CN110314167A - Application and pharmaceutical preparation of the bile acid as preparation treatment PCOS drug - Google Patents

Abstract

The invention discloses the application and pharmaceutical preparation of a bile acid as a medicine for treating PCOS. The present disclosure provides a new field of application of bile acids, which can be used in the preparation of drugs for treating PCOS. The present disclosure helps to develop novel drugs for treating PCOS.

Description

Application and pharmaceutical preparation of bile acid as preparation of medicine for treating PCOS

technical field

The invention relates to the field of treating polycystic ovary syndrome, and more specifically relates to the application and pharmaceutical preparation of a bile acid as a medicine for treating PCOS.

Background technique

Polycystic ovary syndrome, or PCOS for short, is one of the most common causes of anovulatory infertility. PCOS is a syndrome of endocrine and metabolic disorders, characterized by hyperandrogenism, abnormal ovulation, and polycystic ovarian changes, often accompanied by insulin resistance. Studies have shown that 14.2% of PCOS patients in the Chinese population are accompanied by insulin resistance, but the etiology of PCOS is still unclear. Due to the high heterogeneity of its clinical manifestations, the treatment of PCOS has become a hot and difficult issue of common concern in the fields of gynecology and endocrinology. The impact of PCOS on patients is far from limited to reproductive problems, and the time is not limited to women's reproductive age: intrauterine hypertrophy and intrauterine malnutrition in the fetus; early adrenal function in childhood; Menstrual disorders, infertility, endometrial diseases, etc.; even diabetes, cardiovascular and cerebrovascular diseases, and endometrial cancer in middle-aged and elderly people may affect the health of PCOS patients to a certain extent.

For PCOS, the current main drug treatment methods are oral contraceptives, clomiphene, gonadotropins, gonadotropin-releasing analogues, metformin and other drugs to help patients improve menstrual cycle, regulate ovulation abnormalities and metabolic abnormalities.

However, oral contraceptives are often accompanied by early pregnancy reactions such as nausea, vomiting, dizziness, breast swelling, and increased leucorrhea, as well as problems such as fatigue, lethargy, increased pigmentation, hyperphagia, acne, menorrhagia, or bleeding. Clomiphene is associated with hot flashes and an increased risk of multiple pregnancy. Gonadotropins and gonadotropin-releasing analogs increase the risk of multiple pregnancy and ovarian hyperstimulation. Metformin can produce gastrointestinal reactions and even lactic acidosis.

Therefore, how to provide a drug that can effectively treat PCOS has become an urgent technical problem in this field.

Contents of the invention

An object of the present invention is to provide a new technical solution for the application of bile acid as a drug for the preparation of PCOS.

According to the first aspect of the present invention, a kind of bile acid is provided as the application of preparing the medicine for treating PCOS.

Optionally, it is used to prepare a medicament, which is suitable for systemic or local administration, or can be directly injected into the ovary.

Optionally, said administration is by intravenous or intraarterial, intramuscular, transdermal, intraperitoneal, intrathecal, intralymphatic administration, intratracheal instillation, subcutaneous, oral or topical area administration.

Optionally, the bile acids include glycodeoxycholic acid and/or tauroursodeoxycholic acid.

Optionally, the bile acid is glycodeoxycholic acid.

According to a second aspect of the present invention, a pharmaceutical formulation is provided.

The pharmaceutical preparation includes bile acid and one or more pharmaceutically acceptable carriers and/or excipients.

Optionally, the bile acids include glycodeoxycholic acid and/or tauroursodeoxycholic acid.

Optionally, the bile acid is glycodeoxycholic acid.

Optionally, the carrier includes solid excipients or liquid excipients, and the dosage forms of the pharmaceutical preparation include tablets, pills, capsules, ointments, powders, solutions, granules, suspensions, injections, sustained-release preparations and controlled-release preparations. preparation. The liquid adjuvant can be, for example, physiological saline. The concentration of physiological saline can be, for example, 0.9 wt%.

Optionally, the effective dose of the bile acid is 20-100 mg/kg.

The present disclosure provides a new field of application of bile acids, which can be used in the preparation of drugs for treating PCOS. The present disclosure helps to develop novel drugs for the treatment of PCOS.

Other features of the present invention and advantages thereof will become apparent from the following detailed description of exemplary embodiments of the present invention with reference to the accompanying drawings.

Description of drawings

The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate the subject matter of the invention and together with the description serve to explain the principle of the invention.

Figure 1 is the abundance map of different bacterial flora at the family level between the control group and the PCOS group.

Figure 2 is the abundance map of the intestinal flora of the control group and the PCOS group at the genus level.

Figure 3 is a graph showing the abundance of different bacterial flora at the species level in the intestinal flora of the control group and the PCOS group.

Figure 4 is a KEGG analysis chart.

Fig. 5 is a graph showing the quantitative analysis of bile acids in the serum of volunteers in the control group and the PCOS group.

Fig. 6 is a quantitative analysis diagram of bile acids in feces of volunteers in the control group and the PCOS group.

Fig. 7 is a graph showing the correlation analysis between B. vulgatus and serum bile acids.

Fig. 8 is a graph showing the correlation analysis between B. vulgatus and fecal bile acids.

Fig. 9 is a graph showing the efficiency of GDCA hydrolysis in vitro by B. vulgatus bacteria.

Fig. 10 is the expression map of the Bsh gene of B. vulgatus bacteria in the stool of the volunteers in the control group and the PCOS group.

Fig. 11 is a statistical chart of oral glucose tolerance of control group, B. vulgatus mice and DHEA mice.

Fig. 12 is a statistical chart of insulin resistance in the control group, B. vulgatus-administered mice and DHEA mice.

Figure 13 is a statistical chart of the area under the curve of oral glucose tolerance of the control group, B. vulgatus mice and DHEA mice.

Fig. 14 is a statistical chart of the area under the insulin resistance curve of the control group, B. vulgatus orally administered mice and DHEA mice.

Fig. 15 is a statistical chart of fasting blood glucose of the control group, B. vulgatus-administered mice and DHEA mice.

Fig. 16 is a statistical chart of insulin levels in the control group, B. vulgatus-administered mice and DHEA mice.

Fig. 17 is a statistical chart of insulin resistance index of the control group, B. vulgatus orally administered mice and DHEA mice.

Figure 18 is a typical estrous cycle of mice in the control group.

Fig. 19 is a typical estrous cycle of mice in the B. vulgatus group.

Figure 20 is a typical estrous cycle of DHEA mice.

Figure 21 is a statistical chart showing the proportion of each period of the estrous cycle of the control group, B. vulgatus mice and DHEA mice.

Fig. 22 is a typical HE staining image of the ovaries of mice in the control group.

Fig. 23 is a typical HE staining image of the ovaries of mice in the B. vulgatus group.

Figure 24 is a typical HE staining diagram of the ovary of mice in the DHEA group.

Fig. 25 is a statistical diagram of the corpus luteum number of the control group, B.vulgatus-administered mice and DHEA mice.

Figure 26 is a statistical chart of the number of cystic follicles in the control group, B.vulgatus-administered mice and DHEA mice.

Fig. 27 is a statistical chart of testosterone levels in the control group, B. vulgatus-administered mice and DHEA mice.

Fig. 28 is a statistical chart of the levels of luteinizing hormone in the control group, B. vulgatus-administered mice and DHEA mice.

Fig. 29 is a statistical chart of bile acid levels in the intestinal epithelium of the control group, B. vulgatus mice and DHEA mice.

Figure 30 is a statistical chart of oral glucose tolerance of the control group, B.vulgatus-administered mice, and GDCA-treated B.vulgatus-administered mice.

Figure 31 is a statistical chart of insulin resistance in the control group, B.vulgatus-administered mice, and GDCA-treated B.vulgatus-administered mice.

Figure 32 is a statistical chart of the area under the oral glucose tolerance curve of the control group, B.vulgatus-administered mice, and GDCA-treated B.vulgatus-administered mice.

Figure 33 is a statistical chart of the area under the curve of the insulin resistance experiment of the control group, B.vulgatus-administered mice, and GDCA-treated B.vulgatus-administered mice.

Figure 34 is a statistical chart of fasting blood glucose of the control group, B.vulgatus-administered mice, and GDCA-treated B.vulgatus-administered mice.

Figure 35 is a statistical chart of insulin levels in the control group, B.vulgatus-administered mice, and GDCA-treated B.vulgatus-administered mice.

Figure 36 is a statistical chart of insulin resistance index of the control group, B.vulgatus-administered mice, and GDCA-treated B.vulgatus-administered mice.

Figure 37 is a typical estrous cycle of mice in the control group.

Figure 38 is a typical estrous cycle of mice in the B.vulgatus group.

Figure 39 is a typical estrous cycle of GDCA-treated B. vulgatus mice.

Fig. 40 is a statistical chart showing the proportions of the control group, B.vulgatus-administered mice, and GDCA-treated B.vulgatus-administered mice in each stage of each estrous cycle.

Figure 41 is a typical ovary morphology of mice in the control group.

Figure 42 is a typical ovary morphology of mice in the B.vulgatus group.

Fig. 43 is a typical ovary morphology of B. vulgatus-administered mice treated with GDCA.

Figure 44 is a statistical chart of the number of ovarian corpus luteum of the control group, B.vulgatus-administered mice, and GDCA-treated B.vulgatus-administered mice.

Figure 45 is a statistical chart of the number of ovarian corpus luteum and the number of cystic follicles in the control group, B.vulgatus-administered mice, and GDCA-treated B.vulgatus-administered mice.

Fig. 46 is a testosterone level detection chart of the control group, B.vulgatus-administered mice, and GDCA-treated B.vulgatus-administered mice.

Figure 47 is a statistical chart of LH levels in the control group, B.vulgatus-administered mice, and GDCA-treated B.vulgatus-administered mice.

Figure 48 is the algebraic statistical chart of the first litter of the control group, B.vulgatus-administered mice, and GDCA-treated B.vulgatus-administered mice.

Figure 49 is a typical estrous cycle of mice in the control group.

Figure 50 is a typical estrous cycle of GDCA-administered mice.

Figure 51 is a statistical chart showing the proportions of each estrous cycle in the control group and GDCA-administered mice.

Figure 52 is a graph showing the expression levels of molecules related to subcutaneous fat browning in the control group, mice transplanted with B. vulgatus bacteria and DHEA mice.

Figure 53 is a graph showing the expression levels of brown fat browning-related molecules in the control group, mice transplanted with B. vulgatus bacteria, and DHEA mice.

Figure 54 is a graph showing the expression levels of molecules related to subcutaneous fat browning in the control group, B.vulgatus-administered mice, and GDCA-treated B.vulgatus-administered mice.

Figure 55 is a graph showing the expression levels of brown fat browning-related molecules in the control group, B.vulgatus-administered mice, and GDCA-treated B.vulgatus-administered mice.

Detailed ways

Various exemplary embodiments of the present invention will now be described in detail with reference to the accompanying drawings. It should be noted that the relative arrangements of components and steps, numerical expressions and numerical values set forth in these embodiments do not limit the scope of the present invention unless specifically stated otherwise.

The following description of at least one exemplary embodiment is merely illustrative in nature and in no way taken as limiting the invention, its application or uses.

Techniques, methods and devices known to those of ordinary skill in the relevant art may not be discussed in detail, but where appropriate, such techniques, methods and devices should be considered part of the description.

In all examples shown and discussed herein, any specific values should be construed as exemplary only, and not as limitations. Therefore, other instances of the exemplary embodiment may have different values.

It should be noted that like numerals and letters denote like items in the following figures, therefore, once an item is defined in one figure, it does not require further discussion in subsequent figures.

Figures 1 to 3 reflect the differences in intestinal flora between PCOS patients and controls. The results of metagenomic analysis of the intestinal flora of the control group and the PCOS group, the abundance of different bacterial flora of Bacteroides vulgatus at the family (Figure 1), genus (Figure 2) and species levels (Figure 3) between the two groups (wherein, the control n=43 in the group, n=50 in the PCOS group, n is the number of cases in each group, P represents the statistical significance between the two groups, and P<0.05 is considered to be statistically different between the two groups).

Bacteroidaceae in Figure 1 refers to Bacteroidaceae, Lachnospiraceae refers to Lachnospiraceae, Ruminococcaceae refers to Verrucobacteriaceae, Clostridiaceae refers to Clostridiaceae, Eubacteriaceae refers to Eubacteriaceae, and Prevotellaceae refers to Prevotella Bacteriaceae, Veillonellaceae refers to Veillonellaceae, Selenomonadaceae refers to Lunamorphaceae, Rikenellaceae refers to Rikenellaceae, and Tannerellaceae refers to Tannerellaceae. Bacteroides in Figure 2 refers to Bacteroides, Clostridium refers to Clostridium, Faecalibacterium refers to Faecalibacterium, Eubacterium refers to Eubacterium, Roseburia refers to Bacillus, Prevotella refers to Prevotella, Ruminococcus refers to Ruminococcus, Blautia refers to the genus Blautia, Alistipes refers to the genus Mycobacterium, and Megamonas refers to the genus Megamonas. Bacteroides vulgatus in Figure 3 refers to Bacteroides vulgaris.

It can be seen from Figures 1 to 3 that compared with healthy people, the intestinal flora of PCOS patients has significant changes, among which Bacteroidaceae and Bacteroides are the bacterial families with the most obvious differences and the greatest degree of change. Fungus. The level of Bacteroides vulgatus (B.vulgatus) was significantly higher than that of the normal control group.

Figure 4 is a KEGG analysis, that is, an analysis diagram of metabolic pathways related to intestinal flora based on genomics results.

The metabolic activity of intestinal flora was studied by KEGG pathway analysis. As can be seen from Figure 4, there is a significant difference in bile acid metabolism pathways between PCOS and normal control groups, suggesting that intestinal flora-mediated bile acid metabolism Can have a functional regulatory effect on the host. The flora is mainly involved in the regulation of host metabolism through the interaction of its metabolites with the body.

Figures 5 to 10 show the changes in bile acid metabolism between the control group and the PCOS group. The bile acid metabolomics method was used to quantitatively analyze the bile acids in the serum (Figure 5, spearman correlation coefficient refers to the Spearman correlation coefficient) and feces (Figure 6) of two groups of volunteers. Correlation analysis between B.vulgatus and serum (Figure 7) and feces (Figure 8) bile acids. Figure 9 shows the efficiency of B. vulgatus bacteria in vitro hydrolyzing GDCA (Glycodesoxycholic Acid, glycodeoxycholic acid) (n=3/group). Figure 10 shows the expression of the Bsh (bile acid hydrolase) gene in the feces of two groups of volunteers (n=43 in the control group and n=50 in the PCOS group in Figures 5-8 and Figure 10). UDCA refers to ursodeoxycholic acid, HDCA refers to deoxycholic acid, CDCA refers to chenodeoxycholic acid, DCA refers to deoxycholic acid, CA refers to cholic acid, LCA refers to lithocholic acid, GUDCA refers to oxycholic acid GCDCA refers to glycochenodeoxycholic acid, GDCA refers to glycodeoxycholic acid, GCA refers to glycinocholic acid, GLCA refers to glycinolithocholic acid, TUDCA refers to tauroursodeoxycholic acid, TCDCA Taurochenodeoxycholic Acid, TCA Taurodeoxycholic Acid, TCA Taurocholic Acid.

It can be seen from Figure 5 to Figure 10 that Glycodesoxycholic Acid (GDCA) and Tauroursodeoxycholic Acid (TUDCA) in serum and feces were significantly reduced in the PCOS group (Figure 5 and Figure 6). Further analysis of the correlation between B. vulgatus and bile acids showed that there was a strong negative correlation between B. vulgatus and GDCA and TUDCA in serum and feces (Fig. 7 and Fig. 8). More importantly, the hydrolysis process of conjugated bile acids such as GDCA in the intestine is mainly mediated by bile acid hydrolase BSH. B. vulgatus bacteria were cultured in vitro and bacterial protein was extracted for bile acid hydrolysis experiments. The results showed that the efficiency of hydrolyzing GDCA by B. vulgatus bacteria protein extract was as high as 95% ( FIG. 9 ). In order to further confirm the relationship between the reduction of conjugated bile acids in PCOS patients and B.vulgatus, the abundance of Bsh gene was detected, and the results showed that the abundance of Bsh in PCOS patients was significantly increased ( FIG. 10 ). The above results show that B.vulgatus is significantly increased in the intestinal flora of PCOS patients, which can affect the metabolism of host TUDCA or GDCA; the increase in the abundance of B.vulgatus and the increase in the level of Bsh gene lead to the degradation of GDCA and reduce its level the main reason.

Because bile acid hydrolase is a protein substance, which mainly exists in bacterial protein, the main purpose of extracting bacterial protein is to obtain bile acid hydrolase BSH, whose main function is to hydrolyze bile acid. Under normal circumstances, if there are more Bacteroides, the BSH and hydrolyzed bile acids will increase, and the bound bile acids such as GDCA will decrease, so there is a negative correlation between the two. It also shows that the reduction of GDCA is due to the increase of Bacteroidetes, and the increase of Bsh gene abundance also shows the same problem.

Figures 11 to 17 show the glucose tolerance of B. vulgatus-administered mice. Oral glucose tolerance test of control group, B.vulgatus mice and DHEA mice (Figure 11), Figure 12 is the insulin resistance test, Figure 13 is the area under the curve of oral glucose tolerance, Figure 14 is the area under the curve of the insulin resistance test, Figure 15 is the fasting blood glucose, Figure 16 is the insulin level, and Figure 17 is the insulin resistance index (wherein, n=8/group, *P<0.05, **P<0.01vs. the control group, one * represents the P value is less than 0.05, Two ** represent P value less than 0.01, the difference is more significant).

From Figure 11 to Figure 17, it can be seen that the mice fed with B. vulgatus and one of the recognized PCOS mouse models - DHEA mice can show the performance of impaired glucose tolerance: after glucose or insulin injection, the two groups Compared with the control group, the blood glucose level of the mice was significantly higher, and the blood glucose decreased more slowly, and the areas under the curves of the oral glucose tolerance test (GTT) and insulin resistance test (ITT) were significantly increased (Figure 11-Figure 14) . In addition, the fasting blood glucose, fasting insulin and insulin resistance index of the mice were significantly higher than those of the control group. This indicates that intragastric administration of B. vulgatus has a similar effect to DHEA in inducing insulin resistance in mice, and impaired glucose tolerance in mice (Figure 15-Figure 17), which is also one of the common clinical manifestations of PCOS patients.

Figures 18 to 21 show the estrous cycles of the control group, B. vulgatus-administered mice and DHEA mice. The typical estrous cycle of control group mice (Fig. 18), the typical estrous cycle of B.vulgatus group mice (Fig. 19), the typical estrous cycle of DHEA mice (Fig. 20), the control group, B.vulgatus gavage mice and the proportions of each stage of the estrous cycle of DHEA mice ( FIG. 21 ) (wherein P is preestrus, E is estrus, M is postestrus, D is interestrus, n=8/group). The estrous cycle is an important indicator for evaluating the sexual cycle of mice. The estrous period of normal mice is 4-5 days, and preestrus (P), estrus (E), postestrus (M) and interestrus (D) appear repeatedly. , estrous cycle disturbance is an important feature of PCOS mice.

From Figure 18 to Figure 21, it can be seen that the DHEA mice mainly stay in the continuous estrous period, and the estrous cycle of the B.vulgatus-administered mice is dominated by the interval period (Figure 18-20), but both have estrous cycles Disorders are manifested by abnormally high proportions of specific estrous cycles (Fig. 21).

Figure 22 to Figure 26 reflect the ovarian morphology of the control group, B. vulgatus-administered mice and DHEA mice. Figure 22 is the typical HE staining of the ovaries of mice in the control group, Figure 23 is the typical HE staining of the ovaries of mice in the B. The number of corpus luteum in mice and DHEA mice administered by stomach, Fig. 26 is the cystic follicle number of control group, B.vulgatus intragastrically administered mice and DHEA mice (wherein, * represents corpus luteum, # represents cystic follicle, n=8/group ).

The morphological examination of the ovaries from Figure 22 to Figure 26 shows that the number of corpus luteum in normal mice is normal without cystic follicles (Figure 22), and the number of corpus luteum in mice fed with B.vulgatus decreases and the number of cystic follicles increases (Figure 22). 23, 25 and 26), which are typical manifestations of PCOS-like mouse ovarian morphology, similar to the PCOS animal model established by DHEA (Fig. 24-26).

Fig. 27 and Fig. 28 are the testosterone (Testosterone, T) (Fig. 27) and the luteinizing hormone (Luteinizing Hormone, LH) (Fig. 28) levels (wherein, n =8/group).

It can be seen from Figure 27 and Figure 28 that, in terms of hormone levels, testosterone (Testosterone, T) and luteinizing hormone (Luteinizing Hormone, LH) in B. vulgatus mice were significantly higher than those in the control group. In addition, estrogen in B. vulgatus-administered mice did not change significantly compared with the control group, which was similar to the endocrine profile of PCOS patients.

Figure 29 presents mouse intestinal epithelial bile acid levels. The levels of bile acids in the intestinal epithelium of the control group, B. vulgatus mice and DHEA mice were quantitatively analyzed by bile acid metabolomics method (n=8/group). Gut microbiota is closely related to bile acid metabolism, and specific combinations of microbes may cause significant changes in bile acids to regulate host metabolism.

The strongest inverse association of B. vulgatus with GDCA and TUDCA has been found in PCOS patients. Due to the lack of GDCA in mice, GDCA similar to human bile acids cannot be detected in the bile acids detected in the intestinal tissue of mice. However, compared with the control group, it can be seen from Figure 29 that B. vulgatus is less TUDCA was significantly reduced in the intestinal epithelium of mice and DHEA mice.

Figures 30-36 reflect the glucose tolerance of GDCA-treated B. vulgatus model mice. Oral glucose tolerance test (Figure 30), insulin resistance test (Figure 31), area under the oral glucose tolerance curve (Figure 32), insulin resistance test (Figure 32) Area under the experimental curve (Figure 33), fasting blood glucose (Figure 34), insulin level (Figure 35), insulin resistance index (Figure 36) (wherein, n=6/group, *P<0.05, **P<0.01vs . Control group, #P<0.05, ##P<0.01 vs. B. vulgatus).

It can be seen from Figure 30 to Figure 36 that GDCA (30mg/kg gavage) can improve the insulin resistance caused by B.vulgatus gavage, mainly manifested in the increased blood sugar after the B.vulgatus gavage mice are injected with glucose or insulin The level can be suppressed by GDCA (Figure 30 and 31), and the area under the curve of oral glucose tolerance test (GTT) and insulin resistance test (ITT) can also be restored to normal level (Figure 32 and 33). In addition, the elevated fasting blood glucose, fasting insulin, and insulin resistance index of B. vulgatus mice can be restored to normal with the addition of GDCA (Fig. 34-36).

Figure 37 to Figure 40 represent the estrous cycle of GDCA-treated B. vulgatus model mice. The typical estrous cycle of the mice in the control group (Figure 37), the typical estrous cycle of the mice in the B.vulgatus group (Figure 38), the typical estrous cycle of the mice fed with B.vulgatus treated with GDCA (Figure 39), the control group, B. .vulgatus-administered mice, GDCA treatment B.vulgatus-administered mice each estrous cycle proportion (Figure 40) (wherein, P is preestrus, E is estrus, M is late estrus, D is interestrus period, n=6/group).

It can be seen from Fig. 37 to Fig. 40 that the estrous cycle of B. vulgatus mice is disturbed and stays in the estrous period continuously. After giving B.vulgatus mice GDCA treatment (30mg/kg gavage), the disordered estrous cycle of mice can return to normal, with 4-5 days as a cycle (Figure 37-39), the proportion of each period Also returned to normal (Figure 40).

Figure 41 to Figure 45 reflect the ovarian function of GDCA-treated B. vulgatus model mice. The typical ovarian morphology of the mice in the control group (Figure 41), the typical ovarian morphology of the mice in the B.vulgatus group (Figure 42), the typical ovarian morphology of the GDCA-treated B. .vulgatus-administered mice, GDCA treatment B.vulgatus-administered mice ovarian corpus luteum number (Figure 44), control group, B.vulgatus-administered mice, GDCA-treated B.vulgatus-administered mice ovarian corpus luteum number cystic follicles Number ( FIG. 45 ) (where n=6/group).

It can be seen from Figure 41 to Figure 45 that the abnormal ovarian morphology of B.vulgatus mice, including the appearance of cystic follicles and the reduced number of corpora luteum, can be observed after the administration of GDCA (30mg/kg gavage), and the disappearance of cystic follicles , the number of corpus luteum increased, indicating that GDCA can restore the ovarian morphology of B.vulgatus mice to normal.

Figure 46 and Figure 47 reflect the hormone levels of GDCA-treated B. vulgatus model mice. Testosterone ( FIG. 46 ) and LH ( FIG. 47 ) levels in the control group, B. vulgatus-administered mice, and GDCA-treated B. vulgatus-administered mice (wherein, n=6/group).

Elevation of testosterone T and luteinizing hormone LH is a typical manifestation of endocrine abnormalities in PCOS patients, and the decrease of T and LH levels is also considered to be one of the signs of PCOS improvement. It can be seen from Figure 46 and Figure 47 that the elevated T and LH levels of B.vulgatus mice can return to normal after being given GDCA treatment (30mg/kg gavage).

Fig. 48 shows the generation numbers of the first litter of the control group, B. vulgatus-administered mice, and GDCA-treated B. vulgatus-administered mice (wherein, n=6/group).

As can be seen from Figure 48, in terms of fertility, GDCA treatment restored the reduced litter size of B. vulgatus mice to normal levels.

Figures 49 to 51 represent the estrous cycle of the GDCA-treated DHEA mouse model. The typical estrous cycle of mice in the control group (Fig. 49), the typical estrous cycle of GDCA-administered mice (Fig. 50), and the statistical charts of different periods of the estrous cycle of mice (Fig. 51) (wherein, P is preestrus, E is Estrus, M is postestrus, D is interestrus, the control group is DHEA mice, GDCA, DHEA mice were gavaged with GDCA for 3 weeks, n=6/group).

It can be seen from Fig. 49 to Fig. 51 that in the DHEA-induced PCOS mouse model, the disordered estrous cycle of the mice can be restored by administering GDCA (30 mg/kg orally).

Figure 52 and Figure 53 reflect the expression levels of molecules related to mouse fat browning. The expression levels of molecules related to subcutaneous fat browning in the control group, mice transplanted with B. vulgatus bacteria and DHEA mice ( FIG. 52 ), and the expression levels of molecules related to brown fat browning ( FIG. 53 ) (wherein, n=8/group).

The browning of white fat can improve the metabolic level and insulin resistance of PCOS mice, and the transplantation of brown fat to PCOS rats can significantly improve the polycystic ovarian changes and fertility decline of PCOS rats. It can be seen from Figure 52 and Figure 53 that the expression levels of Ucp1, Pgc1a, Cited1, Cox8b in subcutaneous fat and brown fat Ucp1, Pgc1a and Cox8b in B. vulgatus mice were significantly decreased.

Figure 54 and Figure 55 are the expression levels of browning-related molecules in mouse adipose tissue.

It can be seen from Figure 54 and Figure 55 that the expression of subcutaneous fat Ucp1, Pgc1a, Cited1, Cox8b and brown fat Ucp1, Pgc1a, Cox8b of B.vulgatus mice treated with GDCA (30 mg/kg orally) was significantly increased.

Experimental mice: 3-week-old or 6-8-week-old female mice, purchased from the Experimental Animal Center of Peking University Health Science Center, SPF grade. The male mice used for the fertility experiment were 10-week-old male mice with normal fertility, purchased from the Experimental Animal Center of Peking University Health Science Center, SPF grade.

Raising of mice: All mice were raised in the animal room of Peking University Health Science Center, 3-5/cage, the feeding temperature was maintained at 25-25°C, the light time was artificial circadian rhythm of 12L:12D, and they were allowed to eat and drink freely. All animals used in the experiments were approved by Peking University Health Science Center and the Animal Ethics Committee of Peking University Third Hospital.

Experimental bacteria: Bacteroides vulgatus (ATCC8482). All bacteria were purchased from ATCC, USA.

Clinical Sample Collection:

PCOS group: 22-45 years old, female patients who were first diagnosed in the Reproductive Center of the Third Hospital of Peking University from September 2015 to December 2016, and met the Rotterdam PCOS diagnostic criteria.

Control group: 22-45 years old, healthy and normal women with normal physical examination in the physical examination center of our hospital, without insulin resistance, hypertension, hyperlipidemia, and PCOS.

Exclusion criteria:

(1) Have not received treatment three months before enrollment, and have not undergone obesity-related surgery or treatment that affects the immune system;

(2) No history of routine use of antibiotics or treatment, and no participation in other studies within three months;

(3) Have not taken hepatotoxic drugs within three months, and have no arthritis, rheumatoid arthritis, kidney and liver diseases;

(4) No gastrointestinal symptoms such as abdominal pain, constipation or diarrhea;

(5) Non-lactating and pregnant women.

All subjects signed informed consent, filled out questionnaires, and measured height, weight, waist and hip circumference. The experimental process was reviewed by the Ethics Committee of Peking University Third Hospital.

Dietary requirements: normal diet, no medicine, yogurt or other foods containing probiotics within one month before sampling.

Specimen collection: All volunteers collected blood on an empty stomach in the morning of the 2nd to 4th day of menstruation, centrifuged at 1500rpm for 10min, aliquoted into 500ul/tube, and stored at -80°C. Volunteers who are not menstrual period are asked to come to the laboratory to collect appropriate amount of feces, which are immediately divided into 250mg/tubes and stored at -80°C.

Stool DNA Extraction (Kang Wei Century Stool DNA Extraction Kit):

(1) Take 250mg of feces sample, put it in a 1.5ml EP tube, add 1ml BufferSW, vortex for 3-5 minutes, so that the sample is evenly dispersed in the solution. Centrifuge at 12,000rpm for 1min and discard the supernatant.

(2) Add 1ml BufferSL, vortex for 3-5min to disperse the sample evenly in the solution, bathe in 65℃ water for 20min, centrifuge at 12,000rpm for 3min, transfer the supernatant to a new 1.5ml EP tube.

(3) Add an equal volume of BufferGL to the supernatant, mix by inverting for 15-25 times, and place on ice for 5 minutes. Centrifuge at 12,000rpm for 5min, add the obtained supernatant to the adsorption column loaded into the collection tube, centrifuge at 12,000rpm for 1min, discard the waste liquid in the collection tube, and put the adsorption column back into the collection tube.

(4) Add 500 μl BufferGW1 to the adsorption column, centrifuge at 12,000 rpm for 1 min, discard the waste liquid in the collection tube, put the adsorption column back into the collection tube, and repeat.

(5) Add 500 μl BufferGW2 to the adsorption column, centrifuge at 12,000 rpm for 1 min, discard the waste liquid in the collection tube, and put the adsorption column back into the collection tube.

(6) Centrifuge at 12,000 rpm for 2 minutes, and discard the waste liquid in the collection tube. Allow the column to dry completely at room temperature for several minutes.

(7) Put the adsorption column in a new 1.5ml EP tube, add 50-100μl sterilized water dropwise to the middle of the adsorption column, leave it at room temperature for 2-5min, centrifuge at 12,000rpm for 1min, collect the DNA solution, and store at -20℃ DNA is preserved.

Establishment of DHEA animal model: 21-day-old C57BL/6J female mice were subcutaneously injected with DHEA (6 mg/100 g body weight, dissolved in 0.05 ml sesame oil) for 21 consecutive days. The control group received subcutaneous injection of sesame oil for 21 consecutive days.

Oral administration of Bacteroides vulgaris: Centrifuge Bacteroides vulgaris at 5000 rpm for 5 minutes, discard the supernatant, and resuspend the pellet in PBS to 5×108 CFU/ml. Give 21-day-old mice orally, 2×108 CFU/only, twice a week. The control group was given intragastric administration of Bacteroides vulgaris inactivated by high-pressure steam for 3 weeks.

Bile acid gavage experiment: GDCA was gavaged at 30 mg/kg body weight, prepared with 0.9 wt% normal saline, once a day.

Detection of estrous cycle in mice: From the 10th day of drug administration, vaginal smears were taken at 9:00 am every day until the modeling was completed.

Dipping: Wet a sterilized thin cotton swab with physiological saline, gently insert it into the mouse's vagina, and slowly rotate it to take it out.

Smear: Turn the thin cotton swab with vaginal contents evenly on the glass slide to make a smear, and then spread the smear in the air to dry naturally (subject to the whitishness on the slide).

Fixation: Fix the slides in 95% ethanol for 30-60min.

Staining: Put slides in Sauce solution for about 10 minutes, 95% ethanol twice, every few seconds, pure ethanol twice, 3-5min/time; xylene twice, 5min/time.

Observation of results: Place the smear under a microscope, observe the histological changes of the vaginal smear, and determine the different stages of the estrous cycle.

Preestrus (P): Nucleated epithelial cells predominate in vaginal smears under the microscope, some are single, some are in sheets, and may be accompanied by a small amount of white blood cells.

Estrus (E): There are many non-nucleated keratinized squamous cells in the smear, the cells are large and flat, with irregular edges, and no or few white blood cells and epithelial cells can be seen in the field of vision.

Metaestrus (M): There are fewer keratinized epithelial cells in the vaginal cavity, and many white blood cells and nucleated epithelial cells appear.

Diestrus (D): The vaginal mucosa of the mouse is thin, and the white blood cells are released from the mucosa, so that the vaginal smear is almost entirely white blood cells.

Glucose tolerance test:

(1) After the mice were put into a clean cage and fasted for 8 hours (normal drinking water was guaranteed during the fasting period), the weight of each mouse was weighed, and the mice were marked one by one so that they could be quickly identified during the experiment The tested mice.

(2) Use scissors to cut off about 1-2mm of the end of the tail of the mouse, squeeze the tail gently to enrich the blood into a drop, measure the blood glucose level of the mouse with a blood glucose tester, and record it as fasting blood glucose.

(3) Inject glucose solution at a dose of 2 g/kg intraperitoneally into each mouse.

(4) Measuring the fasting blood glucose level and measuring the blood glucose level of the mice by taking blood from the tail vein at 15 min, 30 min, 60 min, and 120 min after the glucose injection.

Insulin resistance test:

(1) After the mice were put into a clean cage and fasted for 4 hours (normal drinking water was guaranteed during the fasting period), the weight of each mouse was weighed, and the mice were marked one by one, so that they could be quickly identified during the experiment The tested mice.

(2) Use scissors to cut off about 1-2mm of the end of the tail of the mouse, squeeze the tail gently to enrich the blood into a drop, measure the blood glucose level of the mouse with a blood glucose tester, and record it as fasting blood glucose.

(3) Insulin at a dose of 1.5 IU/kg was intraperitoneally injected into each mouse.

(4) Measuring the fasting blood glucose level and measuring the blood glucose level of the mice by taking blood from the tail vein at 15min, 30min, 60min, and 120min after the glucose injection.

Blood hormone testing:

The obtained blood was centrifuged at 3000 g for 10 min, and the upper serum was subpackaged and frozen at -80°C. According to the instructions of the insulin immunoassay kit, testosterone quantitative assay kit, and LH quantitative assay kit, the contents of each hormone were analyzed by radioimmunoassay technique.

Ovarian Morphological Detection:

The ovaries were taken and fixed in Bollin's fixative for 24 hours. Paraffin sections were prepared according to the steps of conventional gradient dehydration, transparency, wax immersion, embedding, and sectioning. They were dried at 37°C and stored at room temperature for later use.

Wash twice with xylene, 5min each time

100%, 95%, 90%, 80%, 70% alcohol gradient hydration, each gradient 5min, 2 times, washed twice with distilled water after the end.

After counterstaining with hematoxylin for 15 minutes, wash twice in distilled water for 5 minutes;

Soak in 1% hydrochloric acid alcohol for 30s, wash twice in distilled water for 5min;

After staining with 1% eosin solution for 5 minutes, wash with distilled water for 5 minutes twice;

70%, 80%, 90%, 95%, 100% alcohol gradient dehydration, each gradient 5min, 2 times;

Wash twice with xylene, 5 minutes each time;

Mount with neutral gum.

After the production was completed, the morphology of the ovaries was observed under a light microscope, and the morphological changes of the ovarian tissues of mice in each group were evaluated.

Total RNA extraction:

(1) When preparing cell samples, discard the culture medium, wash with PBS 3 times, add 1ml Trizol to pipette the cells; when preparing tissue samples, put the tissue block directly into the mortar, add a small amount of liquid nitrogen, and grind quickly. The tissue becomes fine powder, add Trizol50-100mg tissue/ml, continue to grind until the Trizol and the tissue are fully mixed, let it stand for 5-10min, wait for it to melt, blow the residue on the wall with a gun, transfer it into a 1.5ml EP tube, add Trizol Bring the total volume to 1 ml.

(2) Add 200 μl of chloroform according to 1/5 volume of the added Trizol, mix by inverting, shake vigorously for 30 seconds to form milk. Place it on ice for 5 minutes, and you can see that the aqueous phase and the organic phase are slowly separated.

(3) Centrifuge at 12,000rpm at 4°C for 10min, and pipette the upper aqueous phase into a new 1.5ml EP tube (do not touch the DNA layer).

(4) Mix by adding an equal volume of isopropanol, mix thoroughly, and place at -20°C overnight or on ice for 10 minutes.

(5) Centrifuge at 12,000 rpm at 4°C for 10 min, discard the supernatant, and leave the bottom of the tube to precipitate RNA.

(6) Add 1 ml of 75% ethanol (prepared with DEPC water) to wash the precipitate, gently shake the centrifuge tube to suspend the precipitate, centrifuge at 12,000 rpm at 4°C for 5 min, discard the supernatant to minimize residual liquid. Repeat 1 time.

(7) Dry at room temperature for 5-10 minutes. Add 20ul sterile DEPC water to dissolve the RNA sample.

(8) Take 1ul to measure the RNA concentration. OD values quantify RNA concentration.

Fertility testing:

(1) Purchase 10-week-old male C57BL/6J mice and house them in a 1:1 cage with sexually mature female mice without experimental use, and separate the male and female mice after seeing the plug;

(2) After confirming that the female mouse is pregnant, it is proved that the male mouse in the same cage has normal sexual ability and can be used. If the female mouse in the same cage cannot be made pregnant, then replace the male mouse;

(3) Select a sufficient number of male mice with normal fertility and put them in a 1:1 cage with the female mice to be tested

(4) Check the litter size of the first litter.

Although some specific embodiments of the present invention have been described in detail through examples, those skilled in the art should understand that the above examples are for illustration only and not intended to limit the scope of the present invention. Those skilled in the art will appreciate that modifications can be made to the above embodiments without departing from the scope and spirit of the invention. The scope of the invention is defined by the appended claims.

Claims (10)

1. a kind of application of bile acid as preparation treatment PCOS drug.

2. the application of bile acid according to claim 1, which is characterized in that be used to prepare medicament, the medicament is suitble to complete Body, local application, or ovary position can be directly injected into.

3. the application of bile acid according to claim 2, which is characterized in that the application by intravenous or intra-arterial, Intramuscular, transdermal, peritonaeum is interior, administration in intrathecal, lymphatic vessel, and subcutaneously, orally or topically regional administration carries out.

4. the application of bile acid according to any one of claim 1 to 3, which is characterized in that the bile acid includes sweet Ammonia deoxycholic acid and/or Tauro ursodesoxy cholic acid.

5. the application of bile acid according to claim 4, which is characterized in that the bile acid is glycodesoxycholic acid.

6. a kind of pharmaceutical preparation, which is characterized in that including bile acid and one or more pharmaceutically acceptable carriers and/or tax Shape agent.

7. pharmaceutical preparation according to claim 6, which is characterized in that the bile acid include glycodesoxycholic acid and/or Tauro ursodesoxy cholic acid.

8. pharmaceutical preparation according to claim 7, which is characterized in that the bile acid is glycodesoxycholic acid.

9. pharmaceutical preparation according to claim 6, which is characterized in that the carrier includes solid adjuvant material or Auxiliary Liquid Material, The dosage form of the pharmaceutical preparation includes tablet, pill, capsule, paste, powder, solution, granule, suspension, injection, delays Release formulation and controlled release preparation.

10. pharmaceutical preparation according to any one of claims 6 to 9, which is characterized in that the effective dose of the bile acid For 20-100mg/kg.