CN118178591A - A Chinese medicine composition for preventing and treating knee osteoarthritis and its application - Google Patents

Abstract

The invention 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 a three-strength prescription, which is composed of the following raw materials in parts by weight: 90-150 parts of eucommia ulmoides leaves, 60-120 parts of papaya, 90-150 parts of astragalus mongholicus, 35-65 parts of gastrodia elata, 35-65 parts of dendrobe, 90-150 parts of radix puerariae, 35-65 parts of turmeric, 35-65 parts of peach kernels, 60-120 parts of coix seeds, 75-125 parts of pulp of dogwood fruit and 9-15 parts of calcium gluconate; the three-strength composition can improve cartilage pathological states and subchondral bone microstructure parameters of knee osteoarthritis, improve antioxidant stress capability, improve quadriceps atrophy caused by the knee osteoarthritis, and has good pharmacological effects, and the action mechanism of the composition is related to activating TGF-beta 1/Smad3, nrf2-Keap1 and PI3K/AKT/FOXO1 channels.

Description

A Chinese medicine composition for preventing and treating knee osteoarthritis and its application

Technical Field

The invention belongs to the technical field of knee osteoarthritis treatment, and in particular relates to a traditional Chinese medicine composition for preventing and treating knee osteoarthritis and application thereof.

Background technique

Knee osteoarthritis (KOA) is a degenerative disease characterized by knee pain, swelling, deformity and functional disability, which seriously affects the quality of life of patients. Currently, there is no specific treatment for KOA. Western medicine has many conservative treatment methods for it, most of which are non-specific treatments, mainly based on analgesics and non-steroidal anti-inflammatory drugs, mainly to relieve symptoms and improve the quality of life. However, long-term use can easily cause gastrointestinal reactions and many other adverse reactions, which greatly limits its clinical application.

Traditional Chinese medicine believes that KOA is mostly caused by liver and kidney deficiency and "bone arthritis" caused by wind evil entering the body. Knee osteoarthritis is mostly suffered by middle-aged and elderly people. The pathogenesis is mostly due to insufficient kidney essence, bone marrow reduction, and malnutrition of bone choroids. The disease is located in the bones and is closely related to the kidney, liver, and spleen. Kidney deficiency is particularly critical. For this reason, the present invention has formulated a three-strength prescription with the main effects of strengthening the spleen, kidney, and liver, and strengthening tendons, bones, and muscles. There is no report on the use of the Chinese medicine composition of the present invention to treat knee osteoarthritis.

The incidence of knee osteoarthritis is increasing year by year, and the pathogenesis is complex. Cartilage damage and changes in subchondral bone structure will occur during the progression of the disease. Chondrocytes are wrapped in an extracellular matrix mainly composed of water, type II collagen and proteoglycans. When knee osteoarthritis occurs, chondrocytes undergo hypertrophy, apoptosis and senescence, resulting in the loss of type II collagen and proteoglycans 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, which damage the microenvironment of cartilage tissue. Anterior cruciate ligament transection is a common modeling method for knee osteoarthritis. After surgery, the damaged tissue inside the knee joint produces a large number of inflammatory factors, including inflammatory factors IL-1, IL-1β, IL-6, IL-17 and TNF-α, thereby destroying joint homeostasis and increasing the risk of knee osteoarthritis. The TGF-β1/Smad3 pathway has been shown to play an important role in maintaining joint homeostasis and promoting the growth and development of chondrocytes. After activation, TGF-β1 promotes the activation and phosphorylation of Smad3, which in turn affects downstream factors, such as promoting the formation of type II collagen and proteoglycan in chondrocytes, inhibiting the expression of type II collagen and MMP13, a marker of cartilage 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.

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 metabolism. However, due to the existence of a complete antioxidant system in the body, the dynamic balance of the two major systems of oxidation and antioxidant can be maintained, so that the antioxidant effect of scavenging free radicals is enhanced, the body returns to normal, and the damage of free radicals is reduced. However, under the stimulation of some adverse external factors, the body's highly active molecules (such as ROS and RNS) are overproduced, causing the oxidation effect to be enhanced and far exceeding the scavenging capacity of the antioxidant effect, showing an imbalance between the oxidation system and the antioxidant system. As a result, ROS and RNS cannot be cleared in time and effectively, and then accumulate in large quantities, resulting in an oxidative stress state, causing various tissue and cell damage. A large number of studies have shown that the level of oxygen free radicals is closely related to the pathogenesis of knee osteoarthritis. ROS mainly includes MDA, nitric oxide (NO), etc. The mechanism of clearing ROS in cells and protecting cells from oxidative stress damage is called the antioxidant system. The antioxidant system includes: superoxide dismutase (SOD), 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. Among them, Nrf2, as an important regulatory factor of the body, can enhance the resistance of cells to oxidative stress and inflammatory response. It belongs to the human body's antioxidant stress defense system. When in an oxidative stress or inflammatory environment, the activated Nrf2 immediately separates from Keap1 and transfers to the nucleus for high expression. Through its highly conserved basic leucine zipper structure, it forms a heterodimer with the small molecule myofibroblast protein and recognizes the Nrf2 antioxidant response element complex ARE, which activates the expression of a series of protective genes including heme oxygenase-1 (HO-1) downstream. In the KOA model, the loss of Nrf2 will aggravate the cartilage damage in osteoarthritis and promote the progression of KOA, while the corresponding activation of the Nrf2/HO-1 pathway can resist the pathological process of KOA. This shows that the activity of Nrf2 is crucial for maintaining cartilage homeostasis. Taking the Nrf2 signaling pathway as a breakthrough, exploring more effective treatment options for the prevention and treatment of KOA is a hot topic in current antioxidant research.

At the same time, the PI3K/Akt signaling pathway is an important signaling pathway that regulates multiple cell functions such as cell proliferation and differentiation, and it regulates muscle growth activities. Studies have found that reduced activity of the PI3K/Akt signaling pathway can lead to muscle atrophy. In muscles, phosphorylation of PI3K/Akt signals can promote net protein accumulation by promoting the phosphorylation of FoxO. Non-phosphorylated FoxO is located in the nucleus and aggravates muscle atrophy by promoting the expression of muscle atrophy genes (Atrogin-1 and MURF1, etc.); when FoxO is phosphorylated by AKT, it will move to the cytoplasm and lose 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, that is, 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 protein). It is usually upregulated in the case of muscle atrophy, such as during inactivity, hunger or disease. Atrogin-1 and MURF1 are both ubiquitin ligases that work by marking specific proteins in muscle cells for attachment of ubiquitin and eventual degradation. This degradation process is an important part of maintaining muscle protein homeostasis, but in muscle atrophy, being overactive can lead to muscle loss. Therefore, understanding and regulating the activity of Atrogin-1 and MURF1 is important for preventing or treating muscle wasting conditions.

The above study found that KOA is closely related to the structural lesions of cartilage and subchondral bone, among which TGF-β/Smad and Keap1-Nrf2 signaling pathways are involved in the growth and development of chondrocytes and the oxidative stress process, 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 quality. 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 cartilage collagen, but also lead to changes in the microstructure of subchondral bone and muscle atrophy. The present invention observes the effects of Sanqiang Fang on the articular cartilage and bone microstructure and quadriceps of KOA rats by designing animal experiments, and explores its therapeutic mechanism. Experimental verification shows that the Sanqiang Fang in the present invention is composed of Chinese medicines with the same origin of medicine and food, with small side effects, and can play a role in treating knee osteoarthritis through multiple pathways.

Summary of the invention

The object of the present invention is to provide a Chinese medicine composition for preventing and treating knee osteoarthritis and its application, so as to solve the problem that there is no specific treatment for knee osteoarthritis in the prior art mentioned in the above background technology.

To achieve the above object, the present invention adopts the following technical solutions:

A first aspect of the present invention provides a traditional Chinese medicine composition for preventing and treating knee osteoarthritis, wherein the traditional Chinese medicine composition is a Sanqiang prescription, which is composed of the following raw materials in parts by weight: 90-150 parts of eucommia leaves, 60-120 parts of papaya, 90-150 parts of astragalus, 35-65 parts of gastrodia, 35-65 parts of dendrobium, 90-150 parts of kudzu root, 35-65 parts of turmeric, 35-65 parts of peach kernel, 60-120 parts of coix seed, 75-125 parts of cornus fruit, and 9-15 parts of calcium gluconate.

Preferably, the weight portions of each raw material are as follows: 120 parts of Eucommia ulmoides leaves, 90 parts of papaya, 120 parts of astragalus, 50 parts of Gastrodia elata, 50 parts of Dendrobium officinale, 120 parts of Pueraria root, 50 parts of Curcuma longa, 50 parts of peach kernel, 90 parts of Coix seeds, 100 parts of Cornus officinalis, and 12 parts of calcium gluconate.

The second aspect of the present invention provides use of the above-mentioned Chinese medicine composition in the preparation of a medicament for treating knee osteoarthritis.

Preferably, a medicament for treating knee osteoarthritis is prepared, and the preparation method thereof comprises the following steps:

S1. Weigh the raw materials of the drug for treating knee osteoarthritis by weight;

S2, soaking Eucommia leaf, Astragalus, Gastrodia elata, Papaya, Pueraria root, Coix seeds, and Cornus officinalis in 70% ethanol solution, using ultrasonic-assisted extraction and filtration and concentration, and retaining the residue separately;

S3, extracting volatile oil from turmeric and peach kernel by distillation, encapsulating them with β-cyclodextrin, and retaining the residue separately;

S4, combining the Chinese medicinal residues of S1 and S2 with Dendrobium officinale for water extraction and concentration;

S5, combining the extracts from S1, S2 and S3, adding calcium gluconate and mixing;

S6. Freeze-dry the mixture in S5 for later use, and dissolve it in distilled water before use.

Preferably, the drug is a drug for repairing chondrocytes and subchondral bone in knee osteoarthritis, reducing oxidative stress of cartilage in knee osteoarthritis, and improving quadriceps femoris in knee osteoarthritis.

Preferably, the drug is a drug for treating knee osteoarthritis by activating TGF-β1/Smad3, Nrf2-Keap1, PI3K/AKT/FOXO1 pathways.

The third aspect of the present invention provides a pharmaceutical composition as described above, comprising the above-mentioned traditional Chinese medicine composition and a pharmaceutically acceptable carrier.

The fourth aspect of the present invention provides use of the above-mentioned Chinese medicine composition in treating knee osteoarthritis.

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

The Sanqiang Recipe in the present invention has the functions of strengthening the spleen and replenishing qi, benefiting the kidney and softening the liver, promoting blood circulation and unblocking collaterals, strengthening tendons, bones and muscles, and can treat knee osteoarthritis through multiple targets and multiple pathways. Studies have shown that the Sanqiang Recipe can improve the cartilage pathological state and subchondral bone microstructure parameters of knee osteoarthritis, improve the ability to resist oxidative stress, and improve quadriceps atrophy caused by knee osteoarthritis. It has good pharmacological effects, and its mechanism of action is related to the activation of TGF-β1/Smad3, Nrf2-Keap1, PI3K/AKT/FOXO1 pathways.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph showing the weight change trend of rats in each group in Example 1 of the present invention ( n = 8);

FIG. 2 is a comparison of body weight before and after administration in Example 1 of the present invention ( n = 8);

FIG. 3 is a graph showing the results of ALT, AST, BUN, and CRE in each group of rats in Example 1 of the present invention ( n = 8);

FIG4 is a micrograph of the pathological morphology of liver tissues of rats in each group in Example 1 of the present invention (HE staining, 200×);

FIG5 is a micrograph of the pathological morphology of renal tissues of rats in each group in Example 1 of the present invention (HE staining, 200×);

FIG6 is a graph showing the slope angle and thermal pain threshold of rats in each group in Example 1 of the present invention;

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

FIG8 is a protein immunoblot of TGF-β1, Smad3, p-Smad3, Col II, Col X, and MMP13 in rats of each group in Example 1 of the present invention;

9 is a graph showing the protein expression levels of TGF-β1, p-Smad3/Smad3, Col II, Col X, and MMP13 in rats of each group in Example 1 of the present invention;

FIG10 is a graph showing the mRNA expression levels of Col II, Col X, and MMP13 in rats of each group in Example 1 of the present invention;

FIG11 is a graph showing the results of SOD, GSH-Px, MDA and NO in each group of rats in Example 1 of the present invention;

FIG. 12 is a graph showing the results of Nrf2, Keap1, HO-1, and NQO1 mRNA in each group of rats in Example 1 of the present invention ( n = 3);

FIG13 is a Micro-CT image of subchondral bone of rats in each group in Example 1 of the present invention;

FIG. 14 is a graph showing the Micro-CT scanning parameters of the subchondral bone of each group of rats in Example 1 of the present invention ( n = 3);

15 is a graph showing the quadriceps weight and quadriceps weight/body weight ratio of rats in each group in Example 1 of the present invention;

FIG16 is a comparison diagram of the range of motion of the knee joints of rats in each group in Example 1 of the present invention;

FIG17 is a protein blot of p-PI3K, PI3K, p-AKT, AKT, p-FOXO1, and FOXO1 in each group of rats in Example 1 of the present invention;

FIG18 is a graph showing the expression levels of PI3K/AKT/FOXO1 pathway proteins in quadriceps tissue of rats in each group in Example 1 of the present invention;

FIG19 is a Western blot of Atrogin-1 and Murf-1 in each group of rats in Example 1 of the present invention;

FIG. 20 is a graph showing the protein expression levels of Atrogin-1 and MuRF1 mRNA in quadriceps muscles of rats in each group in Example 1 of the present invention.

Detailed ways

The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

Embodiment 1:

A traditional Chinese medicine composition for preventing and treating knee osteoarthritis, the traditional Chinese medicine composition is a Sanqiang prescription, which is composed of eucommia leaves, papaya, astragalus, gastrodia, dendrobium, kudzu root, turmeric, peach kernel, coix seed, cornus fruit, and calcium gluconate.

In the Sanqiang prescription of the present invention, the Eucommia leaf in the prescription nourishes the liver and kidney, strengthens the tendons and bones; the Astragalus root nourishes qi and raises yang, promotes the production of body fluid and nourishes blood, and the two are matched, nourishing qi and nourishing the kidney and softening the liver, which are the main medicines. Pueraria root raises yang and relieves muscles, Cornus fruit nourishes the liver and kidney, astringes essence and consolidates the body, papaya relaxes tendons and activates collaterals, Dendrobium nourishes kidney yin, clears away deficiency heat, and helps Eucommia leaf and Astragalus root nourish the liver and kidney, unblock tendons and collaterals, which are all ministerial medicines. Coix seed invigorates the spleen, relaxes tendons and veins and relieves spasm, Gastrodia elata dispels external wind, unblocks meridians, and stops arthralgia and pain, Turmeric promotes qi and blood circulation, unblocks menstruation and relieves pain, Peach kernel promotes blood circulation and removes blood stasis, and calcium gluconate supplements calcium, which are all adjuvants. The whole prescription plays the functions of strengthening the spleen and replenishing qi, nourishing the kidney and softening the liver, activating blood circulation and unblocking collaterals, and strengthening tendons, bones and muscles. Therefore, it is named Sanqiang prescription.

The present invention uses animal experiments to observe the effects of Sanqiang Fang on articular cartilage and bone microstructure and quadriceps femoris in KOA rats, and preliminarily explores its therapeutic mechanism. The experiment specifically includes the following steps:

Step 1: Prepare materials.

Animal preparation: 56 SPF male SD rats, 6 weeks old, weighing 180±20g, were purchased from Beijing Huafukang Biotechnology Co., Ltd. with an experimental animal production license and kept in the clean laboratory of the Animal Experiment Center of North China University of Technology at a constant temperature of 25°C with free access to water and food. This experiment was reviewed by the Experimental Animal Ethics Committee of North China University of Technology (ethics number).

Drug preparation: Sanqiang prescription is composed of 12g Eucommia leaf, 9g papaya, 12g Astragalus, 5g Gastrodia, 5g Dendrobium, 12g Pueraria root, 5g Curcuma, 5g Peach kernel, 9g Coix seed, 10g Cornus fruit, 1.2g calcium gluconate (this is the medium dose, the dose of the prevention group is equal to the medium dose, the low and high doses are 1/2 and 2 times the medium dose respectively), the medicinal materials were purchased from Beijing Tongrentang Pharmacy Tangshan Branch, and identified as authentic by Professor Li Jianan of the School of Traditional Chinese Medicine of North China University of Technology. Celecoxib (National Medicine Standard No. J20140072) was purchased from Pfizer Pharmaceuticals Co., Ltd.

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

Instrument preparation: DB026 intelligent hot plate instrument (Beijing Zhishu Duobao Biotechnology Co., Ltd.), SpectraMaxM3 multi-function microplate reader (Molecular Devices, USA), ViiA-7 real-time fluorescence quantitative PCR instrument (ABI, USA), DYCZ-24DN vertical electrophoresis tank (Beijing Liuyi Instrument Factory), FW606 semi-dry transfer instrument (Nanjing Aisiyi), DS-H200 horizontal shaker (Servicebio), Tissvelyser-24L automatic grinder (Shanghai Jingxin), VNC-102 VENUS Micro CT (Pingsheng Technology), etc.

Step 2: Establish the experimental group and collect samples.

S2.1, model establishment and grouping;

The rat model of knee osteoarthritis was established by transecting the anterior cruciate ligament of the right posterior knee joint of rats, and the "drawer test" was positive and the joint instability was maintained for 6 weeks. After the model was successfully established, the rats were divided into the model group, celecoxib group, Sanqiang prescription low-, medium-, and high-dose groups, and Sanqiang prevention group according to the random number table method, with 8 rats in each group. Another 8 rats were selected as the sham operation group, in which only the skin was cut without cutting the anterior cruciate ligament.

S2.2, drug preparation and administration;

Eucommia ulmoides leaf, astragalus, gastrodia, papaya, kudzu root, coix seed, and cornus fruit in the Sanqiang prescription were soaked in 70% ethanol solution, extracted by ultrasound, filtered and concentrated. Volatile oils were extracted by distillation of turmeric and peach kernel, and then encapsulated with β-cyclodextrin. The residues of the above-mentioned herbs were combined with dendrobium, extracted and concentrated by water. The above extracts were combined, calcium gluconate was added and mixed. Finally, freeze-dried for use, and dissolved in distilled water before use.

The low, medium and high dose groups of Sanqiang Fang were given 3.8, 7.6 and 15.2 g/kg of the drug solution by gavage, the celecoxib group was given 18 mg/kg of the drug solution by gavage, and the sham operation group and the model group were given an equal volume of normal saline by gavage for 8 consecutive weeks. The Sanqiang prevention group was given 7.6 g/kg of the drug solution by gavage, starting from the anterior cruciate ligament transection surgery, and continued for 14 weeks.

S2.3, material collection;

After the administration, the rats were anesthetized by intraperitoneal injection of sodium pentobarbital, and blood was collected from the abdominal aorta. The supernatant was centrifuged at 3000 r/min and stored at -80°C. The muscle tissue around the right hind knee joint of some rats was stripped to obtain a complete right knee joint, which was fixed in paraformaldehyde. The knee cartilage tissue of some rats was placed in a cryopreservation tube and stored at -80°C. The quadriceps femoris was cut at the distal end of the patellar superior margin, and the quadriceps femoris was completely separated along the intermuscular gap and weighed. Part of the quadriceps femoris tissue was fixed in paraformaldehyde; the remaining quadriceps femoris tissue was placed in a cryopreservation tube and stored in a -80°C refrigerator.

Step 3: Experimental testing.

S3.1. Rat behavior and knee passive range of motion test;

Before sampling, rat behavior and passive range of motion of the knee joint were tested. The rats were placed in the intelligent hot plate instrument, the start time of licking the feet 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°, rising or falling 5° each time. The rats stayed on the same slope for more than 10 seconds 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 on the affected side of each group of rats was measured with a medical goniometer, and the maximum flexion angle minus the maximum extension angle was the passive range of motion.

S3.2, serum biochemical index detection;

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 rat serum were detected.

S3.3, Western Blot was used to detect the expression of TGF-β1, Smad3, p-Smad3, Col II, Col X, and MMP13 proteins in cartilage and the expression of PI3K/AKT/FOXO1 signaling pathway and Atrogin-1 and MuRF1 proteins in quadriceps femoris;

Mix the frozen tissue with tissue lysis buffer, protease inhibitors, and phosphatase inhibitors in the corresponding proportions, grind and extract the protein, and determine the protein concentration using the BCA method. Mix the protein sample with the corresponding proportion of loading buffer and double distilled water and place it in a metal bath. After the electrophoresis gel is prepared, perform electrophoresis and transfer the membrane using the semi-dry transfer method. After blocking with rapid blocking solution, primary antibodies were 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) and incubated overnight at 4°C. The membrane was washed with TBST 3 times for 10 min. Diluted goat anti-rabbit secondary antibody (1:10000) or goat anti-mouse secondary antibody (1:10000) was added and incubated at room temperature for 2 h. TBST was used to wash the membrane. The membrane was washed 3 times for 10 min, developed with ECL solution, scanned and archived with Image Lab, and then processed and analyzed with ImageJ for the grayscale value of the target band.

S3.4, Real-time fluorescence quantitative PCR detection of cartilage Col II, Col X, MMP13, Nrf2, Keap1, HO-1, and NQO1 mRNA expressions;

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

Table 1 Primer sequences

S3.5, Micro-CT detection of subchondral bone microstructure;

The knee joint specimen was placed vertically in the sample sleeve and fixed with a paper towel to prevent the specimen from shaking. The sleeve was then fixed in Micro-CT to scan the specimen and analyze bone density (BMD), bone volume fraction (BV/TV), trabecular number (Tb.N), trabecular thickness (Tb.Th), trabecular spacing (Tb.Sp), and trabecular structural model parameters (SMI).

Step 4: Count and analyze the experimental results.

The experimental results were statistically analyzed using SPSS 23.0. The measurement data were in accordance with the normal distribution and were expressed as mean ± standard deviation ( ) indicates that the means of more than three groups were compared by one-way analysis of variance. If the variances were equal, the LSD test was used for comparison between the two groups. If the variances were unequal, the Tamhane's test was used for comparison between the two groups. P<0.05 was considered statistically significant.

The statistical experimental results are as follows:

S4.1. Effects of Sanqiang Formula on body weight of KOA rats;

During the administration period, the body weight of the rats increased steadily. Before and after administration, there was no difference in the body weight of the rats among the groups. The results are shown in Figure 1 and Figure 2. In Figure 2, there was no difference between the NS groups.

S4.2. Effects of Sanqiang Decoction on liver and kidney function in KOA rats:

Effects of Sanqiang Fang on serum liver and kidney function indexes of KOA rats: After administration, there was no difference in ALT, AST, BUN, and CRE among the rats in each group. The results are shown in Figure 3. There was no difference between the NS groups in Figure 3.

Effect of Sanqiang Fang on the liver and kidney morphology of KOA rats: HE staining showed that the hepatocyte structure of rats in each group was intact, the hepatic cords were neatly arranged, there was no significant difference in the size of hepatocytes, and the nucleus was round and located in the center of the hepatocyte. The renal tissue morphology of rats in each group was normal, the glomerulus and renal tubule structure was normal, and no hyperplasia was observed in the renal interstitium and mesangium. The pathological morphological changes of the liver and kidney tissues of rats in each group are shown in Figures 4 and 5.

S4.3, Effects of Sanqiangfang on the behavior of KOA rats;

After the 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 compared with the model group, the slope angle and thermal pain threshold of each drug administration group were significantly higher (P<0.01). The results are shown in Figure 6.

S4.4, Effects of Sanqiang Decoction on serum inflammatory factors in KOA rats;

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 drug-treated group were significantly decreased (P<0.01). The results are shown in Figure 7.

S4.5, Protective effect of Sanqiang prescription on chondrocyte homeostasis in KOA rats;

Effects of Sanqiang Fang on the expression of TGF-β1, Smad3, p-Smad3, Col II, Col X, and MMP13 proteins in cartilage of KOA rats:

Compared with the sham 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 medium and high dose groups of Sanqiang Fang were increased (P＜0.01, P＜0.05). Except that the MMP13 in the low dose group of Sanqiang Fang had a downward trend without statistical significance, the levels of Col X and MMP13 in the celecoxib group and Sanqiang Fang group were decreased (P＜0.01, P＜0.05). The protein immunoblot of each group is shown in Figure 8, and the protein expression level is shown in Figure 9; in Figure 8, A is the sham group, B is the model group, C is the celecoxib group, and D, E, and F are the low, medium, and high dose groups of Sanqiang Fang.

Effects of Sanqiang Fang on the mRNA expression of Col II, Col X and MMP13 in cartilage of KOA rats:

Compared with the sham operation group, the mRNA level of Col II in the model group decreased significantly (P < 0.01), and the mRNA levels of Col X and MMP13 increased significantly (P < 0.01). Compared with the model group, except for the low-dose group of Sanqiang Fang, the mRNA levels of Col II and Col X showed an improvement trend but no statistical significance, the mRNA levels of Col II, Col X, and MMP13 in other drug-treated groups were improved (P < 0.01, P < 0.05). The mRNA levels of each group are shown in Figure 10.

S4.6, Effects of Sanqiang Recipe on Serum Oxidative Stress Factors in KOA Rats;

Effects of Sanqiang Fang on serum oxidative stress factors in KOA rats:

Compared with the sham operation group, the levels of SOD and GSH-Px in the model group decreased significantly (P<0.01), and the levels of MDA and NO increased significantly (P<0.01); compared with the model group, the levels of SOD and GSH-Px in the celecoxib group and the high-dose Sanqiang group increased significantly (P<0.01), and the levels of MDA and NO decreased significantly (P<0.01); the level of SOD in the Sanqiang prevention group increased compared with the model group (P<0.05), the level of GSH-Px increased significantly (P<0.01), and the levels of MDA and NO decreased significantly (P<0.01). The levels of SOD, GSH-Px, MDA, and NO in the low-dose Sanqiang group were slightly improved compared with the model group, but there was no statistical significance. The levels of SOD and NO in the medium-dose Sanqiang group were slightly improved compared with the model group, but there was no statistical significance, and the levels of GSH-Px and MDA were improved compared with the model group (P<0.05). See Figure 11.

Effects of Sanqiang Fang on cartilage Nrf2, Keap1, HO-1, and NQO1 mRNA in KOA rats:

Compared with the sham operation group, the levels of Nrf2, HO-1, and NQO1 in the model group decreased significantly (P<0.01), and the level of Keap1 increased significantly (P<0.01). After treatment, the levels of Nrf2, HO-1, and NQO1 in the celecoxib group, the Sanqiang high-dose group, and the Sanqiang prevention group increased significantly (P<0.01), and the level of Keap1 decreased significantly (P<0.01). The levels of Nrf2 and NQO1 in the Sanqiang low- and medium-dose groups showed an upward trend compared with the model group, but there was no statistical significance. The HO-1 level in the Sanqiang medium-dose group increased significantly compared with the model group (P<0.01), and the Keap1 level decreased significantly compared with the model group (P<0.01). The Keap1 level in the Sanqiang low-dose group increased compared with the model group (P<0.05). See Figure 12.

S4.7, Effects of Sanqiang Recipe on Serum Oxidative Stress Factors in KOA Rats;

Compared with the sham operation group, the BMD, BV/TV, Tb.N, and Tb.Th of the model group decreased (P<0.01), and 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 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 SMI decreased (P<0.05). Micro-CT images showed that the number of trabeculae in the model group was reduced and the distribution was disordered compared with the sham operation group; after treatment, the number of trabeculae increased, the arrangement was tight, and the subchondral bone microstructure was improved. The results are shown in Figures 13 and 14.

S4.8, Effects of Sanqiangfang on quadriceps femoris in KOA rats;

Effects of Sanqiangfang on the weight of quadriceps femoris in KOA rats:

After the administration cycle, the quadriceps muscle weight of the rats in the model group was less than that in the sham operation group, and the difference was statistically significant (P<0.01). The quadriceps muscle weight of the rats in the Sanqiang Fang prevention group and the celecoxib group was significantly increased compared with the model group, which was statistically significant (P<0.01). The low, medium and high dose groups of Sanqiang Fang showed an increasing trend compared with the model group, while there was no significant difference in the body weight of the rats among the groups. See Figure 15.

Effects of Sanqiang Fang on joint mobility of KOA rats:

After the medication cycle was completed, the activity of the model group was significantly lower than that of the sham operation group, and the difference was statistically significant (P<0.01). The activity of the Sanqiang Fang prevention group was higher than that of the model group, and the difference was statistically significant (P<0.05). The activity of the celecoxib group and the low, medium and high dose groups of Sanqiang Fang were all significantly higher than that of the model group, and the difference was statistically significant (P<0.01). See Figure 16.

Effects of Sanqiang Fang on PI3K/AKT/FOXO1 signaling pathway proteins in quadriceps tissue of KOA rats:

Referring to Figures 17 and 18, as shown in Figure 18, compared with the sham operation 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 of rats in the model group were reduced; compared with the model group, the ratios of p-PI3K/PI3K (P<0.001), p-AKT/AKT (P<0.0 01) and p-FOXO1/FOXO1 (P<0.0001) ratios were increased in the Sanqiang low-dose group, p-AKT/AKT (P<0.05) and p-FOXO1/FOXO1 (P<0.0001) ratios were increased in the Sanqiang medium-dose group, and p-PI3K/PI3K (P<0.01), p-AKT/AKT (P<0.001) and p-FOXO1/FOXO1 (P<0.001) ratios were increased in the Sanqiang medium-dose group. In Figure 17, A is the sham operation group, B is the model group, C is the celecoxib group, and D, E, and F are the Sanqiang low-, medium-, and high-dose groups.

Effects of Sanqiangfang on the expression of Atrogin-1 and MuRF1 proteins in quadriceps of KOA rats:

See Figures 19 and 20. As shown in Figure 20, compared with the sham operation group, the expression of Atrogin-1 (P<0.001) and MURF1 (P<0.001) proteins in the quadriceps tissue of rats in the model group 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 Sanqiang Fang were significantly decreased. In Figure 19, A is the sham operation group, B is the model group, C is the celecoxib group, and D, E, and F are the low, medium, and high dose groups of Sanqiang Fang.

Step 5: Verify through experimental results that the Sanqiang formula plays a role in treating KOA through multiple pathways.

S5.1. Sanqiang Formula protects chondrocytes and subchondral bone in knee osteoarthritis;

In the three powerful prescriptions, astragaloside Ⅳ, the active ingredient of astragalus, can increase the expression of TGF-β1 in chondrocytes, reduce the expression of IL-1, promote the expression of type II collagen and proteoglycan in the extracellular matrix of chondrocytes, improve the cartilage environment, and inhibit cartilage degradation; calycosin glucoside can enhance the proliferation of chondrocytes and reduce cell 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 bone loss in subchondral bone, and protect subchondral bone.

The results of this experiment showed that Sanqiang Fang had no damage to the liver and kidney function of rats and had high safety. It can improve the climbing and thermal pain threshold indexes of rats with knee osteoarthritis and reduce the levels of inflammatory factors IL-1, IL-1β, IL-6, IL-17, and TNF-α. Western Blot detection showed that the expression of TGF-β1 and p-Smad3 in the model group rats was reduced, type II collagen was lost, and the expression of type X collagen and MMP13 was enhanced. Sanqiang Fang can activate the TGF-β1/Smad3 pathway, promote the production of type II collagen, downregulate the expression of type X collagen and MMP13, maintain chondrocyte homeostasis, and inhibit chondrocyte hypertrophy. Real-time fluorescence quantitative PCR simultaneously confirmed the expression of type II collagen, type X collagen, and MMP13 in rat cartilage. Micro-CT images showed that the joint space of the rats in the model group was narrowed and the subchondral bone was disordered. The bone microstructure parameters BMD and BV/TV, Tb.N, and Tb.Th decreased, and Tb.Sp and SMI increased. Sanqiang prescription had a repairing effect on the abnormal subchondral bone structure caused by joint injury.

S5.2, Effects of Sanqiang Formula on Oxidative Stress in Cartilage of Knee Osteoarthritis;

Among the three powerful prescriptions, astragaloside IV can increase the activity of antioxidant enzymes, reduce ROS and malondialdehyde in cells in various diseases, increase GSH-Px and SOD, play an antioxidant protective role in injuries, improve the bone environment, and promote the balance between oxidation and anti-oxidation. The phenolic acid components such as chlorogenic acid and flavonoid components such as rutin in Eucommia ulmoides leaves are the material basis for their antioxidant effects. They 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.

The results of this experiment showed that compared with the control group, the levels of SOD and GSH-Px in the KOA model group decreased significantly (P<0.01), and the levels of MDA and NO increased significantly (P<0.01); while in the other experimental groups under the action of Sanqiang Fang, SOD increased significantly, while MDA and NO decreased significantly; this shows that Sanqiang Fang has a certain effect on the body's anti-oxidative stress. Compared with the KOA model, the experimental group using Sanqiang Fang had significantly upregulated mRNA and protein expressions of Nrf2 and Keap1, HO-1 and NQO1, while the mRNA and protein expressions of Keap1 were significantly downregulated. The experiment showed that KOA inhibited the regulation of the Nrf2-Keap1/ARE pathway, while after the action of Sanqiang Fang, it could activate the Nrf2-Keap1 pathway, increase the expression of HO-1 and NQO1, and restore the body's antioxidant capacity against oxidative stress.

S5.3, Effects of Sanqiang Fang on quadriceps femoris in knee osteoarthritis;

Among the three powerful prescriptions, astragaloside, the active ingredient of Astragalus, can regulate protein synthesis and degradation through the PI3K/AKT signaling pathway, reduce the level of skeletal muscle oxidative stress, and improve skeletal muscle atrophy; calycosin has anti-autophagy, anti-apoptosis and anti-inflammatory effects, can upregulate the expression of B-cell lymphoma factor 2-like protein (Bcl-2), inhibit the expression of TNF-α, weaken the expression of Atrogin-1 and MuRF1, block ubiquitin-proteasome-mediated myofiber dissolution, and delay skeletal muscle atrophy. Chlorogenic acid, the active ingredient of Eucommia ulmoides leaves, can upregulate the PI3K-Akt signaling pathway, promote the expression of AKT phosphorylation FoxO and Bcl-2, inhibit cell apoptosis, FoxO loses the regulation of atrophy genes, and slows down the muscle atrophy process.

The results of this experiment showed that Sanqiang Fang can improve the passive range of motion and relative muscle mass of rats with knee osteoarthritis, reduce the connective tissue between muscle cell bundles, and improve muscle atrophy in rats. The protein expressions of p-PI3K/PI3K, p-AKT/AKT, and p-FOXO1/FOXO1 in the quadriceps of the model group were reduced, and the protein and qPCR expressions of Atrogin-1 and MURF1 were increased, indicating that the inhibition of PI3K, AKT, and FOXO1 pathways in the quadriceps of rats in the model group promoted the expression of Atrogin-1 and MURF1, aggravated the degree of fibrosis in the quadriceps, and promoted quadriceps atrophy. The expression of p-PI3K/PI3K, p-AKT/AKT, p-FOXO1/FOXO1 proteins and qPCR in the quadriceps muscle of the Sanqiangfang group was increased, indicating that Sanqiangfang can activate the PI3K, AKT, and FOXO1 pathways, inhibit the translocation of FOXO1 from the cytoplasm to the nucleus, and then inhibit the expression of muscle atrophy genes Atrogin-1 and MURF1, reduce the degree of quadriceps fibrosis, and improve quadriceps atrophy.

Experimental results verify:

The Sanqiang Recipe in the present invention has the functions of strengthening the spleen and replenishing qi, benefiting the kidney and softening the liver, promoting blood circulation and unblocking collaterals, strengthening tendons, bones and muscles, wherein the Eucommia leaf contains phenolic acid components such as chlorogenic acid and flavonoid components such as rutin, the Astragalus contains the effective ingredients astragaloside IV and calycosin isoflavone glucoside, and the papaya contains ursolic acid and oleanolic acid and other components, which cooperate with each other and can treat knee osteoarthritis through multiple targets and multiple pathways. Studies have shown that the Sanqiang Recipe can improve the pathological state of cartilage and subchondral bone microstructure parameters of rats with knee osteoarthritis, improve the anti-oxidative stress ability of rats, and improve quadriceps atrophy caused by knee osteoarthritis in rats, and has good pharmacological effects. Its mechanism of action is related to the activation of TGF-β1/Smad3, Nrf2-Keap1, PI3K/AKT/FOXO1 pathways.

The above description is only used to help understand the method of the present invention and its core essence, but the protection scope of the present invention is not limited thereto. For those skilled in the art in the art, equivalent replacement or change according to the technical solution and inventive concept of the present invention within the technical scope disclosed by the present invention should be included in the protection scope of the present invention. In summary, the content of this specification should not be understood as limiting the present invention.

Claims (8)

1. The traditional Chinese medicine composition for preventing and treating knee osteoarthritis is characterized by being a three-strength prescription, wherein the three-strength prescription is composed of the following raw materials in parts by weight: 90-150 parts of eucommia ulmoides leaves, 60-120 parts of papaya, 90-150 parts of astragalus membranaceus, 35-65 parts of gastrodia elata, 35-65 parts of dendrobium nobile, 90-150 parts of radix puerariae, 35-65 parts of turmeric, 35-65 parts of peach kernels, 60-120 parts of coix seeds, 75-125 parts of pulp of dogwood fruit and 9-15 parts of calcium gluconate.

2. The traditional Chinese medicine composition according to claim 1, wherein the weight parts of the raw materials are as follows: 120 parts of eucommia ulmoides leaf, 90 parts of papaya, 120 parts of astragalus membranaceus, 50 parts of gastrodia elata, 50 parts of dendrobium, 120 parts of radix puerariae, 50 parts of turmeric, 50 parts of peach kernel, 90 parts of coix seed, 100 parts of pulp of dogwood fruit and 12 parts of calcium gluconate.

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

4. The use according to claim 3, wherein the preparation of the medicament for treating knee osteoarthritis comprises the steps of:

S1, weighing raw materials of a medicine for treating knee osteoarthritis according to parts by weight;

S2, soaking eucommia ulmoides leaves, astragalus membranaceus, gastrodia elata, papaya, kudzuvine root, coix seeds and cornus officinalis in 70% ethanol solution, extracting with the aid of ultrasonic waves, filtering, concentrating, and independently retaining dregs;

s3, extracting volatile oil from turmeric and peach kernel by adopting a distillation method, clathrating the volatile oil with beta-cyclodextrin, and independently retaining dregs;

s4, mixing the residues in the steps S1 and S2 with dendrobium, extracting with water and concentrating;

s5, mixing the extracts in the S1, the S2 and the S3, and adding calcium gluconate for uniform mixing;

s6, freeze-drying the uniformly mixed material in the step S5 for standby, and dissolving the uniformly mixed material in distilled water before use.

5. The use according to claim 3, wherein the medicament is a medicament for repairing knee osteoarthritis chondrocytes and subchondral bone, reducing knee osteoarthritis cartilage oxidative stress, improving knee osteoarthritis quadriceps femoris.

6. The use according to claim 5, wherein the medicament is a medicament for activating the TGF- β1/Smad3, nrf2-Keap1, PI3K/AKT/FOXO1 pathway for the treatment of osteoarthritis in the knee.

7. A pharmaceutical composition comprising the traditional Chinese medicine composition according to claim 1 or 2 and a pharmaceutically acceptable carrier.

8. Use of a traditional Chinese medicine composition according to claim 1 or 2 for treating knee osteoarthritis.