Application of a Chinese medicinal composition containing scorpions and centipedes in preparing a medicine for preventing and / or treating osteoarthritis

Abstract

The invention provides an application of a Chinese medicinal composition containing scorpions and centipedes in preparing a medicine for preventing and / or treating osteoarthritis, and belongs to the technical field of medicine. The invention finds through scientific research that the Chinese medicinal composition containing scorpions and centipedes can alleviate pain and inflammation caused by osteoarthritis, reduce cartilage destruction caused by osteoarthritis, and has the effect of treating osteoarthritis. The Chinese medicinal composition containing scorpions and centipedes can be developed into a medicine for preventing and / or treating osteoarthritis.

Description

Technical Field

[0001] The invention belongs to the technical field of medicine, and particularly relates to application of a traditional Chinese medicine composition containing scorpions and centipedes in preparing medicines for preventing and / or treating osteoarthritis. Background Art

[0002] Osteoarthritis (OA) is a chronic degenerative disease characterized by osteophyte formation and cartilage loss on the joint surface. It is the main cause of disability in the elderly, and also causes pain, functional loss, and decreased quality of life (QOL). The main pathological features of OA include progressive loss of articular cartilage, osteophyte formation, and changes in periarticular and subchondral bone. Among them, articular cartilage has received great attention in OA research. The main characteristics of articular cartilage damage are progressive loss of cartilage homeostasis, synovial activation, and subchondral bone reconstruction. Clinically, the main symptoms are knee pain, stiffness, and limited mobility. In severe cases, it can lead to knee swelling and deformity.

[0003] Knee osteoarthritis (KOA) is a chronic joint disease characterized by knee cartilage degeneration and secondary bone hyperplasia. The pathogenesis of KOA is extensive and complex. Its basic principle is the increase of unfavorable factors that lead to the destruction of the balance between the anabolism and catabolism of articular cartilage. Among them, MMP13 is considered to be the key enzyme for pathological destruction of cartilage. It plays an important role in bone development and bone reconstruction. It is the main enzyme for targeted cartilage degradation. It can not only degrade type II collagen in cartilage, but also degrade proteoglycans, type IV collagen and type IX collagen in cartilage. In addition, the activation of the catabolism axis composed of MMP13 plays an important role, mainly through the regulation of growth factors, hormones, cytokines and transcription factors at the transcriptional level. Studies have shown that the levels of TNF-α, IL-8 and IL-18 in serum are positively correlated with MMP13, and other studies have shown that IL-1β can induce the expression of MMP13 in chondrocytes. These results all indicate that MMP13 may play an important role in the progression of OA. With the deepening of research on it, more and more related molecular pathways and targets have been discovered, which lays the foundation for studying the pathogenesis of OA.

[0004] At present, there are various clinical treatment methods. Traditional Chinese medicine treatment mainly includes oral administration of Chinese medicine, acupuncture, massage, moxibustion, etc., and Western medicine treatment includes drug treatment, joint cavity drug injection, endoscopic cleaning, joint replacement and rehabilitation therapy. In the process of Western medicine treatment, drug treatment mainly focuses on oral anti-inflammatory analgesic drugs, but the side effects of anti-inflammatory analgesics, especially intestinal side effects, are very large, and patients find it difficult to take and tolerate them for a long time; and cartilage protection drugs such as glucosamine and hyaluronic acid only temporarily relieve the condition, and have a single effect, a long time to take, and unclear efficacy. Surgical treatment is mainly used for critically ill patients, with large surgical trauma, unclear long-term efficacy, and expensive surgical costs, which are difficult for patients to accept. Compared with Western medicine, traditional Chinese medicine treatment of knee osteoarthritis has the advantages of fewer side effects and low price. As a traditional treatment for osteoarthritis in my country, traditional Chinese medicine external treatment has accumulated rich experience in clinical treatment by successive medical scientists and is deeply trusted by patients. Therefore, it is necessary to develop a drug that can prevent and / or treat osteoarthritis. Summary of the invention

[0005] In view of the shortcomings in the prior art, the present invention provides an application of a traditional Chinese medicine composition containing scorpions and centipedes in preparing a drug for preventing and / or treating osteoarthritis; the present invention finds through scientific research that the traditional Chinese medicine composition containing scorpions and centipedes can alleviate the pain and inflammation caused by osteoarthritis, reduce the cartilage destruction caused by osteoarthritis, and has the effect of treating osteoarthritis; the traditional Chinese medicine composition containing scorpions and centipedes can be developed into a drug for preventing and / or treating osteoarthritis.

[0006] The present invention firstly provides the use of a Chinese medicinal composition containing scorpions and centipedes in preparing a medicine for preventing and / or treating osteoarthritis and diseases caused by osteoarthritis or alleviating the symptoms thereof.

[0007] Preferably, the scorpion and centipede-containing traditional Chinese medicine composition comprises scorpion, centipede, astragalus and black bean skin.

[0008] Preferably, the condition includes chronic progressive polyarticular inflammation, articular cartilage damage, bone hyperplasia, inflammatory cell joint infiltration and synovitis.

[0009] Preferably, the symptoms include pain caused by osteoarthritis, inflammation caused by osteoarthritis, or cartilage destruction caused by osteoarthritis.

[0010] Preferably, the osteoarthritis includes osteoarthritis caused by abnormal expression of MMP13 in osteoarthritic chondrocytes.

[0011] The present invention also provides a medicine for preventing and / or treating osteoarthritis and diseases caused by osteoarthritis or alleviating the symptoms thereof, wherein the active ingredient of the medicine is a traditional Chinese medicine composition containing scorpions and centipedes.

[0012] Preferably, the drug reduces the expression of MMP13 in chondrocytes.

[0013] Preferably, the drug is added with pharmaceutically acceptable excipients to prepare a pharmaceutically acceptable preparation; the pharmaceutically acceptable preparation includes decoction, granules, tablets, capsules, oral liquids, mixtures, syrups or pills.

[0014] Compared with the prior art, the present invention has the following beneficial effects:

[0015] The present invention provides a scientific basis for the development of drugs for treating osteoarthritis using black bean skin, astragalus, scorpion and centipede as raw materials, and has great application value. The scorpion and centipede described in the present invention have the effects of calming wind and relieving spasm, unblocking meridians and relieving pain, and have a good effect on the long-term treatment of wind-cold and dampness arthralgia and stubborn arthralgia caused by joint deformation. Astragalus can replenish the middle earth to support the positive energy on the one hand, and can also alleviate the disadvantages of insect medicine attacking and consuming qi on the other hand. "Ben Cao Qiu Zhen" records that black bean skin can cure water swelling, promote blood circulation and detoxify. Black bean skin can not only help astragalus to strengthen the spleen and stomach in the middle jiao, make the body fluid and blood biochemical active, and remove the evil without hurting the righteousness, but also can cooperate with raw astragalus to control the toxicity of scorpion and centipede. Zhu Liangchun, a master of Chinese medicine, likes to use black bean skin to detoxify, dispel wind, nourish blood and promote diuresis. The above Chinese medicine is based on scientific selection of raw materials and scientific proportioning of raw materials. The various components can coordinate and promote each other to achieve a synergistic effect. They have good effects of searching, removing, penetrating, attacking toxins, dispersing nodules, unblocking meridians and relieving pain. They can effectively relieve symptoms such as knee pain and difficulty in flexion and extension, and have the effects of promoting the metabolism of bone and joint cartilage cells and collagen synthesis.

[0016] The present invention has found through scientific research that the Chinese medicine composition containing scorpions and centipedes can alleviate the pain and inflammation caused by osteoarthritis, reduce the cartilage destruction caused by osteoarthritis, and has the effect of treating osteoarthritis. The Chinese medicine composition containing scorpions and centipedes can be developed into a drug for preventing and / or treating osteoarthritis. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 The effect of the Chinese medicinal composition containing scorpions and centipedes on the cold pain threshold of mice with knee osteoarthritis. Compared with the ACLT model group, ****p<0.001 (n=10).

[0018] Figure 2 Safranin fast green staining images of the knee joints of mice in different groups.

[0019] Figure 3 The OARSI scores of the knee joints in different groups were obtained by safranin fast green staining. Compared with the ACLT model group, *p<0.05 (n=3).

[0020] Figure 4 Immunohistochemical staining of knee joints in different groups. DETAILED DESCRIPTION

[0021] The present invention is further described below in conjunction with the accompanying drawings and specific embodiments, but the protection scope of the present invention is not limited thereto. The experimental methods used in the following embodiments are conventional methods unless otherwise specified; the reagents, biological materials, etc. used in the following embodiments are all commercially available unless otherwise specified.

[0022] The animal health clean grade male C57BL / 6 mice used in the examples of the present invention were purchased from Jiangsu Huachuang Xinnuo Pharmaceutical Technology Co., Ltd.; centipedes, scorpions, astragalus and black bean skins were all sourced from the Chinese medicine pharmacy of Jiangsu Provincial Hospital of Integrated Traditional Chinese and Western Medicine.

[0023] The raw materials of the Chinese medicine composition containing scorpions and centipedes involved in the following embodiments are calculated by weight and include: 60 parts of black bean skins, 40 parts of astragalus, 1 part of scorpions, and 1 part of centipedes.

[0024] Embodiment 1:

[0025] In this example, a mouse anterior cruciate ligament transection (ACLT) model was constructed to investigate the effect of a Chinese medicinal composition containing scorpions and centipedes on osteoarthritis in mice. The specific investigation method is as follows:

[0026] Construction of mouse ACLT model: 40 healthy and clean C57BL / 6 male mice were taken. After one week of adaptive feeding, 5% chloral hydrate was intraperitoneally injected. The mice were anesthetized with a dose of 0.2 ml per mouse. When the mouse's limbs were swinging arbitrarily and unconscious, the right hind limb of the mouse was taken, and the hair around the knee joint was shaved at a radius of 1 cm. After the skin was completely exposed, the surface was disinfected with iodine tincture. The skin on the surface of the knee joint was cut longitudinally with a scalpel. After the knee joint was exposed, the surgical forceps peeled off the connective tissue on the surface of the knee joint, cut the knee joint ligament, exposed the knee joint cavity, cleaned the wound, and the surgical needle was used to separate the anterior cruciate ligament. The anterior cruciate ligament was cut with small surgical scissors under direct vision, and then the knee joint ligament and the skin on the surface of the knee joint of the mouse were sutured in turn. After the operation, the wound surface was disinfected with iodine tincture and the mouse was returned to the mouse cage. After one month of normal feeding, the drug administration began.

[0027] Construction of sham operation group: 10 healthy clean grade C57BL / 6 male mice were selected. After one week of adaptive feeding, the operation was basically the same as that of mouse ACLT model construction, except that the anterior cruciate ligament was separated but not cut after the joint cavity was opened.

[0028] The sham operation group was used as the control group, the mouse ACLT model was used as the model group, and the positive drug group given celecoxib, the low-dose group of the Chinese medicine composition containing scorpions and centipedes (hereinafter referred to as the SF low-dose group), and the high-dose group of the Chinese medicine composition containing scorpions and centipedes (hereinafter referred to as the SF high-dose group) were constructed. The specific construction method is as follows:

[0029] Forty ACLT model mice were randomly divided into four groups, 10 mice in each group, namely the model group, positive drug group, SF low-dose group and SF high-dose group. The model group was given double distilled water with an intragastric volume of 0.2 mL;

[0030] Positive drug group: 10 ACLT model mice were randomly selected and celecoxib was converted into a mouse dosage (37 mg / kg) according to the clinical dosage of humans. The drug was administered once a day with an intragastric volume of 0.2 mL.

[0031] SF low-dose group: 10 ACLT model mice were randomly selected and given 183 mg / kg, which was calculated based on the daily oral dose of the Chinese medicine composition containing scorpions and centipedes for clinical patients. The drug was given once a day with an intragastric volume of 0.2 mL.

[0032] SF high-dose group: 10 ACLT model mice were randomly selected and given 366 mg / kg, twice the daily oral dose of the Chinese medicine composition containing scorpions and centipedes for clinical use, once a day, with an intragastric volume of 0.2 mL.

[0033] Control group: double distilled water was given, and the gavage volume was 0.2 mL.

[0034] All the above groups were given medication continuously for 35 days.

[0035] (1) Effect of a Chinese medicinal composition containing scorpions and centipedes on pain sensitivity in mice:

[0036] In this example, the mice in the control group, model group, positive drug group, SF low-dose group and SF high-dose group were tested for cold pain experiment during the administration period, and the effect of the Chinese medicine composition containing scorpions and centipedes on the pain sensitivity of mice was investigated. The cold pain experiment test process is as follows:

[0037] The cold plate experiment is used to evaluate the cold pain threshold of mice: the mice are placed on a cold plate with a preset temperature of 4°C. When the mice lick their feet, lift their legs, retreat, jump, etc., the time taken is recorded. The mice must stay on the cold plate for no longer than 60 seconds. If the time exceeds 60 seconds and the mice still do not respond, the experiment is stopped immediately. Each cold plate experiment needs to be repeated three times, with a time interval of no less than 10 minutes between each repetition. The average time recorded in the three repeated experiments is taken as the cold pain threshold of the mice. Starting from the day before administration, measurements are taken every 7 days until the end of administration. The test results are as follows: Figure 1 shown.

[0038] Figure 1The effect of the Chinese medicine composition containing scorpions and centipedes on the cold pain threshold of mice with knee osteoarthritis. Compared with the ACLT model group, ****p < 0.001 (n = 10). As can be seen from the figure, starting from the 0th day of administration, the mice that have been modeled began to develop the disease. At this time, the cold pain test results showed that the cold pain threshold of the mice in the model group and the drug group was lower than that of the control group (sham operation group). With the development of arthritis inflammation, the cold pain threshold of the mice in the ACLT model group gradually decreased, indicating that its pain sensitivity increased rapidly and the inflammatory pain intensified. Compared with the sensitivity of ACLT mice to pain, the drug group gradually relieved the pain in the joints of the limbs of the mice, and the cold pain threshold of the mice gradually increased, and it was significantly improved in the first and second weeks, which was significantly different from the model control group (P < 0.001). This shows that oral administration of the Chinese medicine composition containing scorpions and centipedes can significantly alleviate the joint symptoms of ACLT model mice.

[0039] (2) Effect of the Chinese medicinal composition containing scorpions and centipedes on cartilage damage in mice:

[0040] After 35 days of administration, the right hind limbs of the mice in the control group, model group, positive drug group, SF low-dose group and SF high-dose group were respectively fixed with formalin and stained with safranin fast green. The specific method is as follows:

[0041] a. Sampling: The ankle joints of mice were preserved in 4% paraformaldehyde solution;

[0042] b. Dehydration: Place the tissue in a dehydration box and place it in a fully automatic dehydrator for dehydration using gradient ethanol concentrations;

[0043] c. Embedding: After the tissue is dehydrated, immerse it completely in melted paraffin, embed the paraffin in an embedding machine, and then freeze it at -20℃. After trimming the paraffin block, store it at 4℃;

[0044] d. Sectioning and mounting: Slice the wax block into 5-8 μm thick sections and mount them on a glass slide;

[0045] e. Dehydration: Dehydrate with xylene and different concentrations of alcohol, then wash with distilled water several times;

[0046] f. Dewaxing and staining: stain the dehydrated tissue with 0.5% fast green for 20 minutes, wash it with 1% acetic acid for 3 times, and rinse it with running water for 3 seconds; drip 0.5% safranin onto the sample surface, at 37°C for 5 minutes;

[0047] g. Dehydration and sealing: Dehydrate with xylene and different concentrations of alcohol respectively, then place neutral resin on the cover glass, seal and dry;

[0048] h. Microscopic examination and photography: Observe and take photos under a microscope.

[0049] After the sections were stained with safranin fast green, the observer scored each section according to the OARSI scoring principles in Table 1.

[0050] Table 1. OARSI scoring items

[0051] Symptoms Points No wear and tear on cartilage 0 Abnormal safranin loss on the cartilage surface but no structural changes 0.5 Fibrous destruction of the cartilage surface with small cracks but no cartilage loss 1 The fibrous destruction of the cartilage surface is more serious than 1 and there is cartilage loss 2 Cartilage worn down to less than one quarter and destroyed to subchondral bone 3 Cartilage is worn down to less than half and destroyed to the subchondral bone 4 Cartilage worn away by more than half and destroyed to the subchondral bone 5

[0052] Figure 2 The knee joints in different groups were stained with safranin fast green. As can be seen from the figure, the cartilage matrix of the sham operation group was stained evenly, the tide line structure was intact, and the distribution of each layer was clear, while the chondrocytes in the model group were arranged chaotically, there were a large number of necrotic chondrocytes, the cartilage matrix was stained lightly, the cartilage surface was severely damaged, the tide line structure was damaged, and some fiber cracks were visible, indicating that the model was successful. After the positive drug was administered, the surface of the cartilage did not become significantly thinner, the various layers were clearer than the model group, and the tide line structure was basically intact. After the administration of the Chinese medicine composition containing scorpions and centipedes, the cartilage matrix was stained more evenly than the model group, and the tide line structure was slightly damaged, which shows that the Chinese medicine composition containing scorpions and centipedes can improve the cartilage damage of mice with knee osteoarthritis to a certain extent.

[0053] Figure 3 The OARSI scores of the knee joints in different groups were obtained by safranin fast green staining. The results showed that the control group had a significant difference compared with the model group (P<0.01), indicating that the model was successfully established. The high-dose SF group had a significant difference compared with the model group (P<0.05), indicating that the Chinese medicine composition containing scorpions and centipedes reduced the knee osteoarthritis index.

[0054] (3) Effect of the Chinese medicinal composition containing scorpions and centipedes on the expression of MMP13 in chondrocytes of knee osteoarthritis:

[0055] After 35 days of administration, the right hind limbs of the mice in the control group, model group, positive drug group, SF low-dose group and SF high-dose group were respectively taken and fixed with formalin, and immunohistochemical staining was performed. The specific method is as follows:

[0056] a. Dewaxing: Dewax the sections with xylene I, xylene II, xylene III, 100% alcohol I, 100% alcohol II, 95% alcohol, and 85% alcohol, then wash with distilled water several times;

[0057] b. Antigen repair: Add 3% hydrogen peroxide. Wash several times with distilled water, add antigen repair solution, and wash several times with distilled water again; Blocking: Add 5% serum and block for 30 minutes;

[0058] c. Add primary antibody: Add MMP13 primary antibody, incubate at 4°C overnight;

[0059] d. Add secondary antibody: wash with PBS several times, add immunohistochemistry secondary antibody kit, and incubate at room temperature for 30 minutes; add color developer: wash with PBS several times, add DAB color developer;

[0060] e. Restaining: After washing with distilled water several times, stain with hematoxylin, then differentiate and turn blue, and finally rinse with running water;

[0061] f. Dehydration and sealing: Dehydrate with xylene and different concentrations of alcohol respectively, then place neutral resin on the cover glass, seal and dry;

[0062] g. Microscopic examination and photography: Observe and take photos under a microscope. The results are as follows Figure 4 shown.

[0063] Figure 4 The figure shows the immunohistochemical staining of the knee joints in different groups. As can be seen from the figure, compared with the sham operation group, the positive expression rate of MMP-13 in the cartilage tissue of the model group mice increased, mainly concentrated in the area where the cartilage surface degeneration was more serious, and stained dark brown. The distribution was uneven. After the positive drug treatment, a small amount of MMP-13 positive particles were seen, which could be seen in the surface and shallow layers, and the staining was lighter than that of the model group. After the Chinese medicine composition containing scorpions and centipedes was administered, the MMP-13 positive particles were basically evenly distributed, and the staining was lighter than that of the model group. The above results show that the Chinese medicine composition containing scorpions and centipedes can reduce the expression of MMP13 in chondrocytes of knee osteoarthritis.

[0064] In summary, the Chinese medicine composition containing scorpions and centipedes can relieve pain and inflammation caused by osteoarthritis, reduce cartilage destruction caused by osteoarthritis, and reduce the expression of MMP13 in chondrocytes of knee osteoarthritis, and has the effect of treating osteoarthritis. The Chinese medicine composition containing scorpions and centipedes can be developed as a drug for preventing and / or treating osteoarthritis.

[0065] The embodiments are preferred implementations of the present invention, but the present invention is not limited to the above-mentioned implementations. Any obvious improvements, substitutions or modifications that can be made by those skilled in the art without departing from the essential content of the present invention belong to the protection scope of the present invention.

Claims

1. Use of a Chinese medicinal composition containing scorpions and centipedes in the preparation of a medicament for preventing and / or treating osteoarthritis; The Chinese medicine composition containing scorpions and centipedes comprises scorpions, centipedes, astragalus and black bean skin; In parts by weight, the Chinese medicinal composition containing whole scorpion and centipede comprises 60 parts of black bean skin, 40 parts of astragalus, 1 part of whole scorpion and 1 part of centipede; The osteoarthritis is osteoarthritis caused by abnormal expression of MMP13 in osteoarthritic chondrocytes.

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