Mouse polycystic ovarian syndrome model induced by dehydroepiandrosterone under low-dose exposure of bisphenol A

Abstract

The invention belongs to the technical field of medical animal disease model modeling, and particularly discloses a dehydroepiandrosterone-induced mouse polycystic ovarian syndrome model under bisphenol A low-dose exposure, specifically, a preparation method of the model comprises the following steps: continuously injecting a mouse for 21 days according to the injection amount of dehydroepiandrosterone of 6mg / 100g; the method comprises the following steps: carrying out intragastric administration on a mouse by using 10 mg / kg of bisphenol A on the 15th day of injection of dehydroepiandrosterone, and continuously carrying out intragastric administration on the mouse for 7 days to obtain the polycystic ovarian syndrome model of the mouse at the childbearing age. According to the mouse model obtained by DHEA modeling at the early stage of adolescence and BPA exposure modeling at the late stage of adolescence, the disordered mood cycle, obesity and high T level are obviously changed; and the human PCOS disease environment and clinical phenotype can be truly simulated.

Description

Technical Field

[0001] The present invention belongs to the technical field of medical animal disease model establishment, and particularly relates to a mouse polycystic ovary syndrome model induced by low-dose exposure to dehydroepiandrosterone (DHEA) combined with bisphenol A (BPA). Background Art

[0002] Polycystic ovary syndrome (PCOS) is a common endocrine and metabolic disease in women of reproductive age. It has complex etiologies, a high incidence rate, and diverse clinical phenotypes. The main clinical phenotypes are hyperandrogenism, menstrual disorders, and obesity. Its short-term and long-term complications seriously affect the physical and mental health and quality of life of women and their offspring. It is one of the main causes of secondary infertility. Approximately 60%-70% of the female offspring of PCOS patients develop PCOS during puberty and sexual maturity. Its diverse clinical manifestations and long-term complications seriously affect the physical and mental health and quality of life of women and are the main cause of secondary infertility. Establishing a PCOS disease model is a common means of disease research. Due to the complexity of PCOS pathogenesis, there is currently no complete animal model established for studying the pathogenesis and treatment of polycystic ovary syndrome (PCOS).

[0003] Previously, there have been PCOS modeling methods including those for mice, mainly including: androgen modeling method, prenatal androgenized modeling method, prenatal anti-Müllerian hormone (AMH) modeling method, estrogen modeling method, aromatase inhibitor modeling method, models established by specific genes, and light exposure method, etc.

[0004] There are many PCOS modeling methods, but there is a lack of a standard method that meets the PCOS clinical phenotypes. Rats chronically exposed to dehydroepiandrosterone (DHEA) are considered to have induced better PCOS clinical characteristics. Although DHEA can induce polycystic-like changes in the mouse ovaries, typical pathological features of human PCOS (such as thickening of the ovarian tunica albuginea and interstitial fibrosis) are rarely reported in mouse models. And recent studies have found that long-term exposure to DHEA will reduce the ovarian weight of experimental animals, which is not conducive to long-term research. PCOS is a complex endocrine and metabolic disorder disease, and its pathogenesis involves multiple factors, including environmental compounds such as bisphenol compounds (such as BPA, BPS, BPF, etc.), perfluorinated compounds, polychlorinated biphenyls, organophosphate flame retardants and other compounds. Existing studies have reported that the exposure to these compounds is related to the occurrence of PCOS to a certain extent. Therefore, considering multiple factors and constructing a PCOS model that can better meet the clinical phenotypes is of great significance for further studying the mechanism of PCOS occurrence in the real environment. Summary of the Invention

[0005] The main purpose of this invention is to provide a mouse model of polycystic ovary syndrome induced by dehydroepiandrosterone under low-dose exposure to bisphenol A (BPA), which simulates the polycystic ovary syndrome caused by human food-borne BPA exposure in the reproductive age stage of mice, and is a model study close to the reality. In the current animal modeling research, there is no existing evidence and literature reports on the induction of PCOS in mice under the combined influence of BPA food-borne exposure and DHEA. The technical implementation scheme of the present invention is as follows Figure 1 shown.

[0006] Specifically, the technical solution adopted by the present invention is as follows:

[0007] The present invention provides a method for inducing a mouse polycystic ovary syndrome model for observation during childbearing age, comprising the following steps:

[0008] (1) Select female mice aged 4-5 weeks and eliminate mice with obvious poor spirits and slow movements;

[0009] (2) Dissolving dehydroepiandrosterone in 95% ethanol, and then mixing the soybean oil and the dissolved dehydroepiandrosterone in a volume ratio of 9:1 for later use;

[0010] (3) After dissolving bisphenol A in 75% ethanol, soybean oil and the dissolved bisphenol A were mixed in a volume ratio of 9:1 for later use;

[0011] (4) Based on the weight of mice, the mice were injected with 6 mg / 100 g of dehydroepiandrosterone for 21 consecutive days; 10 mg / kg of bisphenol A was administered intragastrically on the 15th day after the injection of dehydroepiandrosterone. After 7 consecutive days of intragastrically administration, a polycystic ovary syndrome model of mice of reproductive age was obtained.

[0012] Furthermore, the present invention provides a polycystic ovary syndrome model of reproductive-age mice prepared by the method.

[0013] Furthermore, the present invention provides the use of the polycystic ovary syndrome model of reproductive-age mice in the preparation of products for studying the pathogenesis of polycystic ovary syndrome.

[0014] Furthermore, the present invention provides the use of the polycystic ovary syndrome model of reproductive-age mice in the preparation of a therapeutic intervention product for evaluating polycystic ovary syndrome.

[0015] Furthermore, the present invention provides the use of the polycystic ovary syndrome model of reproductive-age mice in the preparation of a product for evaluating the efficacy of a drug for treating polycystic ovary syndrome.

[0016] Furthermore, the present invention provides the use of dehydroepiandrosterone and bisphenol A exposure in combination in inducing a polycystic ovary syndrome model in mice of reproductive age.

[0017] Furthermore, the dosage of dehydroepiandrosterone used is 6 mg / 100 g, and female mice at 4-5 weeks of age are selected for continuous injection for 21 days; the dosage of bisphenol A used is 10 mg / kg. On the 15th day of dehydroepiandrosterone injection, the mice are given bisphenol A by gavage for 7 consecutive days.

[0018] Technical effects achieved by the present invention:

[0019] The present invention takes the human dietary BPA exposure characteristics (low-dose exposure) as the main research environment, and constructs a method for establishing a PCOS model in mice and a comprehensive evaluation index system under the combined low-dose exposure of DHEA induction and BPA. The established model can simulate the impact of specific environmental diseases in a more realistic human living environment: polycystic ovary syndrome.

[0020] On the one hand, the technology of the present invention more realistically simulates the human environment of multi-level low-dose exposure to BPA. On the other hand, through technical optimization and reform, it improves the dose-effect control of dietary BPA exposure and DHEA induction. Through pre-pubertal DHEA modeling and late-stage low-dose BPA exposure modeling, the obtained mouse model has obvious changes in estrous cycle disorder, obesity, and high T level, and can more realistically simulate the human PCOS disease environment and clinical phenotypes. The present invention provides a method that can more accurately, objectively, and quantitatively analyze the PCOS modeling process and comprehensive evaluation, and the modeling process is simple and the cycle is short. Description of the drawings

[0021] Figure 1 Technical roadmap;

[0022] Figure 2 Mouse ovarian pathological section diagram;

[0023] Figure 3 Mouse vaginal exfoliated cell smear diagram;

[0024] Figure 4 Mouse apparent characteristic diagram;

[0025] Figure 5 Mouse uterine anatomical diagram;

[0026] Figure 6 Mouse ovarian anatomical diagram; Detailed implementation manners

[0027] The technical solutions of the present invention will be described in detail below in conjunction with the accompanying drawings and embodiments, but the present invention is not limited to the scope of the described embodiments. Unless otherwise specified, the experiments and methods described in the embodiments are basically carried out according to the conventional methods well-known in the art and described in various references. The reagents and raw materials used in the present invention are all commercially available. For those not indicating specific conditions in the embodiments, they are carried out according to the conventional conditions or the conditions recommended by the manufacturer. For those reagents or instruments not indicating the manufacturer, they are all conventional products that can be obtained through commercial purchase.

[0028] Example 1 Modeling with DHEA combined with BPA

[0029] 1. Modeling animals: Kunming mice;

[0030] 2. Modeling method with DHEA 6 mg / 100 g + BPA 10 mg / kg:

[0031] ① For DHEA injection, dissolve the DHEA solid powder with 95% ethanol to obtain a DHEA ethanol solution, add soybean oil and mix well. The volume ratio of soybean oil to the DHEA ethanol solution is 9:1. Weigh the body weight of the mice. According to the injection amount of 6 mg DHEA / 100 g (inject 6 mg DHEA per 100 g of mice), inject 0.1 ml of the solvent per 50 g of mice. Based on the body weight of the mice, calculate the corresponding dose and perform subcutaneous injection behind the neck at the same time period. Inject continuously for 21 days in the same way.

[0032] Dose calculation method: For example, if the body weight of a mouse is 50 g, calculate that 3 mg of DHEA needs to be injected. 3 mg of DHEA needs to be injected, and the solvent is 0.1 ml (that is, dissolve and mix 0.01 ml of ethanol, and then add 0.09 ml of soybean oil). Referring to the density of dehydroepiandrosterone as 1.04 g / ml, the total volume of the solution prepared at this time is 0.10312 ml.

[0033] ② For BPA (bisphenol A) exposure, dissolve the BPA solid with 75% ethanol to obtain a BPA ethanol solution. Add soybean oil and mix well. The volume ratio of soybean oil to the BPA ethanol solution is 9:1. According to the BPA exposure concentration of 10 mg / kg, based on the body weight of the mice, the single - dose gavage for each mouse does not exceed 2 ml, and calculate the corresponding dose. On the 15th day of DHEA injection, use the above - prepared solution for gavage exposure with the BPA solution, and continuously perform gavage for 7 days in the same way.

[0034] Comparative Example 1 Traditional DHEA Modeling

[0035] Use traditional methods to establish a model in Kunming mice with DHEA. For DHEA injection, dissolve the DHEA solid powder with 95% ethanol, add soybean oil and mix well. The ratio of soybean oil to the DHEA ethanol solution is 9:1. Weigh the mice, and according to the injection dose of 6 mg DHEA / 100 g (inject 6 mg DHEA per 100 g of mice), inject 0.1 ml of the solvent per 50 g of mice. Based on the mouse body weight, calculate the corresponding dose and perform subcutaneous injection behind the neck at the same time period. Inject continuously for 21 days in the same way.

[0036] Control Example 2: Establish a model with BPA alone

[0037] Dissolve the BPA solid with 75% ethanol to obtain a BPA ethanol solution. Add soybean oil and mix well. The volume ratio of soybean oil to the BPA ethanol solution is 9:1. According to the BPA exposure concentration of 50 mg / kg, calculate the corresponding dose based on the mouse body weight. Calculate the soybean oil dose according to injecting 0.1 ml of soybean oil per 50 g of mice, and perform subcutaneous injection behind the neck of the mice. On the 15th day after subcutaneous injection of soybean oil behind the neck, use the above-prepared solution to perform gavage exposure with the BPA solution. Gavage continuously for 7 days in the same way.

[0038] 3. Evaluation of model effects

[0039] Compare the effects of the modeling methods in (1)-(3) through the following indicators, and find that the BPA modeling method induced in Kunming mice using DHEA 6 mg / 100 g + BPA 10 mg / kg is better.

[0040] (1) Pathological index: Refer to Figure 2 , under the light microscope, more atretic follicles can be seen in the mice after modeling, the vesicle dilation increases significantly, the granulosa cell layer decreases, and the number of corpora lutea decreases significantly. And it shows the characteristic that the pathological features above a lower dose are more obvious.

[0041] (2) Vaginal exfoliated cells: Refer to Figure 3 , continuously perform vaginal smear examinations on the mice after modeling for 2 cycles, and it is observed that the vaginal epithelium is continuously keratinized. Polycystic ovary syndrome mice induced by dehydroepiandrosterone and bisphenol A have relatively obvious vaginal epithelial keratinization characteristics.

[0042] (3) Biochemical index: Since the GnRH pulse frequency in the hypothalamus is affected, the hormone levels in the modeled animals change, and the levels of androgens and estrogens increase (in this case, because androgen induction is one of the main methods, and androgen is taken as the main detection index, see Table 2).

[0043] (4) Apparent index: Refer to Figure 4 , Figure 5 , Figure 6, in this study, the polycystic ovary syndrome mice induced by dehydroepiandrosterone and bisphenol A showed increased body mass, infertility, slowed down activity and hair loss in the later stage. Anatomically, the ovarian surface was pale, the ovarian tunica albuginea was thickened, the volume was increased, and the wet weight was increased. The uterine surface was pale and congested.

[0044] (5) Success rate of model establishment

[0045] As shown in Table 1, the success rate of model establishment in this invention was consistent with that of using DHEA alone for model establishment, reaching 85.71%, which was higher than that of using BPA alone and exceeded the success rate level of the existing literature. The multi-dimensional index evaluation system was adopted in this invention to judge whether the model establishment was successful, and it had good feasibility.

[0046] Table 1 Success rate of model establishment of mice in each group

[0047]

[0048] Note: For model establishment with BPA alone, the dose was BPA 50 mg / kg; for model establishment with DHEA alone, the dose was DHEA 6 mg / 100 g for conventional model establishment; for model establishment with DHEA + BPA (low dose), the dose was DHEA 6 mg / 100 g + BPA 10 mg / kg

[0049] (6) Comparison of consistency with clinical manifestations of human PCOS

[0050] As shown in Table 2, in this invention, the keratinization of vaginal exfoliated cells, the number of antral follicles, Lee's index, testosterone level, and ovarian wet weight of mice in the estrous cycle after model establishment were taken as the main evaluation indicators, and the clinical phenotypes (menstrual disorders, PCO manifestations, weight gain, hyperandrogenism, ovarian appearance changes) of PCOS in mice under different model establishment methods were compared. The results showed that the mice after model establishment in this invention had more consistent clinical characteristics with human PCOS in the reproductive age under low-dose BPA exposure.

[0051] Table 2 Model establishment indicators of mice in each group

[0052]

[0053] Note: "+" indicates the corresponding model establishment index, and "++" and "+++" indicate more obvious model establishment indexes.

[0054] The diagnostic criteria for human PCOS in the reproductive age are: oligomenorrhea or amenorrhea or ovulatory dysfunctional abnormal uterine bleeding, and any one of the following conditions is met: ① clinical manifestations of elevated androgen or hyperandrogenemia; ② polycystic ovary (PCO) under ultrasound. Human PCOS has clinical phenotype heterogeneity, mainly manifested as menstrual disorders, obesity, hyperandrogenemia or manifestations, and PCOS patients have a widespread BPA exposure. Therefore, selecting a suitable model establishment method is the basis for scientific research on PCOS diseases.

[0055] On the basis of the existing modeling methods, the present invention creatively combines DHEA and BPA for simultaneous induction of modeling, and the obtained model can simulate more realistic disease characteristics of human PCOS. The local high androgen action in the ovary is the main cause of polycystic ovary syndrome, and the disease and pathological characteristics of animals after modeling are highly similar to those of human diseases.

[0056] The present invention takes the human dietary BPA exposure characteristics (low-dose exposure) as the main research environment, and studies the disease characteristics of polycystic ovary syndrome under low-dose BPA exposure and DHEA induction, so as to solve the problem of simulating the impact of environmental specific diseases: polycystic ovary syndrome in a more realistic human living environment. It can be used for the study of polycystic ovary syndrome in a real human environment where it is impossible to carry out multi-dose level and causal inference studies due to widespread BPA exposure.

[0057] The technology of the present invention can save a large amount of manpower, financial resources and material resources for population-based prospective cohort studies, and can better control many confounding factors in population trials, providing a more controllable experimental environment basis for experimental expectations and results. On the basis of this developed technology, the joint research group has carried out research on multiple links including the current situation, etiology, mechanism, etc. Greatly improved the feasibility of experimental animal verification research on related research in the population.

[0058] The above description shows and describes several preferred embodiments of the present invention. However, as mentioned above, it should be understood that the present invention is not limited to the form disclosed herein, should not be regarded as excluding other embodiments, but can be used in various other combinations, modifications, and can be changed within the scope of the present invention's concept through the above teachings or the technology or knowledge in related fields. And any changes and modifications made by those skilled in the art without departing from the spirit and scope of the present invention shall fall within the protection scope of the appended claims of the present invention.

Claims

1. A method for inducing a polycystic ovary syndrome model in mice for observation during the childbearing age, characterized in that, It includes the following steps: (1) Select female mice at 4 - 5 weeks old and exclude those with obvious mental sluggishness and slow movement. (2) After dissolving dehydroepiandrosterone with 95% ethanol, mix soybean oil and the dissolved dehydroepiandrosterone according to a volume ratio of 9:1 for standby. (3) After dissolving bisphenol A with 75% ethanol, mix soybean oil and the dissolved bisphenol A according to a volume ratio of 9:1 for standby. (4) According to the body weight of the mice, inject the mice continuously for 21 days at an injection dose of 6 mg / 100 g of dehydroepiandrosterone; for bisphenol A at 10 mg / kg, on the 15th day of dehydroepiandrosterone injection, conduct intragastric administration of bisphenol A to the mice, and after continuous intragastric administration for 7 days, obtain a polycystic ovary syndrome model of mice in the reproductive age.

2. A polycystic ovary syndrome model of mice in the reproductive age prepared by the method according to claim 1.

3. Use of the polycystic ovary syndrome model of mice in the reproductive age according to claim 2 in the preparation of products for studying the pathogenesis of polycystic ovary syndrome.

4. Use of the polycystic ovary syndrome model of mice in the reproductive age according to claim 2 in the preparation of products for evaluating the therapeutic intervention of polycystic ovary syndrome.

5. Use of the polycystic ovary syndrome model of mice in the reproductive age according to claim 2 in the preparation of products for evaluating the efficacy of drugs for treating polycystic ovary syndrome.

6. Use of the combined use of dehydroepiandrosterone and bisphenol A exposure in inducing a polycystic ovary syndrome model of mice in the reproductive age.

7. The application according to claim 6, wherein The dosage of dehydroepiandrosterone used is 6 mg / 100 g, and female mice at 4 - 5 weeks old are selected for continuous injection for 21 days; the dosage of bisphenol A used is 10 mg / kg, and on the 15th day of dehydroepiandrosterone injection, intragastric administration of bisphenol A is conducted to the mice, and continuous intragastric administration is carried out for 7 days.

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