A method for modeling, evaluating and application of an animal model of menstrual dysmenorrhea

Abstract

The application relates to the technical field of animal models, in particular to a modeling method and an evaluation method of an animal menstrual dysmenorrhea model and application, wherein the animal with removed ovaries is injected with estrogen, progesterone and oxytocin, the pharmacological injection of progesterone can reduce the harm and mortality rate of the animal caused by the implantation and removal of a progesterone release body in the prior art, the surgical stress of the animal is reduced, the injection time of the progesterone in the application is 7-9 days, and the animal menstrual dysmenorrhea model is obtained after the progesterone is withdrawn for 16 hours; after evaluation by the evaluation method, compared with the non-menstrual process of the dysmenorrhea model in the prior art, the animal menstrual dysmenorrhea model can better simulate the pathophysiological process of human dysmenorrhea, promote the development of clinical treatment, and effectively help patients to relieve pain.

Description

Technical Field

[0001] This invention relates to the field of animal model technology, and in particular to a method for establishing, evaluating, and applying an animal menstrual-like dysmenorrhea model. Background Technology

[0002] Primary dysmenorrhea (PD) is a type of menstrual pain without any organic pelvic lesions, typically characterized by spasmodic suprapubic pain. Menstrual pain is a common symptom affecting the quality of life of 50%-90% of young women, impacting interpersonal relationships, studies, and work. Despite this, the prevalence of PD is underestimated because women with the condition generally do not seek medical attention. The pathogenesis of PD is not fully understood, but current views suggest that its development is primarily related to prostaglandins (PGs), leukotrienes, vasopressin, emotions, and the individual's pain threshold. Due to its high incidence and long-term, recurrent nature, PD severely damages patients' physical and mental health and quality of life, becoming a pressing challenge for the medical community.

[0003] In basic experiments, animal experiments are the most commonly used method. Compared to humans, and provided welfare is ensured, laboratory animals are easier to use for experiments in molecular biology and other fields. In current models of primary dysmenorrhea, rats and mice are the most frequently used experimental animals. Rats are rodents, and under natural conditions, the uterine lining of rats does not undergo decidualization, meaning rats do not menstruate naturally. Currently accepted rat models of primary dysmenorrhea all use estrogen to sensitize the rat's uterus, followed by oxytocin to induce pain. Because rats do not menstruate, current models of primary dysmenorrhea are also called dysmenorrhea-like models. However, dysmenorrhea-like models lack menstrual-like processes and cannot accurately simulate the pathophysiological process of human dysmenorrhea.

[0004] For example, CN104524545B discloses a method for establishing an animal model of primary dysmenorrhea caused by cold-induced blood stasis. This method involves inducing cold-induced blood stasis in rats using an ice bath combined with adrenaline injection, followed by the preparation of a primary dysmenorrhea rat model using estrogen and oxytocin. The model is then obtained by measuring relevant biochemical indicators such as β-endorphin, endothelin, and progesterone, and by observing changes in endogenous small molecule metabolites in rat plasma and urine before and after modeling using metabolomics methods.

[0005] Alternatively, CN114848664B discloses a pharmaceutical composition and its use, disclosing FDP and NMN / NAD. + The mechanism of action of combined drug administration in treating a mouse model of primary dysmenorrhea was investigated. A primary dysmenorrhea model was established using continuous subcutaneous injections of estradiol benzoate and intraperitoneal injections of oxytocin. The aim was to demonstrate the effect of FDP and its interaction with NMN / NAD.+ The combination therapy can be used to treat primary dysmenorrhea, reduce gene expression of prostate genes in uterine tissue, and increase the conception rate of female mice.

[0006] In the aforementioned existing technologies, research on dysmenorrhea models is limited to injecting experimental animals with existing estradiol and oxytocin, waiting for the number of writhing responses or recording the writhing response time to establish a model, and then conducting treatment and drug research. The success rate of treatment or drug research depends on whether the constructed dysmenorrhea model is accurate enough.

[0007] Under natural conditions, the endometrium of rodents such as rats does not undergo decidualization, meaning rats do not experience spontaneous menstruation. Therefore, rodents require artificial induction to exhibit a menstrual-like process similar to that in primates; this model is called a menstrual-like model. Current techniques for simulating physiological progesterone withdrawal include castration (removal of both ovaries) → injection of estrogen and peanut oil → scraping the endometrium → implantation of progesterone-releasing bodies → removal of the progesterone-releasing bodies. While this is closer to the human menstrual mechanism, the implantation and removal of progesterone-releasing bodies makes the model establishment process overly complex, resulting in low success rates and a certain mortality rate. Pharmacological injection of progesterone might alleviate the surgical stress on animals; therefore, currently only researchers have induced decidualization in rats, and there is no clear data indicating the successful establishment of a menstrual-like model in rats.

[0008] Based on the above, existing technologies suffer from several technical problems that urgently need to be addressed, such as the lack of accuracy in dysmenorrhea-like models, the absence of a menstrual-like process, the inability to effectively simulate the pathophysiological process of human dysmenorrhea, and the lack of a proven successful establishment of a menstrual-like model. These problems result in low success rates of drug treatment for dysmenorrhea or delayed treatment due to untimely medication. Summary of the Invention

[0009] To solve the above-mentioned technical problems, the present invention provides a method for establishing an animal menstrual-like dysmenorrhea model by pharmacological injection of progesterone. The method involves injecting estrogen, progesterone, and oxytocin into ovariectomized animals. The progesterone is administered for 7-9 days, and the animal menstrual-like dysmenorrhea model is obtained 16 hours after the progesterone is withdrawn.

[0010] Furthermore, the animals selected for the animal menstrual-like dysmenorrhea model are rats.

[0011] Furthermore, the method for establishing the animal menstrual-like dysmenorrhea model includes the following steps:

[0012] Step 1: Select healthy, adult, specific pathogen-free (SPF grade) unmated female Sprague-Dawleg (SD) rats as the model animals;

[0013] Step 2: Select rats in the late estrus and estrus phases using vaginal smear method to prepare for modeling;

[0014] Step 3: Perform ovariectomy on rats to eliminate the influence of endogenous estrogen in rats, thereby simulating the pathophysiological state of primary dysmenorrhea by regulating exogenous hormones;

[0015] Step 4: Supplement rats with exogenous estrogen to simulate changes in estrogen levels in the body, in order to study the role of estrogen in physiological and pathological processes;

[0016] Step 5: After continuous exogenous injection of progestin, the injection is stopped to simulate the progestin withdrawal process in late menstruation. The progestin combined with estrogen induces decidualization of the rat endometrium.

[0017] Step 6: By injecting oxytocin exogenously, the dysmenorrhea process is simulated to obtain a rat model of menstrual-like dysmenorrhea.

[0018] Furthermore, the rats mentioned in step 1 are 2-3 months old, female, non-pregnant, healthy, mature, and unmated.

[0019] Furthermore, the rats described in step 1 should have uniform and glossy fur, no hair loss, no redness or ulceration of the skin, and a symmetrical body shape; they should be active, have normal nerve responses, and no abnormal behavior; their breathing should be stable, and their excretion should be normal.

[0020] Furthermore, in step 3, before performing the ovariectomy, rats were anesthetized by intraperitoneal injection with 2% pentobarbital solution, and the anesthesia injection volume for each rat was calculated at 0.3 ml / 100 g.

[0021] Furthermore, the estrogen mentioned in step 4 is one of estradiol benzoate, estradiol valerate, and diethylstilbestrol.

[0022] Furthermore, the estrogen mentioned in step 4 is preferably estradiol benzoate.

[0023] Furthermore, the estrogen injected in step 4 needs to be prepared as an estrogen injection solution with a concentration of 10 μg / mL.

[0024] Furthermore, the progesterone injections in step 5 are continued for 7-9 days.

[0025] Furthermore, the preferred duration of continuous progesterone injection in step 5 is 8 days.

[0026] Furthermore, the progestin mentioned in step 5 is progesterone.

[0027] Furthermore, the progestin mentioned in step 5 needs to be prepared as a progestin injection solution with a concentration of 2 mg / mL.

[0028] Furthermore, the oxytocin mentioned in step 6 needs to be prepared as an oxytocin injection solution with a concentration of 5 mg / mL.

[0029] The present invention also provides an evaluation method for the above-mentioned animal menstrual-like dysmenorrhea model. The evaluation method includes observing the writhing response, vaginal decidua condition and gross uterine tissue of the animal in the menstrual-like dysmenorrhea model, and obtaining blood and uterine tissue samples from the animal model by collecting blood from the abdominal aorta and removing uterine tissue, so as to perform morphological observation of uterine tissue, ELISA (enzyme-linked immunosorbent assay) detection on serum, and qPCR (quantitative real-time polymerase chain reaction) detection on rat uterine tissue.

[0030] Furthermore, the animals selected for the animal menstrual-like dysmenorrhea model are rats.

[0031] Furthermore, the evaluation method includes the following steps:

[0032] Step 1: Observe the writhing response of rats within 30 minutes after the first injection of oxytocin;

[0033] Step 2: Observe the condition of the vaginal decidua and the gross appearance of the uterine tissue in rats at different times of progesterone withdrawal; the progesterone withdrawal time is calculated from the time after the first injection of oxytocin.

[0034] Step 3: Blood and uterine tissue samples were obtained from the rats by collecting blood from the abdominal aorta and removing uterine tissue.

[0035] Step 4: Perform morphological observation on rat uterine tissue, i.e., perform HE staining (hematoxylin-eosin staining) on ​​uterine tissue sections;

[0036] Step 5: Perform ELISA (Enzyme-Linked Immunosorbent Assay) on rat serum;

[0037] Step 6: Perform qPCR (real-time quantitative polymerase chain reaction) detection on rat uterine tissue.

[0038] Furthermore, the degree of rat writhing response described in step 1 first increases and then decreases as the progesterone withdrawal time increases.

[0039] Furthermore, in step 2, the condition of the vaginal decidua and the gross appearance of the uterine tissue in rats were observed at 0.5h, 12h, 16h, 20h, and 24h after progesterone withdrawal.

[0040] Furthermore, in step 2, the decidualized uterus was significantly congested and swollen compared to the normal uterus, and the frequency of decidualization also increased with the increase of progesterone withdrawal time. The animal menstrual-like dysmenorrhea model was obtained 16 hours after progesterone withdrawal.

[0041] Furthermore, the morphological observations described in step 4 include endometrial degeneration and necrosis, lamina propria edema, increased lamina propria glands, inflammatory cell infiltration of the lamina propria, and myometrial inflammation.

[0042] Furthermore, step 5 includes the detection of PGF2α (prostaglandin F2α) and PGE2 (prostaglandin E2).

[0043] Furthermore, in step 5, the serum PGF2α level was highest 12 hours after progesterone withdrawal; the serum PGE2 level was lowest 16 hours after progesterone withdrawal; and the PGF2α / PGE2 ratio was highest 16 hours after progesterone withdrawal.

[0044] Furthermore, step 6 includes total RNA extraction, reverse transcription, and quantitative PCR experiments to statistically analyze the expression level of NLRP3 in rat uterine tissue.

[0045] The present invention also provides a drug for treating dysmenorrhea, which is prepared based on an animal menstrual-like dysmenorrhea model obtained by the above-described modeling method.

[0046] The beneficial effects of this invention are as follows:

[0047] 1. This invention provides a method for establishing an animal menstrual-like dysmenorrhea model by pharmacologically injecting progesterone. Ovaries-removed animals are injected with estrogen, progesterone, and oxytocin. Pharmacological progesterone injection reduces the harm and mortality caused by implantation and removal of progesterone-releasing bodies in existing technologies, alleviates surgical stress, and, in this invention, the progesterone is administered for 7-9 days. The animal menstrual-like dysmenorrhea model is obtained 16 hours after progesterone withdrawal. After evaluation, compared to existing dysmenorrhea-like models that lack a menstrual-like process, this invention's animal menstrual-like dysmenorrhea model better simulates the pathophysiological process of human dysmenorrhea, promoting the development of clinical treatment and effectively helping patients alleviate pain.

[0048] 2. This invention evaluates the obtained animal menstrual-like dysmenorrhea model from multiple perspectives, including observing the writhing response, vaginal decidua condition, and gross uterine tissue of the animals in the animal menstrual-like dysmenorrhea model, as well as obtaining blood and uterine tissue samples from the animal model by collecting blood from the abdominal aorta and removing uterine tissue, for morphological observation of uterine tissue, detection of serum by ELISA (Enzyme-Linked Immunosorbent Assay), and detection of rat uterine tissue by qPCR (Quantitative Real-Time Polymerase Chain Reaction);

[0049] 3. The writhing response can directly reflect the degree of pain in rats, verify the success of the model, and can be used for further research on pain mechanism, pathophysiology, and pharmacology. The writhing response of rats in group C (16 hours after progesterone withdrawal) was the strongest, indicating that the dysmenorrhea model was successfully established and the experimental data are reliable.

[0050] 4. The SD rat menstrual-like dysmenorrhea animal model was evaluated and verified by observing the presence of decidualization in the vagina and the pathological morphology of the uterine tissue. The comparison showed that the condition of the vaginal decidua in rats increased with the time of progesterone withdrawal. There was no significant change in group A (0.5h withdrawal), slight redness in group B (12h), redness and swelling in groups C (16h) and D (20h), and obvious swelling of decidua in group E (24h). Gross observation of the uterus showed that the decidualized uterus was significantly congested and swollen compared with the normal uterus. The frequency of decidualization also increased with the time of progesterone withdrawal. There was no significant difference among groups C, D, and E. The results indicate that the preliminary experiment of the menstrual-like dysmenorrhea model was successful based on morphological observation, and the experimental data are reliable.

[0051] 5. Blood and uterine tissue samples were obtained from the animal model through two basic procedures: abdominal aortic blood collection and uterine tissue extraction. Auxiliary techniques included serum separation, liquid nitrogen freezing, and fixation with 4% paraformaldehyde. The rat uterine tissue was then subjected to paraffin embedding, sectioning, and staining. Pathological changes under a light microscope were used to score the tissue pathological damage, directly reflecting the degree of inflammation. Morphological observation of the sections revealed extensive vacuolation and necrosis of the rat endometrial epithelial cells, congestion of multispiral arterioles, neutrophil infiltration, and increased glandular tissue in the lamina propria, indicating a basic success in model establishment. Data analysis showed that the pathological change score generally increased with the duration of progesterone withdrawal, suggesting that a longer progesterone withdrawal time resulted in better model establishment.

[0052] 6. By performing ELISA on rat serum, the pathological changes of uterine inflammation in rats were assessed at the molecular level. Specifically, rat serum ELISA (PGF2α) and rat serum ELISA (PGE2) were measured. With the progesterone withdrawal time, the serum inflammatory response increased. The highest serum PGF2α content was observed in group B (12 h after progesterone withdrawal), and the lowest serum PGE2 content was observed in group C (16 h after progesterone withdrawal). The highest PGF2α / PGE2 ratio was observed, indicating a strong inflammatory response. This shows that the changes in PGF2α and PGE2 content directly reflect the occurrence of inflammatory response in rats, further proving the success of the model establishment.

[0053] 7. By performing qPCR detection on rat uterine tissue, the expression of key genes for primary dysmenorrhea in rats was assessed at the molecular level. Compared with group C (progesterone withdrawal 16h), the relative expression level of the target gene in group D (progesterone withdrawal 20h) was significantly lower (P<0.01), and the relative expression level of the target gene in group E (progesterone withdrawal 24h) was significantly lower (P<0.05). Since the target gene is a gene related to the occurrence of primary dysmenorrhea, it indicates that the modeling experiment was successful, the rats have the expression of key genes for primary dysmenorrhea, and the intervention method in group C is more conducive to promoting the modeling of menstrual-like dysmenorrhea in rats.

[0054] 8. This invention creatively explores and establishes an animal menstrual-like dysmenorrhea model that is closer to the clinical pathophysiological process. This model may play an important role in the pathophysiology of primary dysmenorrhea, including endocrine and immune factors, endometrial differentiation and remodeling, uterine vascular pathophysiology, and endometrial apoptosis. It provides more accurate experimental data for clinical primary dysmenorrhea and is of great significance. Attached Figure Description

[0055] Figure 1 Images of the vagina of rats during different estrous cycles according to this invention;

[0056] Figure 2 This is a statistical chart of the duration of the torsional response in this invention;

[0057] Figure 3 This is a statistical chart showing the highest score for the torsional response in this invention.

[0058] Figure 4 This is a diagram showing the rat writhing response of the present invention;

[0059] Figure 5 This is an example diagram of the rat vaginal decidua condition according to the present invention;

[0060] Figure 6 This is a macroscopic comparison image of the isolated rat uterus of the present invention;

[0061] Figure 7 This is a comparative image of the gross uterus of rats in vivo, based on the present invention.

[0062] Figure 8 These are HE-stained sections of rat uterine tissue from different groups according to the present invention.

[0063] Figure 9 The scoring of pathological changes in uterine tissue of rats in different groups according to the present invention;

[0064] Figure 10 HE-stained sections of uterine tissue from group A rats in this invention;

[0065] Figure 11 HE-stained sections of uterine tissue from group B rats in this invention;

[0066] Figure 12 HE-stained sections of uterine tissue from group C rats in this invention;

[0067] Figure 13 HE-stained sections of uterine tissue from group D rats in this invention;

[0068] Figure 14 HE-stained sections of uterine tissue from rats in group E of this invention;

[0069] Figure 15 This invention provides a statistical analysis of serum PGF2α levels in rats from different groups.

[0070] Figure 16 This invention provides a statistical analysis of serum PGF2 levels in rats from different groups.

[0071] Figure 17 This invention provides a statistical analysis of the serum PGF2α / PGF2 ratio in different groups of rats.

[0072] Figure 18 This invention provides a statistical analysis of NLRP3 expression levels in the uterine tissues of rats in each group. Detailed Implementation

[0073] Examples 1-6 illustrate the modeling method for the animal menstrual-like dysmenorrhea model of the present invention, and Examples 7-12 illustrate the model evaluation of the animal menstrual-like dysmenorrhea model obtained in Example 6.

[0074] Example 1

[0075] This embodiment describes step 1 of the modeling method for an animal menstrual-like dysmenorrhea model provided by the present invention, specifically:

[0076] 1. Preparation before the experiment: Experimenters should wear lab coats, hats, masks and gloves, and the experimental area should meet the requirements of SPF (Specific Pathogen Free, SPF) level.

[0077] 2. Selection of rats: 30 healthy adult specific pathogen-free (SPF grade) female Sprague-Dawleg (SD) rats were selected. The rats were 2-3 months old, non-pregnant, healthy, mature and unmated.

[0078] 3. Cage separation and numbering of rats: Randomly place rats into cages, 3 rats per cage, for a total of 10 cages. Gently remove the rats one by one and mark the rats with a marker pen according to cage AE (2 cages per group) and 1-6. Fill in the cage card, put in water and rat food, and put them into the rat rack in order.

[0079] 4. Appearance inspection: Fur: Observe whether the rat's fur color is uniform, whether it is shiny, and whether there is hair loss, bald patches, or parasitic infection; Skin: Check whether the skin is red, swollen, ulcerated, or traumatized; Body shape: Observe whether the rat's body shape is symmetrical and whether there is obvious emaciation or obesity.

[0080] 5. Behavioral observation:

[0081] Activity: Observe the rats' activity in the cage, whether they are active, and whether there is any abnormal quietness or excessive excitement; Responsiveness: Gently tap the cage or touch the rats with tweezers and observe their response to stimuli to determine whether their nervous system is normal; Special behaviors: Record whether the rats self-bite, attack each other, or scratch excessively.

[0082] 6. Physiological examination:

[0083] Respiration: Observe the respiratory rate and breathing pattern of rats to determine whether their respiratory system is normal. Normal rats breathe steadily at a rate of 60-120 breaths / minute. Excretion: Examine the color, texture, and amount of rat feces and urine. Normal feces are granular and dark brown in color. Urine is clear and pale yellow in color.

[0084] 7. Weight Measurement: A plastic bucket was placed on an electronic scale (model ZOGGI Songjing). After pelting, the rats were gently placed in the bucket, and their weights were recorded. It was ensured that the rats were calm during each measurement to avoid inaccurate readings due to struggling. The weight of each rat was recorded in detail in a table, and SPSS 27.0 was used for data analysis. The results are shown in Table 1.

[0085] Table 1. Rat body weight analysis

[0086]

[0087] Before conducting rat experiments, comprehensive observation and recording of the rats is crucial. This not only helps ensure that the rats' health meets the experimental requirements but also provides accurate basic data for subsequent experiments, thereby improving the reliability and reproducibility of the experimental results. In this example, the rats in each group had uniform and glossy fur without hair loss, no skin redness or ulceration, and a symmetrical body shape; they were active, had normal neurological responses, and no abnormal behavior; their breathing was stable, and their excretion was normal. They were ready to proceed to the next stage of the experiment.

[0088] Example 2

[0089] This embodiment describes step 2 of the method for establishing an animal menstrual-like dysmenorrhea model provided by the present invention. Rats with low estradiol levels in the post-estrus and estrus phases are screened using a vaginal smear method. The vaginal smear method, through microscopic observation of cell types and proportions in vaginal secretions, divides the rat's physiological cycle into four phases: proestrus, estrus, metestrus, and diestrus. Specifically:

[0090] 1. Preparation before the experiment: Experiment personnel should wear lab coats, hats, masks and gloves, in accordance with the requirements of SPF-level experimental areas;

[0091] 2. Remove the female SD rat from the cage and place it on the cage lid. Hold the rat's tail with one hand and gently lift it. The rat will grab the cage lid with its front paws, exposing its vaginal opening.

[0092] 3. After wiping the vaginal opening with a sterile cotton swab moistened with physiological saline, insert the prepared cotton swab into the rat's vagina 0.5-1cm, leave it for 1-2 seconds, and after the rat has adapted, gently rotate it 1-2 times to spread the cotton swab evenly on a clean glass slide.

[0093] 4. Preparation of methylene blue staining solution: Add the following to a 500mL wide-mouth bottle in sequence: a. 150mL of 2% methylene blue solution; b. 3g of methylene blue dissolved in 150mL of distilled water; c. 75mL of 95% ethanol; d. Weigh 0.025g of potassium hydroxide using an electronic balance; e. 25mL of distilled water;

[0094] 5. After sampling, dry the sampling slide in the air or with an alcohol lamp. Use a Pasteur pipette to draw an appropriate amount of methylene blue staining solution and drop it into the center of the slide. The staining solution should completely immerse the vaginal smear secretions.

[0095] 6. After staining with methylene blue for 10 minutes, use a pipette to rinse the edges of the slide with water to gently remove the methylene blue. When rinsing, start from the edge and do not use too strong a flow of water to prevent the cells on the smear from being completely washed away (the slide should turn white as a reference).

[0096] 7. Observe the cell morphology, types and numbers of vaginal smears under a microscope to screen out female rats in the late estrus and interestrus stages.

[0097] In this embodiment, images of the rat's vagina during each estrous cycle are shown below. Figure 1 As shown in Table 2, the detailed statistics of the estrous cycle of rats are as follows.

[0098] Table 2. Statistics on the estrous cycle of rats

[0099]

[0100] In this embodiment, vaginal secretions were sampled and stained from 30 rats for identification. The results showed that 23 rats were in the post-estrus and comorbid estrus phases, meeting the modeling criteria; 7 rats were in the proestrus and estrus phases. During this stage, the rats' sex hormone levels were unstable. A second sampling and observation was conducted on the second day before modeling to confirm whether the rats were in the post-estrus or comorbid estrus phase, minimizing hormonal interference. On the second day, vaginal secretions from the previous day's 7 rats were sampled and stained again, and the results showed that all 7 rats were in the post-estrus and comorbid estrus phases. Therefore, the estrous cycles of all 30 rats met the experimental requirements.

[0101] Example 3

[0102] This embodiment describes step 3 of the method for establishing an animal menstrual-like dysmenorrhea model provided by the present invention. Ovariectomy (OVX) in rats is an important experimental technique widely used in endocrinology, reproductive physiology, metabolism, cardiovascular medicine, neuroscience, immunology, and oncology. Surgical removal of the ovary reduces the influence of endogenous estrogen in rats during the experiment, thus more closely resembling the pathophysiological process of primary dysmenorrhea and ensuring the accuracy of experimental data. Specifically:

[0103] 1. Preparation of 2% sodium pentobarbital solution: Dissolve 1g of pure sodium pentobarbital in 50mL of sterile physiological saline to prepare a 2% sodium pentobarbital solution.

[0104] 2. Weight measurement: Place a plastic bucket on the electronic scale, tare the rat, and gently place it into the bucket. Record its weight. Ensure that the rat is quiet during each measurement to avoid inaccurate measurements due to struggling. Accurately record the rat's weight and calculate the anesthesia injection volume for each rat at 0.3ml / 100g.

[0105] 3. Anesthesia and fixation: The rat was anesthetized by intraperitoneal injection of 2% pentobarbital solution, and then fixed in a supine position on the operating table.

[0106] 4. Abdominal preparation: After anesthesia, wipe the abdomen of the rat with povidone-iodine to disinfect it by making a circular motion from the center of the incision outwards, and then remove the iodine with 75% ethanol solution. Repeat this process three times.

[0107] 5. Abdominal opening: After skin preparation, use tissue scissors to cut along the linea alba of the rat's abdomen from about 2-3 cm above the pubic symphysis upwards, successively cutting the skin, superficial fascia, deep fascia, rectus abdominis muscle, and peritoneum. The incision is 1.5cm-2cm.

[0108] 6. Ovarian removal: Locate the two pink, hollow organs wrapped in white adipose tissue on both sides of the linea alba, which are the uterus; at the end of the uterus, a red mulberry-like tissue is seen, which is the ovary; ligate the junction of the uterus and ovary with absorbable sutures, and then remove both ovaries with ophthalmic scissors.

[0109] 7. Incision suturing: Gently place each tissue back into the abdomen, then use a needle holder, forceps, and absorbable sutures (with corner needles) to perform two-layer suturing. Use simple continuous suturing to suture the muscle layer and simple interrupted suturing to suture the epidermis. After tying the knot, cut the suture ends short to prevent the rat from biting.

[0110] 8. Disinfection and nursing care: Wipe the incision with povidone-iodine to remove bloodstains, and apply sulfanilamide powder to prevent postoperative infection;

[0111] 9. Postoperative observation: Place the rats that have undergone surgery in a warm and ventilated place and observe their heart rate, respiration and any abnormal reactions at any time. After the rats have fully recovered, gently place them back into the cage.

[0112] By surgically removing the ovaries of rats, the influence of endogenous estrogen in rats can be effectively eliminated. This allows for the regulation of exogenous hormones to simulate the pathophysiological state of primary dysmenorrhea, thus obtaining a more accurate animal model of menstrual-like dysmenorrhea.

[0113] Example 4

[0114] This embodiment is step 4 of the method for establishing an animal menstrual-like dysmenorrhea model provided by the present invention. In this embodiment, the injected estrogen is estradiol benzoate. The main purpose of its preparation and injection experiment is to simulate changes in estrogen levels in the body by supplementing estrogen exogenously, so as to study the role of estrogen in physiological and pathological processes. By injecting estradiol benzoate, the effects of subsequent injections of progesterone and oxytocin are enhanced, the endometrium is sensitized, and decidualization is induced. This is combined with simulating the pathophysiological environment of primary dysmenorrhea, specifically:

[0115] 1. Preparation of estradiol benzoate injection: Measure 20 mL of physiological saline using a graduated cylinder and pour it into a wide-mouth bottle for later use; take one ampoule of estradiol benzoate injection with a concentration of 2 mg / mL, break open the ampoule, use a pipette to take 0.1 mL of the finished drug solution, dissolve it in the prepared 20 mL of physiological saline, and mix it by shaking on a mixer to prepare estradiol benzoate injection with a concentration of 10 μg / mL;

[0116] 2. Preparation before animal experiments: Experimenters should wear lab coats, hats, masks and gloves, in accordance with SPF-grade experimental area requirements;

[0117] 3. Take the medicine: Take an unopened sterile dosing device, gently pull out the needle, and draw 0.2 mL of the prepared injection solution;

[0118] 4. Estradiol Benzoate Injection: Wearing bite-proof gloves, gently hold the rat and place it on the operating table. Use the pad of your left thumb and the side of your index finger to gently lift the skin behind the rat's neck, while using the other fingers to hold the rat back and down. Disinfect the injection site with 75% alcohol. Hold the syringe in your right hand and insert the needle (bevel facing up) into the central depression of the skin at a 45-degree angle. Gently aspirate; if no blood return is observed, slowly inject the drug solution, ensuring even distribution. After injection, rotate the needle 180 degrees and gently withdraw it. Press the puncture site with a cotton swab to prevent leakage. In this embodiment, all rats were injected with estrogen at fixed times on days 1 and 5 to simulate changes in estrogen levels in the body, for a total of 2 days.

[0119] 5. Observe the rats for any abnormal reactions and ensure they recover to normal.

[0120] In this embodiment, all rats underwent drug administration. A small number of cases involved drug spillage or rats struggling, leading to multiple injections. However, the overall experimental results showed that all rats exhibited no abnormal reactions within half an hour after injection. This indicates that the injection procedure has minimal impact on the rats' physiological state, the experimental results are reliable, and further experiments can proceed.

[0121] Example 5

[0122] This embodiment describes step 5 of the method for establishing an animal menstrual-like dysmenorrhea model provided by the present invention. Under physiological conditions, for most non-menstrual species, decidualization only occurs at the embryo implantation site during normal pregnancy. However, for menstrual species, decidualization does not depend on the presence of a gestational body to begin; instead, it occurs spontaneously during the late luteal phase of the menstrual cycle under the regulation of progesterone. This phenomenon, where stromal cells in the uterus spontaneously produce decidual-like changes without external stimulation, is called spontaneous decidualization. Spontaneous decidualization is a terminal differentiation that is irreversible. This differentiation state requires progesterone to maintain. When the corpus luteum function degenerates in the late menstrual phase and progesterone is withdrawn, the decidualized area of ​​the endometrium will eventually disintegrate, forming menstruation. In this embodiment, the selected SD rats are rodents and do not have a spontaneous menstrual process. This experiment aims to simulate the progesterone withdrawal process by continuously administering exogenous progesterone for 8 days and then stopping the medication, combined with precise estrogen regulation, to induce decidual-like changes in the rat endometrium, in order to create a rodent model that more closely resembles the pathophysiological environment of primary dysmenorrhea. In this embodiment, the progesterone used is progesterone. Specifically:

[0123] 1. Weighing pure progesterone: Turn on the electronic balance and preheat for 30 minutes. Take out the pure progesterone from the -80°C freezer. Take a weighing paper, put it on the balance, and zero it after the value stabilizes. Gently pour in the pure progesterone and weigh 60mg of progesterone. The error should not exceed 0.1mg. After the value stabilizes, take out the weighing paper and the pure progesterone, and turn off the electronic balance.

[0124] 2. Preparation of progesterone injection solution: Gently pour pure progesterone into a wide-mouth bottle. Use a Pasteur dropper and pipette to measure 1.5 mL of dimethyl sulfoxide (DMSO) and add it to the bottle. Place the bottle on a mixer to dissolve it completely. Measure 28.5 mL of sterile corn oil and add it to the bottle. Place the bottle on a mixer and shake to dissolve it, preparing a 2 mg / mL progesterone injection solution.

[0125] 3. Preparation before animal experiments: Experimenters should wear lab coats, hats, masks and gloves, in accordance with SPF-grade experimental area requirements;

[0126] 4. Take the medicine: Take an unopened sterile dosing device, gently pull out the needle, and draw 1 mL of the prepared injection solution;

[0127] 5. Progesterone Injection: Wearing anti-bite gloves, gently hold the rat and place it on the operating table. Use the pad of your left thumb and the side of your index finger to gently lift the skin behind the rat's neck, while using the other fingers to hold the rat back and down. Disinfect the injection site with 75% alcohol. Hold the syringe in your right hand and insert the needle (bevel facing up) into the central depression of the skin at a 45-degree angle. Gently aspirate; if no blood return is observed, slowly inject the medication, ensuring even distribution. After injection, rotate the needle 180 degrees and gently withdraw it. Press the puncture site with a cotton swab to prevent leakage. In this example, progesterone was injected into all animals at a fixed time each day to maintain progesterone levels for a total of 8 days.

[0128] 6. Observe the rats for any abnormal reactions and ensure they recover to normal.

[0129] In this embodiment, all rats underwent drug administration. The overall experimental results showed that the rats had no abnormal reactions within half an hour after injection, indicating that the injection procedure had little impact on the physiological state of the rats and the experimental results were reliable.

[0130] Example 6

[0131] This embodiment describes step 6 of the method for establishing an animal menstrual-like dysmenorrhea model provided by the present invention. In this embodiment, oxytocin is injected into rats to simulate the uterine contractions during childbirth, thereby achieving a pain-inducing effect. That is, by simulating the dysmenorrhea process, combined with estrogen and progesterone, an SD rat model that more closely resembles the pathophysiological process of primary dysmenorrhea is created. Specifically:

[0132] 1. Weighing pure oxytocin: Preheat the electronic balance for 30 minutes, take the pure oxytocin out of the -80°C freezer, take a weighing paper, put it on the balance, and zero the balance after the value stabilizes. Gently pour in the pure oxytocin and weigh 60mg of oxytocin, with an error of no more than 0.1mg. After the value stabilizes, remove the weighing paper and the pure oxytocin, and turn off the electronic balance.

[0133] 2. Preparation of oxytocin injection: Gently pour pure oxytocin into a wide-mouth bottle, use a graduated cylinder to measure 12mL of physiological saline and pour it into the bottle, place it in a mixer and shake to dissolve, and prepare an oxytocin injection solution of 5mg / mL.

[0134] 3. Animal experiment preparation: Experiment personnel should wear lab coats, hats, masks and gloves, in accordance with SPF-grade experimental area requirements.

[0135] 4. Take the medication: Take an unopened sterile dosing device, gently pull out the needle, and draw 0.4 mL of the prepared injection solution;

[0136] 5. Oxytocin Injection: Wearing bite-proof gloves, gently grasp the rat and place it on the operating table. Use your left hand to hold the rat and fix its legs, fully exposing the rat's abdomen. Disinfect the injection site with 75% alcohol. Hold the syringe in your right hand and insert the needle (bevel facing up) into the upper outer part of the pubic symphysis at a 45-degree angle to the skin. Gently aspirate; if no blood return is observed, slowly inject the drug solution, ensuring even distribution. After injection, rotate the needle 180 degrees and gently withdraw it. Press the puncture site with a cotton swab to prevent leakage.

[0137] 6. Observe the rats for any abnormal reactions and ensure they recover to normal.

[0138] In this embodiment, all rats underwent drug administration. The overall experimental results showed that the rats did not have any abnormal reactions after injection, and writhing responses occurred within 0.5-24 hours after injection, indicating that the injection operation had little impact on the physiological state of the rats and the experimental results were reliable.

[0139] In Examples 1-6 above, it is necessary to ensure that estrogen is injected before each progesterone injection and that progesterone is injected at the same time every day. In this example, progesterone is injected at 5 pm every day, and the first oxytocin injection is started 24 hours after the last progesterone injection. The second batch of oxytocin is injected 11.5 hours later, and the third batch of oxytocin is injected 15.5 hours later. The rats after the modeling in Examples 1-6 are evaluated starting from the first injection of oxytocin. For specific evaluation methods, please refer to Examples 7-12 below.

[0140] Example 7

[0141] This embodiment is step 1 of the evaluation method for an animal menstrual-like dysmenorrhea model provided by the present invention. The writhing response is a typical pain behavior. By observing its changes under different conditions, we can understand the generation, transmission, and regulation mechanisms of pain. It is widely used in pharmacological and physiological research and is one of the most common behavioral observations in animal experiments. Embodiments 1-6 above have completed the complete modeling process and entered the model evaluation stage. This embodiment observes the writhing response phenomenon, frequency, and duration in rats and scores them. Through data statistics, the degree of pain in rats can be directly reflected, verifying the success of the modeling. This data can also be used for further research on pain mechanisms, pathophysiology, and pharmacology to further optimize the modeling scheme. Specifically:

[0142] 1. After the oxytocin injection was completed, the rats were placed in an empty rat box and a stopwatch was set for 30 minutes.

[0143] 2. Based on the writhing scoring criteria, the researchers accurately recorded the start and end times, the highest pain level, and the number of writhing episodes of the rats.

[0144] 3. Organize the data and perform data analysis using SPSS 27.0;

[0145] The torsional response scoring criteria are shown in Table 3, the torsional response duration scoring statistics are shown in Table 4, and the torsional response duration statistics chart is shown in [M]. x (P 25 P 75 )]like Figure 2 As shown in the figure, the highest score of the torsional response is statistically analyzed. )like Figure 3 As shown, the rat's writhing response is as follows: Figure 4 As shown:

[0146] Table 3. Scoring Criteria for Torsional Response

[0147]

[0148] Table 4. Statistical Table of Torsional Response Duration Level Scoring

[0149]

[0150] The writhing response observation experiment has important application value in pharmacological and physiological research, providing important experimental evidence for drug development and clinical application. In this example, experimental data shows that the degree of writhing response in rats first increases and then decreases with the increase of progesterone withdrawal time; rats in all groups showed strong and prolonged writhing responses, among which group C rats showed the strongest writhing response; indicating that the animal menstrual-like dysmenorrhea model was initially successfully established and the experimental data are reliable.

[0151] Example 8

[0152] This embodiment describes step 2 of the evaluation method for establishing an animal menstrual-like dysmenorrhea model provided by the present invention. After progesterone withdrawal, apoptosis occurs in the uterine tissue, with a sharp increase in neutrophils in the endometrium, and increases in chemokines and cytokines. These phenomena are also observed in human menstrual uterine tissue. Therefore, decidualization of the vagina and uterus is the most direct way to verify morphological experimental results. The success of establishing the SD rat menstrual-like dysmenorrhea animal model and the effect of different progesterone withdrawal times on uterine decidualization are evaluated and verified by observing whether decidualization occurs in the vagina and the pathological morphology of the uterine tissue. Specifically:

[0153] 1. Prepare a 2% sodium pentobarbital solution: Dissolve 1g of pure sodium pentobarbital in 50ml of sterile physiological saline to prepare a 2% sodium pentobarbital solution.

[0154] 2. Preparation before the experiment: Experiment personnel should wear lab coats, hats, masks and gloves, in accordance with the requirements of the SDF-level experimental area;

[0155] 3. Weight Measurement: Place a plastic bucket on an electronic scale, tare the rat, and gently place it into the bucket. Record its weight. Ensure the rat is calm during each measurement to avoid inaccurate readings due to struggling. Accurately record the rat's weight and calculate the anesthesia injection volume for each rat at 0.3 ml / 100g.

[0156] 4. Anesthesia and fixation: The SD female rat was removed from the cage and anesthetized by intraperitoneal injection of sodium pentobarbital solution. After anesthesia, the rat was fixed in a supine position on the rat operating table.

[0157] 5. Observation of vaginal decidua: Gently clean the rat vagina with a cotton swab moistened with physiological saline and observe the morphological changes of the rat vaginal decidua;

[0158] 6. Abdominal incision: Using tissue scissors, cut along the linea alba of the rat's abdomen from 1 cm above the pubic symphysis upwards, sequentially cutting the skin, superficial fascia, deep fascia, rectus abdominis muscle, and peritoneum. The incision should be 1.5 cm to 2 cm long.

[0159] 7. Observation of uterine tissue appearance: Two pink hollow organs wrapped in white adipose tissue can be found on both sides of the linea alba, namely the uterus. After separating the fat and connective tissue attached to the uterine myometrium, the morphology of the rat uterus is observed.

[0160] like Figure 5 The image shown is an example of the condition of the vaginal decidua in a rat. Figure 6 The image shown is a macroscopic comparison of an isolated rat uterus. Figure 7 The image shown is a comparative macroscopic view of the uterus in vivo from rats.

[0161] In this experiment, vaginal decidua and uterine tissue were observed in 30 rats in batches. Comparison revealed that the condition of the vaginal decidua in rats intensified with increasing progesterone withdrawal time. Group A (0.5 h withdrawal) showed no significant change, Group B (12 h) showed slight redness, Groups C (16 h) and D (20 h) showed redness and swelling, and Group E (24 h) showed significant decidua swelling. Gross uterine observation showed that the decidualized uterus was significantly more congested and swollen than the normal uterus. The frequency of decidualization also increased with increasing progesterone withdrawal time, with no significant difference among Groups C, D, and E. This experiment demonstrates that the preliminary experimental model of menstrual-like dysmenorrhea in animals was successful based on morphological observations, and the experimental data are reliable.

[0162] Example 9

[0163] This embodiment describes step 3 of the evaluation method for an animal menstrual-like dysmenorrhea model provided by the present invention. It involves two basic operations: abdominal aortic blood collection and uterine tissue removal. Auxiliary techniques such as serum separation, liquid nitrogen flash freezing, and 4% paraformaldehyde fixation are used to obtain blood and uterine tissue samples from the animal model. This provides material support for subsequent observations using optical microscopy, ultra-micro transmission electron microscopy of uterine tissue, serum ELISA (Enzyme-Linked Immunosorbent Assay) for PGE2 (prostaglandin E2), PGF2α (prostaglandin F2α), and PCR (polymerase chain reaction) to detect RNA expression of NLRP3 (NOD-like receptor protein 3) inflammasomes in rat uterine tissue. This further verifies the success of model establishment and the impact of progesterone withdrawal time on the overall experimental results from a histological and molecular biological perspective. In this embodiment, the procedures include abdominal aortic blood collection, uterine tissue removal, rat processing, and serum separation, specifically:

[0164] I. Abdominal aortic blood sampling:

[0165] 1. Preparation before the experiment: Experiment personnel should wear lab coats, hats, masks and gloves, in accordance with the requirements of SPF-level experimental areas;

[0166] 2. Anesthesia injection: Remove the SD female rats from the cages and inject 2% sodium pentobarbital solution into the peritoneal cavity of each group of rats for anesthesia. The dosage is calculated as 0.3ml / 100g. During anesthesia, the rats are placed in a supine position with their heads down. The corresponding sodium pentobarbital injection solution is drawn up with a syringe and injected into the peritoneal cavity of the rats for anesthesia.

[0167] 3. Expose the abdominal aorta: Fix the rat on a rat board, cut open the rat's abdomen with a scalpel, gently push aside the internal organs with gauze, locate the abdominal aorta, and use tools to fully expose the abdominal aorta;

[0168] 4. Abdominal aortic blood collection: Insert the lancet into the abdominal aorta in a centripetal direction and collect 5 mL of blood from the abdominal aorta using a blood collection tube for subsequent ELISA testing.

[0169] II. Removal of uterine tissue:

[0170] 1. Uterine removal: Two pink hollow organs wrapped in white adipose tissue can be found on both sides of the linea alba, namely the uterus. After separating the fat and connective tissue attached to the uterine wall, the uterine tissue of each group of rats can be completely removed. After washing in physiological saline, the uterine tissue is divided into six parts with a volume of 5 mm × 5 mm × 5 mm using sterile surgical scissors.

[0171] 2. Tissue fixation: Uterine tissue from each group of rats was quickly removed from an ice tray. A portion of the ipsilateral segment of uterine tissue from each group of rats was rinsed 1-2 times with pre-cooled physiological saline, blotted dry with filter paper, and fixed in 4% paraformaldehyde for HE staining (hematoxylin-eosin staining) and morphological observation under an optical microscope. Tissues prepared for transmission electron microscopy were fixed with electron microscopy fixative. The remaining uterine tissue was temporarily stored in liquid nitrogen for subsequent qPCR detection.

[0172] III. Rat Treatment:

[0173] 1. Observe and confirm death: After the sample is taken, observe the rat's vital signs. The rat will gradually show signs of slowed breathing, weakened heartbeat, until breathing stops, heartbeat disappears, and corneal reflex disappears. Usually, the rat will die 15 minutes after the sample is taken. After confirming death, it is still necessary to continue to observe for a period of time to ensure that the rat has no signs of life.

[0174] 2. Carcass disposal: The dead rats are placed in special biological waste bags and stored at low temperature (-20℃) according to laboratory regulations. They are then disposed of in a unified manner, such as by sending them to a professional institution for incineration, to prevent the spread of disease and environmental pollution.

[0175] IV. Serum Separation

[0176] 1. Allow blood to clot: Place the blood collection tube upright at room temperature and let it stand for 30-60 minutes to allow the blood to clot completely.

[0177] 2. Centrifugation: Place the coagulated blood sample in a centrifuge cooled at 4°C, set it to 1600 rpm for 15 minutes, and after centrifugation, the supernatant clear pale yellow liquid is separated from the red blood clot at the bottom;

[0178] 3. Collection and aliquoting: Carefully aspirate the supernatant serum using a sterile pipette (avoiding the white cell layer and red blood cell clots), aliquot it into clean centrifuge tubes, label them (number, date) during aliquoting, and freeze for storage.

[0179] In this embodiment, blood was collected from the abdominal aorta and uterine tissue was obtained from 30 rats. Different types of storage and fixation were carried out to allow for subsequent observation of uterine inflammatory response and shedding process through optical microscopy, ultra-micro transmission electron microscopy and molecular biology experiments.

[0180] Example 10

[0181] This embodiment describes step 4 of the evaluation method for an animal menstrual-like dysmenorrhea model provided by the present invention. Hematoxylin and eosin (HE) staining of tissue sections is a fundamental experimental technique in histological and pathological research. Through paraffin embedding, sectioning, and staining of rat uterine tissue, and based on the pathological changes in the rat uterus under a light microscope, the tissue is scored for pathological damage. Statistical analysis of the data can directly reflect the degree of inflammatory invasion in rats, verify the success of the model establishment, and can be used for further research on the mechanisms of inflammation, decidualization, pathophysiology, and pharmacology to further optimize the model establishment protocol. Specifically:

[0182] 1. Tissue dehydration: Use a commercial tissue dehydration kit for gradient ethanol dehydration (room temperature): 75% ethanol → 95% ethanol → anhydrous ethanol twice, 2 hours for each step;

[0183] 2. Tissue transparency: Soak twice in xylene, 30 minutes each time, until the tissue becomes transparent;

[0184] 3. Paraffin Impregnation: Transfer the tissue into molten paraffin (60°C) and impregnate for 2 hours. Preheat the embedding mold simultaneously.

[0185] 4. Place the tissue: Use preheated tweezers to remove the tissue from the paraffin stage, select a mold, and quickly place the tissue in the center of the mold (note that the cut surface should be facing downwards).

[0186] 5. Wax Injection and Cooling: Pour in molten paraffin to completely cover the tissue, and move it to a cold table area to cool until the paraffin solidifies (30 minutes).

[0187] 6. Demolding: After solidification, gently push the bottom of the mold to remove the wax block, and trim the edges for later use;

[0188] 7. Fix the wax block: Clamp the wax block onto the sample holder of the microtome, tighten the fixing screws, and adjust the angle of the wax block so that the cut surface is parallel to the blade;

[0189] 8. Rough trimming of tissue: Turn the handwheel to rough trim the paraffin block to a thickness of 20-30μm until the complete tissue section is exposed;

[0190] 9. Install the blade and adjust the blade distance: Install the slicing blade onto the blade holder, adjust the blade angle, tighten the fixing screw, turn the handwheel to bring the wax block closer to the blade, and use the thickness adjustment knob to set the slice thickness (5μm is the usual size).

[0191] 10. Slicing: Rotate the handwheel at a constant speed (avoid sudden changes in speed) to maintain a consistent rhythm; gently support the cut wax strip with a brush to prevent folding or breakage. After cutting out continuous wax strips, use tweezers or a brush to transfer the wax strip to the warm water bath (45℃) of the slicer to flatten it.

[0192] 11. Slide preparation: Tilt a poly-L-lysine slide into water, gently lift the slide, ensuring the tissue is wrinkle-free. Incubate the slides at 37°C overnight to allow them to air dry completely.

[0193] 12. Dewaxing and Staining: Insert the slide containing the tissue into the staining rack, ensuring the tissue sides are facing the same. Place the staining handle into the staining rack, and the instrument will identify the staining program and begin staining (ensure the correct staining rack color is selected for slide staining). The instrument will calculate the start and end times of the staining program, and the staining progress can be viewed on the staining machine's display screen.

[0194] 13. Mounting: After mounting using a fully automatic HE staining and mounting machine (manufacturer: Leica), the robotic arm transfers the staining rack to the download tank and removes the staining rack;

[0195] 14. Section Observation: The sealed sections were removed one by one and observed under a light microscope. The pathological changes were scored based on five aspects: endometrial degeneration and necrosis, edema of the lamina propria, increased glands in the lamina propria, inflammatory cell infiltration of the lamina propria, and inflammation of the myometrium (0 points represent normal, 0.5, 1, 2, 3, and 4 points represent severity from mild to severe). A comprehensive statistical analysis was performed. HE-stained sections of rat uterine tissue from different groups are shown below. Figure 8 As shown, the histopathological changes in uterine tissue of rats in different groups were scored. )like Figure 9 As shown, HE-stained sections of uterine tissue from 30 rats (groups A-E) are as follows. Figure 10-14 As shown in Table 5, the slice statistics results are presented.

[0196] 15. Data Analysis: The recorded scoring data was processed and analyzed using SPSS 27.0.

[0197] Table 5. Statistical Results of Slices

[0198]

[0199] In this embodiment, uterine tissue from 30 rats was paraffin-embedded, sectioned, and stained. The pathological morphological changes in the rat uterine tissue were observed and recorded under an optical microscope, providing a reference for the overall model evaluation. Morphological observation of the sections revealed extensive vacuolation and necrosis of the rat endometrial epithelial cells, congestion of multispiral arterioles, neutrophil infiltration, and increased glandular tissue in the lamina propria, indicating a basic success in model establishment. Data analysis showed that the pathological change score generally increased with the duration of progesterone withdrawal, indicating that a longer progesterone withdrawal time resulted in better model establishment.

[0200] Example 11

[0201] This embodiment describes step 5 of the evaluation method for an animal menstrual-like dysmenorrhea model provided by the present invention. ELISA (Enzyme-Linked Immunosorbent Assay) is a highly sensitive immunoassay technique widely used in biomedicine, clinical diagnosis, and scientific research. The above embodiment has completed the complete modeling process and entered the model evaluation stage. Now, by performing ELISA on rat serum, the pathological changes of uterine inflammation in rats are evaluated at the molecular level. Specifically:

[0202] I. Rat serum ELISA detection (PGF2α):

[0203] 1. Addition of standard samples: Set up standard sample wells and sample wells, and add 50 μL of standard sample of different concentrations to each standard sample well;

[0204] 2. Sample addition: Set up blank wells (blank control wells do not contain sample or enzyme labeling reagent, and the other steps are the same) and sample wells. Add 50 μL of sample to the sample wells on the enzyme-coated plate. Place the sample at the bottom of the well, avoiding touching the well wall as much as possible, and gently shake to mix.

[0205] 3. Add enzyme: Add 100 μl of enzyme-labeled reagent to each well, except for the blank wells. The manufacturer of the enzyme-labeled reader is Rayto / Molecular Devices, model Rayto RT-6100 / MD-SpectraMax 190, with a measurement range of OD: 0-4 and an accuracy of 0.001OD.

[0206] 4. Incubation: After sealing the plate with sealing film, incubate at 37°C for 60 minutes;

[0207] 5. Solution preparation: Dilute the 20-fold concentrated washing solution with distilled water 20 times and set aside.

[0208] 6. Washing: Carefully peel off the sealing film, discard the liquid, shake dry, fill each well with washing solution, let stand for 30 seconds and then discard, repeat this 5 times, and pat dry.

[0209] 7. Color development: Add 50 μl of color developer A to each well, then add 50 μl of color developer B, gently shake to mix, and develop at 37°C in the dark for 15 minutes.

[0210] 8. Termination: Add 50 μl of stop solution to each well to stop the reaction (the blue color will immediately turn yellow).

[0211] 9. Measurement: Zero the instrument with the blank well and measure the absorbance (OD value) of each well in sequence at a wavelength of 450nm. The measurement should be performed within 15 minutes after adding the stop solution.

[0212] 10. Using the concentration of the standard as the x-axis and the corresponding absorbance (OD value) as the y-axis, a standard curve equation is created using computer software with four-parameter Logistic curve fitting (4-pl). The concentration of the sample is then calculated using the equation based on the absorbance (OD value) of the sample.

[0213] In this embodiment, a rat prostaglandin F2α (PGF2α) ELISA kit was used for detection. The kit was manufactured by Zhuocai Biotechnology and the model number was ZC-37104.

[0214] The experimental results are shown in Table 6 and Figure 15 ( As shown in ±SD (6 mice / group):

[0215] Table 6. Statistical table of serum PGF2α content in rats of different groups

[0216]

[0217] The results showed that compared with group A (progesterone withdrawal time 0.5 h), the PGF2α levels in groups B (progesterone withdrawal time 12 h), C (progesterone withdrawal time 16 h), and E (progesterone withdrawal time 24 h) were significantly increased (P < 0.001), and in group D (progesterone withdrawal time 20 h) was significantly increased (P < 0.05). Compared with group B, the PGF2α level in group D was significantly decreased (P < 0.01). Compared with group C, the PGF2α level in group D was significantly decreased (P < 0.05), indicating that the serum inflammatory response increased with the progesterone withdrawal time. Among them, the serum PGF2α level was the highest in group B, i.e., 12 h after progesterone withdrawal, indicating a strong inflammatory response. The changes in PGF2α levels directly reflected the occurrence of the inflammatory response in rats, further proving the success of the model.

[0218] II. Rat serum ELISA detection (PGE2):

[0219] 1. Addition of standard samples: Set up standard sample wells and sample wells, and add 50 μL of standard sample of different concentrations to each standard sample well;

[0220] 2. Sample addition: Set up blank wells (blank control wells do not contain sample or enzyme labeling reagent, and the other steps are the same) and sample wells. Add 50 μL of sample to the sample wells on the enzyme-coated plate. Place the sample at the bottom of the well, avoiding touching the well wall as much as possible, and gently shake to mix.

[0221] 3. Add enzyme: Add 100 μl of enzyme-labeled reagent to each well, except for the blank wells;

[0222] 4. Incubation: After sealing the plate with sealing film, incubate at 37°C for 60 minutes;

[0223] 5. Solution preparation: Dilute the 20-fold concentrated washing solution with distilled water 20 times and set aside.

[0224] 6. Washing: Carefully peel off the sealing film, discard the liquid, shake dry, fill each well with washing solution, let stand for 30 seconds and then discard, repeat this 5 times, and pat dry.

[0225] 7. Color development: Add 50 μl of color developer A to each well, then add 50 μl of color developer B, gently shake to mix, and develop at 37°C in the dark for 15 minutes.

[0226] 8. Termination: Add 50 μl of stop solution to each well to stop the reaction (the blue color will immediately turn yellow).

[0227] 9. Measurement: Zero the instrument with the blank well and measure the absorbance (OD value) of each well in sequence at a wavelength of 450nm. The measurement should be performed within 15 minutes after adding the stop solution.

[0228] 10. Using the concentration of the standard as the x-axis and the corresponding absorbance (OD value) as the y-axis, a standard curve equation is created by using computer software and a four-parameter Logistic curve fitting (4-pl). The concentration of the sample is then calculated using the equation based on the absorbance (OD value) of the sample.

[0229] In this embodiment, a rat prostaglandin E2 (PGE2) ELISA kit was used for detection. The kit was manufactured by Zhuocai Biotechnology and the model number was ZC-37100.

[0230] The experimental results are shown in Table 7 and Figure 16 ( As shown in ±SD (6 mice / group):

[0231] Table 7. Statistical table of serum PGE2 levels in rats from different groups

[0232]

[0233] The results showed that compared with group A (progesterone withdrawal time 0.5 h), the PGE2 levels in groups B (progesterone withdrawal time 12 h), C (progesterone withdrawal time 16 h), D (progesterone withdrawal time 20 h), and E (progesterone withdrawal time 24 h) were significantly lower (P < 0.001). Compared with group B, the PGE2 level in group C was significantly lower (P < 0.001), and the PGE2 level in group D was significantly lower (P < 0.05). Compared with group C, the PGE2 level in group D was significantly higher (P < 0.01), and the PGE2 level in group E (progesterone withdrawal time 24 h) was significantly higher (P < 0.001). This indicates that with the extension of progesterone withdrawal time, the serum inflammatory response first increases and then decreases. Among them, group C, i.e., 16 h after progesterone withdrawal, had the lowest serum PGE2 level and the strongest inflammatory response. The change in PGE2 level directly reflects the occurrence of inflammatory response in rats, further proving the success of the model.

[0234] By performing a division operation on the monitoring data of the two indicators, the PGF2α / PGE2 ratio was obtained, and the results are shown in Table 8. Figure 17 ( As shown in ±SD (6 mice / group):

[0235] Table 8. Statistical table of serum PGF2α / PGE2 ratio in different groups of rats

[0236]

[0237] The results showed that compared with group A (progesterone withdrawal 0.5 h), the PGF2α / PGE2 ratio was significantly increased in groups B (progesterone withdrawal 12 h) and C (progesterone withdrawal 16 h) (P < 0.001), significantly increased in group D (progesterone withdrawal 20 h) (P < 0.01), and significantly increased in group E (progesterone withdrawal 24 h) (P < 0.01); compared with group B, group C P The GF2α / PGE2 ratio was significantly increased (P<0.001); compared with group C, the PGF2α / PGE2 ratio in group D was significantly decreased (P<0.001), and the PGF2α / PGE2 ratio in group E was significantly decreased (P<0.001), indicating that as the progesterone withdrawal time increased, the overall serum inflammatory response first increased and then decreased. Among them, group C had the highest serum PGF2α / PGE2 ratio and the strongest inflammatory response at 16h after progesterone withdrawal.

[0238] Example 12

[0239] This embodiment describes step 6 of the evaluation method for an animal menstrual-like dysmenorrhea model provided by the present invention. The main purpose of the rat uterine tissue qPCR (real-time quantitative polymerase chain reaction) experiment is to analyze the function, regulatory mechanism, or pathological changes of specific genes at the molecular level by detecting the mRNA expression level of specific genes in uterine tissue. The above embodiment has completed the complete modeling process and entered the final stage of model evaluation. Now, by performing qPCR detection on rat uterine tissue, the expression of key genes in primary dysmenorrhea in rats is evaluated at the molecular level. Specifically:

[0240] 1. Total RNA extraction (using sterile, enzyme-free pipette tips and centrifuge tubes)

[0241] 1) Take a grinding tube, add 1 ml of RNA extraction solution, add 3 3 mm grinding beads, and pre-cool on ice;

[0242] 2) Take 5-20 mg of tissue and add it to a grinding tube;

[0243] 3) Use a grinder (manufacturer: Wuhan Saiweier Biotechnology Co., Ltd., model: KZ-5F-3D) to grind thoroughly until no visible tissue blocks remain;

[0244] 4) Centrifuge at 12000 rpm for 10 min at 4℃ and collect the supernatant;

[0245] 5) Add 100 μl of chloroform substitute, invert the centrifuge tube for 15 seconds, mix thoroughly, and let stand for 3 minutes;

[0246] 6) Centrifuge at 12000 rpm for 10 min at 4℃;

[0247] 7) Transfer 400 μl of supernatant to a new centrifuge tube, add 550 μl of isopropanol, and mix by inverting.

[0248] 8) Place at -20℃ for 15 minutes;

[0249] 9) Centrifuge at 12000 rpm for 10 min at 4℃; the white precipitate at the bottom of the tube is RNA.

[0250] 10) Remove the liquid, add 1 ml of 75% ethanol, invert and mix well to wash the precipitate;

[0251] 11) Centrifuge at 12000 rpm for 5 min at 4℃;

[0252] 12) Repeat steps 10)-11) once;

[0253] 13) Remove all liquid and place the centrifuge tube on a clean bench for 3-5 minutes;

[0254] 14) Add 15 μl of RNA dissolving solution to dissolve the RNA;

[0255] 15) Use Nanodrop 2000 to detect RNA concentration and purity: After zeroing the instrument blank, take 2.5 μl of the RNA solution to be tested on the detection base, lower the sample arm, and use the software on the computer to start the absorbance detection;

[0256] 16) Dilute the excessively high concentration of RNA by an appropriate ratio to achieve a final concentration of 200 ng / μl.

[0257] 2. Reverse transcription (using sterile, enzyme-free pipette tips and PCR reaction tubes)

[0258] 1) Preparation of the reverse transcription reaction system (reverse transcription kit catalog number: G3337), detailed parameters are shown in Table 9:

[0259] Table 9. Preparation parameters of the reverse transcription reaction system

[0260]

[0261] 2) Gently mix and centrifuge;

[0262] 3) The reverse transcription program is set as shown in Table 10. The reverse transcription is performed on a PCR instrument (manufacturer: Beijing Dongsheng Innovation Biotechnology Co., Ltd., model: ETC811).

[0263] Table 10 Reverse Transcription Program Settings

[0264]

[0265] 3. Quantitative PCR

[0266] 1) Take 0.1 ml of the reverse transcription product above into a PCR reaction plate (manufacturer: Wuhan Saiweier Biotechnology Co., Ltd., model: PCR-9601-NS), prepare the reaction system as shown in Table 11, prepare 3 tubes for each reverse transcription product, after spotting the samples, use PCR sealing film (manufacturer: Wuhan Saiweier Biotechnology Co., Ltd., model: G6065) with a sealing instrument to complete the sealing, and centrifuge with a microplate centrifuge.

[0267] Table 11 Reaction System

[0268]

[0269] 2) PCR amplification: Amplification was performed on a PCR instrument, and the procedure is shown in Table 12.

[0270] Table 12 PCR Amplification Procedure

[0271]

[0272] 4. Result Processing

[0273] ΔΔCT method:

[0274] A = CT(target gene, sample to be tested) - CT(internal standard gene, sample to be tested)

[0275] B = CT(target gene, control sample) - CT(internal standard gene, control sample)

[0276] K=AB

[0277] Expression multiple = 2 -K

[0278] The experimental results are shown in Table 13, which presents the statistical analysis of NLRP3 expression levels in the uterine tissues of rats in different groups. The statistical analysis of NLRP3 expression levels in the uterine tissues of rats in each group is as follows: Figure 18 ( As shown in ±SD (6 mice / group):

[0279] Table 13 Statistical analysis of NLRP3 expression levels in uterine tissues of rats from different groups

[0280]

[0281] The results showed that compared with group C (progesterone withdrawal 16 h), the relative expression level of the target gene in group D (progesterone withdrawal 20 h) was significantly lower (P < 0.01), and the relative expression level of the target gene in group E (progesterone withdrawal 24 h) was significantly lower (*P < 0.05). Since the target gene is related to the development of primary dysmenorrhea, this indicates that the modeling experiment was successful, the rats expressed the key gene for primary dysmenorrhea, and the intervention method in group C was more conducive to the establishment of a menstrual-like dysmenorrhea model in animals.

[0282] It should be understood that the present invention is not limited to what has been described above and shown in the accompanying drawings, and various modifications and changes can be made without departing from its scope. The scope of the invention is limited only by the appended claims.

Claims

1. A method for modeling a menstrual cramp model in an animal, characterized by, The ovariectomized animals are injected with estrogen, progestogen and oxytocin, wherein the progestogen is injected for 7-9 days, and the menstrual-like dysmenorrhea model of the animals is obtained 16 hours after the progestogen is withdrawn; The animals selected for the menstrual-like dysmenorrhea model are rats; The modeling method of the menstrual-like dysmenorrhea model of animals includes the following steps: Step 1: Select healthy adult specific-pathogen-free virgin female rats as the modeling animal objects; Step 2: Screen the rats in the late estrus and diestrus by the vaginal smear method to prepare for modeling; Step 3: Perform ovariectomy on the rats to exclude the influence of endogenous estrogen of the rats, so as to simulate the primary dysmenorrhea pathophysiological state by regulating exogenous hormones; Step 4: Supplement the estrogen in the rats by exogenous injection; Step 5: Continuously inject progestogen in the rats by exogenous injection; Step 6: Inject oxytocin by exogenous injection.

2. The method for modeling animal menstrual dysmenorrhea according to claim 1, wherein, The time for continuously injecting progestogen in step 5 is 7-9 days.

3. An evaluation method for the modeling method of the animal model of menstrual dysmenorrhea of any one of claims 1-2, characterized in that, The evaluation method includes observing the writhing reaction of the animals in the menstrual-like dysmenorrhea model, the vaginal decidua and the general observation of the uterine tissue, and obtaining the blood and uterine tissue samples of the animal model by abdominal aortic blood sampling and uterine tissue extraction, so as to observe the uterine tissue morphologically, detect the serum by enzyme-linked immunosorbent assay, and detect the rat uterine tissue by real-time quantitative polymerase chain reaction.

4. The method of claim 3, wherein, The evaluation method includes the following steps: Step 1: Observe the writhing reaction of the rats within 30 minutes after the first injection of oxytocin is completed; Step 2: Observe the vaginal decidua and the general observation of the uterine tissue of the rats at different times after the progestogen is withdrawn; Step 3: Obtain the blood and uterine tissue samples of the animal model by abdominal aortic blood sampling and uterine tissue extraction; Step 4: Morphologically observe the rat uterine tissue, that is, perform hematoxylin-eosin staining section on the uterine tissue; Step 5: Detect the serum of the rats by enzyme-linked immunosorbent assay; Step 6: Detect the rat uterine tissue by real-time quantitative polymerase chain reaction.

5. The method of claim 4, wherein, In step 2, the vaginal decidua and the general observation of the uterine tissue of the rats are observed at 0.5h, 12h, 16h, 20h and 24h after the progestogen is withdrawn.

6. The method of claim 4, wherein, In step 5, the detection of prostaglandin F2α and prostaglandin E2 is included.

7. The method of claim 4, wherein, In step 6, total RNA extraction, reverse transcription and quantitative PCR experiment are included, and the expression amount of NOD-like receptor protein 3 in the rat uterine tissue is counted.

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