Preparation method for constructing SHBG-reduced polycystic ovarian syndrome rat model based on ruminococcus lividus

Abstract

The invention discloses a method for constructing a polycystic ovarian syndrome (PCOS) rat model with reduced sex hormone binding globulin (SHBG) on the basis of ruminococcus aureus with the preservation number of ATCC29149, and belongs to the technical field of animal models. According to the present invention, the bacterial strain is subjected to anaerobic culture to prepare the bacterial liquid, the dosage is 0.2-1 mL / 100 g, the intragastric administration frequency is one time per day to two times per week, and the time lasts for 4-8 weeks, such that the SHBG synthesis of the rat liver is induced to decrease, and the PCOS pathological characteristics such as testosterone level increase, estrus cycle disorder, ovarian polycystic change, insulin resistance and the like are initiated; the problem that a traditional hormone induction method does not conform to the physiological status is solved, the modeling period of a high-fat diet induction method is shortened, and an efficient experimental tool is provided for PCOS mechanism research and drug development.

Description

Technical Field

[0001] The present invention relates to the technical field of animal models, and particularly to a preparation method for constructing a rat model of polycystic ovary syndrome with decreased SHBG based on Ruminococcus gnavus. Background Art

[0002] Polycystic ovary syndrome (abbreviated as PCOS) is a complex reproductive endocrine disease. The construction of its animal model plays an important role in studying the pathological mechanism and exploring treatment plans. The principle of constructing a PCOS animal model is to simulate some key characteristics of PCOS, such as ovulation disorder, elevated androgen level, and polycystic ovarian changes.

[0003] Currently, the methods used to construct PCOS animal models mainly include the hormone induction method and the high-fat diet induction method. The former constructs the model from the result rather than the cause of the disease, which does not conform to the physiological state of human PCOS; the latter requires a relatively long time to observe PCOS-like symptoms, and the experimental cycle is long (at least 2 months). Currently, no research has found that Ruminococcus gnavus can cause PCOS. Summary of the Invention

[0004] Aiming at the problems existing in the prior art, the purpose of the present invention is to provide a preparation method for constructing a rat model of polycystic ovary syndrome with decreased SHBG based on Ruminococcus gnavus. By gavage of Ruminococcus gnavus to rats, the synthesis level of SHBG protein in the liver of rats can be reduced, inducing PCOS-related pathological phenotypes.

[0005] In order to achieve the above invention purpose, the present invention provides the following technical solutions.

[0006] The present invention discloses the application of a strain of Ruminococcus gnavus in the construction of a polycystic ovary syndrome animal model, characterized in that: the Ruminococcus gnavus has a preservation number of ATCC29149.

[0007] Further, the application includes the application in constructing a polycystic ovary syndrome animal model.

[0008] The present invention also discloses a method for constructing a polycystic ovary syndrome animal model, characterized in that it is obtained by gavage of the above-mentioned Ruminococcus gnavus strain to experimental animals, and includes the following steps: S1. Take the Ruminococcus gnavus and culture it with a culture medium; S2. Adjust the concentration of the bacterial liquid obtained in S1 to 1×10 9 CFU / mL with a culture medium; S3. Adaptively raise the experimental animals in an SPF environment, and intragastrically administer the bacterial suspension prepared in step S2 at a dose of 0.2 - 1 mL / 100 g, and a polycystic ovary syndrome animal model can be obtained by conventional breeding.

[0009] Further, the experimental animals are female SD rats.

[0010] The present invention also discloses an application of the method as described in any one of the above in constructing polycystic ovary syndrome.

[0011] The present invention also discloses an application of the polycystic ovary syndrome animal model as described above, characterized in that the application includes the application in the research on the pathogenesis of polycystic ovary syndrome.

[0012] The present invention also discloses an application of the polycystic ovary syndrome animal model as described above, characterized in that it is an application in the screening of drugs for polycystic ovary syndrome diseases.

[0013] Compared with the prior art, the beneficial effects of the present invention are as follows.

[0014] The present invention successfully constructs a PCOS rat model with decreased SHBG by intragastric administration of Ruminococcus gnavus. This model can simulate the pathophysiological characteristics of PCOS, avoiding the inconsistency between the model and the physiological state caused by relying on hormone induction, and also avoiding the overly long modeling time caused by relying on a high-fat diet. The present invention establishes a PCOS model based on intestinal microbiota disorder and liver dysfunction, and simulates the pathophysiological state of PCOS by regulating the intestinal flora, providing a new experimental tool for studying the pathogenesis and treatment of PCOS. Specific Embodiments

[0015] The following further describes the present invention in detail with specific embodiments. However, this should not be understood as limiting the scope of the above-mentioned subject matter of the present invention to the following embodiments. All technologies implemented based on the content of the present invention belong to the scope of the present invention.

[0016] Unless otherwise specified, the reagents and materials used in the present invention are all commercially available.

[0017] Example 1. Ruminococcus gnavus has the effect of reducing the synthesis of SHBG in the liver.

[0018] It is found that Ruminococcus gnavus has the effect of reducing the synthesis of SHBG in the liver and inducing PCOS phenotypes. The inventor intragastrically administered the Ruminococcus gnavus bacterial solution to the experimental rats and found that it can induce a decrease in SHBG synthesis, estrous cycle disorder, hormone secretion disorder, and polycystic-like changes in the ovaries. Therefore, the strain can be used to construct a PCOS animal model.

[0019] The present invention uses Ruminococcus gnavus, with the preservation number ATCC 29149. The culturing method of Ruminococcus gnavus is as follows: inoculate Ruminococcus gnavus onto a plate solid medium, and culture it at 37°C for 3 days under anaerobic conditions. After 3 days, pick monoclonal colonies and inoculate them into a liquid medium, and grow them at 37°C for 2 - 3 days under anaerobic conditions.

[0020] The plate solid medium consists of 10 g / L casein tryptic digest, 3 g / L heart infusion tryptic digest, 1 g / L corn starch, 5 g / L meat peptone, 5 g / L yeast extract, 5 g / L sodium chloride, and 15 g / L agar, with a pH value of 7.3 ± 0.2; the liquid medium consists of 17 g / L trypticase peptone, 3 g / L soybean peptone, 2.5 g / L dipotassium hydrogen phosphate, 2.5 g / L glucose, and 5 g / L sodium chloride, with a pH value of 7.3 ± 0.2.

[0021] Example 2: Construction of an animal model.

[0022] The experimental animals used in the present invention were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd., with a temperature of 20 - 24°C, a constant humidity of 50 - 60%, a light duration of 12 h (8:00 - 20:00), sound insulation, free access to food and water, and the experiment was carried out after one week of environmental adaptation. Blood glucose test strips (Roche), blood glucose meters (Roche), and sex hormone ELISA detection kits (Wuhan Huamei).

[0023] Method: Female SD rats, 6 weeks old, weighing 200 - 210 g, 24 in number, were randomly divided into (1) a normal control group, and (2) Ruminococcus gnavus at 1×10 9 CFU / mL, divided into experimental groups 1, 2, and 3, with 6 rats in each group. The control group was given an equal amount of anaerobic PBS. The rats administered with bacteria were given a bacterial solution at 0.2 - 1 mL / 100 g body weight, and the gavage frequency was once a day to twice a week for 4 - 8 weeks. The estrous cycle was detected daily 10 days after modeling; glucose tolerance and insulin tolerance were detected before the last administration; after the last administration, blood was taken, allowed to stand at room temperature for 2 hours, and then centrifuged at 3000 rpm at 4°C to determine the serum sex hormone level; ovarian sections were observed for pathological changes.

[0024] Results.

[0025] Experimental group 1: The gavage amount was 0.2 mL / 100 g body weight, twice a week for 8 weeks.

[0026] Experimental group 2: The gavage amount was 1 mL / 100 g body weight, twice a week for 8 weeks.

[0027] Experimental group 3: The gavage amount was 0.2 mL / 100 g body weight, once a day for 4 weeks.

[0028] Compared with the control group, Ruminococcus gnavus can significantly decrease the SHBG level, increase the testosterone level, disrupt the estrous cycle, cause polycystic ovarian changes, and induce insulin resistance. The results are shown in Tables 1 - 5.

[0029] Table 1 Effects of Ruminococcus gnavus on the estrous cycle of rats 。

[0030] Table 2 Effects of Ruminococcus gnavus on serum sex hormones of rats 。

[0031] Table 3 Effects of Ruminococcus gnavus on GTT of rats 。

[0032] Table 4 Effects of Ruminococcus gnavus on ITT of rats 。

[0033] Table 5 Effects of Ruminococcus gnavus on polycystic ovarian changes in rats 。

[0034] In summary, for the first time, the present invention uses Ruminococcus gnavus ( Ruminococcus gnavus ) to construct a PCOS rat model with decreased SHBG, simulating the natural course of the disease, and solving the key problems of the existing technology models deviating from the physiological state and having a long modeling period. Experimental data confirm that this model highly conforms to the characteristics of PCOS in terms of hormone levels, metabolic indexes, and ovarian pathology, providing an innovative tool for etiological research and drug screening, and having significant scientific research and clinical application value.

[0035] The above are only the preferred embodiments of the present invention and are not used to limit the patent scope of the present invention. For those skilled in the art, the present invention can have various changes and modifications. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. Application of an active Ruminococcus gnavus strain in the construction of an animal model of polycystic ovary syndrome, characterized in that: The active rumenococcus has a deposit number of ATCC29149.

2. The use according to claim 1, characterized in that: The application includes application in constructing an animal model of polycystic ovary syndrome.

3. A method for constructing an animal model of polycystic ovary syndrome, characterized in that: The method is obtained by gavage of experimental animals with the active rumenococcus strain described in claim 1, comprising the following steps: S1, taking the active rumenococci and culturing them in a culture medium; S2, use the culture medium to adjust the concentration of the bacterial solution obtained in S1 to 1×10 9 CFU / mL; S3. The experimental animals are adaptively raised in an SPF environment, and the bacterial suspension prepared in step S2 is gavaged at a rate of 0.2-1 mL / 100 g. The polycystic ovary syndrome animal model can be obtained by conventional feeding.

4. The method for constructing an animal model of polycystic ovary syndrome according to claim 3, characterized in that: The experimental animals were female SD rats.

5. Use of the method according to any one of claims 3 to 4 in constructing polycystic ovary syndrome.

6. A use of the polycystic ovary syndrome animal model according to claim 3, characterized in that: The application includes application in the research on the pathogenesis of polycystic ovary syndrome.

7. A use of the polycystic ovary syndrome animal model according to claim 3, characterized in that: Application in drug screening for polycystic ovary syndrome.