Construction method of osteoarthritis animal model and application thereof

Abstract

The application provides a construction method of an osteoarthritis animal model and application thereof. The construction method of the application selects a standardized experimental Beagle dog and establishes an osteoarthritis model by using a modified Hulth method, and further processing is performed by selecting an induced exercise mode of the experimental dog to construct an animal model close to a clinical actual situation. The animal model obtained by the construction method of the application is close to an injury arthritis caused by an increasingly worn joint in an actual life, and the model has good stability, and the application provides a new animal model with important practical value for research and development of osteoarthritis drugs and medical devices.

Description

Technical Field

[0001] This invention relates to the field of osteoarthritis model establishment and application technology, specifically, to the establishment and application of an animal model that is particularly relevant to the clinical situation of knee osteoarthritis. Background Technology

[0002] Osteoarthritis (OA) is a degenerative joint disease that seriously affects patients' quality of life. It can lead to joint pain, deformity, and impaired mobility, thereby increasing the incidence of cardiovascular events and all-cause mortality, especially osteoarthritis of the knee.

[0003] Currently, animal models of OA mainly include spontaneous, induced, and transgenic animal models. Among them, induced models are more stable and require less time to establish, making them the most commonly used model. Induced models can be divided into surgical and non-surgical categories: surgical models have a high success rate and high reproducibility, making them suitable for research on post-traumatic OA and the best choice for short-term studies, with the widest application.

[0004] Animals used to induce osteoarthritis (OA) models are mostly small animals (rats, rabbits). However, small animals have small joints, which is not conducive to intra-articular manipulation, and their joints differ significantly from those of humans, making the experimental results less suitable for application to humans. In contrast, the joint structures of large animals are more similar to those of humans, making manipulation easier and allowing for a better simulation of the entire process of osteoarthritis development in humans. Furthermore, the effects of drugs can be predicted more accurately when using large animal OA surgical models. Therefore, it is essential to establish large animal OA surgical models and study their mechanisms of action.

[0005] Currently, most international reports on large animal-related research use sheep. However, sheep are not considered laboratory animals and lack standardized genetic background and basic data. Therefore, arthritis models prepared using sheep as experimental subjects cannot be widely adopted or applied. Canines, such as Beagle dogs, have a fixed and superior breed, stable and reproducible genetic performance, strong adaptability to the environment and disease resistance, and a docile temperament. They are widely recognized as ideal laboratory animals and are extensively used in medical research, especially in the development of new drugs and medical devices. However, an internationally recognized, complete, and reliable set of operational procedures and standards for constructing canine osteoarthritis models has not yet been established.

[0006] Therefore, there is an urgent need in this field to obtain a large animal model of osteoarthritis so that it can be applied as soon as possible to the research and development of human osteoarthritis drugs and medical devices to meet clinical needs. Summary of the Invention

[0007] To address the aforementioned technical problems, the purpose of this invention is to provide a method for constructing an animal model of osteoarthritis, wherein the method uses dogs to construct the animal model, thereby overcoming the limitations of current animal models of osteoarthritis.

[0008] Furthermore, another objective of this invention is to provide a method for constructing a canine model of osteoarthritis that is more closely aligned with clinical practice. This method incorporates the activity characteristics of clinical patients and, after constructing the animal model using dogs, further applies movement manipulations so that the resulting animal can simulate the actual clinical situation of osteoarthritis in human patients.

[0009] Furthermore, another object of the present invention is to provide applications for animal models obtained by the two construction methods described above, particularly in testing the effectiveness of compounds in treating osteoarthritis.

[0010] The technical solution of the present invention is as follows.

[0011] Firstly, This invention provides a method for constructing an animal model of osteoarthritis, the method comprising the following steps:

[0012] (1) Take experimental dogs, anesthetize them, shave the hair and prepare the skin in the surgical area of ​​the right hind leg knee joint, and disinfect the skin;

[0013] (2) Make a longitudinal incision on the medial side of the knee joint, cut through the joint cavity layer by layer, and then cut the anterior cruciate ligament.

[0014] (3) Remove the medial meniscus without damaging the articular cartilage surface;

[0015] (4) The joint capsule and skin were closed by suturing layer by layer. Immediately after the operation, penicillin sodium was injected intramuscularly to prevent infection. The injured limb was not fixed afterward, and the experimental dog was allowed to move freely in the cage.

[0016] Preferably, the osteoarthritis is knee osteoarthritis.

[0017] Preferably, the experimental dogs in step (1) are standardized experimental Beagle dogs, preferably 9-12 months old.

[0018] Preferably, in step (2), a 3cm longitudinal skin incision is made on the inner side of the knee joint. This skin incision is located next to the patella on the inner side of the joint capsule at the knee flexion point. After cutting through the joint cavity layer by layer, the patella is turned to the outer side of the knee joint, the knee joint is flexed, the anterior cruciate ligament is found, and it is cut.

[0019] Preferably, the construction method provided by the first aspect of the present invention further includes the following steps:

[0020] (5) Pathological characteristic indicators of the experimental dogs were detected at 6-10 weeks post-surgery, preferably at 8 weeks, to confirm successful modeling.

[0021] Preferably, the pathological characteristic indicators include staining of articular cartilage pathological sections or a Mankin pathological score based on the staining of such sections. The Mankin pathological score is an analytical score based on articular cartilage pathological sections. The constructed model dogs may exhibit elevated Mankin scores in areas such as cartilage appearance and structure.

[0022] Preferably, step (5) further includes detecting one or more of the following: behavioral characteristic indicators, joint swelling and pain characteristic indicators, and biological characteristic indicators of the experimental dog.

[0023] The behavioral characteristic indicators include at least one of lameness, motor coordination, and joint flexion. The constructed model dogs may exhibit at least one of the following: increased lameness score, decreased motor coordination, and reduced joint flexion.

[0024] The joint swelling and pain characteristics include at least one of joint deformity, knee joint circumference, and pain. The constructed model dogs may exhibit at least one of elevated joint deformity scores, increased knee joint circumference, and elevated pain scores.

[0025] The biological indicators include at least one of the following: inflammatory cytokines IL-1β, IL-6, and TNF-α; the free radical NO; the pain factor SP; and the cartilage metabolism marker CTX-II. The levels of these biological indicators refer to serum levels. The constructed dog model may exhibit elevated levels of at least one of the following: inflammatory cytokines IL-1β, IL-6, and TNF-α; the free radical NO; the pain factor SP; and the cartilage metabolism marker CTX-II.

[0026] Secondly, This invention provides a method for constructing an animal model of osteoarthritis that can simulate the clinical condition of arthritis patients. The method includes the following steps:

[0027] (1) Take experimental dogs, anesthetize them, shave the hair and prepare the skin in the surgical area of ​​the right hind leg knee joint, and disinfect the skin;

[0028] (2) Make a longitudinal incision on the medial side of the knee joint, cut through the joint cavity layer by layer, and then cut the anterior cruciate ligament.

[0029] (3) Remove the medial meniscus without damaging the articular cartilage surface;

[0030] (4) Suture layer by layer to close the joint capsule and skin. Immediately after the operation, administer sodium penicillin intramuscularly to prevent infection. After that, do not fix the injured limb. The experimental dog is allowed to move freely in the cage.

[0031] (5) Induce experimental dogs to exercise for 10-30 minutes every day for 6-10 weeks.

[0032] Preferably, the osteoarthritis is knee osteoarthritis.

[0033] Preferably, the experimental dogs in step (1) are standardized experimental Beagle dogs, preferably 9-12 months old.

[0034] Preferably, in step (2), a 3cm longitudinal skin incision is made on the inner side of the knee joint. This skin incision is located next to the patella on the inner side of the joint capsule at the knee flexion point. After cutting through the joint cavity layer by layer, the patella is turned to the outer side of the knee joint, the knee joint is flexed, the anterior cruciate ligament is found, and it is cut.

[0035] Preferably, step (5) lasts for 8 weeks.

[0036] Preferably, in step (5), the daily exercise induction of the experimental dogs is achieved by placing toys and playing music to induce the experimental dogs to exercise.

[0037] Preferably, in step (5), the experimental dogs are induced to exercise for 20 minutes each day.

[0038] Preferably, the construction method provided by the second aspect of the present invention further includes the following steps:

[0039] (6) The pathological characteristics of the experimental dogs were detected at 6-10 weeks post-surgery, preferably at 8 weeks, to confirm successful modeling.

[0040] Preferably, the pathological characteristic indicators include staining of articular cartilage pathological sections or a Mankin pathological score based on the staining of such sections. The Mankin pathological score is an analytical score based on articular cartilage pathological sections. The constructed model dogs may exhibit elevated Mankin scores in areas such as cartilage appearance and structure.

[0041] Preferably, step (6) further includes detecting one or more of the following: behavioral characteristic indicators, joint swelling and pain characteristic indicators, and biological characteristic indicators of the experimental dog.

[0042] The behavioral characteristic indicators include at least one of lameness, motor coordination, and joint flexion. The constructed model dogs may exhibit at least one of the following: increased lameness score, decreased motor coordination, and reduced joint flexion.

[0043] The joint swelling and pain characteristics include at least one of joint deformity, knee joint circumference, and pain. The constructed model dogs may exhibit at least one of elevated joint deformity scores, increased knee joint circumference, and elevated pain scores.

[0044] The biological indicators include at least one of the following: inflammatory cytokines IL-1β, IL-6, and TNF-α; the free radical NO; the pain factor SP; and the cartilage metabolism marker CTX-II. The levels of these biological indicators refer to serum levels. The constructed dog model may exhibit elevated levels of at least one of the following: inflammatory cytokines IL-1β, IL-6, and TNF-α; the free radical NO; the pain factor SP; and the cartilage metabolism marker CTX-II.

[0045] Thirdly, This invention provides a method for identifying whether a compound is effective in treating osteoarthritis, the method comprising the following steps:

[0046] (1) An osteoarthritis animal model dog is constructed by the method provided in the first or second aspect of the present invention, and then the pathological characteristic indicators of the model dog are detected to obtain the first detection data;

[0047] (2) The compound to be identified was administered to the model dog;

[0048] (3) The pathological characteristic indicators of the model dog were detected again to obtain the second detection data;

[0049] (4) Compare the second detection data with the first detection data.

[0050] Preferably, the osteoarthritis is knee osteoarthritis.

[0051] The pathological features include staining of articular cartilage pathological sections or Mankin pathological scores based on such staining. The Mankin pathological score is an analytical score based on articular cartilage pathological sections. The model dogs may exhibit elevated Mankin scores in areas such as cartilage appearance and structure. In the method provided in the third aspect of the invention, after comparing the second detection data with the first detection data in step (4), if a decrease in the Mankin score is found in the model dog, the compound is identified as effective for treating osteoarthritis.

[0052] Preferably, in steps (1) and (3), one or more of the behavioral characteristic indicators, joint swelling and pain characteristic indicators, and biological characteristic indicators of the model dog are also detected.

[0053] The behavioral characteristic indicators include at least one of lameness, motor coordination, and joint flexion. The model dog may exhibit at least one of increased lameness score, decreased motor coordination, and decreased joint flexion. In the method provided in the third aspect of the present invention, after comparing the second detection data with the first detection data in step (4), if it is found that the model dog exhibits at least one of decreased lameness score, increased motor coordination, and increased joint flexion, then the compound is identified as effective for the treatment of osteoarthritis.

[0054] The joint swelling and pain characteristics include at least one of joint deformity, knee joint circumference, and pain. The model dog may exhibit at least one of elevated joint deformity score, increased knee joint circumference, and elevated pain score. In the method provided in the third aspect of the present invention, after comparing the second detection data with the first detection data in step (4), if it is found that the model dog exhibits at least one of decreased joint deformity score, decreased knee joint circumference, and decreased pain score, then the compound is identified as effective for the treatment of osteoarthritis.

[0055] The biological indicators include at least one of the following: inflammatory factors IL-1β, IL-6, and TNF-α; free radical NO; pain factor SP; and cartilage metabolism marker CTX-II. The levels of these biological indicators refer to serum levels. The model dogs may exhibit elevated levels of at least one of the following: inflammatory factors IL-1β, IL-6, and TNF-α; free radical NO; pain factor SP; and cartilage metabolism marker CTX-II. In the method provided in the third aspect of the invention, after comparing the second detection data with the first detection data in step (4), if the model dogs are found to have decreased levels of at least one of the following: inflammatory factors IL-1β, IL-6, and TNF-α; free radical NO; pain factor SP; and cartilage metabolism marker CTX-II, then the compound is identified as effective for the treatment of osteoarthritis.

[0056] This invention provides a method for constructing an animal model of osteoarthritis and its application. Compared with the prior art, this invention has the following technical improvements and effects.

[0057] First, the inventors of this invention comprehensively considered the differences in joints between different animal species and humans, and selected standardized experimental Beagle dogs as the model animal. Compared with existing arthritis models in small animals such as rats and rabbits, the joint structure of Beagle dogs is more similar to that of humans, the modeling time is shorter, the success rate is higher, the model severity is moderate, and the constructed model is more stable. Compared with existing arthritis models in large animals such as sheep, Beagle dogs are standardized experimental animals, meet ethical requirements, and have clear genetic information and background databases.

[0058] Furthermore, after selecting Beagle dogs as the experimental model animal, the inventors of this invention comprehensively considered the effects of various arthritis induction methods on the model animal and chose the modified Hulth method to establish the osteoarthritis model. This method has a short surgical modeling time, is less invasive, can effectively avoid and prevent intraoperative and postoperative infections, and has a higher success rate.

[0059] Furthermore, considering that exercise is a major clinical cause of osteoarthritis and one of the main aggravating factors in the disease process, the inventors of this invention further enhanced the arthritis condition of the experimental dogs by inducing them to exercise, in order to better reflect the actual clinical situation of osteoarthritis. Appropriately increasing exercise can further aggravate joint damage and accelerate the progression of arthritis, but excessive exercise can lead to a severe animal model and even affect health. However, if this factor is not applied and the animals are allowed to move freely in their cages, the limited space does not match the actual clinical situation, and the model progresses slowly with a long modeling time. Therefore, the inventors of this invention chose to induce 20 minutes of exercise in the dogs daily, further optimizing the model construction method, thereby obtaining an animal model that is closer to clinical reality. Furthermore, experiments have shown that the animals already exhibited mild to moderate arthritis 8 weeks after modeling.

[0060] In addition, the model construction process of this invention also includes monitoring animal pathological characteristic indicators, behavioral characteristic indicators, joint swelling and pain characteristic indicators, and biological characteristic indicators.

[0061] (1) Pathological characteristics: Histopathology is the gold standard for assessing the progression of osteoarthritis.

[0062] (2) Behavioral and joint swelling and pain characteristics indicators: These are assessments of pain, joint function, and gait, which can make animal traumatic osteoarthritis models more closely resemble the clinical pathological changes of the disease. They are an indispensable part of the clinical relevance and practicality of translational research models. Small animals are limited by species characteristics, body size, and gait, making it more difficult to assess indicators such as pain and joint dysfunction. However, large animals can be assessed accordingly.

[0063] (3) Biological characteristics indicators (inflammatory factors IL-1β, IL-6 and TNF-α, free radical NO, pain factor SP and cartilage metabolism marker CTX-II levels): 1) Inflammatory response is closely related to the symptoms and disease progression of OA. Among them, inflammatory factors IL-1β, IL-6 and TNF-α participate in and mediate a variety of inflammatory response processes, affecting the degradation process of articular cartilage; 2) Clinically, the NO level in the body of early OA patients is significantly increased. Animal experiments have reported that excessively high concentrations of NO in the body can promote the destruction of articular cartilage. Therefore, reducing the excessive release of NO can delay the destruction of cartilage and thus delay the development of arthritis; 3) Peripheral SP acts as a pain mediator to transmit nociceptive stimuli to the spinal cord. In addition to physical factors, joint pain in OA patients is also related to substance P; 4) Cartilage is composed of special chondrocytes, which produce a large number of collagen extracellular matrix proteins, including proteoglycans and type II collagen, to maintain the homeostasis of articular cartilage and thus maintain its integrity; CTX-II is a type II collagen fragment after cartilage degradation and is a biomarker of cartilage degradation.

[0064] In the embodiments of this invention, meloxicam, a mainstream drug for acute arthritis flare-ups, was used to validate the animal model. Experiments demonstrated that the animal model constructed using the method of this invention closely resembles the traumatic arthritis caused by increasing joint wear in real life, and exhibits good model stability. Therefore, the animal model constructed using the method of this invention can be applied to the research and development of drugs and medical devices, especially providing a new and practically valuable animal model for the development of drugs and medical devices for osteoarthritis. Attached Figure Description

[0065] The embodiments of the present invention will now be described in detail with reference to the accompanying drawings, wherein:

[0066] Figure 1 Histopathological atlas of the model control group at week 8 after modeling.

[0067] Figure 2 Histopathological atlas of the sham surgery group at week 8 after modeling.

[0068] Figure 3 Histopathological atlas of the positive control group at week 12 after modeling (8 weeks of modeling + 4 weeks of drug administration). Detailed Implementation

[0069] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0070] It should be understood that the terminology used in this invention is merely a description of particular embodiments and is not intended to limit the invention. Furthermore, numerical ranges in this invention should be understood to specifically disclose the upper and lower limits of the range and intermediate values ​​between them. Every smaller range between any stated value or intermediate value within a stated range, and any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0071] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation and testing of this invention. All references to this specification are incorporated by way of inclusion and are used to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0072] Those skilled in the art should understand that the numbering of the steps is merely for distinguishing different steps and does not indicate the order of the steps. The order of the steps is not particularly limited as long as the purpose of this invention is achieved. Furthermore, those skilled in the art should understand that other steps or operations may be included between or before any step, for example, to further optimize or improve the method described in this invention.

[0073] Construction of animal models:

[0074] A first aspect of the present invention provides a method for constructing an animal model, comprising the following steps:

[0075] (1) Take a Beagle dog, and after anesthesia, shave the skin of the surgical area of ​​the right hind leg knee joint and disinfect the skin as usual to prepare for the operation.

[0076] (2) Make a 3cm longitudinal skin incision on the medial side of the knee joint (the skin incision is on the medial side of the joint capsule next to the patella at the knee flexion). After cutting through the joint cavity layer by layer, turn the patella to the lateral side of the knee joint, flex the knee joint, expose the anterior cruciate ligament, and cut it.

[0077] (3) Locate the medial meniscus and remove it without damaging the articular cartilage.

[0078] (4) Suture layer by layer, and routinely close the joint capsule and skin. Immediately after the operation, administer an intramuscular injection of sodium penicillin to prevent infection. The injured limb is not immobilized after the operation and can move freely for 8 weeks.

[0079] (5) Detect behavioral characteristic indicators, joint swelling and pain characteristic indicators, biological characteristic indicators and pathological characteristic indicators after step (4).

[0080] In the construction method of this invention, the sex of the animal used to construct the animal model is not particularly limited, and both male and female can be used. Since there are few experimental dog breeds, Beagle dogs are the main type, with Beagle dogs being the preferred choice. Their age is generally 9-12 months.

[0081] In the construction method of this invention, the manner of joint injury is not particularly limited, as long as it can cause joint injury, including drug-based or surgical methods. In an exemplary experimental scheme, this invention is performed surgically, using a modified Hulth method, namely, severing the anterior cruciate ligament and removing the medial meniscus.

[0082] In the construction method of this invention, after the surgical model is completed, the animal's behavioral characteristics, joint swelling and pain characteristics, biological characteristics, and / or pathological characteristics are further tested to confirm and optimize the construction conditions. The behavioral characteristics include the detection of at least one of lameness, motor coordination, and joint flexion. The joint swelling and pain characteristics include the detection of at least one of joint deformity, knee joint circumference, and pain. The biological characteristics include the detection of at least one of the following: inflammatory factors IL-1β, IL-6, and TNF-α; free radical NO; pain factor SP; and cartilage metabolism marker CTX-II levels. The pathological characteristics include the Mankin pathology score. In this invention, the detection time for behavioral characteristics, joint swelling and pain characteristics, and biological characteristics is not particularly limited. For example, detection can be performed before modeling, at week 4, week 8, and week 12 after modeling; pathological characteristics are detected at week 8 and week 12 after modeling.

[0083] Construction of animal models simulating the clinical etiology of arthritis patients:

[0084] A second aspect of the present invention provides a method for constructing an animal model of osteoarthritis capable of simulating the clinical condition of arthritis patients, mainly comprising:

[0085] (a) Construct animal models according to model construction methods (1)-(4).

[0086] (b) In the laboratory anteroom, experimental dogs were induced to exercise for 20 minutes daily for 8 weeks.

[0087] (c) Test behavioral characteristic indicators, joint swelling and pain characteristic detection indicators, biological characteristic indicators, and pathological characteristic indicators after step (b).

[0088] In the simulation method of this invention, exercise is further applied to the animals that have completed the surgical modeling, generally inducing the experimental dogs to exercise for 20 minutes per day. Appropriately increasing exercise can further aggravate joint damage and accelerate the progression of arthritis; excessive exercise may lead to a more severe animal model, even affecting their health. Without this factor, allowing the animals to move freely in their cages results in limited space, which does not match the actual clinical situation of patients, and the model progresses slowly with a long modeling time. Experiments have shown that the pathological histological score at 8 weeks confirms mild to moderate arthritis.

[0089] Methods for identifying compounds effective in treating arthritis:

[0090] A third aspect of the present invention provides a method for identifying whether a compound is effective in treating osteoarthritis, which mainly includes the following steps:

[0091] (i) Test the behavioral characteristics, joint swelling and pain characteristics, biological characteristics and / or pathological characteristics of the animal model to obtain the first test data.

[0092] (ii) The compound to be identified was administered to the animal model;

[0093] (iii) Test the behavioral, joint swelling and pain, biological and / or pathological indicators of the animal model after administration of the compound to be identified to obtain the second test data.

[0094] (iv) Compare the second test data with the first test data.

[0095] In the identification method of this invention, the animal model is obtained by the construction method of the first or second aspect of this invention. In some embodiments, the behavioral characteristic indicators of the animal model include at least one of increased lameness score, decreased motor coordination ability, and decreased joint flexion; if, after administration of the compound to be identified, the animal model exhibits at least one of decreased lameness score, increased motor coordination ability, and increased joint flexion, then the compound is identified as effective in treating arthritis.

[0096] In some implementations, the animal model exhibits at least one of the following joint swelling and pain characteristics: increased joint deformity score, increased knee joint circumference, and increased pain score; if, after administration of the compound to be identified, the animal model exhibits at least one of the following: decreased joint deformity score, decreased knee joint circumference, and decreased pain score, then the compound is identified as effective in treating arthritis.

[0097] In some embodiments, at least one of the biological characteristic indexes of the animal model shows an increase in the levels of inflammatory factors IL-1β, IL-6, and TNF-α, free radical NO, pain factor SP, and cartilage metabolism marker CTX-II; if after administering the compound to be identified, at least one of the levels of inflammatory factors IL-1β, IL-6, and TNF-α, free radical NO, pain factor SP, and cartilage metabolism marker CTX-II in the animal model decreases, then the compound is identified as effective in treating arthritis.

[0098] In some embodiments, the pathological characteristic indexes of the animal model include an increase in the Mankin score such as cartilage appearance and cartilage structure; if after administering the test compound, the Mankin score of the animal model decreases, then the compound is identified as effective in treating arthritis.

[0099] The following are specific embodiments of the present invention.

[0100] Example 1

[0101] I. Experimental methods

[0102] 1. Animals

[0103] The research of the present invention has obtained the approval of the Animal Use and Management Committee of Guangzhou Institute of Pharmaceutical Research, and its animal welfare and ethics have been approved. 9-12-month-old Beagle dogs were purchased from Guangzhou Institute of Pharmaceutical Research Co., Ltd., and the experimental animal production license number: SCXK(Guangdong)2023-0007 (valid until June 26, 2028, issued by the Guangdong Provincial Department of Science and Technology). The experimental animal quality certificate number: 44006900001087. The animals were housed in a conventional environment at a temperature of 18-26 °C, a humidity of 40%-70%, with a 12h / 12h light-dark cycle, and free access to food and water. The experiment started after 2 weeks of quarantine and feeding.

[0104] 2. Grouping. The animals were divided into a sham operation group of 6 and an operation group of 6.

[0105] 3. Operation group: The anterior cruciate ligament was surgically transected and the medial meniscus was resected; Sham operation group: Except for not transecting the anterior cruciate ligament and not resected the medial meniscus, the remaining operations were the same as those in the operation group.

[0106] 4. Selection of exercise intervention factors and research steps

[0107] The present invention selects inducing experimental dogs to exercise as the exercise factor for research. The research steps are as follows:

[0108] 4.1 After the operation, the animals had free access to food and water.

[0109] 4.2 Every day, the animals were moved to the laboratory for induced exercise. The sham surgery group and the surgical group were separated, and music was played. Toy dog ​​bones were placed in the laboratory to encourage the dogs to keep moving.

[0110] 4.3 The induction exercise time is 20 minutes per day.

[0111] 4.4 The duration was 12 weeks. After 8 weeks, 2 dogs in the sham surgery group and 2 dogs in the surgical group were euthanized, and pathological characteristics were used to confirm successful modeling.

[0112] 5. Behavioral characteristic indicator detection

[0113] 5.1 Limping score

[0114] 6. Detection of characteristic indicators of joint swelling and pain

[0115] 6.1 Pain score

[0116] 7. Detection of biological characteristic indicators

[0117] 7.1 Inflammatory factors IL-1β, IL-6, TNF-α

[0118] Inflammatory factor detection kits: IL-1β, purchased from Shanghai Enzyme-Linked Biotechnology Co., Ltd., batch number Jul 2023; IL-6, purchased from Shanghai Enzyme-Linked Biotechnology Co., Ltd., batch number Jul 2023;

[0119] TNF-α, purchased from Shanghai Guduo Biotechnology Co., Ltd., batch number 202307.

[0120] 7.2 Free radical NO

[0121] The NO content determination kit was purchased from Shanghai Varan Biotechnology Co., Ltd., batch number 2023.07.27.

[0122] 7.3 Pain Factor SP

[0123] Pain factor detection kit: SP, purchased from Shanghai Enzyme-Linked Biotechnology Co., Ltd., batch number Jul 2023.

[0124] 7.4 Cartilage Metabolizing Factor CTX-II

[0125] Bone metabolism factor assay kit: CTX-II, purchased from Shanghai Enzyme-Link Biotechnology Co., Ltd., batch number Jul2023.

[0126] 8. Joint pathological characteristic index detection

[0127] Based on scoring indicators (cartilage tissue structure, changes in cell number, HE staining, and tidal line integrity).

[0128] Perform a comprehensive Mankin score.

[0129] 9. Statistical Analysis

[0130] Example 2

[0131] Meloxicam, a treatment drug for osteoarthritis, was administered to dog models obtained using the construction method of this invention, and various characteristic indicators of the dog models were measured. Meloxicam is a mainstream treatment drug for acute flare-ups of osteoarthritis. The research steps are as follows.

[0132] Eighteen Beagle dogs aged 9-12 months were obtained and randomly divided into three groups: a sham surgery group, a model control group, and a positive control group, with six dogs in each group. As described in Example 1, sham surgery was performed on the dogs in the sham surgery group, while surgical modeling was performed on the dogs in the model control group and the positive control group. After modeling, the experimental dogs were induced to exercise for 20 minutes daily.

[0133] Eight weeks after model establishment, two dogs from the "model control group" and "positive control group" were euthanized, and pathological indicators were measured to confirm successful model establishment. Then, the remaining four dogs in the "positive control group" were given meloxicam injection (purchased from Qilu Animal Health Products Co., Ltd., batch number 914301002) for four weeks. The administration method was subcutaneous injection of meloxicam injection, with an initial dose of 0.2 mg / kg and a maintenance dose of 0.1 mg / kg, once daily for 28 consecutive days.

[0134] Behavioral, joint swelling and pain, biological, and pathological indicators of dogs in each group were measured before modeling and at weeks 4, 8, and 12 after modeling.

[0135] The test data of each group of dogs were compared and statistically analyzed. The results of the indicator tests are shown in Tables 1 to 9; the pathological histological results are shown in... Figures 1 to 3 .

[0136] Table 1. Behavioral Characteristic Indicators – Limping Score (x±s)

[0137]

[0138] Note: Compared with the sham surgery group, "*" indicates P<0.05 and "**" indicates P<0.01. Compared with the model control group, "#" indicates P<0.05 and "##" indicates P<0.01.

[0139] Table 2. Characteristic indicators of joint swelling and pain – Pain score (x±s)

[0140]

[0141] Note: Compared with the sham surgery group, "*" indicates P<0.05 and "**" indicates P<0.01. Compared with the model control group, "#" indicates P<0.05 and "##" indicates P<0.01.

[0142] Table 3. Biological characteristic indicators—inflammatory cytokine IL-1β (x±s, pg / mL)

[0143]

[0144] Note: Compared with the sham surgery group, "*" indicates P<0.05 and "**" indicates P<0.01. Compared with the model control group, "#" indicates P<0.05 and "##" indicates P<0.01.

[0145] Table 4. Biomarker – Inflammatory Factor IL-6 (x±s, pg / mL)

[0146]

[0147] Note: Compared with the sham surgery group, "*" indicates P<0.05 and "**" indicates P<0.01. Compared with the model control group, "#" indicates P<0.05 and "##" indicates P<0.01.

[0148] Table 5. Biomarker – Inflammatory Factor TNF-α (x±s, pg / mL)

[0149]

[0150] Note: Compared with the sham surgery group, "*" indicates P<0.05 and "**" indicates P<0.01. Compared with the model control group, "#" indicates P<0.05 and "##" indicates P<0.01.

[0151] Table 6. Biomarker – Free Radical NO (x±s, μmol / L)

[0152]

[0153] Note: Compared with the sham surgery group, "*" indicates P<0.05 and "**" indicates P<0.01. Compared with the model control group, "#" indicates P<0.05 and "##" indicates P<0.01.

[0154] Table 7. Biomarker – Pain Factor SP (x±s, pg / mL)

[0155]

[0156]

[0157] Note: Compared with the sham surgery group, "*" indicates P<0.05 and "**" indicates P<0.01. Compared with the model control group, "#" indicates P<0.05 and "##" indicates P<0.01.

[0158] Table 8. Biomarker – Cartilage Metabolite CTX-II (x±s, ng / mL)

[0159]

[0160] Note: Compared with the sham surgery group, "*" indicates P<0.05 and "**" indicates P<0.01. Compared with the model control group, "#" indicates P<0.05 and "##" indicates P<0.01.

[0161] Table 9. Pathological Feature Indicators – Mankin Pathological Score (x±s, n=4)

[0162]

[0163] Note: Compared with the sham surgery group, "*" indicates P<0.05 and "**" indicates P<0.01. Compared with the model control group, "#" indicates P<0.05 and "##" indicates P<0.01.

[0164] Although the present invention has been described with reference to exemplary embodiments, it should be understood that the invention is not limited to the disclosed embodiments. Various adjustments or changes may be made to the exemplary embodiments described in this specification without departing from the scope or spirit of the invention. The scope of the claims should be interpreted broadly to cover all modifications and equivalent functions.

Claims

1. A method for constructing an animal model of osteoarthritis, wherein the osteoarthritis is knee osteoarthritis, the method comprising the following steps: (1) Take experimental dogs, anesthetize them, shave the hair and prepare the skin in the surgical area of ​​the right hind leg knee joint, and disinfect the skin; (2) Make a 3cm longitudinal skin incision on the medial side of the knee joint. The skin incision is located next to the patella on the medial side of the joint capsule at the knee flexion. After cutting through the joint cavity layer by layer, turn the patella to the lateral side of the knee joint, flex the knee joint, find and cut the anterior cruciate ligament. (3) Remove the medial meniscus without damaging the articular cartilage surface; (4) Suture layer by layer to close the joint capsule and skin. Immediately after the operation, administer an intramuscular injection of sodium penicillin to prevent infection. After that, do not fix the injured limb. The experimental dogs are allowed to move freely in the cage. (5) Pathological feature indicators were detected 6-10 weeks after surgery to confirm successful modeling. The pathological feature indicators included staining of articular cartilage pathological sections or Mankin pathological scores based on the staining of the articular cartilage pathological sections. Furthermore, one or more of the following indicators were detected in the experimental dogs: behavioral characteristics, joint swelling and pain characteristics, and biological characteristics. The behavioral characteristics included at least one of lameness, motor coordination, and joint flexion. The joint swelling and pain characteristics included at least one of joint deformity, knee joint circumference, and pain. The biological characteristics included at least one of the following: inflammatory factors IL-1β, IL-6, and TNF-α, free radical NO, pain factor SP, and cartilage metabolism marker CTX-II levels.

2. The construction method according to claim 1, characterized in that, In step (1), the experimental dogs are standardized experimental Beagle dogs.

3. The construction method according to claim 2, characterized in that, The experimental Beagle dogs were 9-12 months old.

4. The construction method according to any one of claims 1 to 3, characterized in that, In step (5), pathological characteristic indicators are detected at 8 weeks post-surgery to confirm successful modeling.

5. A method for constructing an animal model of osteoarthritis capable of simulating the clinical condition of arthritis patients, wherein the osteoarthritis is knee osteoarthritis, the method comprising the following steps: (1) Take experimental dogs, anesthetize them, shave the hair and prepare the skin in the surgical area of ​​the right hind leg knee joint, and disinfect the skin; (2) Make a 3cm longitudinal skin incision on the medial side of the knee joint. The skin incision is located next to the patella on the medial side of the joint capsule at the knee flexion. After cutting through the joint cavity layer by layer, turn the patella to the lateral side of the knee joint, flex the knee joint, find and cut the anterior cruciate ligament. (3) Remove the medial meniscus without damaging the articular cartilage surface; (4) Suture layer by layer to close the joint capsule and skin. Immediately after the operation, administer an intramuscular injection of sodium penicillin to prevent infection. After that, do not fix the injured limb. The experimental dogs are allowed to move freely in the cage. (5) Induce experimental dogs to exercise for 10-30 minutes every day for 6-10 weeks; (6) The pathological characteristics of the experimental dogs were tested from week 6 to week 10 after surgery to confirm the successful modeling. The pathological characteristics included the staining of articular cartilage pathological sections or the Mankin pathological score based on the staining of the articular cartilage pathological sections. Furthermore, one or more of the following indicators were detected in the experimental dogs: behavioral characteristics, joint swelling and pain characteristics, and biological characteristics. The behavioral characteristics included at least one of lameness, motor coordination, and joint flexion. The joint swelling and pain characteristics included at least one of joint deformity, knee joint circumference, and pain. The biological characteristics included at least one of the following: inflammatory factors IL-1β, IL-6, and TNF-α, free radical NO, pain factor SP, and cartilage metabolism marker CTX-II levels.

6. The construction method according to claim 5, characterized in that, In step (1), the experimental dogs are standardized experimental Beagle dogs.

7. The construction method according to claim 6, characterized in that, The experimental Beagle dogs were 9-12 months old.

8. The construction method according to claim 5, characterized in that, Step (5) lasts for 8 weeks.

9. The construction method according to claim 5, characterized in that, In step (5), the experimental dogs are induced to move by placing toys and playing music every day.

10. The construction method according to claim 5, characterized in that, In step (5), the experimental dogs are induced to exercise for 20 minutes every day.

11. The construction method according to any one of claims 5 to 10, characterized in that, In step (6), the pathological characteristics of the experimental dogs are detected at week 8 post-surgery to confirm successful modeling.

12. A method for identifying whether a compound is effective in treating osteoarthritis, specifically knee osteoarthritis, the method comprising the following steps: (1) An osteoarthritis model dog is constructed by any one of claims 1 to 4 or any one of claims 5 to 11, and then the pathological characteristic indicators of the model dog are detected, and one or more of the behavioral characteristic indicators, joint swelling and pain characteristic indicators and biological characteristic indicators of the model dog are also detected to obtain first detection data; (2) The compound to be identified was administered to the model dog; (3) The pathological characteristic indicators of the model dog are detected again, and one or more of the behavioral characteristic indicators, joint swelling and pain characteristic indicators and biological characteristic indicators of the model dog are also detected to obtain the second detection data; (4) Compare the second detection data with the first detection data; The pathological features mentioned in steps (1) and (3) include staining of articular cartilage pathological sections or Mankin pathological scores based on the staining of articular cartilage pathological sections. In step (4), after comparing the second detection data with the first detection data, if the model dog is found to have a reduced Mankin score, the compound is identified as effective for the treatment of osteoarthritis. The behavioral characteristic indicators mentioned in steps (1) and (3) include at least one of lameness, motor coordination and joint flexion. In step (4), after comparing the second test data with the first test data, if it is found that the model dog has at least one of a decrease in lameness score, an increase in motor coordination ability and an increase in joint flexion, then the compound is identified as effective for the treatment of osteoarthritis. The joint swelling and pain characteristics described in steps (1) and (3) include at least one of joint deformity, knee joint circumference and pain. In step (4), after comparing the second test data with the first test data, if it is found that the model dog has at least one of a reduced joint deformity score, a reduced knee joint circumference and a decreased pain score, then the compound is identified as effective for the treatment of osteoarthritis. The biological characteristic indicators mentioned in steps (1) and (3) include at least one of the following: the levels of inflammatory factors IL-1β, IL-6 and TNF-α, free radical NO, pain factor SP and cartilage metabolism marker CTX-II. In step (4), after comparing the second detection data with the first detection data, if it is found that the model dog has a reduced level of at least one of the following: inflammatory factors IL-1β, IL-6 and TNF-α, free radical NO, pain factor SP and cartilage metabolism marker CTX-II, then the compound is identified as effective for the treatment of osteoarthritis.

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