Animal model construction method for combined diseases of postmenopausal osteoporosis and knee osteoarthritis

Abstract

The invention relates to the field of disease animal model construction, and particularly discloses a postmenopausal osteoporosis and knee osteoarthritis co-disease animal model construction method which comprises the following steps: selecting a female animal as an experimental subject; the selected female animals are injected with a GnRH agonist, meanwhile, low-calcium feed feeding and oxidative stress stimulation are conducted, and feeding is conducted for a preset time; detecting bone mineral density and bone trabecula parameters of the fed female animals, and screening qualified female animals meeting set standards; performing meniscus tearing operation on the qualified female animal, and implanting sustained release microspheres loaded with inflammatory factors at the torn part during the operation; carrying out weight-bearing training on the female animal after the operation, and continuing for a set time length; and performing health detection on the female animals at different time points. According to the method, through multi-factor collaborative induction of the PMOP, minimally invasive construction of the KOA and multi-dimensional health detection, accurate simulation of a co-disease pathological process and clinical characteristics is realized, and the authenticity and stability of the model are improved.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of disease animal model construction, in particular to an animal model construction method for postmenopausal osteoporosis and knee osteoarthritis comorbidity. BACKGROUND

[0002] With the continuous rise of the incidence of postmenopausal osteoporosis (PMOP) and knee osteoarthritis (KOA) comorbidity in the middle-aged and elderly female population, the demand for reliable animal models for related pathological mechanism research and therapeutic drug research and development is increasingly urgent. Current model construction is mostly limited to single factor induction, without fully integrating the postmenopausal osteoporosis risk factors of "estrogen deficiency + low calcium intake + oxidative stress" in the clinic, and without restoring the pathological characteristics of knee osteoarthritis "chronic mechanical damage + local inflammation persistence", resulting in a large difference between the model and the actual pathological process of human comorbidity, making it difficult to support the development of precision research.

[0003] Traditional comorbidity models often induce postmenopausal osteoporosis through ovariectomy surgery, which can easily cause a sudden drop in estrogen, which does not match the gradual hormonal changes of natural menopause in humans, and does not superimpose low calcium, oxidative stress and other common risk factors in the clinic; Knee osteoarthritis induction often uses open surgery to remove the lateral meniscus, which not only causes acute trauma, but also causes cartilage degeneration to concentrate on the lateral platform, which is misplaced from the human characteristic of mainly medial cartilage degeneration, and lacks chronic inflammation control means, with acute resolution of inflammation, and the evaluation index only focuses on bone density and cartilage structure, ignoring function and system correlation, with poor model stability.

[0004] Therefore, it is necessary to design an animal model construction method for postmenopausal osteoporosis and knee osteoarthritis comorbidity to solve the problems of the prior art that the PMOP induction does not match the gradual hormonal changes of humans, the KOA degeneration site and inflammation characteristics deviate from the clinic, the model stability is low, and the evaluation system is single, which cannot accurately simulate the pathology of comorbidity and affects the accuracy of research and drug evaluation. SUMMARY

[0005] In view of this, the present application provides an animal model construction method for postmenopausal osteoporosis and knee osteoarthritis comorbidity to solve the problems of the prior art that the PMOP induction does not match the gradual hormonal changes of humans, the KOA degeneration site and inflammation characteristics deviate from the clinic, the model stability is low, and the evaluation system is single, which cannot accurately simulate the pathology of comorbidity and affects the accuracy of research and drug evaluation.

[0006] In one aspect, the present application provides an animal model construction method for postmenopausal osteoporosis and knee osteoarthritis comorbidity, comprising the following steps: selecting a female animal as the experimental object; injecting a GnRH agonist into the selected female animal, and simultaneously giving low calcium feed and oxidative stress stimulation, and feeding for a predetermined period of time. detect the bone density and trabecular bone parameters of the female animals after feeding, and screen qualified female animals meeting the set standards; perform a meniscus tear operation on the qualified female animals, and implant slow-release microspheres loaded with inflammatory factors at the tear site during the operation; postoperatively, the female animals are subjected to weight-bearing training for a set duration; at different time points, the female animals are subjected to health detection.

[0007] Further, the female animals are 10-12-month-old female SD rats.

[0008] Further, the frequency of injecting the selected female animals with GnRH agonists is once a week, the preset duration of feeding is 6-8 weeks, and the frequency of oxidative stress stimulation is 2-3 times a week.

[0009] Further, the calcium content of the low-calcium feed is 0.1%-0.3%, and the oxidative stress stimulation is to give drinking water containing oxidizing substances.

[0010] Further, the drinking water containing oxidizing substances is a 0.05-0.2 mmol / L H2O2 aqueous solution.

[0011] Further, the set standards of the bone density and trabecular bone parameters are that the bone density of the lumbar vertebrae and the femoral neck of the rats decreases by ≥15% compared with that before treatment, and the number of trabecular bones decreases by ≥10% compared with that before treatment.

[0012] Further, the specific site of the meniscus tear operation is the medial meniscus anterior horn in the knee joint of the rats, and the tear length is 1 / 4-1 / 3 of the total length of the meniscus anterior horn.

[0013] Further, the inflammatory factor is IL-1β, and the loading amount is 30-70 ng / mL; the slow-release microspheres are PLGA microspheres, and the particle size is 30-150 μm.

[0014] Further, the weight-bearing training specifically comprises: daily weight-bearing treadmill training for 0.5-2 hours during the active period of the rats, and the initial load is 3%-7% of the body weight of the rats, and the load is increased by 3%-7% of the body weight of the rats every week; and the set duration is 8-12 weeks.

[0015] Further, the health detection of the female animals at different time points comprises: at the 8th week after modeling, detecting the cartilage state, inflammation-related, and bone metabolism-related; and at the 16th week after modeling, increasing the function-related and intestinal flora-related detection.

[0016] Compared with the prior art, the present application has the following beneficial effects: The present application selects female animals as experimental objects, combines GnRH agonist injection, low calcium feed feeding and oxidative stress stimulation to induce postmenopausal osteoporosis, ensures the consistency of the model through imaging detection screening, and then precisely constructs knee osteoarthritis by combining meniscus tear with slow-release microspheres of inflammatory factors, simulates clinical mechanical load through weight-bearing training after operation, and finally carries out health detection at different time points, which not only avoids the acute trauma and hormone sudden drop of traditional surgery, but also restores the characteristics of "progressive pathology + interaction" of human comorbidity, improves the authenticity and stability of the model, and provides a reliable experimental carrier for comorbidity mechanism research and drug evaluation. BRIEF DESCRIPTION OF DRAWINGS

[0017] Various other advantages and benefits will become apparent to those of ordinary skill in the art upon reading the following detailed description of the preferred embodiments. The drawings are for purposes of illustration only and are not intended to limit the application thereto. Moreover, the use of the same reference symbols in different drawings indicates similar or identical items. In the drawings: Figure 1 The animal model construction method flow chart of postmenopausal osteoporosis and knee osteoarthritis comorbidity provided by the embodiments of the present application. DETAILED DESCRIPTION

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

[0019] In the description of the present application, it should be understood that the terms "center", "upper", "lower", "front", "rear", "left", "right", "vertical", "horizontal", "top", "bottom", "inner", "outer" and the like indicate the orientation or positional relationship shown in the drawings, and are only for the purpose of facilitating the description of the present application and simplifying the description, and do not indicate or imply that the device or element referred to must have a particular orientation, be constructed and operated in a particular orientation, therefore it cannot be understood as a limitation of the present application.

[0020] The terms "first", "second" are only for descriptive purposes, and cannot be understood as indicating or implying relative importance or implicitly indicating the number of the technical features indicated. Therefore, the features defined with "first", "second" can explicitly or implicitly include one or more of the features. In the description of the present application, unless otherwise stated, the meaning of "multiple" is two or more.

[0021] In the description of the present application, it should be noted that unless otherwise explicitly specified and limited, the terms "mounting", "connection", "connecting" should be understood in a broad sense, for example, it can be fixed connection, or detachable connection, or integral connection; it can be mechanical connection, or electrical connection; it can be direct connection, or indirect connection through intermediate medium, or internal communication of two elements. For those skilled in the art, the specific meaning of the above terms in the present application can be understood according to the specific circumstances.

[0022] In one aspect, as Figure 1 shown, in some embodiments of the present application, the method for constructing an animal model of postmenopausal osteoporosis and knee osteoarthritis comorbidity comprises the following steps: S1, selecting female animals as experimental objects; S2, injecting GnRH agonist to the selected female animals, and at the same time, giving low calcium feed for feeding and oxidative stress stimulation, and feeding for a predetermined period of time; S3, detecting the bone mineral density and trabecular bone parameters of the female animals after feeding, and screening qualified female animals meeting the set standard; S4, performing meniscus tear surgery on the qualified female animals, and implanting inflammation factor-loaded slow-release microspheres at the tear site during the surgery; S5, postoperatively, performing weight training on the female animals for a set period of time; S6, at different time points, performing health detection on the female animals.

[0023] Specifically, female animals of the same strain and batch are obtained from an experimental animal breeding center, and individuals with abnormal health are excluded through external observation, such as no hair loss and joint swelling, and basic physiological index detection, such as body weight fluctuation <5% and normal body temperature. Specifically, the GnRH agonist is selected as leuprolide, and the dose of the GnRH agonist is calculated according to the body weight of the female animals, such as 0.1 mg / kg, which is administered by intraperitoneal injection or subcutaneous injection; from the day of injection, low calcium feed is used to replace regular feed, and free feeding is ensured with sufficient drinking water during the period; the screened female animals are randomly divided into experimental and control groups, and are raised in single cages or group cages, 3-5 animals per cage, and after adapting to the environment for 1 week, they enter the subsequent steps.

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

[0025] It can be understood that the above embodiment completes the construction of an animal model of postmenopausal osteoporosis.

[0026] It can be understood that the role of GnRH agonist (gonadotropin-releasing hormone agonist) is to gradually inhibit the function of the "hypothalamic-pituitary-ovarian axis" of rats - initially stimulate the pituitary to secrete gonadotropin (LH, FSH) for a short time, but regular injection for a long time will cause the pituitary to desensitize to GnRH and no longer release LH and FSH; After the ovary loses the stimulation of gonadotropin, follicular development stops, and the amount of estrogen secretion decreases significantly, eventually reaching a state consistent with "natural postmenopausal ovarian function decline and estrogen deficiency" in humans.

[0027] It can be understood that continuous feeding for a predetermined length of time allows the bone metabolism imbalance caused by estrogen deficiency to fully manifest: estrogen deficiency will cause osteoclast activity to increase and osteoblast function to weaken, and calcium and other minerals in the skeleton will gradually be lost. This process is consistent with the clinical process of "slow bone loss" in postmenopausal women, rather than the "rapid hormone drop and bone loss" of traditional ovariectomy, and is closer to the real pathology; Low-calcium feed restricts calcium intake in animals, simulating the situation where the body is short of calcium reserves. When the body is short of calcium reserves, osteoclasts will accelerate the decomposition of bone tissue to release calcium to supplement blood calcium, further exacerbating the imbalance between "bone resorption > bone formation", promoting the proliferation and activity of osteoclasts, and inhibiting the synthesis of bone matrix (such as type I collagen) by osteoblasts, forming a "synergistic effect" with estrogen deficiency, and accelerating the decline in bone density and destruction of bone microstructure.

[0028] It can be understood that the above embodiments induce postmenopausal osteoporosis in female animals through multiple factors in synergy.

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

[0030] Specifically, after feeding for a predetermined length of time, the female animals are fasted for 12 hours to avoid interference from their gastrointestinal contents during imaging. Anesthetic agents such as isoflurane are used for mild anesthesia. The anesthetized female animals are fixed, and the lumbar vertebrae, such as the L3-L5 segments, and the bilateral femoral necks are scanned to obtain three-dimensional images. Image analysis software is used to calculate bone density, trabecular bone number, trabecular bone thickness, and trabecular bone separation. The baseline data of the female animals before feeding are compared. If no baseline data are detected, the female animals can be compared with the same batch of untreated female animals to screen out qualified female animal individuals, thereby excluding female animals with "GnRH resistance" and "low-calcium insensitivity" and ensuring that the starting point of subsequent comorbidity induction is consistent.

[0031] Specifically, the qualified female animals are subjected to preoperative fasting for 6 hours, and after anesthesia, the hair around the knee joints of the female animals is shaved, and the skin is disinfected. Pentobarbital sodium can be used for intraperitoneal injection for anesthesia.

[0032] Specifically, the female animals are subjected to a laparoscope-assisted meniscus tear operation, and a microsyringe is used to inject the slow-release microspheres loaded with inflammatory factors into the meniscus tear site to ensure that the microspheres are uniformly distributed in the damage area.

[0033] Specifically, after the operation, the incision is sutured, an antibiotic ointment is applied to prevent infection, and the female animals are individually raised, and the activity state is observed within 3 days after the operation, intense exercise is avoided, and normal activity is restored after 1 week.

[0034] It can be understood that the "meniscus tear" is selected instead of "meniscectomy" to simulate the chronic damage of the meniscus, such as tear and wear, which is common in human knee osteoarthritis, to avoid "acute joint instability and rapid cartilage degeneration" caused by resection surgery, and to better match the chronic progression characteristics of human knee osteoarthritis.

[0035] Specifically, starting from 1 week after the operation, the female animals are first adapted to the non-weight-bearing treadmill for 3 days, 10 minutes per day, to avoid direct weight-bearing stress; and the formal weight-bearing training starts from the 4th day after the operation.

[0036] It can be understood that the above embodiment completes the construction of the postmenopausal knee osteoarthritis animal model.

[0037] Specifically, in the early and late stages of the comorbidity after modeling, the female animals are subjected to health detection, covering the whole process from early cartilage degeneration to late dysfunction of knee osteoarthritis.

[0038] Specifically, the health detection refers to multidimensional detection of the pathological and functional state of the animals, including structural detection, inflammatory detection, metabolic detection, functional detection, and system detection.

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

[0040] It can be seen that the present application can improve the success rate of constructing the comorbidity model by screening the bone density + trabecular bone parameters to exclude individual differences (such as "resistant" animals); It can be seen that the present application can not only verify whether the model is established, but also reveal the cross-system interaction mechanism of the animal model, to provide a more comprehensive evaluation platform for the research and development of comorbidity treatment drugs; It can be seen that the present application uses GnRH agonist instead of surgical ovariectomy, and uses laparoscope-assisted minimally invasive surgery, which can reduce the risk of animal trauma and infection, and conforms to the "3R principle" (reducing pain and optimizing methods) of experimental animals.

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

[0042] Specifically, the rat weighs 280-330 g.

[0043] It can be understood that a 10-12 month old female SD rat corresponds to a human of 55-60 years old, and at this stage, the ovarian function of the female animal begins to naturally decline, which is highly matched with the postmenopausal physiological state of humans.

[0044] In some embodiments of the present application, the frequency of injecting the selected female animal with a GnRH agonist is once a week, the preset length of feeding is 6-8 weeks, and the frequency of oxidative stress stimulation is 2-3 times a week.

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

[0046] In some embodiments of the present application, the calcium content of the low-calcium feed is 0.1%-0.3%, and the oxidative stress stimulation is to provide drinking water containing oxidizing substances.

[0047] It can be understood that the low-calcium feed is a commercial standardized low-calcium feed, which can be directly purchased on the market.

[0048] In some embodiments of the present application, the drinking water containing oxidizing substances is a 0.05-0.2 mmol / L H2O2 aqueous solution.

[0049] It can be understood that the drinking water of the rat is replaced with a 0.05-0.2 mmol / L H2O2 aqueous solution 2-3 times a week, each time lasts for 24 hours, and ordinary distilled water is restored during the rest of the time, so as to avoid acute oxidative damage.

[0050] In some embodiments of the present application, the set standard of bone mineral density and trabecular bone parameters is that the bone mineral density of the lumbar vertebrae and the femoral neck of the rat decreases by ≥15% compared with that before the treatment, and the number of trabecular bone decreases by ≥10% compared with that before the treatment.

[0051] It can be understood that the bone mineral density of the lumbar vertebrae (a high-risk site of PMOP in humans) and the femoral neck (a site prone to fracture) is measured by Micro-CT, and a decrease of ≥15% corresponds to the bone loss degree of 5-8 years after menopause in humans, so as to exclude individuals with “mild osteopenia” and ensure that the model meets the PMOP diagnostic criteria. It can be understood that the trabecular bone is the main structure of cancellous bone, and a decrease in the number and an increase in the separation degree of the trabecular bone will lead to a decrease in the load-bearing capacity of the bone, which is a key reason why PMOP patients are prone to fracture. Detecting the trabecular bone parameters can further verify the damage to the bone microstructure and avoid “false PMOP model” caused by relying only on bone mineral density, such as a decrease in bone mineral density but normal bone microstructure.

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

[0053] Specifically, a micro-incision of 2-3 mm is made on the lateral side of the knee joint, a laparoscope and surgical instruments are inserted, and the "medial meniscus anterior horn" is accurately positioned, and a microscopical scissors is used to tear it.

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

[0055] It can be understood that after the IL-1β-loaded PLGA microspheres are implanted, IL-1β can be slowly released at the tear site, maintaining a local low-concentration, long-term inflammatory environment, promoting cartilage cell apoptosis and cartilage matrix degradation, and synergistically inducing KOA with mechanical damage, thereby avoiding the problem of acute inflammation that is short-lived and inconsistent with clinical practice caused by traditional "single injection of inflammatory factors".

[0056] In some embodiments of the present application, the weight-bearing training specifically includes: 0.5-2 hours of weight-bearing treadmill training per day during the active period of the rat, with an initial load of 3%-7% of the body weight of the rat, and an incremental load of 3%-7% of the body weight of the rat per week; and the continuous setting duration is 8-12 weeks.

[0057] Specifically, before each training, it is checked whether the rat's weight-bearing vest is fixed, and during the training, it is observed whether the rat has gait abnormalities and fatigue, such as stopping moving, and if gait abnormalities occur, the training is paused; and during the continuous training, the daily training duration and completion of the animal are recorded.

[0058] It can be understood that postoperative weight-bearing training simulates human daily weight-bearing scenarios through progressive loading, allowing animals with existing PMOP (low bone mass and weak joint bearing) to repeatedly move the knee joint under weight-bearing, causing uneven stress on the cartilage at the meniscus tear site and accelerating cartilage degeneration; at the same time, the mechanical stimulation produced by weight-bearing activates local inflammatory responses in the joint, which synergize with the release of IL-1β from the sustained-release microspheres to further exacerbate KOA, ultimately forming a "PMOP+KOA" comorbidity state.

[0059] In some embodiments of the present application, at different time points, the health detection of female animals includes: at 8 weeks after modeling, cartilage status, inflammation-related, and bone metabolism-related detection are performed, and at 16 weeks after modeling, functional-related detection and intestinal flora-related detection are added.

[0060] It can be understood that the 8th week after modeling is the early stage of comorbidity, at which time the detection of cartilage degeneration, such as OARSI score; inflammation level, such as IL-1β level; bone metabolism imbalance, such as OPG / RANKL ratio, is performed to confirm that PMOP and KOA have coexisted and are correlated with each other, thereby proving that the comorbidity model is successfully constructed.

[0061] The 16th week after modeling is the late stage of comorbidity, at which time function-related detection, such as pain: tenderness threshold, joint function: flexion and extension range, fracture risk: vertebral compression strength, and detection of intestinal flora, is increased, which can verify whether the model simulates the characteristics of human comorbidity, i.e., “increased pain, limited function, increased fracture risk, and intestinal-bone-joint interaction”.

[0062] Specifically, in structural detection, the knee joint cartilage of the rat is taken, OARSI score is calculated by tissue staining (such as Safranin O-fast green staining), and the higher the score, the more serious the cartilage degeneration; the bone microstructure parameters of the distal femur of the rat are detected by Micro-CT; in inflammation detection, the knee joint synovial fluid of the rat is extracted, and the IL-1β level is detected by ELISA method; in metabolism detection, the serum of the rat is collected, and the ratio of OPG (osteoprotegerin, which inhibits bone resorption) to RANKL (nuclear factor kappa B receptor activator ligand, which promotes bone resorption) is detected by ELISA method; in intestinal flora detection, the feces of the rat are collected, and the content of short-chain fatty acids (SCFAs) is detected to reflect the metabolic function of intestinal flora; in function detection, the tenderness threshold of the hind limb of the rat is detected by a tenderness instrument to reflect the degree of pain, and gait parameters such as step length and support time are recorded by a gait analysis system to reflect joint function, and the flexion and extension range of the knee joint is measured by a vernier caliper.

[0063] It can be understood that after the 8th week after modeling, the rats are sacrificed, and the cartilage state, inflammation-related, and bone metabolism-related detection are performed; after the 16th week after modeling, the function detection is performed first, then the rats are sacrificed, and the cartilage state, inflammation-related, and bone metabolism-related detection are performed.

[0064] It can be understood that the clinical evaluation of human comorbidity needs to consider “structural abnormalities”, “dysfunction” and “system correlation”, and single detection cannot verify the effectiveness of the model; the present application comprehensively verifies whether the model meets the characteristics of human comorbidity through “multi-time point + multi-dimensional detection”.

[0065] Example 1 S1: 20 SPF female SD rats aged 10 months and weighing 280-320 g are selected, the bone mineral density of the lumbar vertebrae of the rats is detected by Micro-CT as a baseline value before operation, and the OARSI score of the knee joint cartilage is 0.

[0066] S2, subcutaneously inject the rats with leuprolide once a week, and calculate the injection dose of each rat as 0.1 mg / kg; at the same time of injecting leuprolide, feed the rats with 0.2% low-calcium feed, and let the rats drink 0.1 mmol / L H2O2 aqueous solution twice a week, each time for 24 h; continue to feed the rats for 6 weeks.

[0067] S3, after 6 weeks, detect the rats by Micro-CT, and screen out the rats with a decrease of ≥15% in lumbar vertebra (L3-L5) bone density, a decrease of ≥15% in femoral neck bone density, and a decrease of ≥10% in trabecular bone number compared with the baseline, and 18 qualified rats are screened out, with a qualified rate of 90%.

[0068] S4, perform a surgery of tearing the right knee joint medial meniscus anterior horn under laparoscopic assistance on the qualified rats, and the tearing length is 1 / 3 of the total length of the anterior horn; implant PLGA microspheres loaded with 50 ng / mL IL-1β in the surgery, and the particle size of the microspheres is 50-100 μm.

[0069] S5, start the progressive weight-bearing training of the rats one week after the surgery, and perform 1 h weight-bearing treadmill training during the active period of the rats from 19:00 to 20:00 every day: the initial load is 5% of the body weight of the rats, and the body weight load of the rats is increased by 5% every week, and the training is continued for 10 weeks, so as to complete the construction of the postmenopausal osteoporosis and knee osteoarthritis co-morbidity animal model.

[0070] S6, monitor the health of the rats: after 8 weeks of modeling, sacrifice 9 rats, and detect the bone density, cartilage OARSI score, joint fluid IL-1β, serum OPG / RANKL, and intestinal flora Bifidobacterium abundance; after 16 weeks of modeling, first detect the pressure threshold, knee joint flexion range, and open field test activity distance of another 9 rats, then sacrifice the rats, and then detect the bone density, cartilage OARSI score, joint fluid IL-1β, serum OPG / RANKL, and intestinal flora Bifidobacterium abundance, and record the detection data.

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

[0072] Table 1 Control example S1, select 20 SPF female SD rats of 10 months old, with a body weight of 280-320 g, and detect the lumbar vertebra bone density of the rats by Micro-CT as the baseline value, and the knee joint cartilage OARSI score is 0.

[0073] S2, perform a bilateral ovariectomy (OVX) on the rats, and then feed the rats with conventional feed with a calcium content of 1.0% and let the rats drink ordinary distilled water, and continue to feed the rats for 6 weeks.

[0074] S3, 6 weeks later, rats were detected by Micro-CT, rats with lumbar spine (L3-L5) bone mineral density decreased by ≥15% compared with baseline, femoral neck bone mineral density decreased by ≥15% compared with baseline, and trabecular bone number decreased by ≥10% compared with baseline were screened out, 14 qualified rats were screened out, and the qualified rate was 70%.

[0075] S4, open knee surgery was performed on the qualified rats, the right knee joint lateral meniscus was removed by 1 / 2, and no inflammatory factor slow-release microspheres were implanted.

[0076] S5: The rats were subjected to progressive weight-bearing training 1 week after operation, and the rats were subjected to 1h weight-bearing treadmill training at random time every day: the fixed load was 8% of the body weight of the rats, there was no progressive incremental load, and the training lasted for 10 weeks, thus completing the construction of the animal model of postmenopausal osteoporosis and knee osteoarthritis comorbidity.

[0077] S6: The rats were monitored for health: 9 rats were sacrificed 8 weeks after modeling, and the bone mineral density, cartilage OARSI score, joint fluid IL-1β, serum OPG / RANKL, and intestinal flora Bifidobacterium abundance were detected; 9 other rats were detected for pressure threshold, knee joint flexion range, and open field test activity distance 16 weeks after modeling, then the rats were sacrificed, and the bone mineral density, cartilage OARSI score, joint fluid IL-1β, serum OPG / RANKL, and intestinal flora Bifidobacterium abundance were detected, and the detection data were recorded.

[0078] Control example detection data (mean ± standard deviation)

[0079] Table 2 Core difference comparison of example and control example data

[0080] Table 3 Table 1 is the detection data of the example, and Table 2 is the detection data of the control example. As shown in Table 3, it can be found that the example simulates the progressive bone loss of human PMOP by “GnRH agonist + low calcium + oxidative stress” within 16 weeks, with a decrease of 25.0%, and the cartilage degeneration is concentrated on the medial side, which is consistent with human KOA; the control example OVX causes bone loss too fast, with a decrease of 37.0%, and the lateral cartilage degeneration is inconsistent with human clinical pathology.

[0081] The qualified rate of the example rats is 90%, which is significantly higher than that of the control example, which is 70%. The example reduces individual differences due to multiple factors, and the screening standard considers bone mineral density and trabecular bone parameters, and excludes “resistant” rats.

[0082] The IL-1β of the embodiment model continuously increases at 16 weeks, showing chronic inflammation, tenderness, and decreased mobility, which is consistent with the characteristics of human comorbidity "progressive aggravation of pain"; the inflammation of the control example is acute and subsides, and the joint is excessively limited, which cannot simulate the clinical chronic course.

[0083] The abundance of intestinal bifidobacterium of the embodiment model significantly decreases by 38.2%, which reflects the interaction of "gut-bone-joint", and indicates that the clinical research confirms that the disorder of intestinal flora aggravates comorbidity, and the control example has no such correlation and cannot be used for cross-system mechanism research.

[0084] In summary, the comorbidity model constructed in the embodiment is significantly better than the traditional control example in terms of pathological process, stability, and clinical fitting degree, and can be more accurately used for mechanism research and drug evaluation of postmenopausal osteoporosis and knee osteoarthritis comorbidity.

[0085] The above only describes the preferred embodiments of the present application, and it should be noted that, for those skilled in the art, without departing from the principles of the present application, several improvements and refinements can be made, and these improvements and refinements should also be considered within the protection scope of the present application.

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.

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.

8. The method for constructing an animal model according to claim 7, characterized in that, 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.

9. The method for constructing an animal model according to claim 8, characterized in that, 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.

10. The method for constructing an animal model according to claim 9, characterized in that, 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.

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