Animal model for cold-dampness arthralgia type knee osteoarthritis and construction method and application thereof

Abstract

The invention discloses an animal model for cold-dampness arthralgia type knee osteoarthritis as well as a construction method and application of the animal model. The construction method comprises the following steps: placing an experimental animal in a cold-damp environment, and meanwhile, placing hind limbs of the experimental animal in an ice-water mixture for modeling. The method is stable and effective, can be repeatedly verified, fills up the research blank that the existing cold-dampness arthralgia type knee osteoarthritis has diseases without syndromes, is easy and convenient to operate, good in repeatability and low in requirement for processing people, and can be used for constructing animal models on a large scale. The obtained animal model can provide a reliable experimental carrier for new drug research and development, drug effect evaluation, action mechanism research and the like, and has a very good application prospect.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and particularly to an animal model of knee osteoarthritis with cold-damp obstruction syndrome, a construction method thereof, and an application thereof. Background Art

[0002] As the most common degenerative disease in clinical practice, knee osteoarthritis (kOA) is characterized by a high incidence rate and a high disability rate. The main pathological features include articular cartilage degeneration, subchondral bone sclerosis, synovial inflammation, etc., and it often presents symptoms such as knee joint pain, swelling, and dysfunction.

[0003] It is recorded in "Plain Questions - Treatise on Arthralgia Syndromes": "The combination of wind, cold, and dampness leads to arthralgia." Research on the distribution law of TCM syndrome types and syndrome elements of kOA shows that among the most common two syndrome elements of kOA, cold and damp are the most common, and among the three syndrome elements, wind, cold, and damp are the most common. Therefore, cold-damp obstruction is its main syndrome type. TCM clinical treatment of kOA from the perspective of dispelling cold, removing dampness, and relieving pain has achieved positive therapeutic effects. Professor Ding E, a famous TCM doctor across the country, believes that the primary goal of treating kOA with cold-damp obstruction syndrome is to warm the meridians, dispel cold, remove dampness, expel wind, and then relieve arthralgia and pain, which can achieve obvious effects. Although there are currently many research reports on the TCM pathogenesis of kOA and the treatment of corresponding syndromes with traditional Chinese medicine, due to the lack of an effective and unified method for constructing an animal model, in basic experiments, in-depth and effective molecular mechanism and cell experimental studies cannot be carried out for kOA with cold-damp obstruction syndrome, which greatly limits the research and development of new drugs for treating kOA with cold-damp obstruction syndrome. Summary of the Invention

[0004] To overcome the deficiencies of the prior art, the present invention provides an animal model of knee osteoarthritis with cold-damp obstruction syndrome, a construction method thereof, and an application thereof.

[0005] In the first aspect of the present invention, a method for constructing an animal model of knee osteoarthritis is provided, which includes the following steps: placing the experimental animal in a cold-damp environment and simultaneously placing its hind limbs in an ice-water mixture for modeling.

[0006] Specifically, the cold-damp environment is a constant temperature and humidity environment (for example, in a constant temperature and humidity incubator), wherein the temperature is 1 - 10°C (for example, 1, 2, 3, 4, 4.5, 5, 6, 7, 8, 9, 10°C), and the humidity is 80% - 100% (for example, 80%, 85%, 90%, 93%, 94%, 95%, 96%, 97%, 98%, 99%).

[0007] In some embodiments of the present invention, the temperature of the cold-damp environment is 5 ± 2°C.

[0008] In some embodiments of the present invention, the humidity of the cold-damp environment is 95 ± 2%.

[0009] According to the "Specification for Establishing Animal Models of Cold-Dampness Obstruction Syndrome and Damp-Heat Obstruction Syndrome of Rheumatoid Arthritis" issued by the Chinese Association of Traditional Chinese Medicine, the inventors of the present invention have calibrated through multiple experiments and stably controlled the environmental parameters within the range of 5±2°C and relative humidity of 95±2%, which can better simulate the low-temperature and high-humidity environment to construct an animal model of knee osteoarthritis with cold-dampness obstruction syndrome.

[0010] Specifically, the temperature of the ice-water mixture is 0±4°C, such as 0±3°C, 0±2°C, 0±1°C, especially 0±2°C.

[0011] Specifically, the depth of the ice-water mixture is 30-60 mm, such as 30, 35, 40, 45, 50, 55, 60 mm, especially 40-50 mm.

[0012] Specifically, the modeling treatment lasts for 4-16 weeks (such as 4, 6, 8, 10, 12, 14, 16 weeks), for example, 4-12 weeks, 6-10 weeks, and lasts for 1-6 hours per day (such as 1, 1.5, 2, 2.5, 3, 3.5, 4, 5, 6 hours), for example, 2-4 hours; in some embodiments of the present invention, the modeling treatment lasts for 8 weeks and 3 hours per day. To simulate the traditional Chinese medicine pathogenesis characteristics of "cold-dampness invading the collaterals and obstructing the meridians" and its chronic course characteristics of knee osteoarthritis with cold-dampness obstruction syndrome, in the experimental part of the embodiments of the present invention, through an 8-week animal modeling experiment, the experimental animals are exposed to a cold-damp environment (temperature 5±2°C, relative humidity 95±2%) for 3 hours per day, to gradually accumulate stimulation to induce the chronic pathological process of knee osteoarthritis with cold-dampness obstruction syndrome. At the same time, by controlling the duration of a single exposure, non-specific injuries caused by low temperature can be avoided, and an animal model of knee osteoarthritis with cold-dampness obstruction syndrome can be better constructed.

[0013] In some embodiments of the present invention, the method includes: placing the experimental animal cage (with the experimental animal in the cage) in a thermostatic and humidified climate chamber, setting the environmental conditions to a temperature of 5±2°C and a humidity of 95±2%, and at the same time laying an ice-water mixture at the bottom of the cage (due to the freezing and low-temperature stimulation, the experimental animals in the cage cannot keep their four limbs on the ground for a long time and generally show an upright state of the upper and lower limbs to reduce contact with the ice-water mixture. Through the combined "system-local" freezing method, a cold-damp environment is simulated for osteoarthritis modeling), lasting for 6-10 weeks (for example, lasting for 8 weeks), and lasting for 2-4 hours per day (for example, lasting for 3 hours per day).

[0014] Specifically, the experimental animal is a rat.

[0015] Specifically, the rat can be an SD rat or a Wistar rat, especially an SD rat.

[0016] Specifically, the rat is an adult rat.

[0017] Specifically, the rats are healthy, SPF-grade rats.

[0018] Specifically, the rats are male rats.

[0019] Specifically, the method further includes: during the model establishment period, regularly monitoring the physical signs, biochemical indexes, etc. of the animals.

[0020] Specifically, the physical signs include mental state, skin and hair, body weight, foraging, claws, feces, etc.

[0021] Specifically, the method further includes: during the model establishment period, regularly detecting the pain threshold of the animals.

[0022] Specifically, the method further includes a pathological detection step (to verify whether the model construction is successful).

[0023] In some embodiments of the present invention, the method further includes: removing the knee joints of the rats after the model establishment treatment, and performing pathological tissue sectioning, staining, and immunohistochemical index detection.

[0024] Specifically, the immunohistochemical indexes include MMP13 and Col2.

[0025] In the second aspect of the present invention, there is provided an animal model constructed by the method described in the first aspect.

[0026] In the third aspect of the present invention, there is provided the application of the method described in the first aspect and the animal model constructed thereby in drug screening and efficacy evaluation.

[0027] Specifically, the drug is used for the treatment of knee osteoarthritis, especially knee osteoarthritis of the cold-dampness obstruction type.

[0028] In the fourth aspect of the present invention, there is provided a drug screening method, which includes the following steps: administering the drug to the animal model constructed by the method described in the first aspect, and comparing it with the animal model without the drug administration (the drug whose symptoms of knee osteoarthritis are improved or cured after administration is the candidate drug).

[0029] Specifically, the method may further include performing one or more combinations of further cell experiments, animal experiments, and clinical trials on the candidate drug to detect the properties of the candidate drug in terms of efficacy, pharmacokinetics, or toxicology, etc.

[0030] In the fifth aspect of the present invention, there is provided the application of the animal model constructed by the method described in the first aspect in the research on the disease mechanism of knee osteoarthritis of the cold-dampness obstruction type.

[0031] Specifically, the application is for non-diagnostic or therapeutic purposes.

[0032] The present invention provides a method for constructing an animal model of knee osteoarthritis with damp-cold obstruction type, which is stable, effective and can be repeatedly verified. The obtained animal model can provide a reliable experimental carrier for new drug research and development, drug efficacy evaluation, mechanism of action research, etc., and has very good application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 The following shows the pain threshold evaluation results of rats in each group at different modeling time periods, where Figure 1 A shows the mechanical pain threshold and thermal pain threshold of rats in each group at the 8th week of modeling, Figure 1 B shows the mechanical pain threshold and thermal pain threshold of rats in each group at the 12th week of modeling.

[0034] Figure 2 The following shows the histopathological examination results of cartilage tissues of rats in each group, where Figure 2 A shows the SO staining results of knee joint cartilage sections of rats in each group, Figure 2 B shows the Mankin's score results of rats in each group.

[0035] Figure 3 The following shows the influence results of not adding an incubator and adding an incubator on the environment during modeling, where Figure 3 A shows the water temperature detection results without adding an incubator (adding water and ice), Figure 3 B shows the humidity detection results of the climate chamber after adding an incubator (adding ice), Figure 3 C shows the temperature detection results of the climate chamber after adding an incubator (adding ice).

[0036] Figure 4 The following shows the mechanical pain threshold evaluation results of rats in each group at different modeling time periods, where Figure 4 A shows the mechanical pain threshold evaluation results of rats in each group before modeling, Figure 4 B shows the mechanical pain threshold evaluation results of rats in each group at the 4th week of modeling, Figure 4 C shows the mechanical pain threshold evaluation results of rats in each group at the 8th week of modeling.

[0037] Figure 5 The following shows the staining results of knee joint sections of rats in each group.

[0038] Figure 6 The following shows the immunohistochemical index detection results of rats in each group. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0039] Unless otherwise defined, all scientific and technical terms used in the present invention have the same meaning as commonly understood by those skilled in the technical field to which the present invention pertains.

[0040] The diagnostic criteria for knee osteoarthritis of cold-dampness obstruction type in the present invention can be referred to as follows: Diagnostic criteria for traditional Chinese medicine syndromes: Refer to the "Diagnosis and Treatment Plan for Knee Bi Disease (Knee Osteoarthritis) of the Key Specialties Collaboration Group of the State Administration of Traditional Chinese Medicine".

[0041] All publications, patents, and published patent specifications cited herein are incorporated herein by reference in their entirety.

[0042] Next, in combination with the embodiments of the present invention, the technical solutions of the present invention will be clearly and completely described. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without making creative efforts belong to the scope of protection of the present invention.

[0043] Example 1

[0044] 1. Experimental grouping and treatment

[0045] 1.1 Experimental materials

[0046] Adult male Sprague-Dawley (SD) rats (SPF II), weighing 200±2 g, were provided by Shanghai Super B&K Laboratory Animal Co., Ltd.

[0047] 1.2 Experimental grouping

[0048] Forty-five SD rats were randomly divided into 3 groups: normal group, OA surgery group, and cold-dampness (water immersion) group, with 15 rats in each group.

[0049] No treatment was given to the normal group.

[0050] Treatment for the surgery group: Perform DMM surgery (Destabilization of the medial meniscus) for modeling.

[0051] Treatment for the cold-dampness (water immersion) group: Place the rats in water at about 10±2 °C (control the temperature by adding ice boxes). Since rats have a fear of water reaction, they will be in a state of upright upper and lower limbs. Keep the rats in a fixed position in water at about 10±2 °C with both lower limbs upright for about 3 h every day, repeat this for 12 weeks, and take samples for detection at the 8th week and the 12th week respectively.

[0052] 2. Detection methods

[0053] 2.1 The method for measuring the mechanical pain threshold of rats is as follows: Von Frey filaments (measurement range 0.6 - 26 g) that comply with the evaluation specifications of experimental animal ethology and neuroscience are used to detect the mechanical withdrawal latency (MWL) of rats. Measurements are taken before batch sampling. The specific operation is as follows: Place the SD rats on a stainless-steel wire mesh, separate them individually in a transparent acrylic cage to restrict movement, keep the test room quiet, and after the rats are pre-adapted for 1 h and their four limbs are fully in contact with the wire mesh, start the detection. Use nylon fiber filaments with different measurement ranges to vertically press on the central area of the hind paw sole (avoiding the footpad) in turn until the filament bends into a C shape, and hold for 5 seconds. Observe the withdrawal reaction, record and statistically analyze the mechanical withdrawal threshold (g) of the rats to evaluate the mechanics of osteoarthritis rats.

[0054] 2.2 The method for measuring the thermal pain threshold of rats is as follows: The thermal withdrawal latency (TWL) of rats is measured before model establishment and before each batch of sampling. A plantar thermal hyperalgesia tester is used to detect the thermal pain threshold of the bilateral hind toes of rats. During measurement, place the rats in a transparent plexiglass box, and keep the room temperature at 25 ± 2 °C. After the rats are quiet (stop grooming and exploratory activities), place the "cross" mark on the tester at the center of the left / right hind paw sole of the rat and avoid the footpad, start the instrument and observe the reaction of the rats. The time period from starting the instrument until the rats show leg-lifting avoidance is taken as the thermal pain value (s) of the rats. Each rat is detected 3 times, with an interval of 5 - 6 min between each detection. To prevent the rats from being scalded by thermal radiation, the upper limit of the time for thermal pain threshold measurement is set at 20 s, and the upper limit of the temperature is set at 35 °C.

[0055] 2.3 Take the bilateral knee joints of rats, wash them with normal saline, and place them in 4% paraformaldehyde solution. After 1 week, soak them in 10% ethylenediaminetetraacetic acid (EDTA) solution, and change the EDTA solution every 1 week for 4 consecutive weeks until the bone joints are soft and the needle can easily penetrate, and then make pathological sections.

[0056] 3. Experimental Results

[0057] 3.1 Influence on the pain threshold of rats

[0058] As Figure 1 shown, after 8 weeks (W) of model establishment, compared with the normal group, there were no statistically significant differences in the mechanical pain threshold and thermal pain threshold of rats in other groups (p > 0.05). After 12 weeks of model establishment, the OA surgery group showed significant pain sensitization characteristics compared with the normal control group, and its mechanical pain threshold and thermal pain threshold were significantly reduced (p < 0.01), and there were no significant differences in the mechanical pain threshold and thermal pain threshold of the damp-cold (water immersion) group (p > 0.01).

[0059] 3.2 Effects on the histopathology of the knee joints of rats

[0060] The pathological results are as Figure 2 shown by SO staining. After 8 weeks of modeling, compared with the normal group, obvious defects appeared on the cartilage surface of the knee joints of the rats in the OA surgery group, and there was a serious loss of chondrocytes at the defect sites; the cartilage in the cold-dampness (soaked in water) group was basically normal, and there were no obvious changes on the cartilage surface. Quantitative analysis by the Mankin pathological scoring system showed that the OA group presented significant degenerative characteristics at the 8th week, and its score was significantly higher than that of the normal group (p < 0.05); compared with the normal group, there was no significant change in the Mankin's score of the cold-dampness (soaked in water) group (p > 0.05).

[0061] After 12 weeks of modeling, compared with the normal group, obvious defects appeared on the cartilage surface of the knee joints of the rats in the OA surgery group, and there was a serious loss of chondrocytes at the defect sites, and the proteoglycans were significantly degraded; the cartilage in the cold-dampness (soaked in water) group was basically normal, local wear could be seen on the cartilage surface, and there were a small number of hypertrophic chondrocytes. Compared with the normal group, the Mankin's score of the OA surgery group was significantly increased (p < 0.05); compared with the normal group, there was no significant change in the Mankin's score of the cold-dampness (soaked in water) group (p > 0.05).

[0062] The main clinical manifestations of kOA include pain and dysfunction. However, the above experimental results on pain threshold and joint histopathology show that the rats in the cold-dampness (soaked in water) group did not present the characteristic pathological changes of kOA. The above results suggest that the existing cold-dampness (soaked in water) model fails to effectively simulate the pathogenic environment of low temperature and high humidity and stably induce joint cartilage lesions in rats because it is difficult to accurately control temperature and humidity. It is necessary to construct an animal model of osteoarthritis with cold-dampness obstruction by standardizing the control of modeling environment parameters and optimizing the modeling conditions.

[0063] Example 2

[0064] In Example 1, the cold water bath method using water and an ice box was used, but it was found during the modeling process that the rise of the water temperature could not be effectively delayed to simulate a low-temperature environment. The inventor further adopted a high-precision constant temperature and humidity climate chamber and an ice water bath in order to better simulate a low-temperature and high-humidity environment.

[0065] 1. Experimental materials and methods

[0066] 1.1 Experimental materials

[0067] A high-precision constant temperature and humidity climate chamber HWS-600 (hereinafter simply referred to as the constant temperature chamber), purchased from Shanghai Chuanhong Company;

[0068] A snow ice maker AF100, used to prepare crushed ice in the ice water mixture, purchased from SCOTSMAN.

[0069] 1.2 Experimental methods

[0070] After the rats were adaptively fed, they were divided into two groups: 1) The rats were placed in an incubator, and the core parameters were set: temperature 5±2°C, humidity 95±2%, and they were exposed standardized for 3 hours daily in the cold and damp environment. At the same time, a mixture of ice and water (water level about 50 mm, 0±2°C) was laid at the bottom of the rat cage. Due to the freezing and low-temperature stimulation, the rats could not keep all four limbs on the ground for a long time, and generally showed an upright state of the upper and lower limbs to reduce contact with the ice-water mixture. Through the "system-local" combined freezing method, a model of osteoarthritis was established by simulating the cold and damp environment. 2) Without using an incubator, only a mixture of ice and water (water level about 50 mm, 0±2°C) was laid at the bottom of the rat cage.

[0071] Measure the temperature and humidity of the modeling environment. For the group without an incubator, the temperature of the ice-water mixture was measured with a thermometer, and for the incubator group, it was measured through the temperature and humidity control system owned by the machine. The experiment was repeated 4 times.

[0072] 2. Experimental results

[0073] Comparison of the effects of not using an incubator and adding an incubator on the modeling environment

[0074] The results are as Figure 3 shown. Compared with not using an incubator, the incubator can ensure that the temperature inside the box is stable at 4 - 5°C, the water temperature is stable at 0 - 1°C, and the humidity is maintained at about 95%, which can well simulate the cold and damp environment and make the animal model more accurate.

[0075] Example 3

[0076] 1. Experimental grouping and treatment

[0077] 1.1 Experimental materials

[0078] Adult male Sprague-Dawley (SD) rats (SPF II), weighing 200±2 g, were provided by Shanghai Super B&K Experimental Animal Co., Ltd.; after a 1-week adaptation period, they were grouped for experiments;

[0079] High-precision constant temperature and humidity climate chamber HWS-600, used for cold and damp modeling, purchased from Shanghai Chuanhong Company;

[0080] Snowflake ice maker AF100, used for preparing crushed ice in the ice-water mixture, purchased from SCOTSMAN; Plantar tester, used for measuring mechanical withdrawal pain threshold, purchased from Ugo Basile, Italy.

[0081] 1.2 Grouping of experimental animals

[0082] 40 rats were randomly divided into 4 groups: 1) Normal group (NC); 2) OA model group (SS); 3) Cold-damp obstruction syndrome group (CS); 4) OA model + cold-damp obstruction syndrome group (SS + CS).

[0083] The normal group was not given any treatment.

[0084] The OA surgical model group (SS) used the method of anterior cruciate ligament transection (ACLT) of the rat knee joint to surgically induce knee osteoarthritis (kOA). The specific method steps were as follows: After weighing the rats, 3% sodium pentobarbital solution was intraperitoneally injected (0.15 ml was injected per 100 g). After complete anesthesia, a longitudinal incision was made below the patella of the knee joint, and the skin and muscles were gradually dissected layer by layer to fully expose the knee joint cavity. After cutting the anterior cruciate ligament, it was confirmed by drawer test. Finally, the joint capsule and skin were sutured layer by layer, and hemostasis was performed in a timely manner to avoid infection.

[0085] The Cold-Dampness Obstruction Syndrome group (CS): The rats were placed in a constant temperature and humidity chamber, and the environmental conditions were set to a temperature of 5 ± 2°C and a humidity of 95 ± 2%. This was maintained for 3 hours every day. At the same time, a mixture of ice and water (water level about 50 mm, temperature 0 ± 2°C) was laid at the bottom of the rat cage. Due to the cold and low-temperature stimulation, the rats could not keep their four limbs on the ground for a long time and generally showed an upright state of the upper and lower limbs to reduce contact with the ice-water mixture. The osteoarthritis model was established by simulating the cold-dampness environment through a "system - local" combined freezing method, and the modeling lasted for 8 weeks.

[0086] The OA model + Cold-Dampness Obstruction Syndrome group (SS + CS) used the method of anterior cruciate ligament transection (ACLT) of the rat knee joint to surgically induce and combined with the cold-dampness model construction method of the present invention to build an animal model.

[0087] 2. Detection methods

[0088] The main clinical manifestations of kOA include pain and functional impairment. When the KOA of the animal model occurs and develops, the animals will also show corresponding symptoms. Therefore, evaluating the symptoms of the animals can evaluate the modeling effect (Shen Xing, Liu Guangnian, Xiong Huazhang, et al. Research progress on evaluation methods of animal models of knee osteoarthritis [J]. International Journal of Orthopaedics, 2024, 45(01): 19 - 22.).

[0089] In addition, the process of cartilage matrix loss in rats is as follows: First, there is surface fibrosis and matrix loss, then deep fibrosis, matrix loss, and lesion expansion in the middle layer area, and finally, the matrix thickness in the lesion or a large local area is lost to the tibia. The evaluation of the overall cartilage pathology includes important pathological parameters such as collagen matrix fibrosis / loss and chondrocyte death / loss. Among them, chondrocyte loss is the main determinant of osteoarthritis, and proteoglycan loss will occur in the areas of these matrix and chondrocyte losses. The kOA lesions can be evaluated at the cellular and tissue levels by staining methods.

[0090] 2.1 Mechanical pain threshold assessment:

[0091] The "up-down" test method using a series of von Frey filaments (ranging from 0.6 - 26 g, UGO Basile, Italy) was used to measure mechanical allodynia. Briefly, the von Frey filament was vertically pressed onto the mid-plantar surface of the hind paw with sufficient force to bend it, and the 50% paw withdrawal threshold (PWT) was calculated.

[0092] 2.2 Specimen collection:

[0093] After continuous cryo-modeling for 8 weeks (8 weeks after ACLT), the animals were anesthetized by intraperitoneal injection of sodium pentobarbital (3% concentration, 0.15 ml / 100 g). Blood was collected from the heart using a 10-ml syringe. Then, the muscles near the knee joint were removed using scissors, and the knee joint was taken off using bone cutters and fixed in 4% paraformaldehyde solution for 3 days. After 3 days of fixation, the paraformaldehyde on the surface of the joint samples was rinsed off with running water. Then, the samples were placed in embedding cassettes, labeled, and decalcified in EDTA solution. The EDTA decalcifying solution was changed daily for about 2 months until the bone tissue in the joint became soft. The samples were then rinsed under running water overnight. Subsequently, the samples were dehydrated in a dehydrator and finally embedded in paraffin. Each sample was cut into 3-μm-thick sections using a microtome for subsequent staining.

[0094] 2.3 Staining:

[0095] First, the tissue sections were baked in an oven at 60 °C overnight to prevent detachment. Then, the sections were dewaxed in three baths of xylene, 10 min for each bath. Next, they were rehydrated through a gradient of alcohols (alcohol concentrations were 100%, 100%, 95%, 95%, 70% respectively), 5 min for each bath. Finally, they were placed in pure water and left to stand for 3 min. The steps of HE staining were as follows: dewaxing and rehydration; hematoxylin staining for 2 min, rinsed with pure water 3 min each time for a total of three times; differentiated with 1% hydrochloric acid alcohol for 2 - 3 seconds, rinsed with pure water 3 min each time for a total of three times; blued with 0.5% ammonia water for 10 seconds, rinsed with pure water 3 min each time for a total of three times; eosin staining for 1 min, rinsed with pure water 3 min each time for a total of three times; soaked in 95% ethanol, 100% ethanol, xylene I, xylene II, and xylene III for 1 min each for dehydration and clearing, and then sealed with neutral resin.

[0096] The steps of SO staining were as follows: dewaxing and rehydration; stained with fast green solution for 5 min, rinsed with pure water 3 min each time for a total of three times; differentiated with 1% glacial acetic acid for 10 seconds, rinsed with pure water 3 min each time for a total of three times; stained with safranin O solution for 1 min, rinsed with pure water 3 min each time for a total of three times; differentiated with 95% ethanol for several seconds, rinsed with pure water 3 min each time for a total of three times; dried in an oven at 37 °C and then cleared in xylene, and sealed with neutral resin.

[0097] 2.4 Immunohistochemistry:

[0098] The joint tissue sections of rats in each group were dewaxed and rehydrated, and washed with PBS (1×) for 3 min / time, for a total of three times; antigen repair was performed with sodium citrate solution in a 60°C oven for 4 h; the staining jar was taken out and allowed to rewarm at room temperature for 30 min, and washed with PBS (1×) for 3 min / time, for a total of three times; the sections were placed in 0.1% Triton solution to punch the tissues, and washed with PBS (1×) for 3 min / time, for a total of three times; the PBS around the tissues was carefully wiped dry, and a circle was drawn around the tissues with a water blocking pen to fix the range of subsequent reagents; the PBS around the tissues was carefully wiped dry, an appropriate amount of endogenous peroxidase blocker was added, and the sections were incubated at room temperature for 10 min, and washed with PBS (1×) for 3 min / time, for a total of three times; 100 μL of primary antibody (Col2, MMP13, 1:100) diluted with PBS was added to each section and incubated overnight at 4°C , wash with PBS (1×) for 3 min / time, three times in total; carefully wipe off the PBS around the tissue, add about 50 μl of secondary antibody (enhanced enzyme-labeled goat anti-rabbit / anti-mouse IgG polymer), incubate at room temperature for 20 min, wash with PBS (1×) for 3 min / time, three times in total; wipe off the PBS around the tissue, add an appropriate amount of freshly prepared DAB color developing solution, incubate at room temperature for 5-8 min, wash with PBS (1×) for 3 min / time, three times in total; hematoxylin for 2 min, rinse with pure water for 3 min / time, three times in total; differentiate with 1% hydrochloric acid alcohol for 2-3 seconds, rinse with pure water for 3 min / time, three times in total; reverse blue with 0.5% ammonia water for 10 seconds, rinse with pure water for 3 min / time, three times in total; soak in 95% ethanol, 100% ethanol, xylene I, xylene II, and xylene III for 1 min each, dehydrate and make transparent, and seal with neutral resin. After sealing, the sections were placed on a drying rack and ventilated in a fume hood for several hours. After the neutral resin solidified and the xylene evaporated, photos were taken under an optical microscope. The expression of MMP13 was quantified by counting the number of positive cells, and the expression of Col2 was quantified by calculating the positive area. The ratio of the antigen-positive area / cell volume in the selected area to the total area / total cell volume was used as the final statistical indicator.

[0099] 3. Experimental results

[0100] 3.1 Mechanical pain threshold assessment results

[0101] The results are as follows Figure 4As shown, compared with the normal group (NC), the mechanical pain thresholds of rats in each group showed a significant decrease at different modeling time periods. In addition, at the eighth week of modeling, compared with the OA model group (SS), the mechanical pain thresholds of the cold-damp obstruction syndrome group (CS) and the OA model + cold-damp obstruction syndrome group (SS + CS) showed a significant decrease, with a significant difference. However, there was no significant difference between the CS group and the SS + CS group, indicating that the modeling effects of the two groups on rat behavior were basically the same and better than those of the OA model group (SS).

[0102] 3.2 HE staining and SO staining results

[0103] The results are as Figure 5 shown. Compared with the normal group, the cartilage surface was rough and a large number of chondrocytes underwent apoptosis in the OA model group (SS) and the OA model + cold-damp obstruction syndrome group (SS + CS). In the cold-damp obstruction syndrome group (CS) and the OA model + cold-damp obstruction syndrome group (SS + CS), the arrangement of chondrocytes was disordered, irregular, the cells became round, and a large number of cell vacuoles appeared, indicating hypertrophy of chondrocytes. At the same time, the color of the stained area of hyaline cartilage became lighter in both groups, and the cartilage matrix degenerated. At the same time, the OARSI scores of each modeling group were significantly higher than those of the normal group, indicating that the modeling effects of the two groups were basically the same histologically.

[0104] 3.3 Immunohistochemical index detection results

[0105] The results are as Figure 6 shown. Compared with the normal group, the expression of MMP13 increased and the expression of Col2 decreased significantly in the OA model group (SS), the cold-damp obstruction syndrome group (CS), and the OA model + cold-damp obstruction syndrome group (SS + CS), indicating abnormal changes in the cartilage matrix.

[0106] In summary, according to the aforementioned pain behavioral assessment ( Figure 4 ), pathological observation ( Figure 5 ), and immunohistochemical staining of the anabolic index Col2 and catabolic index MMP13 of chondrocytes ( Figure 6 ), it can be seen that compared with the normal group, the pain threshold of the rats with knee osteoarthritis model of cold-damp obstruction type constructed by the present invention decreased significantly, with a significant difference; there were more vacuoles in chondrocytes in the cartilage tissue, confirming damage and apoptosis of chondrocytes; at the same time, the Safranin-O staining of articular cartilage was significantly weakened, indicating degeneration of the cartilage matrix in the freezing group. Thus, it can be judged that the modeling was successful.

[0107] Moreover, compared with the conventional method of modeling with ice-water mixture, the damp-cold model of the present invention can provide a more stable modeling environment. At the same time, compared with the conventional OA surgical model, this method can effectively simulate the damp-cold exogenous pathogenic factors to make rats show more sensitive pain responses, which is in line with the syndrome phenotype of "severe pain upon encountering cold" in traditional Chinese medicine for damp-cold arthralgia obstruction type OA. Constructing a damp-cold specific-guided syndrome model fills the research gap of "having the disease without the syndrome" in the existing OA research. Moreover, its operation is simple, the repeatability is good, the requirements for operators are low, and it can be used for large-scale construction of animal models.

[0108] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent replacements, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

[0109] The foregoing embodiments and methods described in the present invention may vary based on the capabilities, experience, and preferences of those skilled in the art.

[0110] The mere listing of the steps of the method in a certain order in the present invention does not constitute any limitation on the order of the method steps.

Claims

1. A method for constructing an animal model of knee osteoarthritis, which comprises the following steps: placing the experimental animal in a cold and humid environment, and at the same time placing its hind limbs in an ice-water mixture for modeling; wherein, The cold-damp environment is a constant temperature and humidity environment, where the temperature is 1 - 10 °C and the humidity is 80% - 100%.

2. The method according to claim 1, characterized in that, The cold-damp environment is a constant temperature and humidity incubator, where the temperature is set at 3 - 7 °C and the humidity is set at 90% - 100%; Preferably, the temperature is 5 ± 2 °C; Preferably, the humidity is 95 ± 2%.

3. The method according to claim 1, characterized in that, The modeling treatment lasts for 4 - 16 weeks in total, and lasts for 1 - 6 hours every day; Preferably, the modeling treatment lasts for 4 - 12 weeks in total, and lasts for 2 - 4 hours every day; More preferably, the modeling treatment lasts for 8 weeks in total, and 3 hours every day.

4. The method according to claim 1, characterized in that, The method includes: placing the experimental animal cage in a constant temperature and humidity incubator, setting the environmental conditions as temperature 5 ± 2 °C and humidity 95 ± 2%, and at the same time laying ice-water mixture at the bottom of the cage for 6 - 10 weeks, lasting for 2 - 4 hours every day.

5. The method according to any one of claims 1-4, characterized in that, The experimental animal is a rat; Preferably, the rat is an SD rat or a Wistar rat, especially an SD rat; Preferably, the rat is an adult male rat.

6. The method according to claim 5, characterized in that, The method further includes: regularly monitoring the physical signs and biochemical indexes of the animals during the modeling period; Preferably, the method further includes: regularly detecting the pain threshold of the animals during the modeling period.

7. The method according to claim 5, wherein The method further includes a pathological detection step; Preferably, the method further includes: taking the knee joint of the rat after the modeling treatment is over, and performing pathological tissue sectioning, staining, and immunohistochemical index detection.

8. Application of the animal model constructed by the method according to any one of claims 1 - 7 in drug screening and efficacy evaluation.

9. A drug screening method, which includes the following steps: administering a drug to the animal model constructed by the method according to any one of claims 1 - 7, and comparing it with the animal model without the drug administration.

10. Application of the method according to any one of claims 1 - 7 in the research on the disease mechanism of knee osteoarthritis of cold-damp obstruction type, and the application is for non-diagnostic or therapeutic purposes.

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