Method for establishing rat premature ovarian failure model based on tripterygium glycosides

Abstract

The invention relates to the technical field of medical animal models, in particular to a method for establishing a rat premature ovarian failure model based on tripterygium glycosides. The tripterygium glycosides are continuously injected into the stomach of a rat for 4 days, and the intragastric injection amount is 80 mg / kg / day according to the weight of the rat. The molding period is four days; the ovarian injury specificity is high, and the systemic toxicity is controllable; and the pathological mechanism is highly matched with the human iatrogenic POF. The model provides a powerful tool for deeply researching the pathologic mechanism of the premature ovarian failure, is beneficial to discussing the pathologic mechanism, screening and evaluating therapeutic drugs and providing a scientific basis for clinical treatment, can also research the influence of the premature ovarian failure on female reproductive health and overall health, and assists in improving the life quality of a patient.

Description

Technical Field

[0001] The present invention relates to the technical field of medical animal models, and particularly to a method for establishing a rat premature ovarian failure model based on tripterygium glycosides. Background Art

[0002] Clinical premature ovarian failure (POF) is a disorder in which women under 40 years old exhibit characteristics such as amenorrhea, infertility, decreased estrogen levels, and increased gonadotropin levels due to ovarian function decline. This disorder not only causes a reduction in the number and quality of oocytes, but also significantly decreases the pregnancy rate and live birth rate, while increasing the probability of miscarriage, bringing serious negative impacts on women's reproductive health and quality of life. The causes of POF are relatively complex, covering genetic factors, immune abnormalities, iatrogenic injuries (such as radiotherapy and chemotherapy), environmental toxins, and psychological stress, etc. However, its specific molecular mechanism has not been fully clarified yet. To deeply study the pathological mechanism of POF and develop effective intervention measures, it is urgently necessary to construct an animal model highly consistent with human clinical characteristics and etiologies. Currently, the main methods for constructing POF animal models include gene editing models, surgical induction models, and drug toxicity models, etc. Among them, the drug-induced model is widely used because of its relatively simple operation and easy control of pathological manifestations. However, existing methods (such as using chemotherapy drugs like cyclophosphamide and cisplatin for induction) generally have problems such as a relatively long modeling period (the current shortest period is 2 weeks), large systemic toxicity, high animal mortality, and obvious heterogeneity of ovarian damage, which greatly limits its application in mechanism research and drug screening.

[0003] Tripterygium glycosides (TG) is a mixture of terpenoid compounds extracted from the roots of the plant Tripterygium wilfordii in the Celastraceae family. The main active ingredients are triptolide and celastrol. Clinically, TG is widely used to treat autoimmune diseases such as rheumatoid arthritis and systemic lupus erythematosus because of its strong immunosuppressive and anti-inflammatory effects. However, there is no report in the prior art on using tripterygium glycosides to establish a rat premature ovarian failure model. Summary of the Invention

[0004] To solve the above problems, the present invention provides a method for establishing a rat premature ovarian failure model based on tripterygium glycosides, which solves the problems of long modeling period, large systemic toxicity, high animal mortality, and heterogeneous ovarian damage existing in the prior art.

[0005] To achieve the above object, the present invention provides the following technical solutions:

[0006] The present invention provides a method for establishing a premature ovarian failure model in rats based on tripterygium glycosides, comprising the following steps: continuously intragastrically administering tripterygium glycosides to rats for 4 days, and the intragastric administration amount is 80 mg / kg / day according to the body weight of the rats.

[0007] Preferably, the rats are healthy female SD rats at 8 weeks old with a normal estrous cycle.

[0008] Preferably, before intragastric administration, the rats are first raised for 7 days, and the raising conditions include: 12-hour day-night alternation, the environmental temperature is 21-25 °C, and the environmental humidity is 30-40%.

[0009] Preferably, the tripterygium glycosides are mixed with dimethyl sulfoxide and sodium carboxymethylcellulose solution, and the obtained mixed solution is intragastrically administered to the rats.

[0010] Preferably, the mass ratio of the tripterygium glycosides to the volume of dimethyl sulfoxide and the volume of sodium carboxymethylcellulose solution is 640 mg: 1.6 mL: 78.4 mL.

[0011] Preferably, the mass percentage content of the sodium carboxymethylcellulose solution is 0.5%.

[0012] Beneficial effects:

[0013] Simple operation: The method of intragastric administration is adopted, which is simple and easy to perform, and does not require complex surgical operations or special equipment support. Controllable pathological manifestations: By controlling the dosage and administration time, the pathological manifestations of premature ovarian failure in rats can be well controlled, making it highly consistent with human clinical characteristics. Good repeatability: This method can stably construct a premature ovarian failure rat model in multiple experiments, with good repeatability. This model provides a powerful tool for in-depth study of the pathological mechanism of premature ovarian failure, helps to explore the pathogenesis, screen and evaluate therapeutic drugs, provides a scientific basis for clinical treatment, and can also study the impact of premature ovarian failure on female reproductive health and overall health, and helps to improve the quality of life of patients.

[0014] The present invention discovers that TG has a toxic effect on the ovaries, leading to ovarian damage and inducing the occurrence of POF. Based on this, a method for establishing a premature ovarian failure rat model is proposed. Compared with traditional drug models, the TG induction method has the following advantages: (1) The modeling period is short (the research period of the present invention is four days); (2) The ovarian damage is highly specific and the systemic toxicity is controllable; (3) The pathological mechanism is highly consistent with human iatrogenic POF (such as secondary ovarian failure after the treatment of autoimmune diseases). Therefore, establishing a standardized TG-induced premature ovarian failure rat model not only helps to analyze the core molecular mechanism of premature ovarian failure, but also provides an efficient research platform for the development of reproductive protection drugs and alternative therapies. The method provided by the present invention aims to construct a stable, reproducible and clinically relevant premature ovarian failure animal model by optimizing the TG administration dose and schedule, combined with an ovarian function evaluation system (sex hormone detection, follicle counting and ovarian pathological analysis), laying a foundation for promoting the precision medicine research of premature ovarian failure. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required in the embodiments.

[0016] Figure 1 It is a schematic diagram of the changes in ovarian indices of rats in each group in Example 1;

[0017] Figure 2 It is a schematic diagram of the changes in AMH levels of rats in each group in Example 1;

[0018] Figure 3 It is a schematic diagram of the changes in E2 levels of rats in each group in Example 1;

[0019] Figure 4 It is a schematic diagram of the changes in FSH levels of rats in each group in Example 1;

[0020] Figure 5 It is a schematic diagram of the changes in LH levels of rats in each group in Example 1;

[0021] Figure 6 It is a schematic diagram of the changes in Prog levels of rats in each group in Example 1;

[0022] Figure 7 It is a schematic diagram of the changes in estrous cycles of rats in each group in Example 1;

[0023] Figure 8 It is a schematic diagram of ovarian pathological damage of rats in each group in Example 1. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0024] The present invention provides a method for establishing a rat model of premature ovarian failure based on tripterygium glycosides, which includes the following steps: continuously intragastrically administer tripterygium glycosides to rats for 4 days, and according to the body weight of the rats, adopt a gavage dose of 80 mg / kg / day based on the reference data. The present invention has no special limitation on the source of the tripterygium glycosides, and conventional commercially available products can be used, such as Yuanda Feiyun Pharmaceutical Company, Z42021212, Hubei, China.

[0025] In the present invention, the rats are preferably healthy female SD rats at 8 weeks of age with a normal estrous cycle. The ovarian function of 8-week-old rats is relatively stable, and it can better simulate the pathological process of human premature ovarian failure. Preferably, before intragastric administration, the rats are first raised for 7 days, and the raising conditions preferably include: 12-hour day-night alternation, an environmental temperature of 21-25°C, and an environmental humidity of 30-40%. The raising conditions help the rats adapt to the experimental environment and reduce the interference of stress response on the experimental results.

[0026] The present invention preferably mixes the tripterygium glycosides with dimethyl sulfoxide and sodium carboxymethylcellulose solution, and intragastrically administers the obtained mixed solution to rats. Intragastric administration can ensure that the drug directly enters the digestive tract, is absorbed by the intestine and then enters the blood circulation, and exerts its toxic effect on the ovary. In the present invention, the mass ratio of the tripterygium glycosides to the volume of dimethyl sulfoxide and the volume of sodium carboxymethylcellulose solution is preferably 640 mg: 1.6 mL: 78.4 mL. In the present invention, the mass percentage content of the sodium carboxymethylcellulose solution is preferably 0.5%.

[0027] To further illustrate the present invention, the present invention will be described in detail below in conjunction with examples, but they cannot be understood as limiting the protection scope of the present invention.

[0028] Example 1

[0029] In this example, a control experiment is adopted, specifically as follows:

[0030] I. Grouping: Select 20 SPF-grade female SD rats with a body weight of 210 g ± 10 g and a normal estrous cycle. Raise them under 12-hour light-dark alternating illumination, with an environmental temperature of 23°C ± 2°C and an environmental humidity of 30-40%. Diet and water are not restricted. After seven days of adaptive feeding, the rats are randomly divided into 2 groups (10 rats in each group), namely: blank control group (CON), tripterygium glycosides group (TG-80 mg / kg / day).

[0031] II. Drug preparation: The default body weight of SD rats is 200 g, that is, each rat needs 16 mg / d. 10 rats need to be administered drugs for 4 days, and a total of 640 mg of tripterygium glycosides is required; the dosage for each rat is calculated according to the ratio of body weight: dosage = 200 g: 2 ml, that is, a total of 2 ml * 10 * 4 = 80 ml of solution needs to be prepared. The specific preparation process is as follows:

[0032] (1) Raw material treatment: Grind the tripterygium glycoside tablets into powder, and weigh 640 mg of powder with a precision electronic balance (calculated according to 10 rats × 4 days × 16 mg / rat).

[0033] (2) Use a pipette with an appropriate scale to accurately pipette DMSO into a sterile EP tube. The required volume of DMSO is 80 ml * 2% = 1.6 ml;

[0034] (3) Dissolve 640 mg of tripterygium glycosides in the DMSO aliquoted in the previous step, and mix, shake, and centrifuge;

[0035] (4) Transfer the above reagent to a clean reagent bottle, and make up the volume to the required volume with 78.4 ml of 0.5% sodium carboxymethylcellulose (80 ml - 1.6 ml = 78.4 ml), and shake and mix well.

[0036] III. Drug administration: Administer drugs at 9:00 am every day. Weigh the rats, and perform gavage administration according to the ratio of body weight: dosage = 200 g: 2 ml (the specific dosage needs to be determined according to the body weight). The control group is given the same volume of normal saline. The above operations are repeated for 4 days, and samples are taken on the 5th day.

[0037] IV. Estrous cycle: Conduct estrous cycle experiments at 8:00 am every day. Swabbing method: Make a suitable cotton swab, moisten it with normal saline and then squeeze it dry, rotate it into the vagina of the animal, gently rotate it in the vagina for several times and then rotate it out, and evenly rotate the cotton swab with vaginal contents on the glass slide to make a smear, and then let the smear dry naturally in the air (until it turns white on the glass slide). Then stain with Regi's stain for 20 min and examine under the microscope. The characteristics of proestrus are the presence of small, round, nucleated epithelial cells, with relatively uniform appearance and size, and usually no neutrophils can be seen; the characteristics of estrus are the appearance of anuclear keratinized epithelial cells. Many bacteria can be observed attached to the cells or free in the background. The characteristics of metestrus are the observation of anuclear keratinized epithelial cells and neutrophils. The characteristics of diestrus are a sharp decrease in the number of anuclear keratinized epithelial cells, a relatively low overall cell number, and different sizes of neutrophils and nucleated epithelial cells.

[0038] V. Detection indicators and methods: After administration in each group, general status observation, calculation of ovarian organ index, and pathological observation were carried out; General status observation included gastrointestinal reactions such as whether there was diarrhea and the degree of diarrhea, body posture changes such as gait changes, activity frequency, whether there was tremor, etc., and general phenotype observation such as appearance changes, hair luster changes, bleeding conditions, etc., and the body weight, diet, and water intake of rats in each group were evaluated and recorded every 24 h; The ovarian organ index of each rat was calculated (ovarian organ index = ovarian weight (g) / body weight (g)); Hormone detection indicators included serum LH, FSH, E2, AMH, and Prog levels, which were detected using an ELISA kit. Pathological observation was performed using hematoxylin-eosin staining. After treating the rats, the ovarian tissue was removed and fixed in 4% paraformaldehyde solution at room temperature for 48 h, followed by dehydration, embedding, sectioning, staining, and sealing. The sections were viewed under a microscope, and the tissue sections were carefully examined at different magnifications to carefully observe basic pathological changes such as congestion, stasis, hemorrhage, edema, degeneration, necrosis, hyperplasia, fibrosis, organization, granulation tissue, inflammatory changes, etc.; Statistical method: The data were expressed as mean ± standard deviation and statistically processed using GraphPad Prism 10.0 software. When the overall data followed a normal distribution and the variances were homogeneous, a T-test was used for analysis. When the data did not conform to a normal distribution, a rank sum test was used. P < 0.05 represented statistical significance, and P < 0.01 represented a significant difference.

[0039] VI. Experimental results: The mortality rate of rats after TG intervention in this study was 0.

[0040] As Figure 1 shown, compared with the CON group, the ovarian index of the TG group increased significantly, with statistical significance, indicating that the ovarian tissue of the TG group might be damaged, suggesting successful modeling.

[0041] As Figures 2 - 6 shown, compared with the CON group, LH and FSH in the TG group were significantly increased, while E2, AMH, and Prog were significantly decreased; As Figure 7 shown, compared with the CON group, the estrous cycle of SD rats was disrupted after TG treatment, with an increased relative proportion of the estrous interphase and a shortened estrus period. These results indicate that TG intervention directly disrupted the endocrine function and normal reproductive cycle of the rat ovaries, suggesting successful modeling.

[0042] As Figure 8As shown, the ovarian tissues of the CON group showed follicles at different developmental stages, such as primary follicles and secondary follicles, surrounded by a dense network of capillaries and connective tissues. The granulosa cells in the follicles were arranged neatly in layers, and there were no atretic follicles. After TG treatment, obvious damage occurred in the ovarian tissues, with a small number of follicles visible, increased connective tissues, disordered arrangement among corpora lutea, a dense network of capillaries, and an increase in atretic follicles, and the ovarian structure was disordered. These results suggest that TG may cause damage to ovarian tissues, indicating successful model establishment.

[0043] Generally, the pathogenesis of POF is considered to be insufficient reserve of primordial follicle pool, accelerated follicular atresia, changes in recruitment of dominant follicles, and disorders in follicular maturation, etc. After TG treatment, obvious damage occurred in the ovarian tissues, with a small number of follicles visible, increased connective tissues, disordered arrangement among corpora lutea, a dense network of capillaries, and an increase in atretic follicles, and the ovarian structure was disordered. TG acts on follicular granulosa cells, affecting the development and maturation of follicles. The damage of ovarian follicles leads to a decrease in ovarian reserve and irreversible damage, resulting in the occurrence of POF.

[0044] The modeling method of the present invention is through intragastric administration of tripterygium glycosides. The experimental group showed typical characteristics similar to human premature ovarian failure, which is an effective method for establishing premature ovarian failure. The modeling time is short, the model indicators have good stability, high reproducibility, and can be used for exploring the targets of the action mechanism of premature ovarian failure and drug research.

[0045] Although the above embodiments have described the present invention in detail, they are only a part of the embodiments of the present invention, not all embodiments. People can also obtain other embodiments without creative efforts based on these embodiments, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A method for establishing a premature ovarian failure model in rats based on tripterygium glycosides, characterized in that, It includes the following steps: Tripterygium glycosides were continuously intragastrically administered to rats for 4 days, and the intragastric administration dose was 80 mg / kg / day according to the body weight of the rats.

2. The method according to claim 1, characterized in that, The rats were healthy female SD rats at 8 weeks of age with a normal estrous cycle.

3. The method according to claim 1, characterized in that, Before intragastric administration, the rats were first raised for 7 days, and the raising conditions included: 12-hour day-night alternation, an environmental temperature of 21-25 °C, and an environmental humidity of 30-40%.

4. The method according to claim 1, characterized in that, The tripterygium glycosides were mixed with dimethyl sulfoxide and sodium carboxymethylcellulose solution, and the resulting mixture was intragastrically administered to rats.

5. The method according to claim 4, characterized in that, The mass ratio of the tripterygium glycosides to the volume of dimethyl sulfoxide and the volume of sodium carboxymethylcellulose solution was 640 mg: 1.6 mL: 78.4 mL.

6. The method according to claim 4 or 5, characterized in that, The mass percentage content of the sodium carboxymethylcellulose solution was 0.5%.

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