Method for constructing an animal model of co-morbidity of postmenopausal osteoporosis and knee osteoarthritis

Abstract

The present application relates to the field of disease animal model construction, and specifically discloses a method for constructing an animal model of postmenopausal osteoporosis and knee osteoarthritis comorbidity, comprising: selecting female animals as experimental objects; injecting GnRH agonists into the selected female animals, while giving low-calcium feed and oxidative stress stimulation, and feeding for a preset duration; detecting the bone density and trabecular bone parameters of the female animals after feeding, and screening qualified female animals that meet the set standards; performing a meniscus tear operation on the qualified female animals, and implanting inflammation factor-loaded sustained-release microspheres at the tear site during the operation; postoperatively, performing weight-bearing training on the female animals for a set duration; and at different time points, performing health detection on the female animals. The present application realizes precise simulation of the pathological process and clinical characteristics of comorbidity by multi-factor synergistic induction of PMOP, minimally invasive construction of KOA, and multi-dimensional health detection, thereby improving the authenticity and stability of the model.

Description

Technical Field

[0001] This invention relates to the field of animal model construction for diseases, and more specifically, to a method for constructing an animal model of comorbid postmenopausal osteoporosis and knee osteoarthritis. Background Technology

[0002] With the increasing incidence of postmenopausal osteoporosis (PMOP) and knee osteoarthritis (KOA) comorbidities among middle-aged and elderly women, the need for reliable animal models for research on related pathological mechanisms and the development of therapeutic drugs is becoming increasingly urgent. Current model construction is mostly limited to single-factor induction, failing to fully integrate the clinically observed causes of postmenopausal osteoporosis ("estrogen deficiency + low calcium intake + oxidative stress"), nor to recreate the pathological characteristics of knee osteoarthritis ("chronic mechanical damage + prolonged local inflammation"). This results in significant differences between the models and the actual pathological processes of the comorbidity in humans, making it difficult to support precise research.

[0003] Traditional comorbidity models often induce postmenopausal osteoporosis through oophorectomy, which can easily lead to a sharp drop in estrogen levels, inconsistent with the gradual hormonal changes during natural menopause in humans. Furthermore, these models do not incorporate common clinical risk factors such as hypocalcemia and oxidative stress. Knee osteoarthritis induction often involves open surgery to remove the lateral meniscus, causing acute trauma and concentrating cartilage degeneration on the lateral plateau, which is inconsistent with the characteristic of medial cartilage degeneration in humans. In addition, there is a lack of chronic inflammation control mechanisms, resulting in acute resolution of the inflammatory response. Evaluation indicators only focus on bone mineral density and cartilage structure, ignoring function and systemic relationships, leading to poor model stability.

[0004] Therefore, it is necessary to design an animal model for the comorbidity of postmenopausal osteoporosis and knee osteoarthritis to address the problems of existing technologies, such as PMOP induction not conforming to the progressive hormonal changes in humans, KOA degeneration sites and inflammatory characteristics deviating from clinical practice, low model stability, and a single evaluation system, which cannot accurately simulate the comorbid pathology and affect the accuracy of research and drug evaluation. Summary of the Invention

[0005] In view of this, the present invention proposes an animal model for the comorbidity of postmenopausal osteoporosis and knee osteoarthritis, in order to solve the problems of existing technologies such as PMOP induction not conforming to the progressive sex hormone changes in humans, KOA degeneration sites and inflammatory characteristics deviating from clinical practice, low model stability and single evaluation system, which cannot accurately simulate the comorbid pathology and affect the accuracy of research and drug evaluation.

[0006] On the one hand, this invention provides a method for constructing an animal model of comorbid postmenopausal osteoporosis and knee osteoarthritis, comprising the following steps: Female animals were selected as experimental subjects; Selected female animals were injected with GnRH agonists and fed a low-calcium diet and subjected to oxidative stress for a predetermined duration. Bone density and trabecular bone parameters of female animals after rearing were tested, and qualified female animals that met the set standards were selected. Meniscus tears were surgically removed from qualified female animals, and slow-release microspheres loaded with inflammatory factors were implanted at the tear site during the operation. Post-surgery, female animals underwent weight-bearing training for a set duration. Health checks were conducted on the female animals at different time points.

[0007] Furthermore, the female animal is a 10-12 month old female SD rat.

[0008] Furthermore, the selected female animals are injected with GnRH agonists once a week, the preset rearing period is 6-8 weeks, and the oxidative stress stimulation is performed 2-3 times a week.

[0009] Furthermore, the calcium content of the low-calcium feed is 0.1%-0.3%; the oxidative stress stimulus is: providing drinking water containing oxidizing substances.

[0010] Furthermore, the drinking water containing the oxidizing substance is an aqueous solution of H2O2 with a concentration of 0.05-0.2 mmol / L.

[0011] Furthermore, the standards for setting the bone mineral density and trabecular bone parameters are as follows: the bone mineral density of the rat lumbar vertebrae and femoral neck decreased by ≥15% compared with the pretreatment, and the number of trabecular bones decreased by ≥10% compared with the pretreatment.

[0012] Furthermore, the specific location of the meniscus tear surgery is the anterior horn of the medial meniscus of the rat knee joint, and the tear length is 1 / 4 to 1 / 3 of the total length of the anterior horn of the meniscus.

[0013] Furthermore, the inflammatory factor is IL-1β with a loading of 30-70 ng / mL; the sustained-release microspheres are PLGA microspheres with a particle size of 30-150 μm.

[0014] Furthermore, the weight-bearing training specifically involves: 0.5-2 hours of weight-bearing treadmill training daily during the rat's active period, with an initial load of 3%-7% of the rat's body weight, and an increase in load of 3%-7% of the rat's body weight weekly; the duration of this training is set for 8-12 weeks.

[0015] Furthermore, the health checks on female animals at different time points include: cartilage status, inflammation-related, and bone metabolism-related tests at week 8 after modeling, and function-related and gut microbiota-related tests at week 16 after modeling.

[0016] Compared with the prior art, the beneficial effects of the present invention are as follows: This invention selects female animals as experimental subjects and uses a multi-factor synergistic approach to induce postmenopausal osteoporosis through GnRH agonist injection, low-calcium diet feeding, and oxidative stress stimulation. Imaging tests are used to screen and ensure model consistency. Then, knee osteoarthritis is precisely constructed using meniscus tear surgery combined with sustained-release microspheres loaded with inflammatory factors. Postoperatively, weight-bearing training simulates clinical mechanical load, and health monitoring is conducted at different time points. This approach avoids the acute trauma and sudden hormone drop associated with traditional surgery, while also reproducing the "progressive pathology + interaction" characteristics of human comorbidities, improving the model's realism and stability, and providing a reliable experimental platform for comorbidity mechanism research and drug evaluation. Attached Figure Description

[0017] Various other advantages and benefits will become apparent to those skilled in the art upon reading the following detailed description of preferred embodiments. The accompanying drawings are for illustrative purposes only and are not intended to limit the invention. Furthermore, the same reference numerals denote the same parts throughout the drawings. In the drawings: Figure 1 A flowchart illustrating the method for constructing an animal model of postmenopausal osteoporosis and knee osteoarthritis comorbidity provided in this embodiment of the invention. Detailed Implementation

[0018] The technical solutions of the embodiments of this application will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only a part of the embodiments of this application, and not all of the embodiments. Based on the embodiments of this application, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of this application.

[0019] In the description of this application, it should be understood that the terms "center", "upper", "lower", "front", "rear", "left", "right", "vertical", "horizontal", "top", "bottom", "inner", "outer", etc., indicate the orientation or positional relationship based on the orientation or positional relationship shown in the accompanying drawings. They are only for the convenience of describing this application and simplifying the description, and do not indicate or imply that the device or element referred to must have a specific orientation, or be constructed and operated in a specific orientation. Therefore, they should not be construed as limitations on this application.

[0020] The terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of technical features indicated. Therefore, a feature defined as "first" or "second" may explicitly or implicitly include one or more of that feature. In the description of this application, unless otherwise stated, "a plurality of" means two or more.

[0021] In the description of this application, it should be noted that, unless otherwise expressly specified and limited, the terms "installation," "connection," and "linking" should be interpreted broadly. For example, they can refer to a fixed connection, a detachable connection, or an integral connection; they can refer to a mechanical connection or an electrical connection; they can refer to a direct connection or an indirect connection through an intermediate medium; and they can refer to the internal connection between two components. Those skilled in the art can understand the specific meaning of the above terms in this application based on the specific circumstances.

[0022] On the one hand, such as Figure 1 As shown in some embodiments of this application, the method for constructing an animal model of comorbid postmenopausal osteoporosis and knee osteoarthritis includes the following steps: S1. Select female animals as experimental subjects; S2. Inject the selected female animals with GnRH agonists, and feed them with low-calcium diet and oxidative stress for a preset duration. S3. Test the bone density and trabecular bone parameters of female animals after feeding, and screen qualified female animals that meet the set standards. S4. Perform meniscus tear surgery on qualified female animals and implant sustained-release microspheres loaded with inflammatory factors at the tear site during the operation; S5. Post-operatively, female animals undergo weight-bearing training for a set duration. S6. Conduct health checks on female animals at different time points.

[0023] Specifically, female animals of the same strain and batch were obtained from the experimental animal breeding center. Through visual observation, such as no hair loss or joint swelling, and basic physiological indicators such as weight fluctuation <5% and normal body temperature, individuals with abnormal health were excluded. Specifically, leuprorelin was selected as the GnRH agonist. The dosage of the GnRH agonist was calculated based on the weight of the female animals, such as 0.1 mg / kg, and administered via intraperitoneal or subcutaneous injection. From the day of injection, the animals were fed a low-calcium diet instead of the regular diet, with free access to food and adequate drinking water. The selected female animals were randomly divided into experimental and control groups and housed in single or group cages, with 3-5 animals per cage. After acclimatizing to the environment for one week, the animals proceeded to the next steps.

[0024] Specifically, the breeding environment is controlled at a temperature of 22-25℃ and a humidity of 40%-60%, with the preferred temperature being 23℃ and humidity being 50%; the light cycle simulates natural day and night, with 12 hours of light and 12 hours of darkness.

[0025] Understandably, the above embodiments completed the construction of an animal model of postmenopausal osteoporosis.

[0026] Understandably, GnRH agonists (gonadotropin-releasing hormone agonists) work by gradually inhibiting the function of the hypothalamus-pituitary-ovarian axis in rats. Initially, they briefly stimulate the pituitary gland to secrete gonadotropins (LH, FSH), but long-term regular injections cause the pituitary gland to desensitize to GnRH and stop releasing LH and FSH. After the ovaries lose the stimulation of gonadotropins, follicle development stops, and estrogen secretion drops significantly, eventually reaching a state consistent with "ovarian function decline and estrogen deficiency after natural menopause" in humans.

[0027] Understandably, the pre-set duration of continuous feeding is intended to fully manifest the bone metabolic imbalance caused by estrogen deficiency: estrogen deficiency leads to increased osteoclast activity and weakened osteoblast function, resulting in the gradual loss of minerals such as calcium from bones. This process is consistent with the clinical progression of "slow bone loss" after menopause in humans, rather than the "sudden drop in hormones and rapid bone loss" of traditional oophorectomy, and is closer to the actual pathology. Low-calcium diets, by restricting calcium intake in animals, simulate when there is insufficient calcium reserves in the body. Osteoclasts will accelerate the breakdown of bone tissue to release calcium, replenish blood calcium, and further exacerbate the imbalance of "bone resorption > bone formation," promoting osteoclast proliferation and activity, while inhibiting osteoblasts from synthesizing bone matrix (such as type I collagen). This forms a "synergistic effect" with estrogen deficiency, accelerating the decline in bone density and the destruction of bone microstructure.

[0028] It is understandable that the above embodiments induce postmenopausal osteoporosis in female animals through the synergistic effect of multiple factors.

[0029] Specifically, imaging techniques, such as Micro-CT (micro-computed tomography), are used to detect bone density and trabecular bone parameters in female animals.

[0030] Specifically, after being reared for the preset time, the female animals were fasted for 12 hours to avoid interference from their gastrointestinal contents with imaging. They were then lightly anesthetized using an anesthetic such as isoflurane. The anesthetized female animals were then fixed in place, and the lumbar vertebrae, such as the L3-L5 segments, and both femoral necks were scanned to obtain three-dimensional images. Bone density, trabecular bone number, trabecular bone thickness, and trabecular bone separation were calculated using image analysis software. The baseline data of the female animals before rearing were compared. If the baseline data was not tested, the animals were compared with untreated female animals from the same batch to screen out qualified female animals and exclude female animals with "GnRH resistance" or "low calcium insensitivity" to ensure a consistent starting point for subsequent comorbidity induction.

[0031] Specifically, qualified female animals are fasted and deprived of water for 6 hours before surgery. After anesthesia, the hair around the knee joint of the female animal is shaved and the skin is disinfected. Anesthesia can be performed by intraperitoneal injection of sodium pentobarbital.

[0032] Specifically, laparoscopic-assisted meniscus tear surgery was performed on female animals. During the operation, a micro-syringe was used to inject sustained-release microspheres loaded with inflammatory factors into the meniscus tear site to ensure that the microspheres were evenly distributed in the damaged area.

[0033] Specifically, after the surgery, the incision is sutured, and antibiotic ointment is applied to prevent infection. The female animal is kept alone, and its activity level is observed for 3 days after the surgery, avoiding strenuous exercise. Normal activities can resume after 1 week.

[0034] Understandably, the choice of "meniscus tear" instead of "meniscectomy" is to simulate the chronic meniscus damage common in human knee osteoarthritis, such as tearing and wear, and to avoid the "acute joint instability and rapid cartilage degeneration" caused by resection surgery, which is more in line with the chronic progression characteristics of human knee osteoarthritis.

[0035] Specifically, starting one week after surgery, female animals were allowed to acclimatize on a weightless treadmill for 3 days, 10 minutes each day, to avoid stress caused by direct weight-bearing; formal weight-bearing training began on the 4th day after surgery.

[0036] Understandably, the above embodiments completed the construction of an animal model of postmenopausal knee osteoarthritis.

[0037] Specifically, health checks were performed on female animals in the early and late stages of comorbidity after modeling, covering the entire process of knee osteoarthritis from early cartilage degeneration to late-stage functional impairment.

[0038] Specifically, health testing refers to the multi-dimensional detection of an animal's pathological and functional status, including structural testing, inflammation testing, metabolic testing, functional testing, and systemic testing.

[0039] It can be seen that the present invention simulates the multiple risks of human osteoporosis (PMOP) through "GnRH agonist + low calcium + oxidative stress", and simulates the "mechanical damage + chronic inflammation" of knee osteoarthritis (KOA) through "meniscus tear + sustained release of inflammatory factors + weight-bearing training", and the two form a "vicious cycle", which is completely consistent with the pathology of human comorbidity.

[0040] It can be seen that this invention, by screening with bone density and trabecular bone parameters and excluding individual differences (such as "resistant" animals), can improve the success rate of constructing comorbidity models. As can be seen, the multi-time-point and multi-dimensional detection of this invention can not only verify whether the model is valid, but also reveal the cross-system interaction mechanism of animal models, providing a more comprehensive evaluation platform for the development of drugs for the treatment of comorbidities. As can be seen, this invention uses GnRH agonists instead of surgical ovariectomy and employs laparoscopic-assisted minimally invasive surgery, which can reduce animal trauma and infection risks, and conforms to the "3R principle" (reduce pain, optimize methods) for laboratory animals.

[0041] In some embodiments of this application, the female animal is a 10-12 month old female SD rat.

[0042] Specifically, the rats weighed 280-330g.

[0043] It is understandable that female SD rats aged 10-12 months correspond to humans aged 55-60 years, at which stage the ovarian function of female animals begins to decline naturally, which is highly consistent with the physiological state of humans after menopause.

[0044] In some embodiments of this application, the selected female animals are injected with GnRH agonists once a week, the preset feeding period is 6-8 weeks, and the frequency of oxidative stress stimulation is 2-3 times a week.

[0045] Specifically, each oxidative stress stimulus lasts for 24 hours.

[0046] In some embodiments of this application, the calcium content of the low-calcium feed is 0.1%-0.3%; the oxidative stress stimulus is: providing drinking water containing oxidizing substances.

[0047] Understandably, low-calcium feed should be commercially available and standardized, which can be purchased directly from the market.

[0048] In some embodiments of this application, the drinking water containing the oxidizing agent is an aqueous solution of H2O2 with a concentration of 0.05-0.2 mmol / L.

[0049] Understandably, the rats' drinking water was replaced with a 0.05-0.2 mmol / L H2O2 aqueous solution 2-3 times a week for 24 hours each time, while the rest of the time they were given regular distilled water, in order to avoid acute oxidative damage.

[0050] In some embodiments of this application, the standard for setting bone mineral density and trabecular bone parameters is: the bone mineral density of the rat lumbar vertebrae and femoral neck decreased by ≥15% compared with the treatment, and the number of trabecular bones decreased by ≥10% compared with the treatment.

[0051] Understandably, by measuring bone mineral density in the lumbar spine (a common site of PMOP in humans) and femoral neck (a site prone to fracture) using Micro-CT, a decrease of ≥15% corresponds to the degree of bone loss 5-8 years after menopause in humans. Individuals with "mild osteopenia" are excluded to ensure that the model meets the diagnostic criteria for PMOP. It is understandable that trabeculae are the main structure of cancellous bone. A decrease in their number and an increase in their separation will lead to a decrease in the weight-bearing capacity of the bones, which is a key reason why PMOP patients are prone to fractures. Detecting trabecular parameters can further verify the destruction of bone microstructure and avoid the "pseudo-PMOP model" caused by relying solely on bone density, such as decreased bone density but normal bone microstructure.

[0052] In some embodiments of this application, the specific site of the meniscus tear surgery is the anterior horn of the medial meniscus of the rat knee joint, and the tear length is 1 / 4 to 1 / 3 of the total length of the anterior horn of the meniscus.

[0053] Specifically, a tiny 2-3mm incision is made on the outside of the knee joint, a laparoscope and surgical instruments are inserted, the anterior horn of the medial meniscus is precisely located, and it is torn with microsurgical scissors.

[0054] In some embodiments of this application, the inflammatory factor is IL-1β with a loading of 30-70 ng / mL; the sustained-release microspheres are PLGA microspheres with a particle size of 30-150 μm.

[0055] Understandably, after implantation of PLGA microspheres loaded with IL-1β, IL-1β can be slowly released at the tear site, maintaining a low-concentration, long-term inflammatory environment in the local area, promoting chondrocyte apoptosis and cartilage matrix degradation, and synergistically inducing KOA with mechanical damage, thus avoiding the problem of short-lived acute inflammation and clinical inconsistency caused by traditional "single injection of inflammatory factors".

[0056] In some embodiments of this application, the weight training specifically involves: 0.5-2 hours of weighted treadmill training per day during the active period of the rats, with an initial load of 3%-7% of the rat's body weight, and an increase in load of 3%-7% of the rat's body weight per week; the continuous duration is set for 8-12 weeks.

[0057] Specifically, before each training session, check whether the weighted vest is fixed in place. During training, observe whether the rat exhibits abnormal gait or fatigue. If the rat stops moving or exhibits abnormal gait, training should be suspended. During continuous training, record the daily training duration and completion status of the animals.

[0058] Understandably, postoperative weight-bearing training simulates daily weight-bearing scenarios in humans through progressive loading, causing animals with pre-existing PMOP (low bone mass, weak joint load-bearing capacity) to repeatedly move their knee joints under load. This results in uneven stress on the cartilage at the meniscus tear site, accelerating cartilage degeneration. At the same time, the mechanical stimulation generated by the weight-bearing activates the local inflammatory response in the joint, which, in conjunction with the IL-1β released by the sustained-release microspheres, further aggravates KOA, ultimately leading to a comorbid state of "PMOP + KOA".

[0059] In some embodiments of this application, health checks on female animals at different time points include: cartilage status, inflammation-related, and bone metabolism-related tests at week 8 after modeling, and function-related and gut microbiota-related tests added at week 16 after modeling.

[0060] Understandably, week 8 after modeling is considered the early stage of comorbidity. At this time, cartilage degeneration, such as OARSI score; inflammation level, such as IL-1β level; and bone metabolic imbalance, such as OPG / RANKL ratio, are measured to confirm that PMOP and KOA coexist and are related, thus proving that the comorbidity model has been successfully constructed.

[0061] Week 16 after modeling is considered the late stage of comorbidity. At this time, functional-related tests are added, such as pain (tenderness threshold), joint function (flexion and extension range), fracture risk (vertebral compression strength), and gut microbiota testing. This can verify whether the model simulates the characteristics of human comorbidity, such as "increased pain, limited function, increased fracture risk, and gut-bone-joint interaction".

[0062] Specifically, during structural testing, rat knee cartilage was collected, and the OARSI score was calculated using tissue staining (such as Safranin O-Fix Green staining). The higher the score, the more severe the cartilage degeneration. Micro-CT was used to detect the microstructural parameters of the distal femur in rats. During inflammation testing, synovial fluid from the rat knee joint was extracted, and the IL-1β level was detected using ELISA. During metabolic testing, rat serum was collected, and the ratio of OPG (osteopeptide, which inhibits bone resorption) to RANKL (receptor activator of nuclear factor κB, which promotes bone resorption) was detected using ELISA. During gut microbiota testing, rat feces were collected, and the content of short-chain fatty acids (SCFAs) was detected to reflect the metabolic function of gut microbiota. During functional testing, the hindlimb tenderness threshold of rats was detected using a tenderness meter to reflect the degree of pain. Gait parameters, such as stride length and support time, were recorded using a gait analysis system to reflect joint function, and the range of motion of the knee joint was measured using calipers.

[0063] Understandably, rats were sacrificed 8 weeks after modeling to perform cartilage status, inflammation-related, and bone metabolism-related tests; 16 weeks after modeling, functional tests were performed first, then rats were sacrificed, and then cartilage status, inflammation-related, and bone metabolism-related tests were performed again.

[0064] It is understandable that the clinical evaluation of human comorbidities needs to take into account "structural abnormalities", "functional impairments" and "systemic associations", and a single test cannot verify the effectiveness of the model; this invention uses "multi-time point + multi-dimensional testing" to comprehensively verify whether the model conforms to the characteristics of human comorbidities.

[0065] Example 1 S1: Twenty 10-month-old SPF-grade female SD rats, weighing 280-320g, were selected. The lumbar vertebral bone mineral density of the rats was measured by Micro-CT before the operation as the baseline value, and the OARSI score of the knee joint cartilage was 0.

[0066] S2. Rats were subcutaneously injected with leuprorelin once a week, with the injection dose calculated at 0.1 mg / kg per rat. At the same time as the injection of leuprorelin, the rats were fed a low-calcium diet of 0.2% and given 0.1 mmol / L H2O2 aqueous solution twice a week for 24 hours each time. The rats were fed this diet for 6 weeks.

[0067] After 3 and 6 weeks, rats were examined by Micro-CT and screened for rats with a decrease of ≥15% in lumbar spine (L3-L5) bone mineral density, ≥15% in femoral neck bone mineral density, and ≥10% in trabecular bone number compared to baseline. 18 rats were selected as qualified, with a qualification rate of 90%.

[0068] S4. Selected rats underwent laparoscopic-assisted surgery to tear the anterior horn of the medial meniscus of the right knee joint, with the tear length being 1 / 3 of the total length of the anterior horn. During the surgery, PLGA microspheres loaded with 50 ng / mL IL-1β were implanted, with a microsphere particle size of 50-100 μm.

[0069] S5. One week after surgery, rats were subjected to progressive weight-bearing training. During the rats' active period from 19:00 to 20:00 each day, rats underwent 1 hour of weight-bearing treadmill training. The initial load was 5% of the rat's body weight, and the load was increased by 5% of the rat's body weight each week for 10 weeks to complete the establishment of an animal model of postmenopausal osteoporosis and knee osteoarthritis comorbidity.

[0070] S6. Health monitoring of rats: Eight weeks after modeling, nine rats were sacrificed, and bone mineral density, cartilage OARSI score, synovial fluid IL-1β, serum OPG / RANKL, and abundance of bifidobacteria in the gut microbiota were measured. Sixteen weeks after modeling, another nine rats were first tested for tenderness threshold, knee flexion and extension range, and open field distance, and then sacrificed. Bone mineral density, cartilage OARSI score, synovial fluid IL-1β, serum OPG / RANKL, and abundance of bifidobacteria in the gut microbiota were measured again, and the data were recorded.

[0071] Example test data (mean ± standard deviation)

[0072] Table 1 Comparison Example S1. Twenty 10-month-old SPF-grade female SD rats, weighing 280-320g, were selected. The lumbar vertebral bone mineral density of the rats was measured by Micro-CT before the operation as the baseline value, and the OARSI score of the knee joint cartilage was 0.

[0073] S2. Rats underwent bilateral oophorectomy (OVX) and were then fed a standard diet with 1.0% calcium and ordinary distilled water for 6 weeks.

[0074] After 3 and 6 weeks, rats were examined by Micro-CT and screened for rats with a decrease of ≥15% in lumbar spine (L3-L5) bone mineral density, ≥15% in femoral neck bone mineral density, and ≥10% in trabecular bone number compared to baseline. 14 rats were selected as qualified, with a qualification rate of 70%.

[0075] S4. Open knee joint surgery was performed on qualified rats to remove the anterior half of the lateral meniscus of the right knee joint, and microspheres without inflammatory factors were implanted.

[0076] S5: One week after surgery, rats were subjected to progressive weight-bearing training. Rats were given 1 hour of weight-bearing treadmill training at random times each day. The fixed load was 8% of the rat's body weight, with no progressive increase in load, for 10 weeks, thus completing the establishment of an animal model of postmenopausal osteoporosis and knee osteoarthritis comorbidity.

[0077] S6: Health monitoring of rats: Eight weeks after modeling, nine rats were sacrificed, and bone mineral density, cartilage OARSI score, synovial fluid IL-1β, serum OPG / RANKL, and abundance of bifidobacteria in the gut microbiota were measured. Sixteen weeks after modeling, another nine rats were first tested for tenderness threshold, knee flexion and extension range, and open field distance, and then sacrificed. Bone mineral density, cartilage OARSI score, synovial fluid IL-1β, serum OPG / RANKL, and abundance of bifidobacteria in the gut microbiota were measured again, and the data were recorded.

[0078] Control case test data (mean ± standard deviation)

[0079] Table 2 Comparison of core differences between the examples and control examples

[0080] Table 3 Table 1 shows the detection data of the examples, and Table 2 shows the detection data of the control examples. As shown in Table 3, comparing the examples and the control examples, it can be found that the examples simulated the progressive bone loss of human PMOP through "GnRH agonist + low calcium + oxidative stress", which decreased by 25.0% within 16 weeks, and the cartilage degeneration was concentrated on the medial side, consistent with human KOA; the control example OVX caused bone loss to be too rapid, reaching 37.0%, and the cartilage degeneration was on the lateral side, which was inconsistent with human clinical pathology.

[0081] The pass rate of rats in the example was 90%, which was significantly higher than the 70% of the control group. This was because the example reduced individual differences due to the multi-factor induction, and the screening criteria took into account both bone mineral density and trabecular bone parameters, thus excluding "resistant" rats.

[0082] The modeling example showed a sustained increase in IL-1β at 16 weeks, indicating chronic inflammation. Tenderness and decreased range of motion were consistent with the human comorbidity of progressively worsening pain. In contrast, the control group showed acute resolution of inflammation and excessive joint restriction, which could not simulate the clinical chronic disease course.

[0083] The abundance of Bifidobacteria in the gut was significantly reduced to 38.2% in the example, reflecting the interaction between the gut, bone, and joints. This indicates that clinical studies have confirmed that gut microbiota dysbiosis exacerbates comorbidity. The control group did not show this association and could not be used for cross-system mechanism studies.

[0084] In summary, the comorbidity model constructed in the examples is significantly superior to the traditional control model in terms of pathological progression, stability, and clinical fit. It can be used more accurately for mechanism research and drug evaluation of postmenopausal osteoporosis and knee osteoarthritis comorbidity.

[0085] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for constructing an animal model of comorbid postmenopausal osteoporosis and knee osteoarthritis, characterized in that, Includes the following steps: Female animals were selected as experimental subjects; Selected female animals were injected with GnRH agonists and fed a low-calcium diet and subjected to oxidative stress for a predetermined duration. Bone density and trabecular bone parameters of female animals after rearing were tested, and qualified female animals that met the set standards were selected. Meniscus tears were surgically removed from qualified female animals, and slow-release microspheres loaded with inflammatory factors were implanted at the tear site during the operation. Post-surgery, female animals underwent weight-bearing training for a set duration. Health checks were conducted on the female animals at different time points; The inflammatory factor is IL-1β, with a loading of 30-70 ng / mL; the sustained-release microspheres are PLGA microspheres with a particle size of 30-150 μm. The weight training specifically involves: 0.5-2 hours of weight-bearing treadmill training daily during the rat's active period, with an initial load of 3%-7% of the rat's body weight, and an increase in load of 3%-7% of the rat's body weight weekly. The duration of this setting is 8-12 weeks; The health checks on female animals at different time points included: cartilage status, inflammation-related, and bone metabolism-related tests at week 8 after modeling; and functional and gut microbiota-related tests at week 16 after modeling.

2. The method for constructing an animal model according to claim 1, characterized in that, The female animals were 10-12 month old female SD rats.

3. The method for constructing an animal model according to claim 2, characterized in that, The selected female animals were injected with GnRH agonists once a week, the preset rearing period was 6-8 weeks, and the oxidative stress stimulation was performed 2-3 times a week.

4. The method for constructing an animal model according to claim 3, characterized in that, The calcium content of the low-calcium feed is 0.1%-0.3%; the oxidative stress stimulus is: providing drinking water containing oxidizing substances.

5. The method for constructing an animal model according to claim 4, characterized in that, The drinking water containing the oxidizing substance is an aqueous solution of H2O2 with a concentration of 0.05-0.2 mmol / L.

6. The method for constructing an animal model according to claim 5, characterized in that, The standards for setting the bone mineral density and trabecular bone parameters are: the bone mineral density of the lumbar vertebrae and femoral neck of rats decreased by ≥15% compared with the pretreatment, and the number of trabecular bones decreased by ≥10% compared with the pretreatment.

7. The method for constructing an animal model according to claim 6, characterized in that, The specific location for the meniscus tear surgery was the anterior horn of the medial meniscus of the rat's knee joint, with a tear length of 1 / 4 to 1 / 3 of the total length of the anterior horn of the meniscus.

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