Animal model of knee osteoarthritis of cold-dampness obstruction syndrome type, and construction method therefor and use thereof
Patent Information
- Application Number
- PCT/CN2026/080307
- Authority / Receiving Office
- WO · WO
- Patent Type
- Applications
- Current Assignee / Owner
- Priority Date
- 2025-03-26
- Filing Date
- 2026-02-27
- Publication Date
- 2026-10-01
Smart Images
Figure CN2026080307_01102026_PF_FP_ABST
Abstract
Description
An animal model of knee osteoarthritis caused by cold-dampness obstruction, its construction method and application Technical Field
[0001] This invention relates to the field of biotechnology, specifically to an animal model of knee osteoarthritis of the cold-dampness obstruction type, its construction method, and its application. Background Technology
[0002] As the most common degenerative disease in clinical practice, knee osteoarthritis (kOA) is characterized by high incidence and high disability rate. Its main pathological features are degeneration of articular cartilage, subchondral bone sclerosis, and synovitis, which often manifest as knee pain, swelling, and dysfunction.
[0003] The *Suwen* (Plain Questions) chapter on Bi syndrome states, "When wind, cold, and dampness combine, they cause Bi syndrome." Studies on the distribution patterns of KOA syndrome types and syndrome elements in Traditional Chinese Medicine (TCM) show that cold and dampness are the most common two syndrome elements, while wind, cold, and dampness are the most common three syndrome elements. Therefore, cold-dampness obstruction is its main syndrome type. TCM clinical practice treats KOA from the perspective of dispelling cold, eliminating dampness, and relieving pain, achieving positive therapeutic effects. Professor Ding E, a renowned TCM doctor, believes that the primary goal of treating KOA cold-dampness obstruction syndrome is to warm the meridians, dispel cold, eliminate dampness and wind, thereby relieving Bi syndrome and pain, achieving significant results. Although there are many research reports on the TCM pathogenesis of KOA and the treatment with TCM based on syndrome differentiation, the lack of effective and unified methods for constructing animal models hinders in-depth and effective molecular mechanism and cellular experimental research on KOA cold-dampness obstruction syndrome. This significantly limits the development of new drugs for treating KOA cold-dampness obstruction syndrome. Summary of the Invention
[0004] To overcome the shortcomings of the prior art, this invention provides an animal model of knee osteoarthritis of the cold-dampness obstruction type, its construction method, and its application.
[0005] In a first aspect of the present invention, a method for constructing an animal model of knee osteoarthritis is provided, comprising the following steps: placing the experimental animal in a cold and humid environment, while placing its hind limbs in an ice-water mixture to establish the model.
[0006] Specifically, the cold and humid environment is a constant temperature and humidity environment (e.g., in a constant temperature and humidity climate chamber), wherein the temperature is 1-10℃ (e.g., 1, 2, 3, 4, 4.5, 5, 6, 7, 8, 9, 10℃) and the humidity is 80%-100% (e.g., 80%, 85%, 90%, 93%, 94%, 95%, 96%, 97%, 98%, 99%).
[0007] In some embodiments of the present invention, the temperature of the cold and humid environment is 5±2℃.
[0008] In some embodiments of the present invention, the humidity of the cold and humid environment is 95±2%.
[0009] Based on the "Specifications for Establishing Animal Models of Rheumatoid Arthritis with Cold-Dampness Obstruction Syndrome and Damp-Heat Obstruction Syndrome" issued by the Chinese Association of Traditional Chinese Medicine, the inventors of this invention, through multiple experimental measurements and calibrations, stably controlled the environmental parameters within the range of 5±2℃ and 95±2% relative humidity, which can better simulate a 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℃, for example 0±3℃, 0±2℃, 0±1℃, especially 0±2℃.
[0011] Specifically, the depth of the ice-water mixture is 30-60 mm, for example 30, 35, 40, 45, 50, 55, 60 mm, especially 40-50 mm.
[0012] Specifically, the modeling treatment lasts for 4-16 weeks (e.g., 4, 6, 8, 10, 12, 14, 16 weeks), for example, 4-12 weeks, 6-10 weeks, for 1-6 hours daily (e.g., 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, for 3 hours daily. To simulate the TCM pathogenesis characteristics of "cold and dampness entering the collaterals and obstructing the meridians" and its chronic course of osteoarthritis of the cold-dampness obstruction type, the experimental part of the embodiments of the present invention uses an 8-week animal modeling experiment, exposing experimental animals to a cold and damp environment (temperature 5±2℃, relative humidity 95±2%) for 3 hours daily, to gradually accumulate stimulation and induce the chronic pathological process of osteoarthritis of the cold-dampness obstruction type. At the same time, by controlling the duration of a single exposure, non-specific damage caused by low temperature can be avoided, thus better constructing an animal model of knee osteoarthritis of the cold-dampness obstruction type.
[0013] In some embodiments of the present invention, the method includes: placing an experimental animal cage (with the experimental animal in the cage) in a constant temperature and humidity climate chamber, with the environmental conditions set at a temperature of 5±2℃ and a humidity of 95±2%, while laying an ice-water mixture at the bottom of the cage (due to the stimulation of freezing low temperature, the experimental animal in the cage cannot keep its limbs on the ground for a long time, and generally adopts an upright position to reduce contact with the ice-water mixture; by combining "systemic-local" freezing, a cold and humid environment is simulated to model osteoarthritis), for 6-10 weeks (e.g., 8 weeks), for 2-4 hours per day (e.g., 3 hours per day).
[0014] Specifically, the experimental animal is a rat.
[0015] Specifically, the rats may be SD rats or Wistar rats, especially SD rats.
[0016] Specifically, the rats referred to are adult rats.
[0017] Specifically, the rats were healthy, SPF-grade rats.
[0018] Specifically, the rats are male rats.
[0019] Specifically, the method also includes: regularly monitoring the animal's physical signs and biochemical indicators during the modeling period.
[0020] Specifically, physical signs include mental state, skin and hair, body weight, foraging, nails, and stool.
[0021] Specifically, the method further includes periodically detecting the animal's pain threshold during modeling.
[0022] Specifically, the method also includes a pathological testing step (to verify whether the model construction was successful).
[0023] In some embodiments of the present invention, the method further includes: removing the rat knee joint after the modeling process is completed, and performing pathological tissue sectioning, staining, and immunohistochemical index detection.
[0024] Specifically, the immunohistochemical markers include MMP13 and Col2.
[0025] In a second aspect of the invention, an animal model constructed by the method described in the first aspect is provided.
[0026] In a third aspect of the invention, the method described in the first aspect and the animal model constructed therefrom are provided for application in drug screening and efficacy evaluation.
[0027] Specifically, the drug is used to treat knee osteoarthritis, particularly the cold-dampness obstruction type of knee osteoarthritis.
[0028] In a fourth aspect of the present invention, a drug screening method is provided, comprising the following steps: administering a drug to an animal model constructed by the method described in the first aspect, and comparing it with an animal model that has not been administered the drug (the drug that improves or cures the symptoms of knee osteoarthritis after administration is a candidate drug).
[0029] Specifically, the method may further include conducting one or more combinations of cell experiments, animal experiments, and clinical trials on the candidate drug to detect its properties in terms of efficacy, pharmacokinetics, or toxicology.
[0030] In a fifth aspect of the invention, an animal model constructed by the method described in the first aspect is provided for the study of the disease mechanism of knee osteoarthritis of the cold-dampness obstruction type.
[0031] Specifically, the application is for non-diagnostic or therapeutic purposes.
[0032] This invention provides a stable, effective, and reproducible method for constructing an animal model of knee osteoarthritis caused by cold-dampness obstruction. The resulting animal model can provide a reliable experimental vehicle for new drug development, efficacy evaluation, and mechanism of action research, and has very good application prospects. Attached Figure Description
[0033] Figure 1 shows the pain threshold assessment results of rats in each group at different modeling time periods. Figure 1A shows the mechanical pain threshold and thermal pain threshold of rats in each group at week 8 of modeling, and Figure 1B shows the mechanical pain threshold and thermal pain threshold of rats in each group at week 12 of modeling.
[0034] Figure 2 shows the pathological examination results of cartilage tissue in each group of rats. Figure 2A shows the SO staining results of knee joint cartilage sections of rats in each group, and Figure 2B shows the Mankin's score results of rats in each group.
[0035] Figure 3 shows the environmental impact of adding and not adding a constant temperature chamber during modeling. Figure 3A shows the water temperature test results without a constant temperature chamber (with water and ice), Figure 3B shows the humidity test results of the climate chamber after adding a constant temperature chamber (with ice), and Figure 3C shows the temperature test results of the climate chamber after adding a constant temperature chamber (with ice).
[0036] Figure 4 shows the mechanical pain threshold assessment results of rats in each group at different modeling time periods. Figure 4A shows the mechanical pain threshold assessment results of rats in each group before modeling, Figure 4B shows the mechanical pain threshold assessment results of rats in each group at week 4 of modeling, and Figure 4C shows the mechanical pain threshold assessment results of rats in each group at week 8 of modeling.
[0037] Figure 5 shows the staining results of knee joint sections from rats in each group.
[0038] Figure 6 shows the results of immunohistochemical marker detection in each group of rats. Detailed Implementation
[0039] Unless otherwise defined, all scientific and technical terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art.
[0040] The diagnostic criteria for cold-dampness obstruction type knee osteoarthritis described in this invention can be referenced from: Traditional Chinese Medicine syndrome diagnosis criteria: refer to the "Diagnosis and Treatment Plan for Knee Osteoarthritis (Knee Joint Osteoarthritis) of the National Administration of Traditional Chinese Medicine 'Eleventh Five-Year Plan' Key Specialty Collaboration Group".
[0041] All publications, patents, and published patent specifications cited in this article are incorporated herein in their entirety through citation.
[0042] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.
[0043] Example 1
[0044] 1. Experimental grouping and treatment
[0045] 1.1 Experimental Materials
[0046] Male adult Sprague-Dawley (SD) rats (SPF II), weighing 200±2g, 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 three groups: normal group, OA surgery group, and cold and damp (water soaking) group, with 15 rats in each group.
[0049] No action is taken for the normal group.
[0050] Surgical group treatment: DMM surgery (destabilization of the medial meniscus) was performed.
[0051] Cold and damp (water immersion) group treatment: Rats were placed in water at a temperature of about 10±2℃ (with ice packs added to control the temperature) (because rats have hydrophobia, they will be in an upright position with their upper and lower limbs upright). The rats were kept in a fixed position with their lower limbs upright for about 3 hours a day in water at a temperature of about 10±2℃. This was repeated for 12 weeks, and samples were collected and tested at weeks 8 and 12.
[0052] 2. Detection Method
[0053] 2.1 The method for measuring the mechanical pain threshold in rats is as follows: Von Frey wire (measurement range 0.6–26 g) conforming to the standards for evaluation in experimental animal behavior and neuroscience was used to detect the mechanical withdrawal latency (MWL) in rats. Measurements were taken before each batch of samples. Specifically, SD rats were placed on stainless steel wire mesh, individually separated by transparent acrylic cages to restrict movement. The testing room was kept quiet, and after 1 hour of pre-acclimatization, the rats were tested with their limbs fully in contact with the wire mesh. Nylon fibers of different measurement ranges were used to apply vertical pressure sequentially to the central area of the hind limb paw (avoiding the paw pads) until the fibers formed a C-shape, held for 5 seconds. The withdrawal response was observed, and the mechanical withdrawal latency (g) was recorded and statistically analyzed to assess the mechanics of rats with osteoarthritis.
[0054] 2.2 The method for measuring the thermal withdrawal latency (TWL) in rats was as follows: The thermal withdrawal latency (TWL) of rats was measured before model establishment and before each batch of samples were collected. A plantar thermal pain sensitivity tester was used to detect the thermal withdrawal latency of both hind toes of the rats. During measurement, the rats were placed in a transparent plexiglass box, and the room temperature was maintained at 25±2℃. After the rats calmed down (stopping grooming and exploratory activities), the "+" shaped marker on the tester was placed in the center of the left / right hind metatarsal sole of the rat, avoiding the paw pad. The instrument was then activated, and the rat's response was observed. The time period from turning on the instrument until the rat lifted its leg to avoid the test was taken as the rat's thermal pain value (s). Each rat was tested 3 times, with an interval of 5–6 minutes between each test. To prevent burns from heat radiation, the upper limit of the thermal withdrawal latency measurement time was set at 20 seconds, and the upper limit of the temperature was set at 35℃.
[0055] 2.3 Take bilateral knee joints of rats, clean them with physiological saline, place them in 4% paraformaldehyde solution, and after 1 week, soak them in 10% ethylenediaminetetraacetic acid (EDTA) solution. Change the EDTA solution once a week and soak them continuously for 4 weeks until the joints are soft and the needle can be easily penetrated. Prepare pathological sections.
[0056] 3. Experimental Results
[0057] 3.1 Effects on pain threshold in rats
[0058] As shown in Figure 1, after 8 weeks (W) of modeling, there were no statistically significant differences in mechanical pain threshold and thermal pain threshold among rats in other groups compared with the normal group (p > 0.05). After 12 weeks of modeling, the OA surgery group showed significant pain sensitization characteristics compared with the normal control group, with significantly reduced mechanical pain threshold and thermal pain threshold (p < 0.01), while there were no significant differences in mechanical pain threshold and thermal pain threshold among the cold and damp (water immersion) group (p > 0.01).
[0059] 3.2 Effects on the histopathology of rat knee joint tissue
[0060] Pathological results are shown in Figure 2 (SO staining). Eight weeks after modeling, compared with the normal group, the OA surgery group showed obvious defects on the surface of the knee joint cartilage, and severe loss of chondrocytes at the defect site; the cartilage in the cold-dampness (water immersion) group was basically normal, with no obvious changes on the cartilage surface. Quantitative analysis using the Mankin pathological scoring system showed that the OA group had already shown significant degenerative characteristics at week 8, and its score was significantly higher than that of the normal group (p < 0.05); compared with the normal group, the Mankin's score in the cold-dampness (water immersion) group showed no significant change (p > 0.05).
[0061] Twelve weeks after modeling, compared with the normal group, the OA surgery group showed obvious defects on the surface of the knee joint cartilage, with severe loss of chondrocytes and significant degradation of proteoglycans at the defect sites; the cartilage in the cold-dampness (water immersion) group was basically normal, with local wear visible on the cartilage surface and a small number of mast chondrocytes. Compared with normal, the Mankin's score in the OA surgery group was significantly increased (p < 0.05); compared with normal, the Mankin's score in the cold-dampness (water immersion) group showed no significant change (p > 0.05).
[0062] The main clinical manifestations of kOA include pain and functional impairment. However, the experimental results regarding pain threshold and joint histopathology showed that rats in the cold-dampness (water immersion) group did not exhibit the characteristic pathological changes of kOA. These results suggest that the existing cold-dampness (water immersion) model, due to the difficulty in precisely controlling temperature and humidity, fails to effectively simulate the pathogenic environment of low temperature and high humidity, thus failing to stably induce articular cartilage lesions in rats. It is necessary to construct an animal model of cold-dampness obstruction-type osteoarthritis by standardizing and controlling the modeling environment parameters and optimizing the modeling conditions.
[0063] Example 2
[0064] Example 1 uses a cold water bath method with water stacked with ice boxes, but it was found during the modeling process that it could not effectively slow down the rise of water temperature and simulate a low temperature environment. The inventors 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] The high-precision constant temperature and humidity climate chamber HWS-600 (hereinafter referred to as the constant temperature chamber) was purchased from Shanghai Chuanhong Company;
[0068] The AF100 snow ice maker, used to prepare crushed ice in ice-water mixtures, was purchased from SCOTSMAN.
[0069] 1.2 Experimental Methods
[0070] After acclimatization, the rats were divided into two groups: 1) Rats were placed in a constant temperature incubator with core parameters set as follows: temperature 5±2℃, humidity 95±2%, and standardized exposure for 3 hours daily in a cold and humid environment. Simultaneously, an ice-water mixture (approximately 50mm water level, 0±2℃) was placed at the bottom of the cages. Due to the freezing and low temperature stimulation, the rats could not maintain their limbs on the ground for extended periods and generally adopted an upright posture to reduce contact with the ice-water mixture. This "systemic-local" combined freezing method simulated a cold and humid environment to induce osteoarthritis. 2) No constant temperature incubator was used; only an ice-water mixture (approximately 50mm water level, 0±2℃) was placed at the bottom of the cages.
[0071] Measure the temperature and humidity of the modeling environment. Without a constant temperature chamber, measure the temperature of the ice-water mixture using a thermometer; with the constant temperature chamber, measure the temperature and humidity using the machine's built-in temperature and humidity control system. Repeat the experiment four times.
[0072] 2. Experimental Results
[0073] Comparison of the impact of adding and not adding a temperature control chamber on the molding environment
[0074] As shown in Figure 3, compared with the case without a constant temperature chamber, the constant temperature chamber can ensure that the temperature inside the chamber is stable at 4-5℃, the water temperature is stable at 0-1℃, and the humidity is maintained at around 95%, which can well simulate the cold and humid environment and make the animal model more accurate.
[0075] Example 3
[0076] 1. Experimental grouping and treatment
[0077] 1.1 Experimental Materials
[0078] Male adult Sprague-Dawley (SD) rats (SPF II), weighing 200±2g, were provided by Shanghai Super B&K Laboratory Animal Co., Ltd.; grouping experiments were conducted after a 1-week acclimatization period.
[0079] The high-precision constant temperature and humidity climate chamber HWS-600, used for cold and humid modeling, was purchased from Shanghai Chuanhong Company.
[0080] The AF100 snow ice maker, used to prepare crushed ice in ice-water mixtures, was purchased from SCOTSMAN; the plantar apnea tester, used for measuring mechanical foot contraction pain threshold, was purchased from Ugo Basile, Italy.
[0081] 1.2 Grouping of experimental animals
[0082] Forty rats were randomly divided into four groups: 1) normal group (NC); 2) OA model group (SS); 3) cold-dampness obstruction syndrome group (CS); 4) OA model + cold-dampness obstruction syndrome group (SS+CS).
[0083] No action is taken for the normal group.
[0084] The OA surgical model group (SS) used the anterior cruciate ligament transection (ACLT) method to surgically induce knee OA (kOA). The specific steps were as follows: After weighing the rats, they were injected intraperitoneally with 3% sodium pentobarbital solution (0.15 ml per 100g). After complete anesthesia, a longitudinal incision was made on the patellar side of the knee joint. The skin and muscles were dissected layer by layer to fully expose the knee joint cavity. The anterior cruciate ligament was cut and confirmed by the drawer test. Finally, the joint capsule and skin were sutured in layers, and hemostasis was achieved in time to avoid infection.
[0085] Cold-dampness obstruction syndrome group (CS): Rats were placed in a constant temperature and humidity climate chamber with environmental conditions set at 5±2℃ and 95±2% for 3 hours daily. At the same time, an ice-water mixture (water level of about 50mm and temperature of 0±2℃) was laid at the bottom of the rat cage. Due to the freezing low temperature stimulation, the rats could not keep their limbs on the ground for a long time and generally adopted an upright position to reduce contact with the ice-water mixture. The cold-dampness environment was simulated by the "system-local" freezing method to induce osteoarthritis model, which lasted for 8 weeks.
[0086] The OA model + cold-dampness obstruction syndrome group (SS+CS) was constructed by surgical induction using the anterior cruciate ligament transection (ACLT) method of rat knee joint combined with the cold-dampness model construction method of the present invention.
[0087] 2. Detection Method
[0088] The main clinical manifestations of knee osteoarthritis (KOA) include pain and functional impairment. Animals will also show corresponding symptoms when KOA develops. Therefore, assessing the symptoms of animals can evaluate the modeling effect (Chen 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] Furthermore, the process of cartilage matrix loss in rats involves: first, superficial fibrosis and matrix loss, followed by deep fibrosis, matrix loss, and lesion expansion in the middle layer, ultimately leading to matrix thickness loss into the tibia in lesions or large localized areas. Overall cartilage pathological assessment includes important pathological parameters such as collagen matrix fibrosis / loss and chondrocyte death / loss. Chondrocyte loss is a major determinant of osteoarthritis, and areas with matrix and chondrocyte loss show proteoglycan loss. KOA lesions can be evaluated at the cellular and tissue levels using staining methods.
[0090] 2.1 Mechanical pain threshold assessment:
[0091] Mechanical allergic reactions are measured using an “up-down” test method with a range of von Frey yarns (ranging from 0.6-26 grams, UGO Basile, Italy). In short, the von Frey yarn is pressed vertically onto the mid-metaphysis of the hind paw with sufficient force to bend it, and 50% PWT is calculated.
[0092] 2.2 Materials taken:
[0093] Eight weeks after cryo-modeling (eight weeks post-ACLT), the patient was anesthetized with intraperitoneal injection of sodium pentobarbital (3% concentration, 0.15 ml / 100 g). Blood was drawn from the heart using a 10 ml syringe. The muscles near the knee joint were then removed with scissors, and the knee joint was removed using bone forceps. The joint was then fixed in 4% paraformaldehyde solution for 3 days. After 3 days of fixation, the paraformaldehyde on the joint sample surface was rinsed off with running water. The sample was then placed in an embedding cassette, labeled, and placed in EDTA solution for decalcification. The EDTA decalcification solution was changed daily for approximately two months. Once the bone tissue in the joint softened, the sample was rinsed under running water overnight. The sample was then dehydrated and finally embedded in paraffin. Each sample was sectioned to a thickness of 3 μm using a microtome for subsequent staining.
[0094] 2.3 Staining:
[0095] First, the tissue sections were baked overnight in a 60℃ oven to prevent them from falling off. Then, the sections were dewaxed in three xylene solutions for 10 minutes each. Next, they were rehydrated with a gradient of alcohols (100%, 100%, 95%, 95%, and 70% alcohol concentrations) for 5 minutes each. Finally, they were placed in pure water and allowed to stand for 3 minutes. The HE staining procedure was as follows: dewaxing and rehydration; hematoxylin for 2 minutes, followed by rinsing with pure water for 3 minutes each time, for a total of three times; differentiation with 1% hydrochloric acid alcohol for 2-3 seconds, followed by rinsing with pure water for 3 minutes each time, for a total of three times; blue inversion with 0.5% ammonia for 10 seconds, followed by rinsing with pure water for 3 minutes each time, for a total of three times; eosin for 1 minute, followed by rinsing with pure water for 3 minutes each time, for a total of three times; immersion in 95% ethanol, 100% ethanol, xylene I, xylene II, and xylene III for 1 minute each, followed by dehydration and clearing, and mounting with neutral resin.
[0096] SO staining steps are as follows: dewaxing and rehydration; Fast Green staining solution for 5 min, rinse with pure water for 3 min each time, for a total of three times; differentiation with 1% glacial acetic acid for 10 seconds, rinse with pure water for 3 min each time, for a total of three times; Safranin O staining solution for 1 min, rinse with pure water for 3 min each time, for a total of three times; differentiation with 95% ethanol for a few seconds, rinse with pure water for 3 min each time, for a total of three times; drying in an oven at 37℃, then clearing in xylene, and mounting with neutral resin.
[0097] 2.4 Immunohistochemistry:
[0098] Rat joint tissue sections from each group were dewaxed and rehydrated, then washed with PBS (1×) for 3 min each time, for a total of three times. Antigen retrieval was performed using sodium citrate solution in a 60℃ oven for 4 h. The staining tank was then removed and allowed to warm to room temperature for 30 min, followed by washing with PBS (1×) for 3 min each time, for a total of three times. The sections were then perforated in 0.1% Triton solution, and washed with PBS (1×) for 3 min each time, for a total of three times. The PBS around the tissue was carefully dried, and a water-resistant pen was used to draw circles around the tissue to define the area for subsequent reagents. The PBS around the tissue was carefully dried again, and an appropriate amount of endogenous peroxidase inhibitor was added. The sections were incubated at room temperature for 10 min, followed by washing with PBS (1×) for 3 min each time, for a total of three times. 100 μL of primary antibody (Col2, MMP13, 1:100) diluted in PBS was added to each section, and the sections were incubated overnight at 4℃. Wash with PBS (1×) for 3 min each time, for a total of three times; carefully wipe the PBS around the tissue dry, 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 each time, for a total of three times; wipe the PBS around the tissue dry, add an appropriate amount of freshly prepared DAB chromogenic solution, incubate at room temperature for 5-8 min, wash with PBS (1×) for 3 min each time, for a total of three times; hematoxylin for 2 min, rinse with pure water for 3 min each time, for a total of three times; differentiate with 1% hydrochloric acid alcohol for 2-3 seconds, rinse with pure water for 3 min each time, for a total of three times; invert blue with 0.5% ammonia water for 10 seconds, rinse with pure water for 3 min each time, for a total of three times; soak in 95% ethanol, 100% ethanol, xylene I, xylene II, and xylene III for 1 min each, dehydrate and clear, and mount with neutral resin. After mounting, the slides were placed on a drying rack and ventilated in a fume hood for several hours until the neutral resin solidified and the xylene evaporated. Then, they were photographed under an optical microscope. MMP13 expression was quantified by calculating the number of positive cells, and Col2 expression was quantified by calculating the positive area. The ratio of antigen-positive area / cell count in the selected region to the total area / total cell count was used as the final statistical indicator.
[0099] 3. Experimental Results
[0100] 3.1 Results of Mechanical Pain Threshold Assessment
[0101] As shown in Figure 4, compared with the normal group (NC), the mechanical pain threshold of rats in each group was significantly reduced at different modeling time points. In addition, at the eighth week of modeling, compared with the OA model group (SS), the mechanical pain threshold of rats in the cold-dampness obstruction syndrome group (CS) and the OA model + cold-dampness obstruction syndrome group (SS+CS) was significantly reduced, showing a significant difference. However, there was no significant difference between the CS group and the SS+CS group. This indicates that the modeling effect of the two groups in rat behavior was basically the same, and better than that of the OA model group (SS).
[0102] 3.2 Results of HE staining and SO staining
[0103] As shown in Figure 5, compared with the normal group, the OA model group (SS) and the OA model + cold-dampness obstruction syndrome group (SS+CS) showed rough cartilage surface and a large number of chondrocyte apoptosis. In the cold-dampness obstruction syndrome group (CS) and the OA model + cold-dampness obstruction syndrome group (SS+CS), chondrocytes were disordered, irregularly shaped, rounded, and showed numerous vacuoles, indicating chondrocyte hypertrophy. Simultaneously, both groups showed lighter hyaline cartilage staining areas and cartilage matrix degeneration. Furthermore, the OARSI scores of each model group were significantly higher than those of the model group, indicating that the histological modeling effects of the two groups were basically consistent.
[0104] 3.3 Immunohistochemical index detection results
[0105] As shown in Figure 6, compared with the normal group, MMP13 expression increased and Col2 expression decreased significantly in the OA model group (SS), cold-dampness obstruction syndrome group (CS), and OA model + cold-dampness obstruction syndrome group (SS+CS), indicating abnormal changes in the cartilage matrix.
[0106] In summary, based on the aforementioned pain behavior assessment (Figure 4), pathological observation (Figure 5), and immunohistochemical staining results of chondrocyte anabolic metabolic index Col2 and catabolism index MMP13 (Figure 6), it can be seen that, compared with the normal group, the pain threshold of the rat model of knee osteoarthritis of cold-dampness obstruction constructed in this invention was significantly reduced, showing a significant difference; more vacuoles appeared in chondrocytes in the cartilage tissue, confirming chondrocyte damage and apoptosis; at the same time, Safranin-O staining of articular cartilage was significantly weakened, indicating that the cartilage matrix in the frozen group had degenerated, thus indicating that the model was successfully established.
[0107] Furthermore, compared with conventional ice-water mixture modeling methods, the cold-dampness model of this invention can provide a more stable modeling environment. At the same time, compared with conventional OA surgical models, this method can effectively simulate the more sensitive pain response of rats caused by cold-dampness external pathogens, which is consistent with the syndrome phenotype of "severe pain when exposed to cold" in traditional Chinese medicine cold-dampness obstruction type OA. The construction of a cold-dampness specific guided syndrome model fills the research gap of "disease without syndrome" in existing OA research. Moreover, it is simple to operate, has good reproducibility, and has low requirements for the treatment personnel, and can be used to construct animal models on a large scale.
[0108] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.
[0109] The foregoing embodiments and methods described in this invention may vary based on the capabilities, experience, and preferences of those skilled in the art.
[0110] Listing the steps of the method in a certain order in this invention does not constitute any restriction on the order of the method steps.
Claims
1. A method for constructing an animal model of knee osteoarthritis, comprising the following steps: placing the experimental animal in a cold and humid environment, while simultaneously placing its hind limbs in an ice-water mixture to establish the model; wherein, The cold and humid environment is a constant temperature and humidity environment, with a temperature of 1-10℃ and a humidity of 80%-100%.
2. The method as described in claim 1, characterized in that, The cold and humid environment is a constant temperature and humidity climate chamber, in which the temperature is set to 3-7℃ and the humidity is set to 90%-100%. Preferably, the temperature is 5±2℃; Preferably, the humidity is 95±2%.
3. The method as described in claim 1, characterized in that, The molding process lasts for 4-16 weeks, with each day lasting 1-6 hours. Preferably, the molding process lasts for 4-12 weeks, with each day lasting 2-4 hours. More preferably, the molding process lasts for a total of 8 weeks, 3 hours per day.
4. The method as described in claim 1, characterized in that, The method includes: placing the experimental animal cages in a constant temperature and humidity climate chamber, setting the environmental conditions to a temperature of 5±2℃ and a humidity of 95±2%, while laying an ice-water mixture at the bottom of the cages for 6-10 weeks, for 2-4 hours daily.
5. The method according to any one of claims 1-4, characterized in that, The experimental animal was a rat; Preferably, the rat is an SD rat or a Wistar rat, especially an SD rat; Preferably, the rat is a male adult rat.
6. The method as described in claim 5, characterized in that, The method also includes: regularly monitoring the animal's physical signs and biochemical indicators during the modeling period; Preferably, the method further includes periodically detecting the animal's pain threshold during modeling.
7. The method as described in claim 5, characterized in that, The method also includes a pathological testing step; Preferably, the method further includes: removing the rat knee joint after the modeling process and performing pathological tissue sectioning, staining, and immunohistochemical index detection.
8. The 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 comprising the steps of: administering a drug to an animal model constructed by the method of any one of claims 1-7, and comparing it with an animal model not administered the drug.
10. The application of the method according to any one of claims 1-7 in the study of the disease mechanism of knee osteoarthritis of the cold-dampness obstruction type, wherein the application is for non-diagnostic or therapeutic purposes.