A traditional Chinese medicine composition for preventing and treating knee osteoarthritis and application thereof

Abstract

The application discloses a traditional Chinese medicine composition for preventing and treating knee osteoarthritis and application thereof, and relates to the technical field of knee osteoarthritis treatment. The traditional Chinese medicine composition is Sanqiang Decoction which is composed of the following raw materials in parts by weight: Eucommia ulmoides leaves 90-150 parts, Chaenomeles speciosa 60-120 parts, Astragalus membranaceus 90-150 parts, Gastrodia elata 35-65 parts, Dendrobium 35-65 parts, Pueraria lobata 90-150 parts, Curcuma longa 35-65 parts, Persicae Semen 35-65 parts, Coix seed 60-120 parts, Fructus Corni 75-125 parts and calcium gluconate 9-15 parts. The Sanqiang Decoction in the application can improve the pathological state of cartilage and the microstructure parameters of subchondral bone of knee osteoarthritis, improve the antioxidant stress capacity, and improve the quadriceps atrophy caused by knee osteoarthritis, and has good pharmacological effects. The mechanism is related to the activation of TGF-beta 1 / Smad3, Nrf2-Keap1 and PI3K / AKT / FOXO1 pathways.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of knee osteoarthritis treatment, and particularly relates to a traditional Chinese medicine composition for preventing and treating knee osteoarthritis and application thereof. BACKGROUND

[0002] Knee osteoarthritis (KOA) is a degenerative disease characterized by knee joint pain, swelling, deformity and functional disability, which seriously affects the quality of life of patients. At present, there is no specific treatment for KOA, and there are many conservative treatment methods for KOA in Western medicine, most of which are non-specific therapy, mainly using analgesic drugs and non-steroidal anti-inflammatory drugs, mainly to relieve symptoms and improve the quality of life, but long-term use can cause gastrointestinal reactions and other adverse reactions, which greatly limits its clinical application.

[0003] Traditional Chinese medicine believes that KOA is mainly caused by liver and kidney deficiency and wind evil entering the body. Knee osteoarthritis is mostly seen in middle-aged and elderly people, and the pathogenesis is mainly kidney essence deficiency, bone marrow reduction, bone collateral hypotrophy, and the disease is closely related to the kidney, liver and spleen, especially kidney deficiency. Therefore, the present application formulates the three-strong formula with the main effects of invigorating the spleen, tonifying the kidney, soothing the liver, strengthening the tendon, bone and muscle. At present, there is no report on the use of the traditional Chinese medicine composition of the present application to treat knee osteoarthritis.

[0004] The incidence of knee osteoarthritis is increasing year by year, and the pathogenesis is complex. During the progression of the disease, cartilage damage and subchondral bone structure changes occur. The chondrocytes are wrapped by the extracellular matrix mainly composed of water, type II collagen and proteoglycan. When knee osteoarthritis occurs, chondrocytes appear hypertrophy, apoptosis and aging, leading to loss of type II collagen and proteoglycan in the extracellular matrix, weakening the protection and support of chondrocytes; MMP13 and type X collagen are actively expressed, accelerating the degradation of cartilage and extracellular matrix, damaging chondrocytes; and secreting inflammatory factors to damage the microenvironment of cartilage tissue. Anterior cruciate ligament transection is a common method for modeling knee osteoarthritis, and a large amount of inflammatory factors, including inflammatory factors IL-1, IL-1β, IL-6, IL-17 and TNF-α, are produced in the damaged tissue inside the knee joint after the operation, thereby destroying the joint homeostasis and increasing the risk of knee osteoarthritis. The TGF-β1 / Smad3 pathway has been proved to play an important role in maintaining joint homeostasis and promoting the growth and development of chondrocytes. After TGF-β1 activation, it promotes the activation and phosphorylation of Smad3, and then affects downstream factors, such as promoting the formation of type II collagen and proteoglycan of chondrocytes, inhibiting the expression of type II collagen and MMP13, the marker of chondrocyte hypertrophy, and reducing the activity of inflammatory factors. Therefore, regulating the TGF-β1 / Smad3 pathway is a potential target for the treatment of knee osteoarthritis.

[0005] and oxidative stress is one of the main factors in the development of osteoarthritis. Various reactive oxygen species (ROS) and reactive nitrogen species (RNS) are produced during normal metabolic processes, but a complete antioxidant system exists in the body to maintain the dynamic balance between the oxidation and antioxidant systems, enhance the antioxidant effect of free radical clearance, restore normal body function, and thus reduce the damage caused by free radicals. However, under the stimulation of some external adverse factors, the body produces excessive amounts of highly active molecules (such as ROS and RNS), causing oxidative effects to exceed the clearance capacity of antioxidant effects, resulting in an imbalance between the oxidation and antioxidant systems. This leads to the accumulation of ROS and RNS, causing oxidative stress and damage to various tissues and cells. Numerous studies have shown that the level of oxygen free radicals is closely related to the pathogenesis of knee osteoarthritis. ROS mainly includes MDA and nitric oxide (NO). The mechanism of removing ROS and protecting cells from oxidative stress damage in cells is called the antioxidant system. The antioxidant system includes superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px). Therefore, SOD, MDA, and NO can reflect the body's oxygen free radical metabolism. The Nrf2-Keap1 pathway is the most important endogenous antioxidant stress pathway discovered so far. Nrf2, as an important regulator of the body, can enhance the resistance of cells to oxidative stress and inflammatory response, and belongs to the body's antioxidant stress defense system. When in an oxidative stress or inflammatory environment, activated Nrf2 separates from Keap1 and transfers to the nucleus to express highly, forms a heterodimer with a small molecule myoblast fibrosarcoma protein through its highly conserved basic leucine zipper structure, recognizes the Nrf2 antioxidant response element complex ARE, and initiates the expression of a series of downstream protective genes including heme oxygenase-1 (HO-1). In the KOA model, the absence of Nrf2 can exacerbate cartilage damage in osteoarthritis and thus promote the progression of KOA, while the activation of the Nrf2 / HO-1 pathway can inhibit the pathological process of KOA, indicating that the activity of Nrf2 is crucial for maintaining cartilage homeostasis. Exploring more effective treatment options for KOA by targeting the Nrf2 signaling pathway is a hot topic in current antioxidant research.

[0006] Meanwhile, the PI3K / Akt signaling pathway is an important signaling pathway that regulates various cell functions such as cell proliferation and differentiation, and it regulates muscle growth activities. Studies have found that a decrease in the activity of the PI3K / Akt signaling pathway can lead to muscle atrophy. In muscle, the phosphorylation of the PI3K / Akt signal can promote net protein accumulation by promoting the phosphorylation of FoxO. Non-phosphorylated FoxO is located in the nucleus, and by promoting the expression of muscle atrophy genes (Atrogin-1 and MURF1, etc.), it exacerbates muscle atrophy. When FoxO is phosphorylated by AKT, it is transferred to the cytoplasm, losing its regulatory effect on muscle atrophy genes. It is generally believed that Atrogin-1, also known as MAFbx (muscle atrophy F-box protein) and MURF1 (Muscle RING Finger-1), are two proteins involved in the process of muscle atrophy, i.e., the loss of muscle mass and strength. They are both particularly important in the regulation of muscle protein degradation (the process of breaking down muscle proteins). It is usually up-regulated in cases of muscle atrophy, such as during inactivity, hunger, or illness. Atrogin-1 and MURF1 are both ubiquitin ligases that mark specific proteins in muscle cells by attaching ubiquitin to them and ultimately degrading them. This degradation process is an important part of maintaining muscle protein balance, but over-activity in muscle atrophy can lead to muscle loss. Therefore, understanding and regulating the activity of Atrogin-1 and MURF1 is very important for preventing or treating muscle atrophy conditions.

[0007] The above study found that KOA is closely related to structural lesions of cartilage and subchondral bone, and the TGF-β / Smad and Keap1-Nrf2 signaling pathways are involved in the growth and development of chondrocytes and the process of oxidative stress, and play an important role in maintaining the homeostasis of chondrocytes. The PI3K / AKT / FOXO1 pathway plays an important role in regulating muscle atrophy and maintaining muscle mass. These pathways play an important role in the development of knee osteoarthritis. IL-1, IL-1β, IL-6, IL-17, and TNF-α, as important inflammatory factors, not only accelerate the dissolution of collagen in cartilage, but also cause changes in the microstructure of subchondral bone and muscle atrophy. The present application observes the effects of Sanqiang Decoction on the articular cartilage and bone microstructure of KOA rats and the quadriceps femoris muscle, and explores its mechanism of action. Experimental verification shows that Sanqiang Decoction in the present application is composed of Chinese medicines with medicinal and edible properties, has few side effects, and can exert a therapeutic effect on knee osteoarthritis through multiple pathways. SUMMARY

[0008] The present application aims to provide a traditional Chinese medicine composition for preventing and treating knee osteoarthritis and its application, in order to solve the problems in the prior art mentioned in the background, such as the lack of specific treatment for knee osteoarthritis.

[0009] To achieve the above object, the present application adopts the following technical solutions:

[0010] The first aspect of the present application provides a traditional Chinese medicine composition for preventing and treating knee osteoarthritis, which is Sanqiang Decoction, and the Sanqiang Decoction is composed of the following raw materials in parts by weight: Eucommia ulmoides leaves 90-150 parts, Chaenomeles fruit 60-120 parts, Astragalus root 90-150 parts, Gastrodia 35-65 parts, Dendrobium 35-65 parts, Pueraria 90-150 parts, Curcuma 35-65 parts, Peach kernel 35-65 parts, Coix seed 60-120 parts, Fructus Corni 75-125 parts, and calcium gluconate 9-15 parts.

[0011] Preferably, the parts by weight of each raw material are as follows: Eucommia ulmoides leaves 120 parts, Chaenomeles fruit 90 parts, Astragalus root 120 parts, Gastrodia 50 parts, Dendrobium 50 parts, Pueraria 120 parts, Curcuma 50 parts, Peach kernel 50 parts, Coix seed 90 parts, Fructus Corni 100 parts, and calcium gluconate 12 parts.

[0012] The second aspect of the present application provides the use of the above traditional Chinese medicine composition in the preparation of a medicine for treating knee osteoarthritis.

[0013] Preferably, the preparation method of the medicine for treating knee osteoarthritis comprises the following steps:

[0014] S1, raw materials of the medicine for treating knee osteoarthritis are weighed according to the parts by weight;

[0015] S2, Eucommia ulmoides leaves, Astragalus root, Gastrodia, Chaenomeles fruit, Pueraria, Coix seed, and Fructus Corni are soaked in 70% ethanol solution, and then extracted and filtered and concentrated under the assistance of ultrasonic waves, and the residues are separately reserved;

[0016] S3, Curcuma and Peach kernel are extracted by distillation to obtain volatile oil, and then the volatile oil is wrapped with β-cyclodextrin, and the residues are separately reserved;

[0017] S4, the residues in S2 and S3 are combined with Dendrobium, and then extracted with water and concentrated;

[0018] S5, the extracts in S2, S3, and S4 are combined, and then calcium gluconate is added and uniformly mixed;

[0019] S6, the uniformly mixed material in S5 is freeze-dried for standby, and then dissolved with distilled water before use.

[0020] Preferably, the medicine is a medicine for repairing knee osteoarthritis chondrocytes and subchondral bone, reducing knee osteoarthritis cartilage oxidative stress, and improving knee osteoarthritis quadriceps femoris.

[0021] Preferably, the medicine is a medicine for activating TGF-β1 / Smad3, Nrf2-Keap1, and PI3K / AKT / FOXO1 pathways to treat knee osteoarthritis.

[0022] The third aspect of the present application provides the above-mentioned pharmaceutical composition, which comprises the above-mentioned traditional Chinese medicine composition and a pharmaceutically acceptable carrier.

[0023] The fourth aspect of the present application provides the use of the above-mentioned traditional Chinese medicine composition in the treatment of knee osteoarthritis.

[0024] Compared with the prior art, the present application has the following beneficial effects:

[0025] The Sanqiang Formula in the present application has the functions of invigorating the spleen and tonifying qi, benefiting the kidney and softening the liver, promoting blood circulation to remove meridian obstruction, and strengthening the tendon, bone and muscle, and can treat knee osteoarthritis through multiple targets and multiple pathways. Research shows that the Sanqiang Formula can improve the pathological state of cartilage and the microstructure parameters of subchondral bone of knee osteoarthritis, improve the antioxidant stress capacity, and improve the quadriceps muscle atrophy caused by knee osteoarthritis, and has good pharmacological effects. The mechanism of action is related to the activation of TGF-β1 / Smad3, Nrf2-Keap1 and PI3K / AKT / FOXO1 pathways. BRIEF DESCRIPTION OF DRAWINGS

[0026] Figure 1 Figure 1 is a graph showing the body weight change trend of each group of rats in Example 1 of the present application (n=8);

[0027] Figure 2 Figure 2 is a graph showing the comparison of body weight before and after administration in Example 1 of the present application (n=8);

[0028] Figure 3 Figure 3 is a graph showing the results of ALT, AST, BUN and CRE of each group of rats in Example 1 of the present application (n=8);

[0029] Figure 4 Figure 4 is a micrograph showing the histopathology of liver tissue of each group of rats in Example 1 of the present application (HE staining, 200x);

[0030] Figure 5 Figure 5 is a micrograph showing the histopathology of kidney tissue of each group of rats in Example 1 of the present application (HE staining, 200x);

[0031] Figure 6 Figure 6 is a graph showing the results of ramp angle and thermal pain threshold of each group of rats in Example 1 of the present application;

[0032] Figure 7 Figure 7 is a graph showing the results of IL-1, IL-1β, IL-6, IL-17 and TNF-α of each group of rats in Example 1 of the present application (n=8);

[0033] Figure 8 ​​​​Figure 1 is a TGF-β1, Smad3, p-Smad3, Col II, Col X, MMP13 protein immunoblotting chart of each group of rats in Example 1 of the present application;

[0034] Figure 9 Figure 2 is a TGF-β1, p-Smad3 / Smad3, Col II, Col X, MMP13 protein expression level chart of each group of rats in Example 1 of the present application;

[0035] Figure 10 Figure 3 is a Col II, Col X, MMP13 mRNA expression level chart of each group of rats in Example 1 of the present application;

[0036] Figure 11 Figure 4 is a SOD, GSH-Px, MDA, NO result chart of each group of rats in Example 1 of the present application;

[0037] Figure 12 Figure 5 is a Nrf2, Keap1, HO-1, NQO1 mRNA result chart of each group of rats in Example 1 of the present application (n=3);

[0038] Figure 13 Figure 6 is a Micro-CT chart of subchondral bone of each group of rats in Example 1 of the present application;

[0039] Figure 14 Figure 7 is a Micro-CT scanning parameter level chart of subchondral bone of each group of rats in Example 1 of the present application (n=3);

[0040] Figure 15 Figure 8 is a quadriceps muscle weight, quadriceps muscle weight / body weight ratio chart of each group of rats in Example 1 of the present application;

[0041] Figure 16 Figure 9 is a knee joint range of motion comparison chart of each group of rats in Example 1 of the present application;

[0042] Figure 17 Figure 10 is a p-PI3K, PI3K, p-AKT, AKT, p-FOXO1, FOXO1 protein immunoblotting chart of each group of rats in Example 1 of the present application;

[0043] Figure 18 Figure 11 is a PI3K / AKT / FOXO1 pathway protein expression level chart of quadriceps muscle tissue of each group of rats in Example 1 of the present application;

[0044] Figure 19 Figure 12 is an Atrogin-1, Murf-1 protein immunoblotting chart of each group of rats in Example 1 of the present application;

[0045] ​​Figure 20 Figure of protein expression level of Atrogin-1 and MuRF1 mRNA in quadriceps of each group of rats in embodiment 1 of the present application. DETAILED DESCRIPTION

[0046] The technical solutions in the embodiments of the present application will be clearly and completely described in combination with the drawings in the embodiments of the present application. Obviously, the described embodiments are only part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor fall within the protection scope of the present application.

[0047] Embodiment 1

[0048] The Chinese medicinal composition for preventing and treating knee osteoarthritis is Sanqiang Decoction, which is composed of Eucommia ulmoides leaf, Chaenomeles fruit, Astragalus root, Gastrodia tuber, Dendrobium, Pueraria root, Curcuma longa, Semen Persicae, Coix seed, Fructus Corni, and calcium gluconate.

[0049] In the present application, Sanqiang Decoction is composed of Eucommia ulmoides leaf, Chaenomeles fruit, Astragalus root, Gastrodia tuber, Dendrobium, Pueraria root, Curcuma longa, Semen Persicae, Coix seed, Fructus Corni, and calcium gluconate. Eucommia ulmoides leaf tonifies liver and kidney and strengthens bones and muscles; Astragalus root tonifies qi, raises yang, generates fluid and nourishes blood. When the two are combined, they can tonify qi, tonify kidney and soften liver, and are the monarch drug. Pueraria root raises yang and resolves muscle, Fructus Corni tonifies liver and kidney, astringes essence and consolidates, Chaenomeles fruit relaxes sinews and collaterals, Dendrobium nourishes kidney yin and clears heat, and assists Eucommia ulmoides leaf and Astragalus root in tonifying liver and kidney and dredging collaterals, and are the minister drugs. Coix seed invigorates spleen, relaxes sinews and relieves convulsion, Gastrodia tuber expels external wind, dredges meridians and collaterals and stops pain, Curcuma longa promotes blood circulation and relieves pain, Semen Persicae promotes blood circulation and removes stasis, and calcium gluconate supplements calcium. They are the auxiliary drugs. The whole prescription can invigorate spleen, tonify qi, tonify kidney, soften liver, promote blood circulation, dredge collaterals, strengthen bones and muscles, and has the effect of invigorating spleen, tonifying qi, tonifying kidney, softening liver, promoting blood circulation and dredging collaterals. Therefore, it is named Sanqiang Decoction.

[0050] The present application observes the influence of Sanqiang Decoction on articular cartilage and bone microstructure and quadriceps of KOA rats through animal experiments, and preliminarily discusses the mechanism of its curative effect. The experiment specifically includes the following steps:

[0051] Step one: material preparation.

[0052] Animal preparation: 56 SPF male SD rats, 6 weeks old, body weight 180±20g, purchased from Beijing Huafukang Biotechnology Co., Ltd., experimental animal production license, raised in the clean laboratory of the animal experiment center of Huabei Polytechnic University, constant temperature 25℃, free water and food. The experiment is reviewed by the experimental animal ethics committee of Huabei Polytechnic University (ethics number).

[0053] Drug preparation: Sanqiang Decoction was composed of Euonymus alatus 12 g, Chaenomeles lagenaria 9 g, Astragalus membranaceus 12 g, Gastrodia elata 5 g, Dendrobium nobile 5 g, Pueraria lobata 12 g, Curcuma longa 5 g, Semen Persicae 5 g, Coix lachryma-jobi 9 g, Fructus Corni 10 g, and calcium gluconate 1.2 g (this is the medium dose, the dose of the prevention group is equal to the medium dose, and the low and high doses are 1 / 2 and 2 times of the medium dose, respectively). The medicinal materials were purchased from Beijing Tong Ren Tang Pharmacy Co., Ltd. Tangshan Branch and identified as authentic by Professor Li Ji'an of Huabei University of Chinese Medicine.

[0054] Reagent preparation: Serum biochemical reagent kits were purchased from Nanjing Jiancheng, which were alanine aminotransferase (ALT) test kit, aspartate aminotransferase (AST) test kit, urea nitrogen (BUN) test kit, creatinine (CRE) test kit, and the order numbers were C009-2-1, C010-2-1, C013-2-1, C011-2-1, respectively. ELISA KIT was purchased from Xiamen Lunchangshuo, which were rat interleukin 1 (IL-1), rat interleukin 1β (IL-1β), rat interleukin 6 (IL-6), rat interleukin 17 (IL-17), rat tumor necrosis factor alpha (TNF-α), rat glutathione peroxidase (GSH-Px), rat superoxide dismutase (SOD), nitric oxide (NO) content determination kit, malondialdehyde (MDA) content determination kit, and the order numbers were ED-30193, ED-30206, ED-30219, ED-30201, ED-31063, ED-35362, ED-34817, LCSSH-0132W, LCSSH-0109W, respectively. Rabbit polyclonal transforming growth factor-β1 (TGF-β1), rabbit monoclonal cell signal transduction molecule 3 (Smad3), rabbit polyclonal type II collagen (Col II), mouse monoclonal matrix metalloproteinase 13 (MMP13), rabbit monoclonal (PI3K), rabbit monoclonal (AKT), rabbit monoclonal (FOXO1) were purchased from Dr. Deki Biological, and the order numbers were Ba0290, Bm3919, Ba0533, Ma00420, Bm5187, Bm4400, Bm4249, respectively. Rabbit polyclonal p-Smad3, rabbit polyclonal X collagen (Col X), rabbit polyclonal (p-AKT) were purchased from Bioo Sun Biological, and the order numbers were Bs5616r, Bs0554r, Bs0876r, respectively. Mouse monoclonal β-actin, rabbit polyclonal (p-PI3K), rabbit polyclonal (p-FOXO1) were purchased from Affinity, and the order numbers were T0022, Af3241, Af3416. HRP-labeled goat anti-rabbit secondary antibody was purchased from Biyun Tian Biological, and the order number was A0208. Mouse monoclonal (Atrogin-1), rabbit polyclonal (MuRF1), HRP-labeled goat anti-mouse secondary antibody were purchased from Sanying Biological, and the order numbers were 67172-1-ig, 55456-1-ap, SA00001-1. Trizol was purchased from Ambion, and the order number was 15596-026. HiScript® II Q Select RT SuperMix for qPCR was purchased from VAZYME, and the order number was R233.

[0055] Instrument preparation: DB026 intelligent hot plate instrument (Beijing Zhi Shu Duobao Biological Technology Co., Ltd.), SpectraMax M3 multifunctional microplate reader (Molecular Devices, USA), ViiA-7 real-time fluorescent quantitative PCR instrument (ABI, USA), DYCZ-24DN vertical electrophoresis tank (Beijing Liuyi Instrument Factory), FW606 semi-dry transfer instrument (Nanjing Aiseyi), DS-H200 horizontal shaker (Servicebio), Tissuelyser-24L automatic grinder (Shanghai Jingxin), VNC-102 VENUS Micro CT (Ping Sheng Technology), etc.

[0056] Step two, establish experimental groups and take samples.

[0057] S2.1, model establishment and grouping;

[0058] The rat model of knee osteoarthritis was prepared by transecting the right posterior cruciate ligament of the knee joint, positive "drawer test" and maintaining joint instability for 6 weeks. After successful modeling, the rats were randomly divided into model group, celecoxib group, Sanqiang prescription low, medium and high dose groups, and Sanqiang prevention group, 8 rats in each group. Another 8 rats were used as sham operation group, only the skin was incised, and the anterior cruciate ligament was not cut.

[0059] S2.2, drug preparation and administration;

[0060] The leaves of Eucommia ulmoides, Astragalus membranaceus, Gastrodia elata, Chaenomeles speciosa, Pueraria lobata, Coix lacryma-jobi, and Fructus Corni were soaked in 70% ethanol solution, then extracted by ultrasonic assistance and filtered and concentrated. Curcuma longa and Semen Persicae were distilled to extract volatile oil, and then wrapped with β-cyclodextrin. The residues of the above-mentioned drugs were combined with Dendrobium officinale and water-extracted and concentrated. The above-mentioned extracts were combined, and calcium gluconate was added and mixed. Finally, freeze-drying was used for standby, and distilled water was used for dissolution before use.

[0061] The Sanqiang prescription low, medium and high dose groups were given 3.8, 7.6 and 15.2 g / kg of drug solution by gavage, the celecoxib group was given 18 mg / kg of drug solution by gavage, the sham operation group and the model group were given the same volume of normal saline by gavage, and the continuous administration lasted for 8 weeks. The Sanqiang prevention group was given 7.6 g / kg of drug solution by gavage, and the administration started immediately after the anterior cruciate ligament transection, and the continuous administration lasted for 14 weeks.

[0062] S2.3, sample collection;

[0063] After the end of administration, the rats were anesthetized by intraperitoneal injection of sodium pentobarbital, blood was taken from the abdominal aorta, centrifuged at 3000 r / min to obtain the supernatant, and stored at -80°C. Some rats were stripped of the right knee joint around the muscle tissue, and the right knee joint was obtained intact and fixed in paraformaldehyde. Some rats were taken to the knee cartilage tissue into a cryopreservation tube and stored at -80°C. The quadriceps femoris muscle was cut at the distal end of the femur at the patellar margin stop point, completely separated along the intermuscular space and weighed. Some of the quadriceps femoris muscle tissue was fixed in paraformaldehyde; the remaining quadriceps femoris muscle tissue was placed in a cryopreservation tube and stored in a -80°C refrigerator.

[0064] Step three, experimental detection.

[0065] S3.1, detection of rat behavior and knee joint passive range of motion;

[0066] Before sampling, the rat behavior and knee joint passive range of motion were detected. The rats were placed in an intelligent hot plate instrument, the licking foot start time was observed, and they were quickly taken out, and the thermal pain threshold time was recorded. The rats were placed one by one on a slope with a slope of 25°-45°, each time the slope was increased or decreased by 5°, and the rats stayed on the same slope for more than 10 seconds for three times in a row, and this slope was recorded as the stable slope of the rat. The passive range of motion of the knee joint of the rats in each group was measured with a medical protractor, and the maximum flexion angle minus the maximum extension angle was the passive range of motion.

[0067] S3.2, detection of serum biochemical indicators;

[0068] According to the kit instructions, the levels of ALT, AST, BUN, CRE, IL-1, IL-1β, IL-6, IL-17, TNF-α, GSH-Px, SOD, NO, and MDA in the rat serum were detected.

[0069] S3.3, Western Blot detection of cartilage TGF-β1, Smad3, p-Smad3, Col II, Col X, MMP13 protein expression and quadriceps femoris PI3K / AKT / FOXO1 signal pathway and Atrogin-1 and MuRF1 protein expression;

[0070] The frozen tissue was mixed with the corresponding proportion of tissue lysis solution, protease inhibitor and phosphatase inhibitor, ground and extracted protein, and the protein concentration was determined by BCA method. The protein sample was mixed with the corresponding proportion of loading buffer and double distilled water, and then subjected to metal bath. After the electrophoresis gel was prepared, electrophoresis was performed, and semi-dry transfer method was used for membrane transfer. After rapid blocking solution blocking, the primary antibody was added: TGF-β1 (1:1000), Smad3 (1:1000), p-Smad3 (1:1000), Col II (1:1000), Col X (1:1000), MMP13 (1:1000), β-actin (1:20000), PI3K (1:1000), p-PI3K (1:1000), AKT (1:1000), p-AKT (1:1000), FOXO1 (1:1000), p-FOXO1 (1:1000), Atrogin-1 (1:10000), MuRF1 (1:2000) 4°C overnight incubation, TBST membrane washing 3 times x 10 min; add diluted goat anti-rabbit secondary antibody (1:10000) or goat anti-mouse secondary antibody (1:10000), incubate at room temperature for 2h; TBST membrane washing 3 times x 10 min, ECL color development, Image Lab scanning and archiving, and ImageJ processing and analysis of the gray value of the target band.

[0071] S3.4, Real-time fluorescent quantitative PCR detection of cartilage Col II, Col X, MMP13, Nrf2, Keap1, HO-1, NQO1 mRNA expression;

[0072] Take 100 mg of fresh frozen cartilage tissue, add 1 ml of Trizol reagent, homogenize, add chloroform, centrifuge, and then aspirate the upper colorless liquid and mix with isopropanol, centrifuge to obtain the precipitate, mix the precipitate with 75% ethanol, centrifuge to discard the supernatant, add DEPC water to dissolve and store in the freezer. Use the HiScript® II Q Select RT SuperMix for qPCR kit to reverse transcribe total RNA to cDNA, and then perform quantitative PCR reaction on the PCR instrument. The primer sequences are shown in Table 1.

[0073] Table 1 Primer sequences

[0074]

[0075] S3.5, Micro-CT detection of cartilage subchondral bone microstructure;

[0076] The knee joint specimen was placed vertically in the sample sleeve, and the specimen was fixed with a paper towel to prevent the specimen from shaking. Then the sleeve was fixed in the Micro-CT for scanning the specimen, and the bone mineral density (BMD), bone volume fraction (BV / TV), trabecular number (Tb.N), trabecular thickness (Tb.Th), trabecular spacing (Tb.Sp), and trabecular structure model parameter (SMI) were analyzed.

[0077] Step four, statistical analysis of experimental results.

[0078] The experimental results were statistically analyzed using SPSS 23.0. The measurement data met the normal distribution, and were expressed as mean ± standard deviation (x±s). The comparison of means among three or more groups was performed using one-way ANOVA. If the variances were equal, the comparison between two groups was performed using LSD test. If the variances were not equal, the comparison between two groups was performed using Tamhane's test. P<0.05 was considered statistically significant.

[0079] The statistical results of the experiment are as follows:

[0080] S4.1, the effect of Sanqiangfang on the body weight of KOA rats;

[0081] During the administration period, the body weight of rats increased steadily. There was no difference in body weight between rats before and after administration. The results are shown in Figure 1 , Figure 2 , Figure 2 There was no difference between the NS groups.

[0082] S4.2, the effect of Sanqiangfang on the liver and kidney function of KOA rats:

[0083] Effect of Sanqiangfang on serum liver and kidney function in KOA rats: After administration, there was no difference in ALT, AST, BUN and CRE between rats in each group. The results are shown in Figure 3 , Figure 3 There was no difference between the NS groups.

[0084] Effect of Sanqiangfang on liver and kidney morphology in KOA rats: HE staining showed that the liver cell structure of rats in each group was complete, the liver cord was arranged in order, there was no obvious difference in the size of liver cells, and the nucleus was round and located in the center of liver cells. The kidney tissue morphology of rats in each group was normal, the structure of glomerulus and renal tubule was normal, and the interstitium and mesangium showed no hyperplasia. The pathological changes of liver and kidney tissue in rats in each group are shown in Figure 4 , Figure 5 .

[0085] S4.3, the effect of Sanqiangfang on the behavior of KOA rats;

[0086] ​After the end of administration, the slope angle and thermal pain threshold of the model group were significantly lower than those of the sham operation group (P<0.01), and were significantly higher than those of the model group (P<0.01). The results are shown in Figure 6 .

[0087] S4.4, Effect of Sanqiang Decoction on Serum Inflammatory Factors in KOA Rats

[0088] Compared with the sham operation group, the levels of IL-1, IL-1β, IL-6, IL-17, and TNF-α in the model group were significantly increased (P<0.01). Compared with the model group, the levels of IL-1, IL-1β, IL-6, IL-17, and TNF-α in each administration group were significantly decreased (P<0.01). The results are shown in Figure 7 .

[0089] S4.5, Protective Effect of Sanqiang Decoction on Cartilage Cell Homeostasis in KOA Rats

[0090] Effect of Sanqiang Decoction on Protein Expression of TGF-β1, Smad3, p-Smad3, Col II, Col X, and MMP13 in KOA Rat Cartilage

[0091] Compared with the sham operation group, the levels of TGF-β1, p-Smad3 / Smad3, and Col II in the model group were significantly decreased (P<0.01), and the levels of Col X and MMP13 were significantly increased (P<0.01). Compared with the model group, the levels of TGF-β1, p-Smad3 / Smad3, and Col II in the celecoxib group and the high-dose Sanqiang Decoction group were increased (P<0.01, P<0.05). Except for the trend of decreased MMP13 in the low-dose Sanqiang Decoction group, which was not statistically significant, the levels of Col X and MMP13 in the celecoxib group and the Sanqiang Decoction groups were decreased (P<0.01, P<0.05). The Western blot results of each group are shown in Figure 8 , and the protein expression levels are shown in Figure 9 ; Figure 8 Group A is the sham operation group, group B is the model group, group C is the celecoxib group, groups D, E, and F are the low-, medium-, and high-dose Sanqiang Decoction groups.

[0092] Effect of Sanqiang Decoction on mRNA Expression of Col II, Col X, and MMP13 in KOA Rat Cartilage

[0093] Compared with the sham operation group, the Col II mRNA level of the model group was significantly decreased (P<0.01), and the Col X and MMP13 mRNA levels were significantly increased (P<0.01). Compared with the model group, the Col II, Col X and MMP13 mRNA levels of the other groups were improved (P<0.01, P<0.05). The mRNA levels of each group are shown in Table 2. Figure 10 .

[0094] S4.6, Effect of Sanqiang Formula on Serum Oxidative Stress Factors of KOA Rats

[0095] Effect of Sanqiang Formula on Serum Oxidative Stress Factors of KOA Rats

[0096] Compared with the sham operation group, the SOD and GSH-Px levels of the model group were significantly decreased (P<0.01), and the MDA and NO levels were significantly increased (P<0.01). Compared with the model group, the SOD and GSH-Px levels of the celecoxib group and the high-dose Sanqiang group were significantly increased (P<0.01), and the MDA and NO levels were significantly decreased (P<0.01). The SOD level of the Sanqiang prevention group was slightly increased (P<0.05), the GSH-Px level was significantly increased (P<0.01), and the MDA and NO levels were significantly decreased (P<0.01). The SOD, GSH-Px, MDA and NO levels of the low-dose Sanqiang group were slightly improved but not statistically significant. The SOD and NO levels of the medium-dose Sanqiang group were slightly improved but not statistically significant, and the GSH-Px and MDA levels were improved (P<0.05). See Table 3. Figure 11 .

[0097] Effect of Sanqiang Formula on Nrf2, Keap1, HO-1 and NQO1 mRNA in KOA Rat Cartilage

[0098] Compared with the sham operation group, the Nrf2, HO-1 and NQO1 levels of the model group were significantly decreased (P<0.01), and the Keap1 level was significantly increased (P<0.01). After treatment, the Nrf2, HO-1 and NQO1 levels of the celecoxib group, the high-dose Sanqiang group and the Sanqiang prevention group were significantly increased (P<0.01), and the Keap1 level was significantly decreased (P<0.01). The Nrf2 and NQO1 levels of the low- and medium-dose Sanqiang groups had an upward trend but were not statistically significant. The HO-1 level of the medium-dose Sanqiang group was significantly increased (P<0.01), and the Keap1 level was significantly decreased (P<0.01). The Keap1 level of the low-dose Sanqiang group was slightly increased (P<0.05). See Table 4. Figure 12 .

[0099] S4.7, Effect of Sanqiang Decoction on Serum Oxidative Stress Factors in KOA Rats

[0100] Compared with the sham operation group, the BMD, BV / TV, Tb.N and Tb.Th of the model group decreased (P<0.01), and the Tb.Sp and SMI increased (P<0.01). Compared with the model group, the BMD, BV / TV and Tb.Th of the celecoxib group increased (P<0.01), and the SMI decreased (P<0.05); the BMD, BV / TV and Tb.Th of the Sanqiang medium dose group increased (P<0.05); the BMD, BV / TV, Tb.N and Tb.Th of the Sanqiang high dose group increased (P<0.01), and the SMI decreased (P<0.05). The Micro-CT images showed that the trabecular bone number decreased and the distribution was disordered in the model group compared with the sham operation group; after treatment, the trabecular bone number increased and the arrangement was close, and the subchondral bone microstructure was improved. The results are shown in Figure 13 , Figure 14 .

[0101] S4.8, Effect of Sanqiang Decoction on the Quadriceps Muscle of KOA Rats

[0102] Effect of Sanqiang Decoction on the Weight of Quadriceps Muscle of KOA Rats

[0103] After the administration period was completed, the weight of the quadriceps muscle of the model group was less than that of the sham operation group, and the difference was statistically significant (P<0.01). The weight of the quadriceps muscle of the Sanqiang Decoction prevention group and the celecoxib group was significantly higher than that of the model group, and the difference was statistically significant (P<0.01). The weight of the quadriceps muscle of the Sanqiang Decoction low, medium and high dose groups had an increasing trend compared with the model group, and there was no significant difference in the body weight of the rats among the groups. See Figure 15 .

[0104] Effect of Sanqiang Decoction on the Joint Range of Motion of KOA Rats

[0105] After the administration period was completed, the range of motion of the model group was significantly lower than that of the sham operation group, and the difference was statistically significant (P<0.01). The range of motion of the Sanqiang Decoction prevention group was higher than that of the model group, and the difference was statistically significant (P<0.05). The range of motion of the celecoxib group and the Sanqiang Decoction low, medium and high dose groups was significantly higher than that of the model group, and the difference was statistically significant (P<0.01). See Figure 16 .

[0106] Effect of Sanqiang Decoction on the PI3K / AKT / FOXO1 Signaling Pathway Protein in the Quadriceps Muscle Tissue of KOA Rats

[0107] See Figure 17-18 , for example Figure 18As shown, compared with the sham-operated group, the ratios of p-PI3K / PI3K (P<0.001), p-AKT / AKT (P<0.001), and p-FOXO1 / FOXO1 (P<0.001) in the quadriceps femoris muscle of rats in the model group were decreased; compared with the model group, the ratios of p-PI3K / PI3K (P<0.001), p-AKT / AKT (P<0.001), and p-FOXO1 / FOXO1 (P<0.001) in the celecoxib group and the high-dose triple-strength formula group were significantly increased. The ratio of OXO1 / FOXO1 (P<0.0001) was increased in the low-dose group of the three-strength group, and the ratios of p-AKT / AKT (P<0.05) and p-FOXO1 / FOXO1 (P<0.0001) were increased in the medium-dose group of the three-strength group. Figure 17 In the group, A represents the sham surgery group, B represents the model group, C represents the celecoxib group, and D, E, and F represent the low, medium, and high dose groups of the three-strength formula.

[0108] Effects of the three-pronged approach on the expression of Atrogin-1 and MuRF1 proteins in the quadriceps femoris muscle of KOA rats:

[0109] See Figure 19-20 ,like Figure 20 As shown, compared with the sham-operated group, the expression of Atrogin-1 (P<0.001) and MURF1 (P<0.001) proteins in the quadriceps femoris muscle tissue of the model group rats was significantly increased; compared with the model group, the expression of Atrogin-1 (P<0.01) and MURF1 (P<0.001) proteins in the celecoxib group and the medium and high dose groups of the three-strong formula was significantly decreased. Figure 19 In the group, A represents the sham surgery group, B represents the model group, C represents the celecoxib group, and D, E, and F represent the low, medium, and high dose groups of the three-strength formula.

[0110] Step 5: Verify the effect of the three-strong formula in treating KOA through multiple pathways by using experimental results.

[0111] S5.1, the protection of chondrocytes and subchondral bone by the three-pronged approach in knee osteoarthritis;

[0112] In the three-component formula, astragaloside, the active ingredient in Astragalus membranaceus, can increase the expression of TGF-β1 in chondrocytes, decrease the expression of IL-1, promote the expression of type II collagen and proteoglycans in the extracellular matrix of chondrocytes, improve the cartilage environment, and inhibit cartilage degradation; verrucoside isoflavone glucoside can enhance the proliferation capacity of chondrocytes and reduce apoptosis. Chlorogenic acid, the active ingredient in Eucommia ulmoides leaves, can inhibit the expression of MMP13, reduce the levels of inflammatory factors such as IL-1, IL-17, and TNF-α, reduce subchondral bone loss, and protect subchondral bone.

[0113] The experimental results show that Sanqiang Decoction has no damage to the liver and kidney function of rats, and has high safety. It can improve the indicators of knee osteoarthritis rats climbing and thermal pain threshold, and reduce the levels of inflammatory factors IL-1, IL-1β, IL-6, IL-17 and TNF-α. Western Blot detection found that the expression of TGF-β1 and p-Smad3 in the model group of rats decreased, type II collagen was lost, and the expression of type X collagen and MMP13 was enhanced. Sanqiang Decoction can activate the TGF-β1 / Smad3 pathway, promote the generation of type II collagen, down-regulate the expression of type X collagen and MMP13, maintain the homeostasis of chondrocytes, and inhibit chondrocyte hypertrophy. Real-time fluorescent quantitative PCR synchronously confirmed the expression of type II collagen, type X collagen and MMP13 in the cartilage of rats. Through Micro-CT image observation, it was found that the joint space of the model group of rats was narrowed, the subchondral bone was disordered, and the bone microstructure parameters BMD and BV / TV, Tb.N, Tb.Th decreased, and Tb.Sp, SMI increased, while Sanqiang Decoction had a repairing effect on the abnormal subchondral bone structure caused by joint damage.

[0114] S5.2, Effect of Sanqiang Decoction on oxidative stress of knee osteoarthritis cartilage

[0115] In Sanqiang Decoction, astragaloside A can increase the level of antioxidant enzyme activity, reduce ROS and malondialdehyde in cells, increase GSH-Px and SOD in various diseases, and play an antioxidant protective role in injury, improve the bone environment, and promote the balance between oxidation and antioxidant. The phenolic acid components such as chlorogenic acid in Eucommia ulmoides leaf and the flavonoid components such as rutin are the material basis for its antioxidant effect. It can increase the level of antioxidant factors in serum, reduce oxidative damage, prevent inflammation, reduce the release of pro-inflammatory cytokines such as NO and interleukin IL-6, and TNF-α, thereby reducing the level of inflammatory factors in knee osteoarthritis.

[0116] The experimental results show that compared with the control group, the levels of SOD and GSH-Px in the KOA model group significantly decreased (P<0.01), and the levels of MDA and NO significantly increased (P<0.01); while in the remaining experimental groups treated with Sanqiang Decoction, SOD significantly increased, and MDA and NO significantly decreased; this indicates that Sanqiang Decoction has a certain effect on the antioxidant stress of the body. Compared with the KOA model, the mRNA and protein expressions of Nrf2 and Keap1, HO-1 and NQO1 in the experimental groups treated with Sanqiang Decoction were significantly up-regulated, while the mRNA and protein expressions of Keap1 were significantly down-regulated. The experiment shows that KOA can inhibit the Nrf2-Keap1 / ARE pathway, and after the action of Sanqiang Decoction, the Nrf2-Keap1 pathway can be activated, the expressions of HO-1 and NQO1 can be increased, and the antioxidant capacity of the body to oxidative stress can be restored.

[0117] S5.3, Effect of Sanqiang Decoction on Quadriceps Muscle of Knee Osteoarthritis Rats;

[0118] In Sanqiang Decoction, the effective component of Huangqi, Huangqiaosuan, can regulate protein synthesis and degradation through PI3K / AKT signaling pathway, reduce the level of oxidative stress of skeletal muscle, and improve the atrophy of skeletal muscle. The calycosin in Sanqiang Decoction has the effects of anti-autophagy, anti-apoptosis and anti-inflammation, can up-regulate the expression of B-cell lymphoma 2-like protein (Bcl-2), inhibit the expression of TNF-α, weaken the expression of Atrogin-1 and MuRF1, block the ubiquitin-proteasome-mediated muscle fiber dissolution, and delay the atrophy of skeletal muscle. The effective component of leaves of Eucommia ulmoides Oliv., chlorogenic acid, can up-regulate the PI3K-Akt signaling pathway, promote the expression of AKT phosphorylation FoxO and Bcl-2, inhibit the apoptosis of cells, and FoxO loses the regulation of atrophy genes, thus slowing down the atrophy process of muscle.

[0119] The experimental results show that Sanqiang Decoction can improve the passive activity of knee osteoarthritis rats, the relative muscle mass of rats, reduce the connective tissue between muscle cell bundles, and improve the atrophy of muscle of rats. The protein expression of p-PI3K / PI3K, p-AKT / AKT and p-FOXO1 / FOXO1 in the quadriceps muscle of the model group is reduced, and the protein and qPCR expression of Atrogin-1 and MURF1 is increased, which indicates that the PI3K, AKT and FOXO1 pathways of the quadriceps muscle of the model group are inhibited, the expression of Atrogin-1 and MURF1 is promoted, the degree of fibrosis of the quadriceps muscle is increased, and the atrophy of the quadriceps muscle is promoted. The protein and qPCR expression of p-PI3K / PI3K, p-AKT / AKT and p-FOXO1 / FOXO1 in the quadriceps muscle of the Sanqiang Decoction group is increased, which indicates that Sanqiang Decoction can activate the PI3K, AKT and FOXO1 pathways, inhibit the translocation of FOXO1 from cytoplasm to nucleus, inhibit the expression of muscle atrophy genes Atrogin-1 and MURF1, reduce the degree of fibrosis of the quadriceps muscle, and improve the atrophy of the quadriceps muscle.

[0120] The experimental results verify that:

[0121] Sanqiang Decoction has the effects of invigorating the spleen and tonifying qi, benefiting the kidney and soothing the liver, promoting blood circulation to remove meridian obstruction, and strengthening the sinew, bone and muscle. The leaves of Eucommia ulmoides Oliv. contain phenolic acid components such as chlorogenic acid and flavonoid components such as rutin, Huangqi contains effective components of astragaloside A and calycosin glucoside, and papaya contains ursolic acid and oleanolic acid and other components, which can treat knee osteoarthritis through multiple targets and multiple pathways. Research shows that Sanqiang Decoction can improve the pathological state of cartilage and the microstructure parameters of subchondral bone of knee osteoarthritis rats, improve the antioxidant stress capacity of rats, and improve the atrophy of quadriceps muscle caused by knee osteoarthritis of rats, and has good pharmacological effects. The mechanism is related to the activation of TGF-β1 / Smad3, Nrf2-Keap1 and PI3K / AKT / FOXO1 pathways.

[0122] The above description is only used to help understand the method of the present application and its core idea, but the protection scope of the present application is not limited thereto. For those skilled in the art, according to the technical scheme and the inventive concept of the present application, equivalent replacement or change within the technical range disclosed by the present application should be covered within the protection scope of the present application. In summary, the content of the present specification should not be understood as a limitation of the present application.

Claims

1. A traditional Chinese medicine composition for preventing and treating knee osteoarthritis, characterized in that, The traditional Chinese medicine composition is Sanqiang Decoction, which is composed of the following raw materials in parts by weight: Eucommia ulmoides leaf 90-150 parts, Chaenomeles fruit 60-120 parts, Astragalus root 90-150 parts, Gastrodia 35-65 parts, Dendrobium 35-65 parts, Pueraria 90-150 parts, Curcuma 35-65 parts, Peach kernel 35-65 parts, Coix seed 60-120 parts, Cornus 75-125 parts, and calcium gluconate 9-15 parts.

2. The traditional Chinese medicine composition according to claim 1, characterized in that, The parts by weight of each raw material are as follows: Eucommia ulmoides leaf 120 parts, Chaenomeles fruit 90 parts, Astragalus root 120 parts, Gastrodia 50 parts, Dendrobium 50 parts, Pueraria 120 parts, Curcuma 50 parts, Peach kernel 50 parts, Coix seed 90 parts, Cornus 100 parts, and calcium gluconate 12 parts.

3. Use of the traditional Chinese medicine composition of claim 1 or 2 in the preparation of a medicine for treating knee osteoarthritis.

4. Use according to claim 3, characterized in that, The preparation method of the medicine for treating knee osteoarthritis comprises the following steps: S1. Weighing the raw materials of the medicine for treating knee osteoarthritis according to the parts by weight; S2. Soaking Eucommia ulmoides leaf, Astragalus root, Gastrodia, Chaenomeles fruit, Pueraria, Coix seed, and Cornus in 70% ethanol solution, and then extracting and filtering under ultrasonic assistance, and separately retaining the residues; S3. Distilling volatile oil from Curcuma and Peach kernel, and then using β-cyclodextrin to include the volatile oil, and separately retaining the residues; S4. Water-extracting and concentrating the residues of S2 and S3 together with Dendrobium; S5. Mixing calcium gluconate into the extracts of S2, S3, and S4; S6. Freeze-drying the mixture of S5 for standby, and dissolving it in distilled water before use.

5. Use according to claim 3, characterized in that, The medicine is a medicine for repairing knee osteoarthritis cartilage cells and subchondral bone, reducing knee osteoarthritis cartilage oxidative stress, and improving knee osteoarthritis quadriceps.

6. Use according to claim 5, characterized in that, The medicine is a medicine for activating TGF-β1 / Smad3, Nrf2-Keap1, and PI3K / AKT / FOXO1 pathways to treat knee osteoarthritis.

7. A pharmaceutical composition, characterized by, The traditional Chinese medicine composition as claimed in claim 1 or 2 and a pharmaceutically acceptable carrier.

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