Traditional Chinese medicine composition for preventing and / or treating knee osteoarthritis as well as preparation method and application thereof

Abstract

The invention discloses a traditional Chinese medicine composition for preventing and / or treating knee osteoarthritis as well as a preparation method and application thereof, and belongs to the technical field of traditional Chinese medicines. The traditional Chinese medicine is characterized by being prepared from the following components in parts by weight: 10 parts of salted eucommia ulmoides, 15 parts of radix rehmanniae recen, 20 parts of astragalus membranaceus, 12 parts of angelica sinensis, 12 parts of radix achyranthis bidentatae, 9 parts of ligusticum wallichii, 12 parts of steamed fructus corni, 10 parts of bran Qu fructus aurantii, 15 parts of bran radix paeoniae alba and 5 parts of honey-fried licorice root. The traditional Chinese medicine composition provided by the invention not only can delay the progress of knee osteoarthritis, but also can reduce the pain of knee osteoarthritis. In-vivo and in-vitro experiments prove that the OARSI score of knee osteoarthritis treated by the kidney marrow anglerkang potion is reduced, the expression of type II collagen and matrix metalloproteinase-13 is obviously changed, and the volume fraction of subchondral bone is reduced; the expression of peripheral nerve secretion pain sensitizing factors is improved, and the pain threshold value is increased. Therefore, the kidney-marrow health drink has a good application prospect in treatment of knee osteoarthritis.

Description

Technical Field

[0001] The present invention relates to the technical field of traditional Chinese medicine, and in particular to a traditional Chinese medicine composition for preventing and / or treating knee osteoarthritis, and a preparation method and application thereof. Background Art

[0002] Osteoarthritis (OA) is an age-related degenerative disease, and joint pain is its key clinical symptom. OA is characterized by pathological features such as cartilage degeneration, abnormal subchondral bone remodeling, and osteophyte formation at the joint margins. OA often accumulates in the weight-bearing joints of the human body, and knee osteoarthritis (KOA) is one of the most common types of OA. In view of the continuous advancement of the global aging trend, the incidence of OA has been increasing year by year. The latest epidemiological data show that approximately 250 million people suffer from OA worldwide. In China, the proportion of people suffering from KOA reaches 8.1%, and the number of patients exceeds 120 million. However, the current clinical treatment of KOA drugs is mainly based on analgesic symptomatic treatment, and long-term use is often accompanied by obvious toxic and side effects.

[0003] Traditional Chinese medicine has unique advantages in the prevention and treatment of KOA, and it is believed that its symptoms are similar to "knee arthritis". Based on the classic Chinese medicine theory of "kidney controls bone and produces marrow", traditional medicine believes that "marrow" is an important fat-like substance in the bones, which is homologous to the kidney and has the effects of promoting sperm, producing blood, and nourishing bones. Contemporary research has found that the function of bone marrow mesenchymal stem cells is highly similar to the "marrow" in traditional Chinese medicine. As progenitor cells, they can differentiate into cartilage and osteoblasts to promote bone repair. On this basis, the "Zhejiang School of Traditional Chinese Medicine" bone injury established the "marrow bone disease" theory, pointing out that the fundamental pathogenesis of KOA is "kidney deficiency, marrow atrophy, bone dryness and pain", and then proposed the method of "tonifying the kidney and strengthening bones, benefiting the marrow and relieving pain". Therefore, it is crucial to develop effective drugs that have the functions of tonifying the kidney and marrow, unblocking the meridians and relieving pain, and can significantly relieve the symptoms of joint swelling, pain, and functional limitation in osteoarthritis during the attack period, improve the quality of life of patients, and delay the progression of the disease. Summary of the invention

[0004] The purpose of the present invention is to provide a Chinese medicine composition for preventing and / or treating knee osteoarthritis and its preparation method and application, so as to solve the problems existing in the above-mentioned prior art. The present invention systematically explores the possible mechanism of Shen Sui An Kang Yin in treating KOA through animal experiments, aiming to provide a new scientific basis for the clinical treatment of KOA.

[0005] To achieve the above object, the present invention provides the following solutions:

[0006] One of the technical solutions of the present invention is a traditional Chinese medicine composition for preventing and / or treating knee osteoarthritis, which is prepared from the following components in parts by weight: 6-10 parts of salt eucommia, 9-15 parts of raw rehmannia, 15-20 parts of astragalus, 9-12 parts of angelica, 9-12 parts of achyranthes, 6-9 parts of chuanxiong, 9-12 parts of steamed cornus, 6-10 parts of bran quzhi fructus aurantii, 9-15 parts of white peony root, and 3-5 parts of roasted liquorice.

[0007] The second technical solution of the present invention is a method for preparing the Chinese medicine composition, which comprises weighing the medicinal materials by weight, soaking them and decocting them by reflux method, and concentrating the medicinal liquid to obtain the Chinese medicine composition.

[0008] A third technical solution of the present invention is the use of the Chinese medicine composition in the preparation of a drug for preventing and / or treating knee osteoarthritis.

[0009] A fourth technical solution of the present invention is the use of the traditional Chinese medicine composition in the preparation of a drug for reducing OARSI scores and regulating the expression of type II collagen and matrix metalloproteinase-13.

[0010] A fifth technical solution of the present invention is the use of the Chinese medicine composition in the preparation of a drug for improving the expression of pain sensitizing factor NGF secreted by peripheral nerves and increasing the pain threshold.

[0011] The sixth technical solution of the present invention is the use of the Chinese medicine composition in the preparation of a drug for reducing the medullary cavity space in the subchondral bone area.

[0012] Based on the above technical solution, the present invention has the following technical effects:

[0013] 1) It can not only delay the progression of knee osteoarthritis, but also reduce the pain of knee osteoarthritis. 2) Through in vivo and in vitro experiments, it was verified that after treatment with Shen Sui An Kang Yin, the OARSI score of knee osteoarthritis was reduced, the expression of type II collagen and matrix metalloproteinase-13 was significantly changed, and the subchondral bone volume fraction was decreased; the expression of pain sensitization factors secreted by peripheral nerves was improved, the pain threshold was increased, and the safety of Shen Sui An Kang Yin in treating KOA was verified. Therefore, Shen Sui An Kang Yin has a good application prospect for the treatment of knee osteoarthritis. Because the current clinical drugs for the treatment of knee osteoarthritis are mostly based on analgesic symptomatic treatment, there is a lack of effective treatment methods. Therefore, the present invention can not only delay the progression of knee osteoarthritis, but also alleviate the peripheral pain sensitization of knee osteoarthritis. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0015] Figure 1 These are the Micro-CT three-dimensional reconstruction images of the subchondral bone of the articular joints of each group of mice after continuous intervention with the Shen Sui An Kang Yin of the present invention for 8 weeks; wherein A is the Micro-CT scanning result, and B is the relative volume result of the bone tissue.

[0016] Figure 2 These are the ABH / OG staining images of the knee cartilage and bone morphometric analysis images of each group of mice after continuous intervention with the Shen Sui An Kang Yin of the present invention for 8 weeks; wherein A is the ABH / OG staining scan result image, and B is the OARSI scoring result image of each knee joint.

[0017] Figure 3 These are the immunohistochemical staining images of Col2a1 and MMP-13 in the knee cartilage of mice in each group after continuous intervention with the Shen Sui An Kang Yin of the present invention for 8 weeks; wherein, A is the Col2a1 and MMP-13 staining result image, B is the Col2a1 relative quantitative analysis result image, and C is the MMP-13 relative quantitative analysis result image.

[0018] Figure 4 The graphs of pain-related behavioral tests of mice in each group after continuous intervention with Shen Sui An Kang Yin for 8 weeks are shown in the figure. A is the result graph of mechanical pain threshold, B is the result graph of hot plate test, C is the result graph of gait test, D is the result graph of the right lower limb stance phase time of gait test, E is the result graph of the right lower limb stride length of gait test, and F is the result graph of the right lower limb plantar contact area of ​​gait test.

[0019] Figure 5 These are the immunofluorescence staining images of NGF in the subchondral bone and DRG of each group of mice after continuous intervention with the Shen Sui An Kang Yin of the present invention for 8 weeks, wherein A is the immunofluorescence staining image of NGF in the subchondral bone and DRG, B is the relative quantitative analysis result image of NGF in DRG, and C is the relative quantitative analysis result image of NGF in the subchondral bone.

[0020] Figure 6 H&E staining of the viscera of mice in each group after continuous intervention with Shen Sui An Kang Yin of the present invention for 8 weeks.

[0021] Figure 7 The results of the effects of different concentrations of Shen Sui An Kang Yin-containing serum on chondrocyte survival are shown in Figure 1. A is a 24-hour CCK8 result diagram, and B is a 48-hour CCK8 result diagram.

[0022] Figure 8 This is a picture of Alcian blue staining of chondrocytes by the medicinal serum of Shen Sui An Kang Yin of the present invention.

[0023] Fig. 9 The figure is a Real-time PCR analysis result diagram of the mRNA expression of cartilage degeneration related genes by the Shen Sui An Kang Yin medicated serum of the present invention; wherein A is the analysis result diagram of Col2a1 and B is the analysis result diagram of Mmp13. DETAILED DESCRIPTION

[0024] 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 should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0025] It should be understood that the terms described in the present invention are only for describing special embodiments and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. 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.

[0026] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. 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 or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0027] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present application description and examples are exemplary only.

[0028] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0029] The technical solutions described in the present invention, unless otherwise specified, are all conventional solutions in the art, and the reagents or raw materials used, unless otherwise specified, are purchased from commercial channels or have been disclosed.

[0030] The embodiment of the present invention provides a traditional Chinese medicine composition for preventing and / or treating knee osteoarthritis, which is prepared from the following components in parts by weight: 6-10 parts of salt eucommia, 9-15 parts of raw rehmannia, 15-20 parts of astragalus, 9-12 parts of angelica, 9-12 parts of achyranthes, 6-9 parts of chuanxiong, 9-12 parts of steamed cornus, 6-10 parts of bran quzhi fructus aurantii, 9-15 parts of white peony root, and 3-5 parts of roasted liquorice.

[0031] In some specific embodiments, the composition is made of the following components in parts by weight: 10 parts of salt eucommia, 15 parts of raw rehmannia, 20 parts of astragalus, 12 parts of angelica, 12 parts of Achyranthes bidentata, 9 parts of chuanxiong, 12 parts of steamed cornus fruit, 10 parts of bran quzhi fructus aurantii, 15 parts of white peony root, and 5 parts of roasted liquorice.

[0032] The embodiment of the present invention also provides a method for preparing the Chinese medicine composition. The medicinal materials are weighed by weight, soaked and decocted by a reflux method, and the medicinal liquid is concentrated to obtain the Chinese medicine composition.

[0033] In some specific embodiments, the soaking time is 1 to 2 hours.

[0034] In some specific embodiments, the decoction method is: decoct twice in total, each decoction is 1 to 2 hours.

[0035] In some specific embodiments, the method for concentrating the drug solution is: concentrating to a crude drug content of 1.6 g·mL -1 .

[0036] The embodiment of the present invention also provides the use of the traditional Chinese medicine composition in preparing a medicine for preventing and / or treating knee osteoarthritis.

[0037] The embodiment of the present invention also provides the use of the traditional Chinese medicine composition in preparing a drug for reducing OARSI score and regulating the expression of type II collagen and matrix metalloproteinase-13.

[0038] The embodiment of the present invention also provides the use of the traditional Chinese medicine composition in the preparation of a drug for improving the expression of pain sensitization factor NGF secreted by peripheral nerves and increasing the pain threshold.

[0039] The embodiment of the present invention also provides the use of the traditional Chinese medicine composition in preparing a medicine for reducing the medullary cavity space in the subchondral bone area.

[0040] Shen Sui An Kang Yin was formed based on clinical experience. The whole prescription uses salt Eucommia ulmoides and raw Rehmannia as the main medicines. Salt Eucommia ulmoides nourishes the kidney and fills the marrow, strengthens the tendons and bones, and raw Rehmannia nourishes yin and replenishes blood, removes numbness and fills the marrow. The two medicines work together to nourish yin and assist yang, and replenish the kidney and fill the marrow; Astragalus and Angelica sinensis are used to replenish qi and generate blood, and Achyranthes bidentata is used to replenish the kidney and remove blood stasis and dredge the menstruation. The three are ministerial medicines to dispel evil and strengthen the body, dredge the menstruation and remove numbness; Chuanxiong is a blood-qi medicine, which can promote qi and break blood stasis, dissipate cold and soften the tendons; Cornus officinalis enters the Jueyin meridian, nourishes the liver and kidney, and relieves ventilating numbness; Citrus aurantium regulates qi and relieves the middle, so as to consolidate the acquired foundation; White Peony Root and Glycyrrhizic Acid sweeten Yin, soften tendons and relieve pain. The combination of these medicines not only has the functions of nourishing the liver and kidney and benefiting the essence and marrow, but also has the effects of dredge the menstruation and relieve pain, and promote qi and blood circulation. At the same time, modern pharmacology has also proved that the raw Rehmannia glutinosa, Astragalus membranaceus, Angelica sinensis, and White Peony Root in this prescription can promote immune regulation, inhibit inflammation, and relieve OA pain.

[0041] The Chinese medicine composition provided by the present invention is a medicine for preventing and treating musculoskeletal disease knee osteoarthritis prepared by using Shen Sui An Kang Yin; the Shen Sui An Kang Yin can delay the progression of knee osteoarthritis and relieve the peripheral pain sensitization of knee osteoarthritis. The Shen Sui An Kang Yin reduces the OARSI score and regulates the expression of type II collagen and matrix metalloproteinase-13, thereby delaying the progression of knee osteoarthritis. The Shen Sui An Kang Yin relieves the peripheral pain sensitization of knee osteoarthritis by improving the expression of pain sensitization factor NGF secreted by peripheral nerves and increasing the pain threshold.

[0042] The drug is administered orally.

[0043] The drug can reduce OARSI score, increase the expression of type II collagen, reduce the expression of matrix metalloproteinase-13, and reduce the subchondral bone volume fraction; improve the expression of pain sensitization factor NGF secreted by peripheral nerves, increase the pain threshold, and verify the safety of the drug. The dosage form of the drug can be any one of tablets, capsules, oral liquids, lozenges, granules, granules, pills, powders, ointments, pills, suspensions, powders, solutions, injections, suppositories, creams, sprays, drops or patches. The dosage of the drug in animals is 1.6g·mL -1 .

[0044] The Shen Sui An Kang Yin used in the embodiment of the present invention is composed of 10 grams of salt Eucommia ulmoides, 15 grams of raw Rehmannia root, 20 grams of Astragalus, 12 grams of Angelica sinensis, 12 grams of Achyranthes bidentata, 9 grams of Chuanxiong, 12 grams of steamed Cornus officinalis, 10 grams of Fructus Aurantii Immaturus, 15 grams of Radix Paeoniae Rubrae, and 5 grams of Radix Glycyrrhizae Radix et Rhizoma. The above Chinese medicine pieces are purchased from Zhejiang University of Chinese Medicine Chinese Medicine Piece Co., Ltd. After soaking the above pieces in distilled water for 1-2 hours, the pieces are decocted by reflux method for a total of 2 decoctions, each time for 1 hour. Then the medicine is filtered and evaporated and concentrated to a crude drug content of 1.6 g·mL -1 , and then stored at -20°C. The distilled water refers to distilled water at standard atmospheric pressure, with a boiling point of 100°C and a density of 1 g / cm3 (at 4°C). Unless otherwise specified, the percentage concentrations described in the embodiments of the present invention are percentage concentrations by mass.

[0045] Example 1

[0046] Effect of Shen Sui An Kang Yin on the microstructure of joints in KOA mice: Verify that Shen Sui An Kang Yin can reverse the lesions in bone microstructure in KOA mice and clarify the progression of the disease in Shen Sui An Kang Yin KOA mice.

[0047] 1. Methods

[0048] 1 Experimental Grouping

[0049] C57BL / 6J mice were used in this experiment. Thirty C57BL / 6J mice were randomly divided into three groups using the random number table method, namely, a sham operation group, a model group, and a Shen Sui An Kang Yin group, with 10 mice in each group.

[0050] 2 Establishment of KOA mouse model

[0051] The KOA model was established by destabilization of medial meniscus (DMM) in both the model group and the Shen Sui An Kang Yin group. First, the mice were anesthetized with Shu Tai 50 (dose 50 mg / kg), and the knee joint was locally prepared in a sterile environment. Then a longitudinal incision was made along the distal patella and the proximal tibial plateau, with an incision of about 1 cm, and the patellar ligament was pulled to one side. The fat pad was bluntly separated with ophthalmic forceps, and the medial meniscus was loosened with a suture needle. Finally, the patella was repositioned and the wound was closed. For the sham group, a similar operation was performed, the only difference being that no damage was caused to the joint tissue.

[0052] 3 Drug preparation and intervention

[0053] 3.1 Drug preparation

[0054] Shen Sui An Kang Yin is composed of: 10g of salt Eucommia ulmoides, 15g of raw Rehmannia root, 20g of Astragalus, 12g of Angelica sinensis, 12g of Achyranthes bidentata, 9g of Chuanxiong, 12g of steamed Cornus officinalis, 10g of Fructus Aurantii Immaturus, 15g of Radix Paeoniae Rubra and 5g of Radix Glycyrrhizae Radix. The above Chinese herbal medicines were purchased from the Chinese Medicine Ginseng and Antler Branch of East China Pharmaceutical Co., Ltd. After soaking the above-mentioned herbal medicines in distilled water for 1 hour, they were decocted by reflux method for a total of 2 times, each time for 1 hour. The medicine was then filtered and evaporated, and the two filtrates were combined and concentrated to a crude drug content of 1.6g·mL -1 The preparation of the intervention drugs required by the present invention is carried out using the same method.

[0055] 3.2 Drug intervention

[0056] Starting from the second day after surgery, mice in the Shensui Ankangyin group were given a crude drug concentration of 1.6 g mL-1 The mice in the model group and sham operation group were gavaged with 0.5 mL of Shen Sui An Kang Yin, once a day, for 8 weeks; the mice in the model group and sham operation group were gavaged with 0.5 mL of normal saline, with the same frequency.

[0057] 4 indicators detection

[0058] 4.1 Micro-CT detection

[0059] On the day after the end of oral gavage, mice in each group were anesthetized by intraperitoneal injection of Shutai 50 (dose 50 mg / kg), and samples were collected after being killed by cervical dislocation. The knee joint samples were placed in 4% paraformaldehyde, fixed at 4°C for 48 hours, and then transferred to 75% ethanol. Micro-CT scanning was used to perform three-dimensional imaging of the knee joint using a micro-tomography imaging system. The parameters were 75 kV voltage and 200 μA current, and the resolution was 18 μm. The three-dimensional reconstruction processing software NRecon and DataViewer were used for reconstruction and selection, and CTAnalyzer was used to quantitatively analyze the cartilage and subchondral bone areas at the same position in the image. The results are shown in Figure 1 A. The bone volume fraction (BV / TV: the percentage of bone tissue volume to subchondral bone volume) was calculated, and the structural changes of the knee joint bone tissue were evaluated based on the morphometric parameters of the subchondral bone. The results are shown in Figure 1 Middle B.

[0060] Depend on Figure 1 It can be seen that the Micro-CT three-dimensional reconstruction image of the tibial subchondral bone ( Figure 1 The results in the middle (A) showed that the abnormal bone remodeling of the subchondral bone in the model group was significantly increased compared with the sham operation group. The DMM surgical modeling caused subchondral bone sclerosis, and the subchondral bone BV / TV increased significantly (p<0.001). Compared with the model group, the medullary cavity space in the subchondral bone area of ​​the mice in the Shen Sui An Kang Yin group was significantly reduced, and the subchondral bone BV / TV decreased (p<0.001). For details, see Figure 1 .

[0061] 4.2 Histological and bone morphometric analysis

[0062] 4.2.1 Obtaining KOA Mouse Knee Tissue and Preparing Sections

[0063] After the Micro-CT scan, the knee joint samples of each group of mice were taken out, rinsed with tap water for 1 hour, and then decalcified with 14% EDTA solution for 12-14 days. The solution was changed every day, and the decalcification was observed every day. The tissue was easily broken through with a pin. After decalcification, it was rinsed with tap water for 4-6 hours, and then processed by a fully automatic tissue dehydrator. All samples were embedded in paraffin to prepare sample wax blocks and sliced. When slicing, all slices were 3μm thick slices, and each slice was based on meniscus separation.

[0064] 4.2.2 ABH / OG staining

[0065] 1) Take slices of knee joint tissue from each group (take slices with the same number from each sample) and bake them at 65°C overnight;

[0066] 2) On the second day, remove the sections, wait for them to return to room temperature, and then dewax and rehydrate them step by step (dewax with xylene 3 times, 15 minutes each time; 100% and 95% alcohol 2 times, 8 minutes each time; 70% alcohol 1 time, 8 minutes each time; deionized water 3 times, 3 minutes each time);

[0067] 3) Place the slices in hydrochloric acid alcohol for 30 seconds and briefly absorb excess water on paper;

[0068] 4) After staining the sections in alcian blue hematoxylin solution for 1 hour, stain with deionized water three times, 3 minutes each time;

[0069] 5) Place the slices in hydrochloric acid alcohol for 3 seconds, then wash with deionized water 3 times, 3 minutes each time;

[0070] 6) Place the slices in 0.5% ammonia water for 20 seconds, then wash with deionized water three times, 1 minute each time;

[0071] 7) Place the sections in 95% alcohol for 1 minute, then transfer to Eosin / Orange G working solution for staining for 2 minutes;

[0072] 8) Dehydrate and make transparent step by step (95% alcohol 3 times, 1 minute / time; 100% alcohol 2 times, 1 minute / time; xylene dewaxing 3 times, 1 minute / time), and seal with sealing medium;

[0073] 9) Bake the slides at 37℃ overnight, observe and complete the slide collection under the digital pathology slide scanning system, and the results are shown in Figure 2 Middle A.

[0074] 4.2.3 Evaluation of KOA cartilage tissue according to OARSI scoring criteria

[0075] Slices of knee joint tissues were taken from each group (slices with the same number were taken from each sample) and the severity of KOA cartilage damage was evaluated using the histological scoring system developed by the Osteoarthritis Research Society International (OARSI) (OARSI scores are shown in Table 1). Two people other than the project team members performed the grading and the average of the scores was taken. The results are shown in Table 1. Figure 2 Middle B.

[0076] Table 1 OARSI scoring details

[0077]

[0078] Depend on Figure 2 The results of ABH / OG staining of the knee cartilage of mice showed that the joints of the sham-operated group had only slight KOA changes, the articular cartilage was relatively normal, and the cartilage matrix morphology was relatively uniform; the articular cartilage of the model group was severely worn, the cartilage layer was obviously missing and thinned, and the cracks were deep into the subchondral bone; the Shen Sui An Kang Yin group showed significant improvement compared with the model group, with partial damage on the cartilage surface and a relatively intact cartilage matrix structure ( Figure 2 The morphology of articular cartilage tissue in each group of KOA mice was scored according to the OARSI score. It was found that the OARSI score of the model group was significantly higher than that of the sham operation group (p<0.001), while the OARSI score of the Shen Sui An Kang Yin group was significantly lower than that of the model group (p<0.001) ( Figure 2 (shown in B).

[0079] 4.3 Immunohistochemical staining

[0080] 1) Take slices of knee joint tissue from each group (take slices with the same number from each sample) and bake them at 65°C overnight;

[0081] 2) On the second day, remove the sections, wait for them to return to room temperature, and then dewax and rehydrate them step by step (dewax with xylene 3 times, 15 minutes each time; 100% and 95% alcohol 2 times, 8 minutes each time; 70% alcohol 1 time, 8 minutes each time; deionized water 3 times, 3 minutes each time);

[0082] 3) All antibodies were heat-fixed as follows: heat-fixed with sodium citrate (pH 6.0) (temperature 65°C), and the sections were returned to room temperature and washed three times with 1× PBS, 3 minutes each time;

[0083] 4) After the repair is completed, wait for it to cool naturally, incubate it with 50-100u endogenous peroxidase blocker for 15 minutes, and wash it 3 times with 1×PBS, 3 minutes each time;

[0084] 5) Col2a1 (1:200) and Mmp13 (1:200) primary antibodies (diluted in PBS containing 5% NGS and 0.1% Triton X-100 detergent) were added dropwise, and the sections were placed in a humidified box and incubated overnight at 4°C;

[0085] 6) The next day, remove the wet box, and wash the sections 3 times with 1×PBS after returning to room temperature naturally, 5 minutes each time; add the corresponding secondary antibody (1×PBS diluted 1:1000), incubate at room temperature for 1 hour, and then wash 3 times with 1×PBS, 5 minutes each time; then perform DAB color development, and the color development time of the same antibody is consistent;

[0086] 7) Nuclear counterstaining with hematoxylin for 10 seconds, washing with deionized water 3 times, 5 minutes each time; dehydration and transparency step by step (95% alcohol 3 times, 1 minute each time; 100% alcohol 2 times, 1 minute each time; xylene dewaxing 3 times, 1 minute each time), and sealing with sealing medium;

[0087] 8) Bake the slides at 37℃ overnight, observe and complete the slide collection under the digital pathology slide scanning system, and the results are shown in Figure 3 Middle A.

[0088] Depend on Figure 3 It can be seen that the results of immunohistochemistry of Col2a1 in the knee cartilage of mice showed that after DMM modeling, the expression of Col2a1 in the cartilage tissue of mice was significantly reduced compared with the sham operation group (p<0.001), while the expression of Col2a1 in the cartilage of mice treated with Shen Sui An Kang Yin was reversed compared with the model group (p<0.01) ( Figure 3 In addition, the results of immunohistochemistry for MMP-13 showed that compared with the sham operation group, the model group could significantly increase the expression of MMP-13 in the cartilage area (p<0.001). At the same time, after the intervention of Shen Sui An Kang Yin, the expression of MMP-13 was downregulated to a certain extent (p<0.001) ( Figure 3 (as shown in C).

[0089] The above results indicate that Shen Sui An Kang Yin can effectively improve cartilage degeneration, improve abnormal bone remodeling of subchondral bone, and delay the progression of KOA.

[0090] Example 2

[0091] Effect of Shen Sui An Kang Yin on peripheral pain in KOA mice: It was verified that Shen Sui An Kang Yin can reduce the peripheral pain sensitivity of KOA mice and it was confirmed that Shen Sui An Kang Yin can relieve the pain in KOA mice.

[0092] 1. Methods

[0093] 1 Experimental Grouping

[0094] C57BL / 6J mice were used in this experiment. Thirty C57BL / 6J mice were randomly divided into three groups using the random number table method, namely, a sham operation group, a model group, and a Shen Sui An Kang Yin group, with 10 mice in each group.

[0095] 2 Establishment of KOA mouse model

[0096] The KOA model was established by destabilization of medial meniscus (DMM) in both the model group and the Shen Sui An Kang Yin group. First, the mice were anesthetized with Shu Tai 50 (dose 50 mg / kg), and the knee joint was locally prepared in a sterile environment. Then a longitudinal incision was made along the distal patella and the proximal tibial plateau, with an incision of about 1 cm, and the patellar ligament was pulled to one side. The fat pad was bluntly separated with ophthalmic forceps, and the medial meniscus was loosened with a suture needle. Finally, the patella was repositioned and the wound was closed. For the sham group, a similar operation was performed, the only difference being that no damage was caused to the joint tissue.

[0097] 3. Drug intervention

[0098] Starting from the second day after surgery, mice in the Shensui Ankangyin group were given a crude drug concentration of 1.6 g mL -1 The mice in the model group and sham operation group were gavaged with 0.5 mL of Shen Sui An Kang Yin, once a day, for 8 weeks; the mice in the model group and sham operation group were gavaged with 0.5 mL of normal saline, with the same frequency.

[0099] 4 indicators detection

[0100] 4.1 Behavioral testing

[0101] Gait detection: After intragastric administration, the DigGait imaging system was used to track and analyze the gait of the mice. The mice were placed on a moving track at a speed of 18 cm / s, and the camera recorded the movement status of the mice within a specified time for subsequent data analysis;

[0102] The mechanical withdrawal threshold (MWT) of mice was measured by the Vonfrey fiber pain meter after different fiber stimulations were applied to both lower limbs according to the up&down method. The mice were first placed in a quiet glass partition to adapt to the environment. Paw withdrawal or licking the paw was considered positive when data was collected. The test was repeated 3 times for both lower limbs, and the average value was taken, which was measured before modeling and 2, 4, and 8 weeks after administration.

[0103] Hot plate test: Use YLS-6B intelligent hot plate instrument to record the thermal pain response of mice. Place the mouse on a hot plate at 50°C and start timing. Stop timing when the mouse licks its right hind foot or jumps. Record and measure the reaction time of each mouse 5 times, with an interval of 5 minutes between each measurement, and finally take the average value for subsequent analysis.

[0104] The results of behavioral tests showed that there was no significant statistical difference in MWT among the mice in each group before modeling (p<0.001). Compared with the model group, the MWT of the mice in the sham operation group was significantly different from that in the 2nd to 8th week (p<0.001), indicating that the mice were sensitive to pain after DMM modeling; after intervention with Shen Sui An Kang Yin, the peripheral pain sensitivity of the mice decreased from the 2nd to the 8th week (p<0.001, Figure 4 A). The hot plate method was then used to assess the pain threshold of the mice. Compared with the sham operation group, the reaction time of the right hind foot in the model group was shortened, indicating that the pain sensitivity of the model group increased and the pain threshold decreased (p<0.001). After the intervention of Shen Sui An Kang Yin, the pain threshold of the mice increased (p<0.001), indicating that the pain of the mice was relieved ( Figure 4 Finally, the Dig Gait imaging system was used to evaluate the walking gait of KOA mice. Compared with the sham operation group, the swing phase time of the right lower limb of the model group mice increased (p<0.001), while the stance phase time, stride length, and plantar contact area decreased (p<0.001, Figure 4 After receiving Shen Sui An Kang Yin treatment, the stride length increased, the swing time decreased, and the plantar contact area increased (p<0.001, Figure 4 CF). The above results indicate that Shen Sui An Kang Yin treatment can effectively reduce the pain threshold of DMM mice.

[0105] 4.2 Obtaining and Slicing Knee Tissue and Right L4 Dorsal Ganglion of KOA Mice

[0106] After completing the Micro-CT scan, remove the knee joint samples of each group of mice, rinse with tap water for 1 hour, and then decalcify with 14% EDTA solution for 7 days. Change the solution every day and observe the decalcification every day. The tissue can be easily penetrated with a pin. Then dehydrate in 30% sucrose solution. After the tissue is completely dehydrated, embed and slice with OCT freezing embedding medium. All slices are 10μm thick. Carefully peel off the right L4 dorsal root ganglion (DRG) under a stereo microscope, fix it in 4% paraformaldehyde for 1 hour, and then dehydrate in 30% sucrose solution. After the tissue is completely dehydrated, embed and slice with OCT freezing embedding medium.

[0107] 4.3 Immunohistofluorescence staining

[0108] 1) Take slices of knee joint tissue and DRG from each group (take slices with the same number from each sample) and rewarm at 37°C for 30 minutes;

[0109] 2) After returning to room temperature (wash 3 times with 1× PBS, 3 minutes each time);

[0110] 3) All antibodies were repaired with frozen section repair solution as follows: repair with 1X frozen section repair solution for 5 minutes, and washed 3 times with special washing solution, 5 minutes each time;

[0111] 4) After repair, add NGF (1:200) primary antibody (diluted with PBS containing 5% NGS and 0.1% Triton X-100 detergent), and then place the slices in a humidified box and incubate at 4°C overnight;

[0112] 5) The next day, remove the wet box, restore to room temperature naturally, wash the sections 3 times with 1×PBS, 5 minutes each time; add the corresponding secondary antibody (1×PBS diluted 1:1000), incubate at 37°C for 1 hour, wash 3 times with 1×PBS, 5 minutes each time; then perform DAPI color development, incubate at 37°C for 10 minutes, and wash 3 times with 1×PBS, 5 minutes each time;

[0113] 6) The slides were sealed with anti-fluorescence attenuation sealing agent and observed under a Zeiss upright microscope. The results are shown in Figure 5 .

[0114] After 8 weeks of DMM modeling, the expression of NGF (green fluorescent marker), a key factor in pain sensitization, in the subchondral bone and DRG of mice was significantly increased compared with the sham operation group (p<0.001, Figure 5 ); After treatment with Shen Sui An Kang Yin, the expression level of NGF in subchondral bone was moderately reduced, and the difference was significant compared with the model group (p<0.001, Figure 5 C), and NGF expression in mouse DRG was also significantly downregulated ( Figure 5 (B) Thus, Shen Sui An Kang Yin can effectively improve KOA pain.

[0115] The above results indicate that Shen Sui An Kang Yin can increase the pain threshold of KOA mice and relieve KOA peripheral pain.

[0116] Example 3

[0117] Safety evaluation of Shensui Ankangyin in treating KOA mice: Verify the safety evaluation of Shensui Ankangyin in treating KOA mice and clarify the drug safety of Shensui Ankangyin in treating KOA mice.

[0118] 1. Methods

[0119] 1 Experimental Grouping

[0120] C57BL / 6J mice were used in this experiment. Thirty C57BL / 6J mice were randomly divided into three groups using the random number table method, namely, a sham operation group, a model group, and a Shen Sui An Kang Yin group, with 10 mice in each group.

[0121] 2 Establishment of KOA mouse model

[0122] The KOA model was established by destabilization of medial meniscus (DMM) in both the model group and the Shen Sui An Kang Yin group. First, the mice were anesthetized with Shu Tai 50 (dose 50 mg / kg), and the knee joint was locally prepared in a sterile environment. Then a longitudinal incision was made along the distal patella and the proximal tibial plateau, with an incision of about 1 cm, and the patellar ligament was pulled to one side. The fat pad was bluntly separated with ophthalmic forceps, and the medial meniscus was loosened with a suture needle. Finally, the patella was repositioned and the wound was closed. For the sham group, a similar operation was performed, the only difference being that no damage was caused to the joint tissue.

[0123] 3. Drug intervention

[0124] Starting from the second day after surgery, mice in the Shensui Ankangyin group were given a crude drug concentration of 1.6 g mL -1 The mice in the model group and sham operation group were gavaged with 0.5 mL of Shen Sui An Kang Yin, once a day, for 8 weeks; the mice in the model group and sham operation group were gavaged with 0.5 mL of normal saline, with the same frequency.

[0125] 4 indicators detection

[0126] 4.1 H&E staining

[0127] 1) Take slices of visceral tissues (heart, liver, spleen, lung, kidney) from each group (take slices with the same number from each sample) and bake them at 65°C overnight;

[0128] 2) On the second day, remove the sections, wait for them to return to room temperature, and then dewax and rehydrate them step by step (dewax with xylene 3 times, 15 minutes each time; 100% and 95% alcohol 2 times, 8 minutes each time; 70% alcohol 1 time, 8 minutes each time; deionized water 3 times, 3 minutes each time);

[0129] 3) Place the sections in hematoxylin solution for 1 minute and then in deionized water 3 times, 3 minutes each time;

[0130] 4) Acidify the slices in 1% hydrochloric acid alcohol solution for 10 seconds, then rinse with deionized water 3 times, 3 minutes each time;

[0131] 5) Place the slices in 0.5% ammonia water for 20 seconds, then wash with deionized water three times, 1 minute each time;

[0132] 6) Place the sections in eosin stain for 1 minute and 30 seconds, then wash with deionized water three times, 1 minute each time;

[0133] 7) Wash with deionized water 3 times, 1 minute each time; dehydrate and make transparent step by step (95% alcohol 3 times, 1 minute each time; 100% alcohol 2 times, 1 minute each time; xylene dewaxing 3 times, 1 minute each time), and seal with sealing medium;

[0134] 8) Bake the slides at 37℃ overnight, observe and complete the slide collection under the digital pathology slide scanning system, and the results are shown in Figure 6 .

[0135] The results of H&E staining proved that Shen Sui An Kang Yin did not cause any damage to these organs. Shen Sui An Kang Yin had no drug toxicity when treating KOA mice and did not damage the internal organs of the animals.

[0136] Example 4

[0137] In vitro study on the effect of Shen Sui An Kang Yin on preventing chondrocyte degeneration: In vitro verification of Shen Sui An Kang Yin's ability to regulate changes in anabolic indicators in chondrocytes was conducted, and the mechanism of Shen Sui An Kang Yin in preventing chondrocyte degeneration was elucidated.

[0138] 1. Methods

[0139] 1 Experimental Grouping

[0140] 1) Control serum group, model group, and Shensui Ankangyin medicated serum group. The control group and model group were added with 10% blank serum, and the model group and Shensui Ankangyin medicated serum group were added with a concentration of 10 ng·ml -1 IL-1β, 20% Shensui Ankangyin medicated serum was added to the Shensui Ankangyin medicated serum group.

[0141] 2 Isolation and culture of mouse articular chondrocytes

[0142] 1) Two-week-old C57BL / 6J mice were killed with CO2 and then immersed in 75% alcohol for 30 minutes.

[0143] 2) The mouse was removed, the skin was removed, the femoral head cartilage layer was grasped with forceps, and the attached ligaments and soft tissues were cut with scissors. The cartilage tissue was placed in a 1.5 mL centrifuge tube containing PBS.

[0144] 3) Remove the absorbent pad, clean the clean bench, change gloves, and preheat the primary digestion medium (F12 medium + collagenase P 0.5 mg / mL) at 37°C for 30 minutes.

[0145] 4) Discard the supernatant, place the cartilage tissue in a new 10 cm culture dish, and place it in a 37°C incubator for digestion for 4-6 hours. Every 1 hour, blow the cartilage cap in the primary digestion medium, and blow with the pipette tip for more than 1 minute each time.

[0146] 5) After the digestion of the cartilage cap is completed, the digestion medium in the culture dish is transferred to a 15 mL centrifuge tube and continued to be pipetted (30-40 times), filtered through a 40 μm cell strainer, and centrifuged at 1000 rpm for 5 minutes.

[0147] 6) Gently shake the culture dish, mark it, and place it in a 37℃ incubator for digestion for 4-6 hours.

[0148] 7) Resuspend with 5 mL of normal culture medium (F12 medium + 10% FBS + 1% P / S), count and adjust the appropriate concentration to inoculate in the well plate, place in a 37°C, 5% CO2 constant temperature incubator for 12 hours, and continue with subsequent experiments after the cells are in good growth state.

[0149] 3 Extraction of drug-containing serum

[0150] 1) Ten 2-month-old male SD rats were randomly divided into two groups, namely, the Shen Sui An Kang Yin-containing serum group and the blank serum group. -1 The mice were given Shen Sui An Kang Yin by gavage and the same volume of normal saline by gavage, both once a day for 1 week.

[0151] 2) On the 7th day, the rats were fasted and anesthetized by intraperitoneal injection of Zotal 50 (dose 50 mg / kg) 2 hours after morning gavage. Blood was collected from the abdominal aorta. The blood samples were left at room temperature for 1 hour and rotated at 3000 r / min. -1 Centrifuge for 15 minutes, take the upper serum and divide it into centrifuge tubes, inactivate it in a 56℃ water bath for 30 minutes, sterilize it through a 0.22μm sterile filter membrane, and store it in a -80℃ refrigerator for later use.

[0152] Effect of Shen Sui An Kang Yin on the survival of primary articular chondrocytes in mice

[0153] After the cells adhered to the wall, the blank group (no chondrocytes and no Shen Sui An Kang Yin medicated serum) and the control group (chondrocytes without Shen Sui An Kang Yin medicated serum) were replaced with serum-free medium (F12 medium), and the drug group was added with the following final concentrations of serum-free medium (5%, 10%, 20%, 40%), and cultured for 24 hours and 48 hours respectively. The culture medium was aspirated at the corresponding time, washed twice with DPBS, and 100 μL of F12 culture medium containing 10 μL CCK8 was added to each well. After continuing to culture for 2 hours, the supernatant was collected, and the average optical density (OD value) of each well was measured at 450nm, and repeated 3 times independently. Calculate the survival rate of chondrocytes. Chondrocyte survival rate (%) = [(OD value of drug group - OD value of blank group) / (OD value of control group - OD value of blank group)] × 100%.

[0154] In this experiment, the CKK8 method was used to determine the effects of different concentrations of Shen Sui An Kang Yin-containing serum on chondrocyte survival. The survival rate of primary mouse articular chondrocytes under the action of different concentrations of Shen Sui An Kang Yin-containing serum was as follows: Figure 7 As shown in , the survival rate of mouse primary articular chondrocytes was not affected and was the most stable under the action of 20% concentration of Shen Sui An Kang Yin medicated serum. Therefore, in subsequent experiments, 20% Shen Sui An Kang Yin was used for subsequent research.

[0155] 5. Evaluation of the effect of Shen Sui An Kang Yin on chondrocyte matrix by Alcian blue staining

[0156] 1) After the cells adhere to the wall, add trypsin for 1 minute and place in a 37°C incubator; then add complete culture medium to terminate the incubation, blow with a pipette tip, and transfer to a 15 mL centrifuge tube at 3000 r / min -1 , 5 minutes.

[0157] 2) Add 1 mL of complete culture medium and repeatedly pipette to count the cells, then draw 20 μL and drop it in the center of the well plate. After 4-6 hours, when the cells adhere to the wall, add 10% blank serum to the control group and model group, and add 10 ng·ml to the model group and Shen Sui An Kang Yin drug-containing serum group. -1 IL-1β, 20% Shensui Ankangyin medicated serum was added to the Shensui Ankangyin medicated serum group.

[0158] 3) Cultivate the cells for 7 days using the above method, wash once with 1×PBS for 3 minutes, fix overnight, wash 3 times with 1×PBS for 3 minutes, add Alcian blue for 30 minutes, wash 3 times with 1×PBS for 3 minutes. Observe and complete the slide collection under a Zeiss upright microscope. The results are shown in the figure. Figure 8 .

[0159] Figure 8 Compared with the control group, 10ng·ml -1 The staining in the chondrocytes of the IL-1β-induced model group was uneven and the staining became lighter in some areas. These differences intuitively reflected the pathological changes in the chondrocytes of the model group. Compared with the model group, the staining in the chondrocytes became uniform and the color deepened after treatment with the Shen Sui An Kang Yin medicated serum, indicating that the Shen Sui An Kang Yin medicated serum can reverse the changes in the extracellular matrix composition and structure of chondrocytes.

[0160] 6Real-time PCR analysis of mRNA expression levels of cartilage degeneration-related genes in chondrocytes of each group

[0161] 6.1 Extraction of mRNA from primary chondrocytes

[0162] 1) Aspirate the original culture medium, wash once with 1× PBS, add 0.5 mL Trizol directly to the culture plate, pipette and vortex to mix evenly, then transfer to a separation gel tube (centrifuge at 12,000 rpm for 1 minute before use), and let stand at room temperature for 5 minutes.

[0163] 2) Add 0.1 mL of chloroform (1 / 5 of the total volume of Trizol) to the gel tube in step 1), mix by inverting for 15-30 seconds, and let stand at room temperature for 2-3 minutes; after completion, centrifuge at 12,000 rpm at 4°C for 15 minutes, carefully aspirate the supernatant, and transfer it to another 1.5 mL centrifuge tube.

[0164] 3) Add 1 volume of 70% alcohol to the supernatant in step 2), shake and mix for 30 seconds, then add it to the 2 mL collection tube provided by the RNA extraction kit, and further purify the mRNA according to the operating instructions in the kit.

[0165] 6.2 mRNA concentration determination and reverse transcription

[0166] 1.5 μL mRNA was taken from each group of samples, and the RNA concentration was measured using an ultra-micro UV spectrophotometer; 500 ng mRNA was taken from each group of samples to prepare the reaction system (10 μL) according to the operating instructions of the reverse transcription kit to synthesize cDNA, and the remaining mRNA was stored in a -80°C refrigerator.

[0167] The reverse transcription reaction system was as follows: 500 ng mRNA, 2 μL 5x Mix, and RNase-free water to a volume of 10 μL.

[0168] The reverse transcription procedure was as follows: reverse transcription reaction at 37°C for 15 minutes, reverse transcriptase inactivation (termination reaction) at 85°C for 5 seconds.

[0169] 6.3 Real-time PCR quantitative analysis of genes

[0170] Take the complementary deoxyribonucleic acid (cDNA) obtained by reverse transcription in each group, add 40 μL of RNase-free water and dilute it 5 times as a PCR reaction template. Take 500 ng of mRNA from each group of samples according to the instructions in the SYBR kit to prepare the reaction system (10 μL) to amplify the target gene, and store the remaining mRNA in a -80℃ refrigerator. In this experiment, Mmp13 and Col2a1 were selected as target genes, Actb was used as the internal reference gene (primer sequences are shown in Table 2), and the target gene copy number / Actb ratio was used as the statistical value. 2 -ΔΔCT Real-time PCR was used to analyze the expression level of the target gene in each group of primary chondrocytes. Fig. 9 shown.

[0171] The real-time PCR reaction system is as follows:

[0172] 2 μL of cDNA template, 0.5 μL of upstream primer (10 μM), 0.5 μL of downstream primer (10 μM), 5 μL of 2x Sybr green mix, and RNase-free water to make up to 10 μL.

[0173] Real-time PCR reaction procedure is as follows:

[0174] Initial denaturation at 95°C for 30 seconds, denaturation at 95°C for 10 seconds, annealing and extension at 60°C for 60 seconds (40 cycles of denaturation, annealing and extension), and melting curve at 60-95°C for 5 seconds hold / 1°C.

[0175] Table 2 Primer sequences

[0176]

[0177]

[0178] The test results showed that compared with the control group, 10ng·ml -1 The Mmp13 mRNA level in chondrocytes of the IL-1β-induced model group was significantly increased (p<0.001) ( Fig. 9 (B), while the Col2a1 level decreased significantly, and the differences were statistically significant (p<0.01) ( Fig. 9 Middle A). Compared with the model group, the Mmp13 mRNA level decreased in a dose-dependent manner after treatment with Shensui Ankangyin-containing serum (p<0.01) ( Fig. 9 In B), Col2a1 mRNA level increased in a dose-dependent manner, and the differences were statistically significant (p<0.01) ( Fig. 9 Middle A). This indicates that Shen Sui An Kang Yin-containing serum can delay IL-1β-induced KOA chondrocyte degeneration.

[0179] Obviously, the above embodiments of the present invention are merely examples for clearly illustrating the present invention, and are not intended to limit the implementation methods of the present invention. For ordinary technical users in the relevant field, other different forms of changes or modifications can be made on the basis of the above description. It is not necessary and impossible to list all the implementation methods here. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the claims of the present invention.

Claims

1. A Chinese medicine composition for preventing and / or treating knee osteoarthritis, characterized in that: The invention is prepared from the following components in parts by weight: 6-10 parts of salt eucommia bark, 9-15 parts of raw rehmannia root, 15-20 parts of astragalus, 9-12 parts of angelica, 9-12 parts of achyranthes bidentata, 6-9 parts of ligusticum chuanxiong, 9-12 parts of steamed cornus fruit, 6-10 parts of fructus aurantii, 9-15 parts of white peony root and 3-5 parts of roasted liquorice.

2. The Chinese medicine composition according to claim 1, characterized in that: The invention is prepared from the following components in parts by weight: 10 parts of salt eucommia bark, 15 parts of raw rehmannia root, 20 parts of astragalus, 12 parts of angelica, 12 parts of achyranthes bidentata, 9 parts of ligusticum chuanxiong, 12 parts of steamed cornus fruit, 10 parts of fructus aurantii, 15 parts of white peony root, and 5 parts of roasted liquorice.

3. The method for preparing the Chinese medicine composition according to claim 1 or 2, characterized in that: The medicinal materials are weighed according to the weight proportions in claim 1 or 2, soaked and decocted by reflux method, and the medicinal liquid is concentrated to obtain the Chinese medicine composition.

4. The preparation method according to claim 3, characterized in that: The soaking time is 1 to 2 hours.

5. The preparation method according to claim 3, characterized in that: The decoction method is: decoct twice in total, each decoction is 1 to 2 hours.

6. The preparation method according to claim 3, characterized in that: The method for concentrating the medicinal solution is: concentrating to a crude drug content of 1.6 g·mL -1 .

7. Use of the Chinese medicine composition according to claim 1 or 2 in the preparation of a medicament for preventing and / or treating knee osteoarthritis.

8. Use of the Chinese medicine composition according to claim 1 or 2 in the preparation of a drug for reducing OARSI scores and regulating the expression of type II collagen and matrix metalloproteinase-13.

9. Use of the Chinese medicine composition as claimed in claim 1 or 2 in the preparation of a drug for improving the expression of pain sensitizing factor NGF secreted by peripheral nerves and increasing the pain threshold.

10. Use of the Chinese medicine composition as claimed in claim 1 or 2 in the preparation of a medicament for reducing the medullary cavity space in the subchondral bone region.

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