A method for establishing a premature ovarian failure rat model

Abstract

The application relates to a method for establishing an early ovarian failure rat model, which comprises the following steps: taking healthy rats, and performing gavage on the rats according to a dosage of 10-20 mg / Kg of a phytostilbene preparation, wherein the dosage is the ratio of phytostilbene to the weight of the rats, and obtaining the early ovarian failure rat model after four days of administration. The early ovarian failure rat model is obtained by performing gavage on the rats after the phytostilbene preparation, the model is simple to establish, and the model establishment time is short. Through ovarian pathological observation, it can be seen that the simulated animal model has obvious ovary tissue damage, follicular granulosa cell destruction and apoptosis, and follicle atresia and other symptoms. The obtained model has good stability and repeatability. The phytostilbene is used to successfully establish the early ovarian failure rat model, lays a good foundation for establishing a stable early ovarian failure model for basic research, and provides a reliable method for the construction of the early ovarian failure model.

Description

Technical Field

[0001] This invention relates to the field of animal model technology, and specifically to a method for establishing a rat model of premature ovarian failure. Background Technology

[0002] Premature ovarian failure (POF) in clinical practice refers to the premature decline in ovarian function, commonly manifesting as amenorrhea in women before the age of 40. It is characterized by amenorrhea, infertility, estrogen deficiency, and elevated gonadotropin levels. POF not only affects the quantity and quality of oocytes but also reduces pregnancy and live birth rates, increases miscarriage rates, and significantly impacts women's quality of life. The etiology of POF is diverse, and its specific pathogenesis remains unclear. To identify the causes and effective treatments, it is essential to establish animal models that closely resemble human clinical manifestations and pathogenesis. The etiology of POF is closely related to genetics, immune diseases, drugs, surgery, and psychological factors. Therefore, in the development of related drugs and the application of research into the mechanisms of premature ovarian failure, models of premature ovarian failure caused by similar factors should be used as much as possible, based on actual purposes and requirements. Examples include autoimmune POF models constructed based on immune factors, stress-induced POF models constructed based on psychological factors, and galactose-induced POF models constructed based on genetic factors. Premature ovarian failure (POF) can also be drug-induced, and drug-induced POF models are currently the classic animal models for studying POF. However, existing methods for drug-induced POF modeling suffer from problems such as long treatment cycles, significant adverse reactions, high animal mortality, high costs, and low success rates.

[0003] Podophyllotoxin (PPT) belongs to the aryltetrahydronaphthalene lignan class, and is mostly derived from plants in the genera *Polygonum*, *Diphylleia*, and *Pholiota*. Currently, it is mainly used clinically for its antiviral effects in treating diseases such as condyloma acuminata and herpes, and can also be used for antibacterial, antiviral, and antioxidant purposes. In addition, podophyllotoxin also has cell division inhibitory effects, and its derivatives VP-16 and VM-26 are representative antitumor drugs. This application found that it has ovarian toxicity, leading to ovarian damage and inducing premature ovarian failure (POF), and based on this, proposes a method for establishing a rat model of premature ovarian failure. Summary of the Invention

[0004] To address the problems of long cycles, significant adverse reactions, high animal mortality, high costs, and low success rates in existing drug-induced premature ovarian failure (POF) modeling methods, this invention proposes a method for establishing a rat model of POF based on podophyllotoxin. The specific technical solution is as follows:

[0005] A method for establishing a rat model of premature ovarian failure includes the following steps: taking healthy rats, administering podophyllotoxin preparations by gavage according to a daily dose of 10-20 mg / kg of podophyllotoxin to rat body weight, and repeating the administration for four days to obtain a rat model of premature ovarian failure.

[0006] Furthermore, the healthy rats were female SD rats aged 6-8 weeks in their estrous cycle.

[0007] Furthermore, healthy rats were acclimatized for one week before drug administration, with a 12-hour day-night cycle, an ambient temperature of 21-25℃, and a humidity of 30-40%.

[0008] Furthermore, in the formulation process, podophyllotoxin is dissolved in a 2% DMSO solvent to prepare a podophyllotoxin reagent, which is then applied.

[0009] Furthermore, the preparation of the podophyllotoxin reagent includes the following steps:

[0010] S1. Calculate the amount of podophyllotoxin reagent based on the ratio of rat body weight to daily podophyllotoxin reagent dose of 200g: 2ml. Calculate the total amount of podophyllotoxin used over four days and the total amount of podophyllotoxin reagent based on the daily podophyllotoxin dosage and daily podophyllotoxin reagent dose.

[0011] S2. Weigh the total amount of solid powdered podophyllotoxin used over four days using an analytical balance.

[0012] S3. Use a pipette to transfer DMSO into an EP tube. The DMSO is 2% of the total podophyllotoxin reagent dose. Dissolve the weighed podophyllotoxin in the EP tube containing DMSO, mix well, shake, and centrifuge.

[0013] S4. Transfer the mixed podophyllotoxin-DMSO mixture to a centrifuge tube, bring the volume up to the total podophyllotoxin reagent dose with 0.5% CMC-Na solvent, and then shake to mix.

[0014] The beneficial effects of this invention are as follows:

[0015] The model is simple to establish. After administering podophyllotoxin by gavage for 4 days, pathological observation of the ovaries showed that the simulated animal model had obvious damage to the ovarian tissue, destruction of granulosa cells in follicles, and symptoms such as follicular atresia. The model has good stability and strong reproducibility. The rat premature ovarian failure model was successfully established using podophyllotoxin, which lays a good foundation for establishing a stable premature ovarian failure model in basic research and provides a reliable method for constructing premature ovarian failure models. Attached Figure Description

[0016] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0017] Figure 1 This is a schematic diagram illustrating the changes in body weight of rats in each group in the examples;

[0018] Figure 2 This is a schematic diagram illustrating the dietary changes of rats in each group in the examples;

[0019] Figure 3 This is a schematic diagram illustrating the changes in water intake among the rats in each group in the example;

[0020] Figure 4 This is a schematic diagram of the ovarian organ index of each group of rats in the examples;

[0021] Figure 5 This is a schematic diagram of the pathological damage to the ovaries of rats in each group in the examples;

[0022] Figure 6 This is a schematic diagram of the pathological damage scores of rats in each group in the examples. Detailed Implementation

[0023] The present invention will be further described below with reference to embodiments. However, the scope of the present invention is not limited to the following embodiments. Those skilled in the art will understand that various changes and modifications can be made to the present invention without departing from the spirit and scope thereof.

[0024] Unless otherwise specified, the instruments, reagents, and materials used in the following embodiments are all conventional instruments, reagents, and materials already available in the prior art and can be obtained through legitimate commercial channels. Unless otherwise specified, the experimental methods and detection methods used in the following embodiments are all conventional experimental methods and detection methods already available in the prior art.

[0025] It should be noted that podophyllotoxin belongs to the aryltetrahydronaphthalene lignan class, and is mostly derived from plants of the genera *Podophyllum*, *Symplocos*, and *Pholiota*. Currently, it is mainly used clinically for its antiviral effects in treating diseases such as condyloma acuminata and herpes, and can also be used for antibacterial, antiviral, and antioxidant purposes. Furthermore, podophyllotoxin also has cell division inhibitory effects, and its derivatives VP-16 and VM-26 are representative antitumor drugs. Previous research by the inventors of this application found that it has ovarian toxicity, leading to ovarian damage and inducing POF (Polyovarian Failure). Specifically, this invention provides the following embodiments:

[0026] This invention provides a method for establishing a rat model of premature ovarian failure, comprising the following steps:

[0027] Step 1, Selection Stage: Select female SD mice aged 6-8 weeks with normal estrous cycles and acclimatize them for one week with a 12-hour day-night cycle, an ambient temperature of 21-25℃, and a humidity of 30-40%.

[0028] Step Two, Cultivation Stage: Following a daily podophyllotoxin to rat body weight ratio of 10-20 mg / kg, rats were administered the podophyllotoxin preparation via gavage for four consecutive days to obtain a rat model of premature ovarian failure. Specifically, the podophyllotoxin reagent was prepared by dissolving podophyllotoxin in 2% DMSO before administration. The preparation of the podophyllotoxin reagent includes the following steps:

[0029] S1. Calculate the amount of podophyllotoxin reagent based on the ratio of rat body weight to daily podophyllotoxin reagent dose of 200g: 2ml. Calculate the total amount of podophyllotoxin used over four days and the total amount of podophyllotoxin reagent based on the daily podophyllotoxin dosage and daily podophyllotoxin reagent dose.

[0030] S2. Weigh the total amount of solid powdered podophyllotoxin used over four days using an analytical balance.

[0031] S3. Use a pipette to transfer DMSO into an EP tube. The DMSO is 2% of the total podophyllotoxin reagent dose. Dissolve the weighed podophyllotoxin in the EP tube containing DMSO, mix well, shake, and centrifuge.

[0032] S4. Transfer the mixed podophyllotoxin-DMSO mixture to a centrifuge tube, bring the volume up to the total podophyllotoxin reagent dose with 0.5% CMC-Na solvent, and then shake to mix.

[0033] This embodiment uses a control experiment, as detailed below:

[0034] I. Grouping: Sixty SPF-grade male SD rats, weighing 190-230g, were selected and housed under alternating light and dark conditions for 12 hours, at an ambient temperature of 21-25℃ and an ambient humidity of 30-40%. Food and water were not restricted, and the rats were acclimatized for one day. The rats were then randomly divided into four groups (n=15 per group): blank control group (CON), low-dose PPT group (PPT-10mg / kg / day), high-dose PPT group (PPT-20mg / kg / day), and positive control group for cyclophosphamide (CTX-70mg / kg / day).

[0035] II. Drug Preparation: Taking the preparation of the low-dose PPT group as an example, the default weight of the SD rats is 200g, meaning each rat needs 2mg / day. 15 rats need to be administered the drug for 4 days, requiring a total of 2mg * 15 * 4 = 120mg of podophyllotoxin. The dosage per rat is based on a weight:dose ratio of 200g:2ml, meaning a total of 2ml * 15 * 4 = 120ml of solution needs to be prepared. The specific preparation process is as follows:

[0036] (1) 120 mg of solid powdered podophyllotoxin was accurately weighed using an analytical balance;

[0037] (2) Use a pipette to precisely transfer the required 120ml*2%=2.4ml of DMSO into a 5ml EP tube;

[0038] (3) Dissolve the weighed podophyllotoxin in DMSO, mix well, shake, and centrifuge;

[0039] (4) Transfer the above reagents to a clean reagent bottle, and make up to the required volume with 120ml-2.4ml=117.6ml of 0.5% sodium carboxymethyl cellulose, and shake to mix.

[0040] III. Administration: Administer the medication daily at 10:00 AM. Weigh the rats and administer the medication by gavage at a ratio of body weight:dose = 200g:2ml (the specific dosage needs to be determined based on body weight). Repeat the above procedure for 4 days, and collect samples on the 5th day.

[0041] IV. Detection Indicators and Methods: After drug administration, general condition observation, ovarian organ index calculation, and pathological observation were performed on each group. General condition observation included gastrointestinal reactions such as diarrhea and its severity, body changes such as gait changes, activity frequency, and tremors, as well as general phenotypic observations such as changes in appearance, changes in hair luster, and bleeding. The body weight, diet, and water intake of each group of rats were assessed and recorded every 24 hours. The ovarian organ index (ovarian organ index = ovarian weight (g) / body weight (g)) was calculated for each rat. Pathological observation was performed using hematoxylin-eosin staining. After treatment, the rats were harvested. Ovarian tissue was fixed in 4% paraformaldehyde solution at room temperature for 48 hours, then dehydrated, embedded, sectioned, stained, and mounted. The sections were examined under a microscope at different magnifications to observe basic pathological changes such as congestion, ecchymosis, hemorrhage, edema, degeneration, necrosis, hyperplasia, fibrosis, organization, granulation tissue, and inflammatory changes. As shown in Table 1, a four-level grading system was used to quantify the pathological changes in rats, with scores of 0 (normal), 1 (very mild), 2 (mild), 3 (moderate), and 4 (severe). Statistical methods: Data are expressed as mean ± standard deviation. Graph Pad Prism 8 software was used for statistical processing. If the population followed a normal distribution, one-way ANOVA was used for homogeneous variances. LSD test was used for pairwise comparisons between groups, and rank-sum test was used for heterogeneous variances. P < 0.05 was considered statistically significant, and P < 0.01 was considered statistically significant.

[0042] Table 1. Level 4 Grading System

[0043]

[0044] V. Experimental Results: (e.g.) Figure 1 As shown, there was no significant change in weight in the CON group, while both the PPT-10 and PPT-20 groups experienced varying degrees of weight loss. The weight in the PPT intervention group varied with the intervention dose, and the CTX group also showed a weight decrease. Figure 2-3 As shown, the food and water intake of the PPT intervention group was significantly lower than that of the other two groups; observation revealed that 72 hours after drug administration, rats in the PPT intervention group showed varying degrees of surface ecchymosis and petechiae, including on the face, mouth, nose, and forepaws, and their fur turned dark red; Figure 4 As shown, comparing organ indices reveals a statistically significant increase in organ indices in the PPT-20 intervention group; for example... Figure 5As shown, rat ovarian tissue was stained with hematoxylin and eosin (HE). No obvious necrosis or inflammatory cell infiltration was observed in the CON control group; punctate necrosis of granulosa cells (black arrows) was observed in the PPT intervention group, with condensed and deeply stained or fragmented nuclei, indicating significant tissue damage; punctate necrosis of follicular granulosa cells (black arrows) was occasionally observed in the positive control drug CTX group, with condensed and deeply stained or fragmented nuclei, indicating mild pathological damage. Pathological damage scores showed that ovarian tissue damage significantly worsened with increasing podophyllotoxin dosage.

[0045] Following PPT treatment, significant damage was observed in ovarian tissue, including destruction of granulosa cells in follicles and follicular atresia. The mechanism of POF (Potentially Ovulated Failure) is generally believed to include insufficient primordial follicle reserve, accelerated follicular atresia, altered recruitment of dominant follicles, and impaired follicular maturation. PPT toxicity affects granulosa cells in follicles, impacting follicular development and maturation. Damage to ovarian follicles leads to irreversible damage and a reduction in ovarian reserve, resulting in POF.

[0046] The modeling method of this invention involves treatment with podophyllotoxin via gavage. The experimental group exhibits typical characteristics similar to human premature ovarian failure, making it an effective method for establishing premature ovarian failure. The modeling time is short, the model indicators are stable, and the reproducibility is strong. It can be used for target and drug research to explore the mechanism of action of premature ovarian failure.

[0047] The foregoing has shown and described the basic principles, main features, and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The embodiments and descriptions in the specification are merely illustrative of the principles of the invention. Various changes and modifications can be made to the invention without departing from its spirit and scope, and all such changes and modifications fall within the scope of the claimed invention.

Claims

1. A method for establishing a rat model of premature ovarian failure, characterized in that: The procedure includes the following steps: female SD rats aged 6-8 weeks in estrous cycles are selected as healthy rats. The rats are administered podophyllotoxin at a daily dose of 10-20 mg / kg of body weight. The podophyllotoxin preparation is administered by gavage and repeated for four days. Ovarian tissue shows significant damage, granulosa cells of follicles are destroyed and follicular atresia occurs, thus obtaining a rat model of premature ovarian failure.

2. The method for establishing a rat model of premature ovarian failure according to claim 1, characterized in that: Healthy rats were acclimatized for one week before drug administration, with a 12-hour day-night cycle, an ambient temperature of 21-25℃, and a humidity of 30-40%.

3. The method for establishing a rat model of premature ovarian failure according to claim 1, characterized in that: In formulation, podophyllotoxin is dissolved in 2% DMSO to prepare a podophyllotoxin reagent, which is then applied.

4. The method for establishing a rat model of premature ovarian failure according to claim 3, characterized in that: The preparation of podophyllotoxin reagent includes the following steps: S1. Calculate the amount of podophyllotoxin reagent based on the ratio of rat body weight to daily podophyllotoxin reagent dose of 200g:2ml. Calculate the total amount of podophyllotoxin used over four days and the total amount of podophyllotoxin reagent based on the daily dose of podophyllotoxin and the daily dose of podophyllotoxin reagent. S2. Weigh the total amount of solid powdered podophyllotoxin used over four days using an analytical balance. S3. Use a pipette to transfer DMSO into an EP tube. The DMSO is 2% of the total podophyllotoxin reagent dose. Dissolve the weighed podophyllotoxin in the EP tube containing DMSO, mix well, shake, and centrifuge. S4. Transfer the mixed podophyllotoxin-DMSO mixture to a centrifuge tube, bring the volume up to the total podophyllotoxin reagent dose with 0.5% CMC-Na solvent, and then shake to mix.

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