Construction method and application of osteoarthritis animal model

Abstract

The invention provides a construction method and application of an osteoarthritis animal model. According to the construction method, a standardized experimental Beagle dog is selected, an improved Hulth method is used for establishing the osteoarthritis model, a mode of inducing the experimental dog to move is optionally adopted for further treatment, and the animal model close to the clinical actual situation is constructed. The animal model obtained through the construction method fits traumatic arthritis caused by increasingly worn joints in actual life, the model stability is good, and a new animal model with important practical value is provided for research and development of osteoarthritis drugs and medical instruments.

Description

Technical Field

[0001] The present invention relates to the technical field of osteoarthritis model establishment and application, and in particular, to the establishment and application of an animal model that is particularly suitable for the clinical situation of knee osteoarthritis. Background Art

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

[0003] At present, animal models of OA mainly include spontaneous, induced and transgenic animal models; among them, the induced model is more stable and takes less time to establish, so it is the most commonly used model. Induced models can be divided into surgical and non-surgical models: surgical models have a high success rate and high repeatability, are suitable for the study of post-traumatic OA, are the best choice for short-term research, and are the most widely used.

[0004] Animals used to induce OA models are mostly small animals (rats, rabbits), but small animals have small joints, which is not conducive to intra-articular operations, and they are quite different from humans, so their experimental results are not suitable for human application; while large animals have joint structures that are more similar to humans, which is convenient for operation and can better simulate the entire process of occurrence and development of human osteoarthritis, and can more accurately predict the effect of drugs when used. Therefore, it is very necessary to establish a large animal OA surgical model and study its mechanism of action.

[0005] At present, most of the literature on large animals reported internationally uses sheep, which are not experimental animals and have no standardized genetic background and basic data. Therefore, the arthritis model prepared with sheep as the experimental subject cannot be promoted and applied. Canines such as Beagle dogs are of fixed and excellent breeds, stable genetic performance, good repeatability, strong adaptability to the environment and disease resistance, and docile temperament. They are recognized as ideal experimental animals and are widely used in medical research and other fields, especially in the field of new drug and medical device research and development. They are recognized as experimental animals in the world. However, the world has not yet formed a complete and reliable set of operating technical procedures and specifications for constructing canine osteoarthritis models.

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

[0007] In view of the above technical problems, the purpose of the present invention is to provide a method for constructing an osteoarthritis animal model, wherein the method uses dogs to construct the animal model to solve the limitations of the current osteoarthritis animal model.

[0008] Furthermore, another object of the present invention is to provide a method for constructing a canine osteoarthritis model that is more in line with the actual clinical situation. This method combines the activity characteristics of clinical patients. After using dogs to construct the animal model, further movement operations are applied so that the constructed animal can simulate the actual clinical situation of osteoarthritis in human patients.

[0009] In addition, another object of the present invention is to provide applications of the animal models obtained by the two construction methods described above, particularly in testing whether a compound is effective in treating osteoarthritis.

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

[0011] First, The present invention provides a method for constructing an osteoarthritis animal model, the method comprising the following steps:

[0012] (1) Take the experimental dog, shave the hair and prepare the skin at the right hind limb knee joint surgery area after anesthesia, and disinfect the skin;

[0013] (2) Make a longitudinal incision on the inner side of the knee joint, cut into the joint cavity layer by layer, and cut the anterior cruciate ligament;

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

[0015] (4) The joint capsule and skin were closed by suturing layer by layer, and sodium penicillin was injected intramuscularly immediately after the operation to prevent infection. The injured limb was not fixed afterwards, and the experimental dogs were free to move 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 3 cm skin incision is made on the inner side of the knee joint. The skin incision is located next to the patella on the inner side of the joint capsule at the flexion point of the knee joint. After cutting layer by layer into the joint cavity, the patella is turned toward the outside 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 in the first aspect of the present invention further comprises the following steps:

[0020] (5) Detect pathological characteristic indicators of the experimental dogs 6-10 weeks after surgery, preferably 8 weeks, to confirm the success of the modeling.

[0021] Preferably, the pathological characteristic index includes articular cartilage pathological section staining or Mankin pathological score based on the articular cartilage pathological section staining. Mankin pathological score is an analysis score based on articular cartilage pathological section. The constructed model dog may have an elevated Mankin score for cartilage appearance, cartilage structure, etc.

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

[0023] The behavioral characteristic index includes at least one of lameness, motor coordination and joint flexion. The constructed model dog may have at least one of increased lameness score, decreased motor coordination ability and decreased joint flexion.

[0024] The joint swelling and pain characteristics include at least one of joint deformity, knee joint circumference and pain. The constructed model dog may have at least one of increased joint deformity score, increased knee joint circumference and increased pain score.

[0025] The biological indicators include 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. Wherein, the level of the biological indicator refers to the level in serum. The constructed model dog may have an increased level of at least one of the inflammatory factors IL-1β, IL-6 and TNF-α, free radical NO, pain factor SP and cartilage metabolism marker CTX-II.

[0026] Second, The present invention provides a method for constructing an osteoarthritis animal model capable of simulating the clinical condition of an arthritis patient, the method comprising the following steps:

[0027] (1) Take the experimental dog, shave the hair and prepare the skin at the right hind limb knee joint surgery area after anesthesia, and disinfect the skin;

[0028] (2) Make a longitudinal incision on the inner side of the knee joint, cut into the joint cavity layer by layer, and cut the anterior cruciate ligament;

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

[0030] (4) The joint capsule and skin were closed by suturing layer by layer, and penicillin sodium was injected intramuscularly immediately after the operation to prevent infection. The injured limb was not fixed afterwards, and the experimental dogs were allowed to move freely in the cage;

[0031] (5) The experimental dogs were induced 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 3 cm skin incision is made on the inner side of the knee joint. The skin incision is located next to the patella on the inner side of the joint capsule at the flexion point of the knee joint. After cutting layer by layer into the joint cavity, the patella is turned toward the outside 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), inducing the experimental dog to exercise every day comprises placing toys and playing music to induce the experimental dog to exercise.

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

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

[0039] (6) Detect pathological characteristic indicators of the experimental dogs 6-10 weeks after surgery, preferably 8 weeks, to confirm the success of the modeling.

[0040] Preferably, the pathological characteristic index includes articular cartilage pathological section staining or Mankin pathological score based on the articular cartilage pathological section staining. Mankin pathological score is an analysis score based on articular cartilage pathological section. The constructed model dog may have an elevated Mankin score for cartilage appearance, cartilage structure, etc.

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

[0042] The behavioral characteristic index includes at least one of lameness, motor coordination and joint flexion. The constructed model dog may have at least one of increased lameness score, decreased motor coordination ability and decreased joint flexion.

[0043] The joint swelling and pain characteristics include at least one of joint deformity, knee joint circumference and pain. The constructed model dog may have at least one of increased joint deformity score, increased knee joint circumference and increased pain score.

[0044] The biological indicators include 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. Wherein, the level of the biological indicator refers to the level in serum. The constructed model dog may have an increased level of at least one of the inflammatory factors IL-1β, IL-6 and TNF-α, free radical NO, pain factor SP and cartilage metabolism marker CTX-II.

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

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

[0047] (2) administering the compound to be identified to the model dog;

[0048] (3) detecting the pathological characteristic indexes of the model dog again to obtain 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 characteristic index includes articular cartilage pathological section staining or Mankin pathological score based on the articular cartilage pathological section staining. Mankin pathological score is an analytical score based on articular cartilage pathological section. The model dog may have an elevated Mankin score for cartilage appearance, cartilage structure, etc. In the method provided in the third aspect of the present invention, after comparing the second test data with the first test data in step (4), if it is found that the model dog has a reduced Mankin score, the compound is identified as being effective for the treatment of osteoarthritis.

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

[0053] The behavioral characteristic index includes at least one of lameness, motor coordination and joint flexion. The model dog may have at least one of an increased lameness score, decreased motor coordination ability and decreased joint flexion. In the method provided in the third aspect of the present invention, after comparing the second test data with the first test data in step (4), if it is found that the model dog has at least one of a decreased lameness score, an increased motor coordination ability and an increased joint flexion, the compound is identified as being 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 have at least one of an increased joint deformity score, an increased knee joint circumference and an increased pain score. In the method provided in the third aspect of the present invention, after comparing the second test data with the first test data in step (4), if it is found that the model dog has at least one of a decreased joint deformity score, a decreased knee joint circumference and a decreased pain score, the compound is identified as being effective for the treatment of osteoarthritis.

[0055] The biological indicators include 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. Wherein, the level of the biological indicator refers to the level in serum. The model dog may have an increased level of at least one of the 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 present invention, after comparing the second test data with the first test data in step (4), if it is found that the model dog has a reduced level of at least one of the inflammatory factors IL-1β, IL-6 and TNF-α, free radical NO, pain factor SP and cartilage metabolism marker CTX-II, the compound is identified as being effective for the treatment of osteoarthritis.

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

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

[0058] Moreover, after selecting experimental Beagle dogs as model animals, the inventors of the present invention comprehensively considered the effects of various arthritis induction methods on model animals and selected the modified Hulth method to establish an osteoarthritis model. This method has a short surgical modeling time, is less invasive, can effectively avoid and prevent infections caused during and after surgery, and has a higher success rate.

[0059] Furthermore, considering that exercise is the main clinical cause of osteoarthritis patients and one of the main aggravating factors in the pathogenesis, in order to be more in line with the actual clinical situation of osteoarthritis, the inventors of the present invention further strengthened the arthritis condition of the experimental dogs by inducing dog exercise. Appropriate increase in exercise can further increase joint damage and accelerate the progression of arthritis, but excessive exercise will cause the animal model to be serious and even affect health; however, if this factor is not applied, the animals are allowed to move freely in the cage with limited space for activities, which is inconsistent with the actual clinical situation, and the model progresses slowly and the modeling time is long. Therefore, the inventors of the present invention chose to induce dogs to exercise for 20 minutes a day, and further optimized the model construction method, thereby obtaining an animal model that is closer to clinical practice; and experiments have shown that the animals have mild to moderate arthritis 8 weeks after modeling.

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

[0061] (1) Pathological characteristic indicators: Histopathology is the gold standard for evaluating the progression of osteoarthritis.

[0062] (2) Behavioral and joint swelling and pain characteristic indicators: This is an assessment of pain, joint function and gait. It can make the animal traumatic osteoarthritis model closer to the clinical pathological changes of the disease and is an indispensable part of the clinical relevance and practicality of the translational research model. Small animals are subject to species characteristics, body size and gait, and it is difficult to assess indicators such as pain and joint dysfunction, but large animals can be assessed accordingly.

[0063] (3) Biological characteristic indicators (inflammatory factors IL-1β, IL-6 and TNF-α, free radical NO, pain factor SP and cartilage metabolism marker CTX-II level): 1) Inflammatory response is closely related to the symptoms and 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 patients with early OA is significantly increased. Animal experiments have reported that excessive concentrations of NO in the body can promote the destruction of articular cartilage. Therefore, reducing excessive NO release can delay the destruction of cartilage and thus delay the development of arthritis; 3) Peripheral SP acts as a pain transmitter to transmit noxious stimulation signals to the spinal cord. In addition to physical factors, the joint pain of OA patients is also related to substance P; 4) Cartilage is composed of special chondrocytes, which produce a large amount of collagen extracellular matrix proteins, including proteoglycans and type II collagen, to maintain the homeostasis of articular cartilage and thus keep it intact; CTX-II is a type II collagen fragment after cartilage degradation and is a biomarker of cartilage degradation.

[0064] In the embodiments of the present invention, the animal model was verified using meloxicam, a mainstream drug for acute attacks of arthritis. Experiments have shown that the animal model obtained by the construction method of the present invention fits the traumatic arthritis caused by the increasing wear of joints in real life, and the model has good stability. It can be seen that the animal model obtained by the construction method of the present invention can be applied to the research and development of drugs and medical devices, especially providing a new animal model with important practical value for the research and development of osteoarthritis drugs and medical devices. BRIEF DESCRIPTION OF THE DRAWINGS

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

[0066] Figure 1 : Histopathological images of the model control group at 8 weeks after modeling.

[0067] Figure 2 : Histopathological images of the sham-operated group at 8 weeks after modeling.

[0068] Figure 3 : Histopathological images of the positive control group at week 12 after modeling (8 weeks of modeling + 4 weeks of drug administration). DETAILED DESCRIPTION

[0069] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting 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 terms described in the present invention are only to describe specific embodiments and are not intended to limit the present invention. In addition, the numerical range in the present invention should be understood to specifically disclose the upper and lower limits of the scope and the intermediate values ​​therebetween. Each smaller range between the intermediate value in any stated value or stated range and any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0071] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as commonly understood by those skilled in the art to which the invention belongs. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation and testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials related to the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0072] It will be understood by those skilled in the art that the numbering of the steps is only for distinguishing different steps and does not mean the order of the steps. As long as the purpose of the present invention can be achieved, the order of the steps is not particularly limited. In addition, it will be further understood by those skilled in the art that other steps or operations may be included between or before and after any steps, such as further optimizing or improving the method described in the present invention.

[0073] Construction of animal model:

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

[0075] (1) A Beagle dog was selected. After anesthesia, the hair and skin of the surgical area of ​​the right hind knee joint were shaved and the skin was routinely disinfected for preoperative preparation.

[0076] (2) Make a 3 cm skin incision on the inner side of the knee joint (the skin incision is made next to the patella on the inner side of the joint capsule at the flexion point of the knee joint). After cutting through the layers and entering the joint cavity, turn the patella to the outside of the knee joint, flex the knee joint, expose the anterior cruciate ligament, and cut it.

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

[0078] (4) Suture layer by layer, and close the joint capsule and skin routinely. Immediately inject sodium penicillin intramuscularly to prevent infection. Do not fix the injured limb after surgery, and allow free movement for 8 weeks.

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

[0080] In the construction method of the present invention, the sex of the animal used to construct the animal model is not particularly limited, and both males and females can be used. Since there are fewer types of experimental dogs, Beagle dogs are mainly used, and Beagle dogs are preferred. Their age is generally 9-12 months old.

[0081] In the construction method of the present invention, the method of joint injury is not particularly limited, as long as it can cause joint injury, including drug method or surgical method. In an exemplary experimental scheme, the present invention is performed by surgery, and the present invention adopts a modified Hulth method, that is, cutting the anterior cruciate ligament and removing the medial meniscus.

[0082] In the construction method of the present invention, after completing the surgical modeling, the behavioral characteristic indicators, joint swelling and pain characteristic indicators, biological characteristic indicators and / or pathological characteristic indicators of the animal are further detected to confirm and optimize the construction conditions. The behavioral characteristic indicators include at least one of lameness, motor coordination, and joint curvature. The joint swelling and pain characteristic indicators include at least one of joint deformity, knee joint circumference, and pain. The biological characteristic indicators include 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. Pathological characteristic indicators include Mankin pathological scores. In the present invention, the detection time of behavioral characteristic indicators, joint swelling and pain characteristic indicators, and biological characteristic indicators is not particularly limited. For example, they can be detected before modeling, 4 weeks, 8 weeks, and 12 weeks after modeling; pathological characteristic indicators are detected at 8 weeks and 12 weeks after modeling.

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

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

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

[0086] (b) In the laboratory antechamber, the experimental dogs were induced to exercise for 20 min every day for 8 weeks.

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

[0088] In the simulation method of the present invention, the factor of exercise is further applied to the animals that have completed the surgical modeling, generally inducing the experimental dogs to exercise for 20 minutes every day. Appropriate increase in exercise can further increase joint damage and accelerate the progression of arthritis. Excessive exercise may cause serious degree of animal model and even affect health. If this factor is not applied, the animals are allowed to move freely in the cage with limited activity space, which is inconsistent with the actual clinical patients, and the model progresses slowly and the modeling time is long. Experiments have shown that the pathological histological score of 8 weeks has been confirmed as mild to moderate arthritis.

[0089] Methods for Identifying Compounds Effective for Treating Arthritis:

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

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

[0092] (ii) administering the compound to be identified to the animal model;

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

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

[0095] In the identification method of the present invention, the animal model is obtained by the construction method of the first aspect or the second aspect of the present invention. In certain embodiments, the behavioral characteristic index of the animal model includes at least one of an increased lameness score, decreased motor coordination ability, and decreased joint flexion; if after administering the compound to be identified, the animal model has at least one of a decreased lameness score, an increased motor coordination ability, and an increased joint flexion, the compound is identified as effective in treating arthritis.

[0096] In certain embodiments, the characteristic indicators of joint swelling and pain in the animal model include at least one of an increased joint deformity score, an increased knee joint circumference, and an increased pain score; if after administering the compound to be identified, the animal model has at least one of a decreased joint deformity score, a decreased knee joint circumference, and a decreased pain score, the compound is identified as being effective in treating arthritis.

[0097] In certain embodiments, the biological characteristic indicators of the animal model include at least one of increased 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, the animal model has at least one of decreased levels of 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 in treating arthritis.

[0098] In certain embodiments, the pathological characteristic indicators of the animal model include an increased Mankin score such as cartilage appearance and cartilage structure; if the Mankin score of the animal model decreases after administration of the test compound, the compound is identified as effective in treating arthritis.

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

[0100] Example 1

[0101] 1. Experimental Methods

[0102] 1. Animals

[0103] The research of the present invention has been approved by the Animal Use and Management Committee of Guangzhou General Research Institute for Pharmaceutical Research, and its animal welfare and ethics have been approved. Beagle dogs aged 9-12 months were purchased from Guangzhou General Research Institute for Pharmaceutical Research Co., Ltd., with experimental animal production license number: SCXK (Guangdong) 2023-0007 (valid until June 26, 2028, issued by Guangdong Provincial Department of Science and Technology). Laboratory animal quality certificate number: 44006900001087. Animals were raised in an ordinary environment with a temperature of 18-26°C, a humidity of 40%-70%, alternating light and dark for 12h / 12h, and free food and water. The experiment began after 2 weeks of quarantine.

[0104] 2. Grouping: The animals were divided into a sham operation group (n=6) and an operation group (n=6).

[0105] 3. Surgery group: the anterior cruciate ligament was cut and the medial meniscus was removed; sham operation group: except that the anterior cruciate ligament was not cut and the medial meniscus was not removed, the rest of the operations were the same as those of the surgery 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 were free to eat and drink.

[0109] 4.2 The animals were moved to the laboratory for induced exercise every day, with the sham operation group and the operation group being carried out separately. Music was played and toy dog ​​bones were placed in the laboratory to induce the dogs to exercise continuously.

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

[0111] 4.4 The duration was 12 weeks. After 8 weeks, 2 dogs in the sham operation group and 2 dogs in the operation group were euthanized, and pathological characteristic indicators were performed to confirm the success of the model.

[0112] 5. Behavioral characteristic indicator detection

[0113] 5.1 Lameness score

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

[0115] 6.1 Pain score

[0116] 7. Biological characteristic index detection

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

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

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

[0120] 7.2 Free Radical NO

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

[0122] 7.3 Pain factor SP

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

[0124] 7.4 Cartilage Metabolism Factor CTX-II

[0125] Bone metabolic factor detection kit: CTX-II, purchased from Shanghai ELISA Biotechnology Co., Ltd., batch number Jul2023.

[0126] 8. Detection of joint pathological characteristic indicators

[0127] According to the scoring indicators (cartilage tissue structure, cell number changes, HE staining, tide line integrity)

[0128] A comprehensive Mankin score was performed.

[0129] 9. Statistical Analysis

[0130] Example 2

[0131] Meloxicam, a drug for treating osteoarthritis, was administered to the model dogs obtained by the construction method of the present invention, and various characteristic indicators of the model dogs were detected. Meloxicam is a mainstream drug for treating acute attacks of osteoarthritis. The research steps are as follows.

[0132] A total of 18 Beagle dogs aged 9-12 months were obtained and randomly divided into a "sham operation group", a "model control group" and a "positive control group", with 6 dogs in each group. As described in Example 1, sham operation was performed on the dogs in the "sham operation group", and 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 every day.

[0133] At 8 weeks after modeling, two dogs in the "model control group" and "positive control group" were euthanized, and the pathological characteristic indicators were detected to confirm the success of the modeling. Then, the remaining four dogs in the "positive control group" were given the test compound meloxicam injection (purchased from Qilu Animal Health Products Co., Ltd., batch number 914301002) for 4 weeks. The administration method was subcutaneous injection of meloxicam injection, the first dose was 0.2 mg / kg, the maintenance dose was 0.1 mg / kg, once a day, and the administration was continued for 28 days.

[0134] Before modeling, and at the 4th, 8th, and 12th weeks after modeling, the behavioral characteristic indices, joint swelling and pain characteristic indices, biological characteristic indices, and pathological characteristic indices of dogs in each group were tested.

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

[0136] Table 1. Behavioral characteristic index - lameness score (x±s)

[0137]

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

[0139] Table 2. Characteristic indexes of joint swelling and pain - pain score (x±s)

[0140]

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

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

[0143]

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

[0145] Table 4. Biological characteristic index - inflammatory factor IL-6 (x±s, pg / mL)

[0146]

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

[0148] Table 5. Biological characteristic indexes - inflammatory factor TNF-α (x±s, pg / mL)

[0149]

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

[0151] Table 6. Biological characteristic index - free radical NO (x±s, μmoL / L)

[0152]

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

[0154] Table 7. Biometric indicators - pain factor SP (x±s, pg / mL)

[0155]

[0156]

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

[0158] Table 8. Biological characteristic index - cartilage metabolism factor CTX-II (x±s, ng / mL)

[0159]

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

[0161] Table 9. Pathological characteristic index - Mankin pathological score (x±s, n=4)

[0162]

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

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

Claims

1. A method for constructing an osteoarthritis animal model, the method comprising the following steps: (1) Take the experimental dog, shave the hair and prepare the skin at the right hind limb knee joint surgery area after anesthesia, and disinfect the skin; (2) Make a longitudinal incision on the inner side of the knee joint, cut into the joint cavity layer by layer, and cut the anterior cruciate ligament; (3) Resection of the medial meniscus without damaging the articular cartilage surface; (4) The joint capsule and skin were closed by suturing layer by layer, and sodium penicillin was injected intramuscularly immediately after the operation to prevent infection. The injured limb was not fixed afterwards, and the experimental dogs were free to move in the cage.

2. The construction method according to claim 1, characterized in that: The osteoarthritis is knee osteoarthritis; Preferably, the experimental dog in step (1) is a standardized experimental Beagle dog, preferably 9-12 months old; Preferably, in step (2), a 3 cm skin incision is made on the inner side of the knee joint. The skin incision is located next to the patella on the inner side of the joint capsule at the flexion point of the knee joint. After cutting layer by layer into the joint cavity, the patella is turned toward the outside of the knee joint, the knee joint is flexed, the anterior cruciate ligament is found, and it is cut.

3. The construction method according to claim 1 or 2, characterized in that: The construction method further comprises the following steps: (5) Detect pathological characteristic indicators at 6-10 weeks after surgery, preferably at 8 weeks, to confirm the success of the model; Preferably, step (5) further comprises detecting one or more of the behavioral characteristic index, joint swelling and pain characteristic index and biological characteristic index of the experimental dog; Preferably, the pathological characteristic indicators include articular cartilage pathological section staining or Mankin pathological score based on the articular cartilage pathological section staining; and / or, the behavioral characteristic indicators include at least one of lameness, motor coordination and joint flexion; and / or, the joint swelling and pain characteristics include at least one of joint deformity, knee joint circumference and pain; and / or, the biological indicators include at least one of the inflammatory factors IL-1β, IL-6 and TNF-α, free radical NO, pain factor SP and cartilage metabolism marker CTX-II level.

4. A method for constructing an osteoarthritis animal model capable of simulating the clinical condition of an arthritis patient, the method comprising the following steps: (1) Take the experimental dog, shave the hair and prepare the skin at the right hind limb knee joint surgery area after anesthesia, and disinfect the skin; (2) Make a longitudinal incision on the inner side of the knee joint, cut into the joint cavity layer by layer, and cut the anterior cruciate ligament; (3) Resection of the medial meniscus without damaging the articular cartilage surface; (4) The joint capsule and skin were closed by suturing layer by layer, and penicillin sodium was injected intramuscularly immediately after the operation to prevent infection. The injured limb was not fixed afterwards, and the experimental dogs were allowed to move freely in the cage; (5) The experimental dogs were induced to exercise for 10-30 minutes every day for 6-10 weeks.

5. The construction method according to claim 4, characterized in that: The osteoarthritis is knee osteoarthritis; Preferably, the experimental dog in step (1) is a standardized experimental Beagle dog, preferably 9-12 months old; Preferably, in step (2), a 3 cm skin incision is made on the inner side of the knee joint. The skin incision is located next to the patella on the inner side of the joint capsule at the flexion point of the knee joint. After cutting layer by layer into the joint cavity, the patella is turned toward the outside of the knee joint, the knee joint is flexed, the anterior cruciate ligament is found, and it is cut.

6. The construction method according to claim 4 or 5, characterized in that: Step (5) lasts for 8 weeks; Preferably, in step (5), inducing the experimental dog to exercise every day comprises placing toys and playing music to induce the experimental dog to exercise; Preferably, in step (5), the experimental dogs are induced to exercise for 20 minutes every day.

7. The construction method according to any one of claims 4 to 6, characterized in that: The construction method further comprises the following steps: (6) Detecting the pathological characteristic indicators of the experimental dogs at 6-10 weeks after surgery, preferably at 8 weeks, to confirm the success of the modeling; Preferably, step (6) further comprises detecting one or more of the behavioral characteristic index, joint swelling and pain characteristic index and biological characteristic index of the experimental dog; Preferably, the pathological characteristic indicators include articular cartilage pathological section staining or Mankin pathological score based on the articular cartilage pathological section staining; and / or, the behavioral characteristic indicators include at least one of lameness, motor coordination and joint flexion; and / or, the joint swelling and pain characteristics include at least one of joint deformity, knee joint circumference and pain; and / or, the biological indicators include at least one of the inflammatory factors IL-1β, IL-6 and TNF-α, free radical NO, pain factor SP and cartilage metabolism marker CTX-II level.

8. A method for identifying whether a compound is effective in treating osteoarthritis, the method comprising the following steps: (1) constructing a dog osteoarthritis animal model by the method of any one of claims 1 to 3 or any one of claims 4 to 7, and then detecting pathological characteristic indicators of the model dog to obtain first detection data; (2) administering the compound to be identified to the model dog; (3) detecting the pathological characteristic indexes of the model dog again to obtain second detection data; (4) Compare the second detection data with the first detection data.

9. The method according to claim 8, characterized in that The osteoarthritis is knee osteoarthritis; Preferably, in step (1) and step (3), one or more of the behavioral characteristic indexes, joint swelling and pain characteristic indexes and biological characteristic indexes of the model dog are also detected.

10. The method according to claim 8 or 9, characterized in that: The pathological characteristic index includes articular cartilage pathological section staining or Mankin pathological score based on the articular cartilage pathological section staining. If, after comparing the second test data with the first test data in step (4), it is found that the Mankin score of the model dog is reduced, the compound is identified as being effective for the treatment of osteoarthritis; and / or The behavioral characteristic index includes at least one of lameness, motor coordination and joint flexion. If, after comparing the second test data with the first test data in step (4), it is found that the model dog has at least one of a decreased lameness score, an increased motor coordination ability and an increased joint flexion, the compound is identified as being effective for the treatment of osteoarthritis; and / or The joint swelling and pain characteristics 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 decreased joint deformity score, a decreased knee joint circumference and a decreased pain score, the compound is identified as being effective for the treatment of osteoarthritis; The biological indicator includes 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. After comparing the second test data with the first test data in step (4), if it is found that the level of at least one of the inflammatory factors IL-1β, IL-6 and TNF-α, free radical NO, pain factor SP and cartilage metabolism marker CTX-II in the model dog is reduced, the compound is identified as effective for the treatment of osteoarthritis.

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