for 10 min → 95% alcohol for 1-2 min → 85% alcohol for 1-2 min → 75% alcohol for 1-2 min) → rinse the sections with distilled water for 5 min → stain with hematoxylin solution for 5 min, rinse with running water → differentiate with 5% acetic acid for 1 min, rinse with running water → stain with eosin for 1 min, rinse with running water → dehydrate (80% alcohol for 2 seconds, 90% alcohol I for 3 seconds, 90% alcohol II for 5 seconds, 100% alcohol for 5 minutes, carbolic acid xylene for 10 minutes, xylene I, II, III for 2 minutes each) → dry for 5 min → seal the sections with neutral gum and observe under a microscope.

[0090] Test results: WT-Con group, Mas - / - -Con group, WT-PCOS group and Mas - / - -Ovarian morphology of PCOS mice, the results are as follows Figure 1 As shown. - / - There was no significant difference in the ovaries of the mice induced by DHEA between the WT-PCOS group and the Mas - / - Cystic dilatation of follicles was observed in all mice in the -PCOS group, but fewer cystic dilatation of follicles was observed in the WT-PCOS group. - / - -PCOS group) The ovaries showed more cystic dilated follicles, and the ovaries also showed masculinization, larger ovaries and pale surface.

[0091] 1.3 Hormone level detection

[0092] Detection method: On the 22nd day after mice were enrolled in the experiment, ELISA kit was used to detect the - / - -Con group, WT-PCOS group and Mas - / - -PCOS group underwent serum testosterone (Testo) (CSB-E05101m), follicle-stimulating hormone (FSH) (CSB-E06871m), and luteinizing hormone (LH) (CSB-E12770m) hormone level testing.

[0093] Test results: WT-Con group, Mas - / - -Con group, WT-PCOS group and Mas - / - -The levels of serum testosterone, follicle-stimulating hormone, and luteinizing hormone in mice in the PCOS group were as follows Figure 2 As shown. Among them, A is the test result of serum testosterone content; B is the test result of follicle stimulating hormone content; C is the test result of luteinizing hormone content.

[0094] - / -

[0095] As can be seen from the figure, there was no significant difference in the hormone levels of mice in the Mas-Con group compared with the WT-Con group. The DHEA-induced WT-PCOS group mice had significantly higher levels of follicle-stimulating hormone than the WT-Con group; - / - -The levels of serum testosterone, follicle-stimulating hormone, and luteinizing hormone in mice in the PCOS group were significantly increased.

[0096] 1.4 Blood lipid test

[0097] Detection method: On the 22nd day after mice were enrolled in the experiment, the WT-Con group and Mas - / - -Con group, WT-PCOS group and Mas - / - -PCOS group mice were anesthetized, and blood samples were collected from the inferior vena cava and placed in 1.5 mL EP tubes. Serum was separated by centrifugation at 2500 rpm for 15 min, and the supernatant was collected and packaged, stored at -80°C, and triglyceride (TG) and total cholesterol (TC) levels were detected using relevant kits of Bio-Time.

[0098] Test results: WT-Con group, Mas - / - -Con group, WT-PCOS group and Mas - / - -Total cholesterol and serum triglyceride levels in PCOS mice. Figure 3 As shown in the figure, A is the total cholesterol content test result; B is the serum triglyceride content test result.

[0099] As can be seen from the figure, compared with the WT-Con group mice, Mas - / - -Con group mice, Mas - / - -PCOS group mice had significantly increased total cholesterol levels. - / - The total cholesterol level of mice in the -PCOS group was significantly higher than that in the WT-PCOS group. There was no significant difference in the serum triglyceride level among the four groups of mice.

[0100] 1.5 Body weight and fasting blood sugar test

[0101] Detection method: On the 22nd day after mice were enrolled in the experiment, the mice were changed in bedding and fasted for 16 hours overnight (water is not prohibited). Their body weights were weighed and recorded at 7:00 the next day. Then the mice were taken out of the cages and gently placed on the iron grid of the cages. The end of the tail of the mice was cut off by about 1-2 mm with sterilized scissors, and the tail vein of the mice was gently squeezed to enrich the blood into a drop. Blood was taken from the tail vein in a quiet state and fasting blood glucose was tested with a blood glucose meter.

[0102] Test results: WT-Con group, Mas - / - -Con group, WT-PCOS group and Mas - / - - Body weight and fasting blood glucose of PCOS group mice. Figure 4 As shown in the figure, C is the weight test result; D is the fasting blood sugar test result.

[0103] As can be seen from the figure, the weight of mice in the WT-PCOS group was significantly higher than that in the WT-Con group. - / - -Con group mice and Mas - / - There was no significant difference in the body weight of mice in the WT-Con group and the WT-PCOS group. There was no significant difference in the fasting blood glucose of mice in the WT-Con group and the WT-PCOS group. - / - -Con group mice fasting blood glucose was significantly lower than Mas - / - -PCOS group mice.

[0104] 1.6 Estrous cycle detection

[0105] Detection method: Starting from the 22nd day of the mice entering the experiment, the WT-Con group, Mas - / - -Con group, WT-PCOS group and Mas - / - -The estrous cycle of PCOS group mice was tested for 8 consecutive days. The mice were fixed, the vaginal opening of the mice was exposed, and about 30μl of phosphate buffered saline (PBS) was sucked with a pipette. The pipette tip was placed at the vaginal opening and aspirated repeatedly for 3 times. The aspirated PBS was dripped onto a glass slide and observed under an optical microscope. A small number of epithelial cells and a large number of multinuclear leukocytes were visible in the estrus; a large number of epithelial cells with granular cytoplasm, no leukocytes, and a small number of anuclear keratinized epithelial cells were seen in the proestrus; a large number of irregular keratinized epithelial cells and larger cells were seen in the estrus; a small number of fused keratinized epithelial cells and a large number of leukocytes were seen in the metestrus.

[0106] Test results: WT-Con group, Mas - / - -Con group, WT-PCOS group and Mas - / - -Estrous cycle of PCOS mice. Figure 5 Among them, A is the estrous cycle monitoring results of WT-Con group mice, B is the Mas - / - -Con group mice estrous cycle monitoring results; C is WT-PCOS group mice estrous cycle monitoring results; D is Mas - / - -Estrous cycle monitoring results of PCOS group mice.

[0107] As can be seen from the figure, Mas - / -There was no significant difference in the estrous cycle between the -Con group and the WT-Con group. Compared with the WT-Con group, the Mas - / - The estrous cycles of both the -PCOS and WT-PCOS mice were disordered, but in the DHEA-induced PCOS model, Mas - / - The estrous cycle disorders of the -PCOS group mice were more severe than those of the WT-PCOS group mice.

[0108] 1.7 Fertility Assessment

[0109] Detection method: 21 days after DHEA injection or sesame oil injection, 9 female mice in each group were randomly selected from the 22nd day and mated with healthy male mice in a 1:1 ratio for 7 days. The four groups exchanged male mice once a day. Successful mating was determined by the presence of a vaginal plug. The number of newborn pups was counted and recorded after natural delivery of female mice.

[0110] Test results: The results of the fertility test are shown in Table 1:

[0111] Table 1 Fertility test results

[0112] Group Quantity / pcs Number of pregnancy and births / Number of young mice WT-Con group 9 9 62 <![CDATA[Mas - / - -With]]> 9 9 59 WT-PCOS group 9 6 42 <![CDATA[Mas - / - -PCOS group]]> 9 2 8

[0113] WT-Con mice and Mas - / - There was no significant difference in fertility in the -Con group of mice. - / - The number of mice in the -PCOS group and the WT-PCOS group was significantly reduced; moreover, Mas - / - -PCOS mice gave birth to significantly fewer mice than WT-PCOS mice.

[0114] The above experiments show that MAS receptor affects the occurrence and development of PCOS mice.

[0115] Experimental Example 2: AVE0991 intervention in PCOS mice

[0116] 1. Establishment of PCOS mouse model

[0117] 1.1 Experimental method: Wild-type C57BL / 6 female mice (3 weeks old) were randomly divided into three groups, 20 mice in each group; normal feed feeding group (Con group) and HFD combined with DHEA subcutaneous injection group (HFD+DHEA).

[0118] The Con group consisted of wild-type mice fed with a 60% high-fat diet and injected subcutaneously with sesame oil at the back of the neck every day (the daily injection volume of mice was 0.1 ml / 100 g of mouse body weight) for 21 days each;

[0119] The HFD+DHEA group consisted of wild-type mice fed a 60% high-fat diet and treated with a dehydroepiandrosterone mixture subcutaneously at the back of the neck (the daily injection volume for mice was 6 μL / g mouse body weight) for 21 days each;

[0120] Dehydroepiandrosterone mixed solution: 60 mg of dehydroepiandrosterone (brand MCE, model HY-14650, abbreviated as DHEA) is dissolved in 6 ml of sesame oil to form a dehydroepiandrosterone mixed solution.

[0121] 2. AVE0991 intervention

[0122] 2.1 Experimental method: 9 rats were randomly selected from the Con group and recorded as the Control group; 18 rats were selected from the HFD+DHEA group and divided into two groups, 9 rats in each group, recorded as the DHEA group and the DHEA+AVE0991 group. A total of 27 rats in the three groups were subjected to the AVE0991 intervention test, as follows:

[0123] The mice in the control group were fed with a 60% high-fat diet and injected subcutaneously with sesame oil (McLean, CAS: 8008-7400, 0.1 ml / 100 g mouse body weight) at the back of the neck from the 22nd day. The subsequent functional test was performed after 28 days of treatment. The sesame oil intervention was continued until the mice were killed for sampling.

[0124] The mice in the DHEA group were fed with a 60% high-fat diet and were subcutaneously injected with a dehydroepiandrosterone mixture at the back of the neck for 21 days. From the 22nd day, they were fed with a 60% high-fat diet and were subcutaneously injected with a dehydroepiandrosterone mixture at the back of the neck every day. They were also subcutaneously injected with an equal amount of corn oil at the back of the neck every day (the daily injection volume was 0.1 ml / 100 g of mouse body weight). They served as the control group for the AVE0991 intervention experiment. After 28 days of combined treatment (49 days of enrollment), subsequent functional tests could be performed. At the same time, the DHEA+corn oil intervention was continued until the mice were killed for sampling.

[0125] The mice in the DHEA+AVE0991 group were fed a 60% high-fat diet and were intervened with a dehydroepiandrosterone mixture subcutaneously in the back of the neck for 21 days. From the 22nd day, they were fed a 60% high-fat diet and were injected with a dehydroepiandrosterone mixture subcutaneously in the back of the neck every day, combined with daily subcutaneous injections of the AVE0991 mixture in the back of the neck (the daily injection volume for mice was 2 μL / g mouse body weight). After 28 days of combined treatment (the 49th day of the trial), subsequent functional tests could be performed; during the experiment, DHEA+AVE0991 intervention continued until the mice were killed and samples were collected.

[0126] AVE0991 mixed solution: 3 mg AVE0991 (brand MCE, model HY-15778) was dissolved in 6 ml corn oil to form AVE0991 mixed solution.

[0127] 2.2 Estrous cycle monitoring methods and results

[0128] The same method as in 1.6 estrous cycle detection in Example 1 was used to monitor the control group mice, DHEA group mice and DHEA+AVE0991 group mice for 8 days starting from the 50th day of the mouse enrollment experiment. The results are as follows: Figure 6 Among them, A is the estrous cycle monitoring result of mice in the Control group, B is the estrous cycle monitoring result of mice in the DHEA group; C is the estrous cycle monitoring result of mice in the DHEA+AVE0991 group.

[0129] As can be seen from the figure, compared with the mice in the DHEA group, the estrous cycle of the mice in the DHEA+AVE0991 group tended to be normal.

[0130] 2.3 Body weight and fasting blood glucose testing methods and results

[0131] The weight and fasting blood glucose of the mice in the Control group, DHEA group and DHEA+AVE0991 group were tested by the same method as in 1.5 of Example 1. The results are as follows: Figure 7 Among them, C is the weight test results of the three groups of mice; D is the fasting blood glucose test results of the three groups of mice.

[0132] As can be seen from the figure, compared with the mice in the DHEA group, the fasting blood glucose and body weight of the mice in the DHEA+AVE0991 group were lower and significantly decreased, both tending to normal.

[0133] 2.4 Detection methods and results of ovarian sex hormone-related genes, liver, brown fat and subcutaneous fat metabolism-related genes expression

[0134] Detection method: RNA extraction and RT-PCR

[0135] Total RNA was extracted from cultured GLUTag cells using the RNA Preparation Pure Tissue Kit (Tingen) for the control group, DHEA group, and DHEA+AVE0991 group, and then converted into cDNA using the RevertAidTM First-Strand cDNA Synthesis Kit (Fermentas K1622) according to the manufacturer. Real-time qPCR was performed using SYBR Green I Master Mix reagent (Roch) and primers on a real-time PCR system (Roche) and using GAPDH as the housekeeping gene.

[0136] Test results:

[0137] like Figure 8 As shown in the data, the detection results of ovarian sex hormone-related genes (Lhegr, P450scc, StAR, Cyp19a1, Fshr, Hsd17b2) of mice in each group showed that compared with the mice in the DHEA group, the expression of ovarian sex hormone-related genes Cyp19a1 and Hsd17b2 in the DHEA+AVE0991 group of mice intervened by AVE0991 was significantly decreased (P < 0.05).

[0138] like Fig. 9 As shown, compared with the DHEA group mice, the expression of liver (AOX, HSL, Fabpl) metabolism-related genes in the DHEA+AVE0991 group mice intervened by AVE0991 was significantly improved (P < 0.05 or P < 0.01).

[0139] like Fig.10 As shown, compared with the DHEA group mice, the expression of brown fat (COX4, UQCRC2) metabolism-related genes in the DHEA+AVE0991 group of mice intervened by AVE0991 was significantly improved.

[0140] like Fig.11 As shown in the figure, compared with the DHEA group mice, the expression of genes related to subcutaneous fat metabolism (PPARγ, ATGL, PGC1α, AGPAT and SDHB) in the DHEA+AVE0991 group mice treated with AVE0991 was significantly improved. Among them, A is the expression of PPARγ; B is the expression of ATGL; C is the expression of PGC1α; D is the expression of AGPAT; E is the expression of SDHB.

[0141] 2.5 Fertility Assessment

[0142] The fertility of the Control group mice, the DHEA group mice and the DHEA+AVE0991 group mice was evaluated using the same method as in 1.7 Fertility Evaluation of Example 1.

[0143] As shown in Table 2, compared with the DHEA group mice, the AVE0991-intervention DHEA+AVE0991 group mice significantly improved the fertility of the DHEA group model mice.

[0144] Table 2 Fertility test results

[0145] Group Quantity / pcs Number of pregnancy and births / Number of young mice Control Group 9 8 45 DHEA group 9 3 21 DHEA+AVE0991 group 9 7 39

[0146] The above experiments proved that the intervention of MAS receptor agonist AVE0991 in PCOS group mice made the estrous cycle of mice tend to normal; fasting blood sugar decreased, and body weight decreased significantly; the expression of ovarian sex hormone-related genes (Cyp19a1, Hsd17b2) decreased; in addition, the expression of metabolism-related genes in liver (AOX, HSL, Fabpl), brown fat (COX4, UQCRC2) and subcutaneous fat (PPARγ, ATGL, PGC1α, AGPAT and SDHB) of PCOS group (DHEA+AVE0991 group) intervened by AVE0991 was significantly improved; most notably, AVE0991 significantly improved the fertility of PCOS group mice, and the number of mice born in DHEA+AVE0991 group was significantly higher than that in DHEA group.

[0147] The present invention also uses Ang-(1-7) as a MAS receptor agonist to intervene in DHEA group model mice for experiments, and the results are basically the same as the experimental results of using AVE0991 as a MAS receptor agonist to intervene in DHEA group model mice in the above experiment.

[0148] Obviously, the above embodiments are merely examples for the purpose of clear explanation, and are not intended to limit the implementation methods. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation methods here. The obvious changes or modifications derived therefrom are still within the scope of protection of the invention.

Claims

1. Use of a MAS receptor agonist in the preparation of a drug for preventing or treating polycystic ovary syndrome, characterized in that: The MAS receptor agonist is AVE0991.

2. The use according to claim 1, characterized in that: Symptoms of PCOS include multiple cystic dilated follicles visible within the ovaries.

3. The use according to claim 1, characterized in that: The symptoms of PCOS include at least one of enlarged ovarian volume, pale ovarian surface, increased serum testosterone level, increased follicle stimulating hormone level, increased luteinizing hormone level, increased total cholesterol level, and estrous cycle disorder.

4. The use according to claim 1, characterized in that: The MAS receptor agonist is used in preparing a medicine for preventing or treating polycystic ovary syndrome combined with hypertension.

5. The use according to claim 4, characterized in that: The symptoms of polycystic ovary syndrome with hypertension include multiple cystic dilated follicles visible in the ovaries.

6. The use according to claim 4, characterized in that: The symptoms of polycystic ovary syndrome combined with hypertension include at least one of enlarged ovarian volume, pale ovarian surface, increased serum testosterone level, increased follicle-stimulating hormone level, increased luteinizing hormone level, increased total cholesterol level, and estrous cycle disorder.

7. The use according to any one of claims 1 to 6, characterized in that: The drug has at least one of the following functions (1)-(7): (1) reducing the expression of ovarian sex hormone-related genes, wherein the ovarian sex hormone-related genes include Cyp19a1 and / or Hsd17b2; (2) improving the expression of genes related to liver metabolism, wherein the improved liver metabolism-related genes include one or a combination of two or more of AOX, HSL and Fabpl; (3) improving the expression of genes related to brown fat metabolism, including COX4 and / or UQCRC2; (4) improving the expression of genes related to subcutaneous fat metabolism, wherein the genes related to subcutaneous fat metabolism include one or a combination of two or more of PPARγ, ATGL, PGC1α, AGPAT and SDHB; (5) Regulate the estrous cycle; (6) Lowering blood sugar; (7) Improve fertility.

8. The use according to any one of claims 1 to 6, characterized in that: The drug also includes pharmaceutically acceptable excipients.

9. The use according to any one of claims 1 to 6, characterized in that: The medicine is in the form of tablets, capsules, oral liquids, granules, pills or injections.

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