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

Abstract

The application discloses a traditional Chinese medicine composition for preventing and / or treating knee osteoarthritis and a preparation method and application thereof, and belongs to the technical field of traditional Chinese medicine. The traditional Chinese medicine composition is prepared from the following components in parts by weight: salt eucommia 10 parts, raw rehmannia 15 parts, astragalus 20 parts, angelica 12 parts, cyathula 12 parts, chuanxiong 9 parts, steamed lycium 12 parts, bran quince 10 parts, bran white peony root 15 parts and fried licorice 5 parts. The traditional Chinese medicine composition can not only delay the progression of knee osteoarthritis, but also reduce the pain of knee osteoarthritis. Through in-vivo and in-vitro experiments, it is verified that the OARSI score of knee osteoarthritis is reduced after the treatment of the kidney marrow Ankang drink, the expression of type II collagen and matrix metalloproteinase-13 is obviously changed, the bone volume fraction of subchondral bone is reduced, the expression of pain sensitization factors secreted by peripheral nerves is improved, and the pain threshold is improved. Therefore, the kidney marrow Ankang drink has a good application prospect in the treatment of knee osteoarthritis.

Description

TECHNICAL FIELD

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

[0002] Osteoarthritis (OA) is an age-related degenerative disease, and joint pain is its key clinical symptom. OA is mainly characterized by cartilage degeneration, abnormal bone remodeling under the cartilage, and osteophyte formation at the joint margin. OA often affects weight-bearing joints of the human body, and knee osteoarthritis (KOA) is one of the most common types of OA. Given the global trend of population aging, the incidence of OA is increasing year by year. The latest epidemiological data shows that there are about 250 million people worldwide suffering from OA, and in China, the proportion of people suffering from KOA reaches 8.1%, with more than 120 million people suffering from the disease. However, the current drugs for treating KOA in clinical practice are mainly for symptomatic treatment of pain, and long-term use is often accompanied by significant side effects.

[0003] Traditional Chinese medicine has unique advantages in the prevention and treatment of KOA, and considers it as "knee numbness". Based on the classical theory of traditional Chinese medicine that "the kidney governs bone marrow", traditional medicine believes that "marrow" is an important grease-like substance in the skeleton, which is homologous to the kidney, and has the effects of promoting sperm, generating blood, and nourishing bone. Contemporary research has found that bone marrow mesenchymal stem cells have high similarity to traditional Chinese medicine "marrow", and as progenitor cells, they can differentiate into cartilage and osteoblasts to promote bone repair. On this basis, the "Zhejiang School of Traditional Chinese Medicine" bone injury established the "marrow system bone disease" theory, pointing out that the fundamental pathogenesis of KOA is "kidney deficiency, marrow atrophy, and bone dry numbness and pain", and then proposed the method of "tonifying the kidney, strengthening the bone, benefiting the marrow, and relieving pain". Therefore, it is crucial to develop an effective drug with the functions of tonifying the kidney, benefiting the marrow, dredging the collaterals, and relieving pain, which can significantly relieve the symptoms of joint swelling, pain, and functional limitation of osteoarthritis in the acute stage, improve the quality of life of patients, and delay the progression of the disease. SUMMARY

[0004] The purpose of the present application is to provide a traditional Chinese medicine composition for preventing and / or treating knee osteoarthritis and a preparation method and application thereof, in order to solve the problems existing in the prior art. Through animal experiments, the present application systematically explores the possible mechanism of kidney marrow anhe drink in the treatment of KOA, aiming to provide new scientific basis for the clinical treatment of KOA.

[0005] To achieve the above-mentioned purpose, the present application provides the following solutions:

[0006] One of the technical solutions of the present application is a traditional Chinese medicine composition for preventing and / or treating knee osteoarthritis, which is prepared from the following components in parts by weight: salt of eucommia ulmoides 6-10 parts, radix rehmanniae 9-15 parts, radix astragali 15-20 parts, angelica 9-12 parts, radix cyathulae 9-12 parts, chuanxiong 6-9 parts, steamed lycium 9-12 parts, bran of citri fructus 6-10 parts, bran of radix paeoniae alba 9-15 parts, and fried licorice 3-5 parts.

[0007] The second technical solution of the present application is a preparation method of the traditional Chinese medicine composition, which comprises the following steps: taking the medicinal materials according to the weight parts, soaking, and decocting by a reflux method, and concentrating the medicinal liquid to obtain the traditional Chinese medicine composition.

[0008] The third technical solution of the present application is an application of the traditional Chinese medicine composition in preparing a medicine for preventing and / or treating knee osteoarthritis.

[0009] The fourth technical solution of the present application is an application of the traditional Chinese medicine composition in preparing a medicine for reducing OARSI score, regulating the expression of type II collagen and matrix metalloproteinase-13.

[0010] The fifth technical solution of the present application is an application of the traditional Chinese medicine composition in preparing a medicine for improving the expression of peripheral nerve secretion pain sensitization factor NGF and increasing the pain threshold.

[0011] The sixth technical solution of the present application is an application of the traditional Chinese medicine composition in preparing a medicine for reducing the marrow cavity gap of the subchondral bone region.

[0012] Based on the above technical solutions, the present application has the following technical effects:

[0013] 1) It can not only delay the progression of knee osteoarthritis, but also reduce the pain of knee osteoarthritis. 2) Through in vivo and in vitro experiments, it is verified that after the treatment of kidney marrow Ankang drink, the OARSI score of knee osteoarthritis is reduced, the expression of type II collagen and matrix metalloproteinase-13 is obviously changed, the bone volume fraction of subchondral bone is decreased, the expression of peripheral nerve secretion pain sensitization factor is improved, the pain threshold is increased, and the safety of kidney marrow Ankang drink in treating KOA is verified. Therefore, kidney marrow Ankang drink has good application prospect in treating knee osteoarthritis. At present, the drugs for treating knee osteoarthritis in clinic are mainly for pain relief, and there is a lack of effective treatment. Therefore, the present application not only can delay the progression of knee osteoarthritis, but also can relieve the peripheral pain sensitization of knee osteoarthritis. BRIEF DESCRIPTION OF DRAWINGS

[0014] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the accompanying drawings needed in the embodiments will be briefly introduced as follows. Obviously, the accompanying drawings in the following description only constitute some embodiments of the present application, and for those skilled in the art, other drawings can also be obtained from these drawings without creative labor.

[0015] Figure 1 Micro-CT three-dimensional reconstruction images of subchondral bone of mice in each group after continuous intervention of the kidney marrow Kangkang drink for 8 weeks; wherein, A is the Micro-CT scanning result, and B is the bone tissue relative volume result image.

[0016] Figure 2 ABH / OG staining images and bone morphometry analysis images of knee joint cartilage of mice in each group after continuous intervention of the kidney marrow Kangkang drink for 8 weeks; wherein, A is the ABH / OG staining scanning result image, and B is the OARSI score result image of each knee joint.

[0017] Figure 3 Col2a1 and MMP-13 immunohistochemical staining images of knee joint cartilage of mice in each group after continuous intervention of the kidney marrow Kangkang drink for 8 weeks; wherein, A is the Col2a1 and MMP-13 staining result image, B is the Col2a1 relative quantitative analysis result image, and C is the MMP-13 relative quantitative analysis result image.

[0018] Figure 4 Pain-related behavior detection images of mice in each group after continuous intervention of the kidney marrow Kangkang drink for 8 weeks. Wherein, A is the mechanical pain threshold result image, B is the hot plate test result image, C is the gait detection, D is the right lower limb support phase time result image of gait detection, E is the right lower limb stride length result image of gait detection, and F is the right lower limb foot contact area result image of gait detection.

[0019] Figure 5 Immunofluorescence staining images of subchondral bone and NGF of mice in each group after continuous intervention of the kidney marrow Kangkang drink for 8 weeks, wherein, A is the immunofluorescence staining image of NGF in subchondral bone and DRG, B is the relative quantitative analysis result image of NGF in DRG, and C is the relative quantitative analysis result image of NGF in subchondral bone.

[0020] Figure 6 H&E staining images of internal organs of mice in each group after continuous intervention of the kidney marrow Kangkang drink for 8 weeks.

[0021] Figure 7 Influence result images of different concentrations of the kidney marrow Kangkang drink containing serum on the survival of chondrocytes. Wherein, A is the 24-hour CCK8 result image, and B is the 48-hour CCK8 result image.

[0022] Figure 8 Figure 4 is a chart of the effect of the Shensuankang drink-containing serum of the present application on the alcian blue staining of chondrocytes.

[0023] Figure 9 Figure 5 is a chart of the results of real-time PCR analysis of mRNA expression of chondrocyte degeneration-related genes by the Shensuankang drink-containing serum of the present application; wherein A is Col2al and B is Mmp13. DETAILED DESCRIPTION

[0024] The various illustrative embodiments of the present application will now be described in detail below. The detailed description is made with reference to the drawings, wherein like reference numerals refer to like elements throughout the several views. The present application is not limited to the embodiments described herein, but can be practiced with modification and alteration within the scope of the present application.

[0025] It should be understood that the terms used herein are merely for describing particular embodiments and are not intended to limit the present application. Also, for the numerical ranges of the present application, it should be understood that every intermediate value or range between the upper and lower limits of the range is also specifically disclosed. Each smaller range between any stated value or stated range, as well as every intermediate value or range between the stated value or range, is also encompassed. The upper and lower limits of these smaller ranges can independently be included or excluded in the range.

[0026] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present application, the preferred methods and materials are described. All publications mentioned herein are incorporated by reference to disclose and describe the methods and / or materials in connection with which the publications are cited. The citation of any reference is not an admission that it is prior art with respect to the present application. All documents mentioned herein are incorporated herein by reference.

[0027] Many modifications and variations of this application can be made in the light of the above teachings without departing from the spirit and scope thereof. Other implementations of this application will be apparent to those skilled in the art from consideration of the specification and practice of the application disclosed herein. The examples and embodiments described herein are exemplary and are not intended to be limiting.

[0028] As used herein, the terms "comprise", "comprising", "include", "including", "have", "having" and the like are open-ended terms that are intended to mean "including but not limited to".

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

[0030] The embodiment of the present application provides a traditional Chinese medicine composition for preventing and / or treating knee osteoarthritis, which is prepared from the following components in parts by weight: salt eucommia 6-10 parts, rehmannia 9-15 parts, astragalus 15-20 parts, angelica 9-12 parts, cyathula 9-12 parts, chuanxiong 6-9 parts, steamed lycium 9-12 parts, bran pummelo fruit 6-10 parts, bran white peony root 9-15 parts, and fried licorice 3-5 parts.

[0031] In some specific embodiments, the traditional Chinese medicine composition is prepared from the following components in parts by weight: salt eucommia 10 parts, rehmannia 15 parts, astragalus 20 parts, angelica 12 parts, cyathula 12 parts, chuanxiong 9 parts, steamed lycium 12 parts, bran pummelo fruit 10 parts, bran white peony root 15 parts, and fried licorice 5 parts.

[0032] The embodiment of the present application also provides a preparation method of the traditional Chinese medicine composition, which comprises the following steps: taking the medicinal materials in parts by weight, soaking, and decocting by a reflux method, and concentrating the medicinal liquid to obtain the traditional Chinese medicine composition.

[0033] In some specific embodiments, the soaking time is 1-2 h.

[0034] In some specific embodiments, the decocting method comprises the following steps: decocting for 1-2 h for two times.

[0035] In some specific embodiments, the concentration method of the medicinal liquid comprises the following steps: concentrating the medicinal liquid to a crude drug content of 1.6 g·mL -1 .

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

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

[0038] The embodiment of the present application also provides an application of the traditional Chinese medicine composition in preparing a medicine for improving the expression of peripheral nerve secretion pain sensitization factor NGF and increasing the pain threshold.

[0039] The embodiment of the present application also provides an application of the traditional Chinese medicine composition in preparing a medicine for reducing the marrow cavity gap of the subchondral bone region.

[0040] According to clinical experience, the kidney marrow health drink is formed, and all parties take salt eucommia and raw rehmannia as the monarch drug. Salt eucommia fills the marrow of the kidney, strengthens the muscles and bones, and raw rehmannia nourishes yin and blood. The two drugs are used together to nourish yin and assist yang, and to benefit the kidney and fill the marrow. Huangqi and Danggui are used to supplement qi and blood, and are supplemented by Niuqi to supplement the kidney, remove blood stasis, and unblock the channels. The three drugs expel evil and support the healthy, unblock the channels and remove arthralgia, and are the ministerial drugs. Chuanxiong is a blood and qi drug, which can unblock blood stasis, dispel cold and soften the tendons. Shanzhuye enters the pericardium meridian, supplements the liver and kidney, unblocks the wind and arthralgia, and Jichu is used to regulate qi and expand the middle to consolidate the root of the afterworld. Baishao and Ganzao can turn yin into acid and turn yin into acid, soften the tendons and relieve pain. The combination of various drugs has the functions of supplementing the liver and kidney, benefiting essence and marrow, unblocking the channels and relieving pain, and activating blood and regulating qi. Modern pharmacology also proves that raw rehmannia, astragalus, angelica and white peony in the prescription can promote immune regulation, inhibit inflammation and relieve OA pain.

[0041] The traditional Chinese medicine composition provided by the present application is a medicine for preventing and treating musculoskeletal diseases and knee osteoarthritis prepared by kidney marrow health drink. The kidney marrow health drink can delay the progression of knee osteoarthritis and relieve peripheral pain sensitization of knee osteoarthritis. The kidney marrow health drink can delay the progression of knee osteoarthritis by reducing the OARSI score, regulating the expression of type II collagen and matrix metalloproteinase-13. The kidney marrow health drink can relieve peripheral pain sensitization of knee osteoarthritis by improving the expression of peripheral nerve secretion pain sensitization factor NGF and increasing the pain threshold.

[0042] The administration route of the medicine is oral administration.

[0043] The medicine is a medicine for reducing the OARSI score, increasing the expression of type II collagen, reducing the expression of matrix metalloproteinase-13, reducing the volume fraction of subchondral bone, improving the expression of peripheral nerve secretion pain sensitization factor NGF, increasing the pain threshold, and verifying the safety. The dosage form of the medicine can be any one of tablets, capsules, oral liquids, oral preparations, granules, decoctions, pills, powders, pastes, dan preparations, suspensions, powders, solutions, injections, suppositories, creams, sprays, drops or patches. The dose of the medicine in animals is 1.6 g·mL -1 .

[0044] The kidney marrow health drink used in the embodiment of the present application is composed of salt eucommia 10g, raw rehmannia 15g, astragalus 20g, angelica 12g, milkweed 12g, chuanxiong 9g, steamed meat 12g, bran jujube 10g, bran peony 15g and fried licorice 5g. The above-mentioned traditional Chinese medicine decoction pieces are purchased from Zhejiang University of Traditional Chinese Medicine. The above-mentioned decoction pieces are soaked in distilled water for 1-2 hours, and then refluxed for 2 times, 1 hour each time. Then the medicine is filtered and evaporated, and then concentrated to the content of crude drug 1.6 g·mL -1, and then stored at -20 ℃. The distilled water refers to the boiling point of distilled water is 100 ℃ and the density is 1 g / cm 3 The percentage concentration in the embodiments of the present application is mass percentage concentration unless otherwise specified.

[0045] Example 1

[0046] Effect of Shen Su An Kang Yin on the joint microstructure of KOA mice: to verify that Shen Su An Kang Yin can reverse the pathological changes of bone microstructure in KOA mice, and to clarify the progression of KOA mice disease treated with Shen Su An Kang Yin.

[0047] I. Method

[0048] 1. Experimental grouping

[0049] In this experiment, C57BL / 6J mice were used, and then 30 C57BL / 6J mice were randomly divided into 3 groups by random number table method, namely sham operation group, model group and Shen Su An Kang Yin group, 10 mice in each group.

[0050] 2. Establishment of KOA mouse model

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

[0052] 3. Drug preparation and intervention

[0053] 3.1 Drug preparation

[0054] Shen Su An Kang Yin composition: salt of eucommia 10 g, raw rehmannia 15 g, astragalus 20 g, angelica 12 g, cyathula 12 g, chuanxiong 9 g, steamed meat 12 g, bran qu jujube 10 g, bran peony 15 g and fried licorice 5 g. The above traditional Chinese medicine decoction pieces are purchased from the Medicinal Materials and Antler Branch Company of East China Pharmaceutical Co., Ltd. After soaking the above decoction pieces in distilled water for 1 hour, reflux method is used for decoction, a total of 2 times, 1 hour each time. Then filter the medicine and evaporate, combine the two filtrates and concentrate to a crude drug content of 1.6 g·mL -1 The preparation of the required intervention drug in the present application is the same method.

[0055] 3.2 Drug intervention

[0056] Starting from the second day after surgery, mice in the kidney marrow health-preserving decoction group were administered a crude drug concentration of 1.6 g / mL. -1 Mice in the kidney marrow health-preserving drink were administered 0.5 mL of the drink via gavage once a day for 8 weeks. Mice in the model group and sham-operated group were administered 0.5 mL of physiological saline via gavage at the same frequency.

[0057] 4. Indicator Testing

[0058] 4.1 Micro-CT Detection

[0059] The day after gavage, mice in each group were anesthetized by intraperitoneal injection of Sutacetin 50 (50 mg / kg), euthanized by cervical dislocation, and their tissues were collected. Knee joint samples were fixed in 4% paraformaldehyde at 4°C for 48 hours, then transferred to 75% ethanol. Three-dimensional imaging of the knee joint was performed using a micro-CT scanning system with parameters of 75 kV voltage, 200 µA current, and 18 µm resolution. Reconstruction and region selection were performed using the three-dimensional reconstruction software NRecon and Data Viewer. Quantitative analysis of cartilage and subchondral bone regions at the same locations in the images was performed using CTAnalyzer. Results are shown in [Figure number missing]. Figure 1 In section A, the bone volume fraction (BV / TV: the percentage of bone tissue volume to subchondral bone volume) was calculated. Based on the morphological parameters of the subchondral bone, the structural changes of the knee joint bone tissue were evaluated. The results are shown in [Table A]. Figure 1 B.

[0060] Depend on Figure 1 It can be seen that the Micro-CT three-dimensional reconstruction image of the subchondral bone of the tibia ( Figure 1 The results showed that abnormal bone remodeling in the subchondral bone of the model group mice was significantly increased compared with that in the sham-operated group. DMM surgery induced subchondral bone sclerosis, and the subchondral bone BV / TV ratio was significantly increased. p <0.001); Compared with the model group, the medullary cavity space in the subchondral bone region of mice in the Shen Sui An Kang Yin group was significantly reduced, and the subchondral bone BV / TV ratio was decreased ( p <0.001), see details. Figure 1 .

[0061] 4.2 Histological and Bone Morphometric Analysis

[0062] 4.2.1 Obtaining and preparing sections of KOA mouse knee tissue

[0063] After the completion of the Micro-CT scanning, the knee joint samples of each group of mice were taken out, washed with tap water for 1 hour, and then treated with 14% EDTA solution for 12-14 days, with liquid changed every day, and the decalcification condition was observed every day. The tissue was easily broken through with a large needle. After the completion of the decalcification, the samples were washed with tap water for 4-6 hours, and then treated by the automatic tissue dehydrator. All the samples were embedded with paraffin to prepare sample wax blocks and sections. During the sectioning, all the sections were 3 μm thick sections, and each section was taken as the standard for separating the meniscus.

[0064] 4.2.2 ABH / OG staining

[0065] 1) The sections of the knee joint tissues of each group (the same numbered sections were taken from each sample) were baked at 65°C overnight;

[0066] 2) The next day, the sections were taken out and gradually deparaffinated and rehydrated after they were restored to room temperature (deparaffinated with xylene for 3 times, 15 minutes / time; 100%, 95% alcohol for 2 times, 8 minutes / time; 70% alcohol for 1 time, 8 minutes / time; deionized water for 3 times, 3 minutes / time);

[0067] 3) The sections were placed in hydrochloric acid alcohol for differentiation for 30 seconds, and the excess water was briefly absorbed on paper;

[0068] 4) The sections were placed in Alcian blue hematoxylin solution for 1 hour, and then deionized water for 3 times, 3 minutes / time;

[0069] 5) The sections were placed in hydrochloric acid alcohol for differentiation for 3 seconds, and then washed with deionized water for 3 times, 3 minutes / time;

[0070] 6) The sections were placed in 0.5% ammonia water for 20 seconds, and then washed with deionized water for 3 times, 1 minute / time;

[0071] 7) The sections were placed in 95% alcohol for 1 minute, and then transferred to the Eosin / Orange G working solution for staining for 2 minutes;

[0072] 8) Gradually dehydrated and transparent (95% alcohol for 3 times, 1 minute / time; 100% alcohol for 2 times, 1 minute / time; deparaffinated with xylene for 3 times, 1 minute / time), and then completed with the mounting medium;

[0073] 9) Baked at 37°C overnight, observed under the digital pathology slide scanning system, and completed with the sampling, and the results are shown in Figure 2 A.

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

[0075] Sections of knee joint tissue were taken from each group (sections with the same number were taken from each sample). The severity of KOA cartilage damage was assessed using the histological scoring system developed by the Osteoarthritis Research Society International (OARSI) (OARSI scores are shown in Table 1). The severity was determined by two individuals outside the project team, and the average score was taken. The results are shown in Table 1. Figure 2 B.

[0076] Table 1 OARSI Scoring Details

[0077]

[0078] Depend on Figure 2 The results of ABH / OG staining of mouse knee cartilage showed that the sham-operated group mice had only slight KOA changes in their joints, with relatively normal articular cartilage and a more uniform cartilage matrix morphology; the model group had severe articular cartilage wear, with significant loss and thinning of the cartilage layer, and fissures extending to the subchondral bone; the Shen Sui An Kang Yin group showed significant improvement compared to the model group, with some damage visible on the cartilage surface and a more intact cartilage matrix structure. Figure 2 (As shown in Figure A). The morphology of articular cartilage tissue in each group of KOA mice was scored according to the OARSI score, and it was found that the OARSI score of the model group was significantly higher than that of the sham-operated group. p <0.001), while the OARSI score of the Shensui Ankang Decoction component was significantly lower than that of the model group ( p <0.001) Figure 2 (As shown in B).

[0079] 4.3 Immunohistochemical staining

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

[0081] 2) On the second day, remove the sections and allow them to return to room temperature before dewaxing and rehydrating them step by step (xylene dewaxing 3 times, 15 minutes each time; 100% and 95% alcohol 2 times each, 8 minutes each time; 70% alcohol 1 time, 8 minutes each time; deionized water 3 times, 3 minutes each time).

[0082] 3) All antibodies were heat-retarded using the following method: heat retardation was performed with sodium citrate (pH 6.0) (temperature 65℃), and after the sections returned to room temperature, they were washed 3 times with 1×PBS, 3 minutes each time;

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

[0084] 5) Add Col2a1 (1:200) and Mmp13 (1:200) primary antibodies dropwise (diluted separately with PBS containing 5% NGS and 0.1% Triton X-100 detergent), then place the sections in a wet box and incubate overnight at 4°C;

[0085] 6) The next day, take out the wet box. After naturally restoring to room temperature, wash the sections 3 times with 1×PBS for 5 minutes each; add the corresponding secondary antibody (diluted 1:1000 with 1×PBS), incubate at room temperature for 1 hour, then wash 3 times with 1×PBS for 5 minutes each; then perform DAB color development, with the same antibody color development time;

[0086] 7) Counterstain the nuclei with hematoxylin for 10 seconds, wash 3 times with deionized water for 5 minutes each; dehydrate and clear step by step (3 times with 95% alcohol for 1 minute each; 2 times with 100% alcohol for 1 minute each; 3 times with xylene for dewaxing for 1 minute each), and complete the mounting with a mounting medium;

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

[0088] As can be seen from Figure 3 the immunohistochemical results of Col2a1 in the knee joint cartilage of mice, after DMM modeling, the expression of Col2a1 in the cartilage tissue of mice was significantly decreased compared with the sham operation group ( p <0.001), while the expression of Col2a1 in the cartilage of mice treated with Shensuiankangyin was reversed compared with the model group ( p <0.01) ( Figure 3 shown in B below). In addition, the immunohistochemical results of MMP-13 showed that compared with the sham operation group, the model group could significantly increase the expression of MMP-13 in the cartilage area ( p <0.001), and at the same time, the expression of MMP-13 was downregulated to a certain extent after the intervention of Shensuiankangyin ( p <0.001) ( Figure 3 shown in C below).

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

[0090] Example 2

[0091] Effect of Shensuiankangyin on peripheral pain in KOA mice: Verify that Shensuiankangyin can reduce peripheral pain sensitivity in KOA mice and clarify that Shensuiankangyin can relieve pain in KOA mice.

[0092] I. Methods

[0093] 1. Experimental grouping

[0094] C57BL / 6J mice were used in this experiment, and then 30 C57BL / 6J mice were randomly divided into 3 groups by random number table method, namely sham operation group, model group and Shenshui An Kang Yin group, 10 mice in each group.

[0095] 2 KOA mouse model was established

[0096] The model group mice and the Shenshui An Kang Yin group mice were all constructed KOA model by destabilization of medial meniscus (DMM). First, the mice were anesthetized with su-tai 50 (dose 50 mg / kg), and the knee joint was prepared for local skin in a sterile environment. Then, a longitudinal incision was made along the distal patella and proximal tibial platform, about 1 cm in length, and the patellar ligament was pulled to one side. The fat pad was bluntly separated with an ophthalmic forceps, and the medial meniscus was loosened with a suture needle. Finally, the patella was reduced, and the wound was closed. For the sham operation group, a similar operation was performed, the only difference was that no damage was caused to the joint tissue.

[0097] 3 Drug intervention

[0098] From the 2nd day after operation, the Shenshui An Kang Yin group mice were given Shen-shui An Kang Yin with a crude drug concentration of 1.6 g·mL -1 , 0.5 mL per mouse each time, 1 time / day, for 8 weeks; the model group and sham operation group mice were given 0.5 mL of normal saline by gavage, with the same frequency.

[0099] 4 Index detection

[0100] 4.1 Behavioral detection

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

[0102] The mechanical pain withdrawal threshold (MWT) of mice was determined by Vonfrey fiber pain tester after different fiber stimulation of both lower limbs according to the up&down method. The mice were placed in a quiet glass partition to adapt to the environment. Paw withdrawal or licking was taken as a sign of positive. Each lower limb was repeated 3 times, and the average value was taken, which was measured before modeling and 2, 4, 8 weeks after administration, respectively;

[0103] Hot plate test: The thermal nociceptive response of mice was recorded using a YLS-6B intelligent hot plate instrument. The mice were placed on a hot plate with a temperature of 50 °C to start timing, and the timing was stopped when the mice licked the right hind foot or jumped. The reaction time of each mouse was recorded and measured 5 times, with an interval of 5 minutes each time, and the average value was taken for subsequent analysis.

[0104] The results of behavioral tests showed that there was no significant statistical difference in MWT of mice in each group before modeling p <0.001). Compared with the model group, the MWT of mice in the sham operation group showed significant statistical difference from the 2nd to the 8th week p <0.001), indicating that the mice were sensitive to pain after DMM modeling; after the intervention of Shen Su An Kang Yin, the peripheral pain sensitivity of mice decreased from the 2nd to the 8th week p <0.001, Figure 4 Fig. 1A). Then the hot plate method was used to evaluate the pain threshold of mice. Compared with the sham operation group, the right hind foot reaction time of the model group was shortened, indicating that the model group had increased pain sensitivity and decreased pain threshold p <0.001); after the intervention of Shen Su An Kang Yin, the pain threshold of mice increased p <0.001), indicating that the pain of mice was relieved Figure 4 Fig. 1A). Finally, the Dig Gait imaging system was used to evaluate the walking gait of KOA mice. Compared with the sham operation group, the swing phase time of the right lower limb of the model group increased p <0.001) and the support phase time, stride length, and foot contact area decreased p <0.001, Figure 4 Fig. 1C-F). After receiving Shen Su An Kang Yin treatment, they all showed increased stride length, decreased swing time, and increased foot contact area p <0.001, Figure 4 C-F). The above results showed that Shen Su An Kang Yin treatment could effectively reduce the pain threshold of DMM mice.

[0105] 4.2 Acquisition of KOA mouse knee tissue and right L4 dorsal root ganglion and preparation of sections

[0106] After completing the Micro-CT scan, knee joint samples from each group of mice were removed, rinsed with tap water for 1 hour, and then decalcified with 14% EDTA solution for 7 days, changing the solution daily and observing the decalcification process daily. The decalcification was considered complete when the tissue could be easily penetrated with a pin. Next, the tissue was dehydrated in 30% sucrose solution. After complete dehydration, the tissue was embedded and sectioned using OCT cryoemulation medium, with all sections being 10 μm thick. The right L4 dorsal root ganglion (DRG) was carefully dissected under a stereomicroscope, fixed in 4% paraformaldehyde for 1 hour, and then dehydrated in 30% sucrose solution. After complete dehydration, the tissue was embedded and sectioned using OCT cryoemulation medium.

[0107] 4.3 Immunohistofluorescence staining

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

[0109] 2) After it returns to room temperature, wash 3 times with 1×PBS, 3 minutes each time.

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

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

[0112] 5) The next day, remove the humidified chamber and allow it to return to room temperature naturally. Wash the slides three times with 1×PBS, 5 minutes each time. Add the corresponding secondary antibody (1×PBS diluted 1:1000), incubate at 37°C for 1 hour, then wash three times with 1×PBS, 5 minutes each time. Then perform DAPI staining, incubate at 37°C for 10 minutes, and wash three times with 1×PBS, 5 minutes each time.

[0113] 6) After mounting with anti-fluorescence attenuation mounting medium, the slides were observed and collected under a Zeiss upright microscope. The results are shown in [see attached image]. Figure 5 .

[0114] Eight weeks after DMM modeling, the expression of NGF (green fluorescent marker), a key pain sensitization factor, in the subchondral bone and DRG of mice was significantly increased compared with the sham-operated group. p <0.001, Figure 5 After treatment with Shen Sui An Kang Yin (a traditional Chinese medicine formula), the expression level of NGF in subchondral bone was moderately reduced, showing a significant difference compared with the model group. p <0.001, Figure 5C) and NGF expression in mouse DRG was also significantly down-regulated Figure 5 C) and NGF expression in mouse DRG was also significantly down-regulated

[0115] The above results show that Shensui Ankang Decoction can improve the pain threshold of KOA mice and relieve peripheral pain in KOA mice.

[0116] Example 3

[0117] Safety evaluation of Shensui Ankang Decoction in treating KOA mice: to verify the safety of Shensui Ankang Decoction in treating KOA mice and determine the drug safety of Shensui Ankang Decoction in treating KOA mice.

[0118] I. Methods

[0119] 1. Experimental grouping

[0120] In this experiment, C57BL / 6J mice were used, and then 30 C57BL / 6J mice were randomly divided into 3 groups by random number table method, namely sham operation group, model group and Shensui Ankang Decoction group, 10 mice in each group.

[0121] 2. Establishment of KOA mouse model

[0122] The model group mice and the Shensui Ankang Decoction group mice were all constructed KOA model by destabilization of medial meniscus (DMM). First, the mice were anesthetized with su-tai 50 (dose 50 mg / kg), and the knee joint was prepared for local skin in a sterile environment. Then make a longitudinal incision, along the distal patella and proximal tibial platform, about 1 cm, and pull the patellar ligament to one side. Use ophthalmic forceps to bluntly separate the fat pad, and loosen the medial meniscus with a suture needle. Finally, reduce the patella and close the wound. For the sham operation group, a similar operation was performed, the only difference was that no damage was caused to the joint tissue.

[0123] 3. Drug intervention

[0124] From the 2nd day after operation, the Shensui Ankang Decoction group mice were given Shensui Ankang Decoction with a crude drug concentration of 1.6 g·mL -1 , 0.5 mL per mouse each time, 1 time / day, for 8 weeks; the model group and sham operation group mice were given 0.5 mL of normal saline by gavage, with the same frequency.

[0125] 4. Index detection

[0126] 4.1 H&E staining

[0127] 1) Take the sections of internal organs (heart, liver, spleen, lung, kidney) of each group (take the same number of sections of each sample) and bake at 65°C overnight;

[0128] 2) On the second day, the sections were removed, and after they returned to room temperature, they were deparaffinated and rehydrated step by step (xylene deparaffination for 3 times, 15 minutes / time; 100%, 95% alcohol for 2 times, 8 minutes / time; 70% alcohol for 1 time, 8 minutes / time; deionized water for 3 times, 3 minutes / time);

[0129] 3) The sections were placed in hematoxylin staining solution for differentiation for 1 minute, and deionized water for 3 times, 3 minutes / time;

[0130] 4) The sections were placed in 1% hydrochloric acid alcohol solution for acidification for 10 seconds, and deionized water for 3 times, 3 minutes / time;

[0131] 5) The sections were placed in 0.5% ammonia water for 20 seconds, and washed with deionized water for 3 times, 1 minute / time;

[0132] 6) The sections were placed in eosin staining solution for 1 minute and 30 seconds, and washed with deionized water for 3 times, 1 minute / time;

[0133] 7) Washed with deionized water for 3 times, 1 minute / time; dehydrated and transparented step by step (95% alcohol for 3 times, 1 minute / time; 100% alcohol for 2 times, 1 minute / time; xylene deparaffination for 3 times, 1 minute / time), and completed mounting with mounting medium;

[0134] 8) Baked at 37°C overnight, observed and completed sampling under the digital pathology slide scanning system, and the results are shown in Figure 6 .

[0135] The H&E staining results prove that kidney marrow health drink does not cause any damage to these organs, and kidney marrow health drink has no drug toxicity while treating KOA mice, and no damage to the internal organs of animals.

[0136] Example 4

[0137] In vitro study of kidney marrow health drink preventing chondrocyte degeneration: verify the change of kidney marrow health drink regulating the anabolism index in chondrocytes in vitro, and clarify the mechanism of kidney marrow health drink preventing chondrocyte degeneration.

[0138] I. Method

[0139] 1 Experimental grouping

[0140] 1) Control serum group, model group, kidney marrow health drink drug-containing serum group. The control group and the model group were added with 10% blank serum, the model group and the kidney marrow health drink drug-containing serum group were added with IL-1 -1 at a concentration of 10 ng·ml β , and the kidney marrow health drink drug-containing serum group was added with 20% kidney marrow health drink drug-containing serum.

[0141] 2 Isolation and culture of mouse articular chondrocytes

[0142] 1)Take 2 weeks C57BL / 6J mice, after killing with CO2, soak them in 75% alcohol for 30 minutes.

[0143] 2)Take out the mouse, remove the skin, clamp the femoral head cartilage layer with forceps, and cut the attached ligament and soft tissue with scissors. Place the cartilage tissue in a 1.5 mL centrifuge tube containing PBS.

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

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

[0146] 5)After the cartilage cap is digested, transfer the digestion medium in the culture dish to a 15 mL centrifuge tube and continue to blow (30-40 times). After filtering with a 40 µm cell filter, centrifuge at 1000 rpm for 5 minutes.

[0147] 6)Gently shake the culture dish, label it, and incubate it in a 37°C incubator for 4-6 hours.

[0148] 7)Resuspend with 5 mL normal medium (F12 medium + 10% FBS + 1% P / S), count and adjust the appropriate concentration for inoculation in the well plate. Incubate in a 37°C, 5% CO2 incubator for 12 hours. After the cells grow well, continue the subsequent experiment.

[0149] 3 Drug-containing serum extraction

[0150] 1)Take 10 2-month-old male SD rats, and randomly divide them into two groups: kidney marrow Kangyin drug-containing serum group and blank serum group. The former was given 1.6 g·mL -1 of crude drug by gavage, and the latter was given the same volume of normal saline, once a day for 1 week.

[0151] 2)On the 7th day, the rats were fasted, and 2 hours after morning gavage, they were anesthetized by intraperitoneal injection of su-tai 50 (dose 50 mg / kg). The abdominal aortic blood was collected. The blood sample was incubated at room temperature for 1 hour, centrifuged at 3000 r·min -1 for 15 minutes, and the upper serum was taken and divided into centrifuge tubes. It was inactivated in a 56°C water bath for 30 minutes, sterilized through a 0.22 µm sterile filter, and stored in a -80 ℃ refrigerator for standby.

[0152] 4 Effect of kidney marrow Kangyin on the survival of primary articular chondrocytes in mice

[0153] After the cells adhere, the blank group (without chondrocytes and without kidney marrow Kangrongyin drug-containing serum), the control group (with chondrocytes and without kidney marrow Kangrongyin drug-containing serum) is replaced with serum-free medium (F12 medium), and the drug group is added with serum-free medium (5%, 10%, 20%, 40%) at the final concentration. The culture is continued for 24 hours and 48 hours, respectively. At the corresponding time, the culture solution is aspirated, washed twice with DPBS, and 100 μL of F12 medium containing 10 μL of CCK8 is added to each well. After 2 hours of continuous culture, the supernatant is collected, and the average optical density (OD value) of each well is measured at 450 nm. The experiment is repeated independently for 3 times. The survival rate of chondrocytes is calculated. Chondrocyte survival rate (%) = [(drug group OD value - blank group OD value) / (control group OD value - blank group OD value)] x 100%.

[0154] In this experiment, the effect of different concentrations of kidney marrow Kangrongyin drug-containing serum on the survival of chondrocytes was determined by CCK8 method. The survival rate of mouse primary articular chondrocytes under the action of different concentrations of kidney marrow Kangrongyin drug-containing serum is shown in Table 1. Figure 7 The survival rate of mouse primary articular chondrocytes under the action of 20% concentration of kidney marrow Kangrongyin drug-containing serum is not affected and is the most stable, so in the follow-up experiment, 20% kidney marrow Kangrongyin is used for follow-up study.

[0155] 5 Alcian blue staining to evaluate the effect of kidney marrow Kangrongyin on chondrocyte matrix

[0156] 1) After the cells adhere, add trypsin for 1 minute and put it in a 37°C incubator; then add complete medium to stop, blow with a gun head, and transfer to a 15 mL centrifuge tube at 3000 r·min -1 for 5 minutes.

[0157] 2) Add 1 mL of complete medium and blow repeatedly for cell counting, then aspirate 20 μL and drop in the center of the well plate. After 4-6 hours, the cells adhere, the control group and the model group are added with 10% blank serum, the model group and the kidney marrow Kangrongyin drug-containing serum group are added with IL-1 -1 at a concentration of 10 ng·ml β , and the kidney marrow Kangrongyin drug-containing serum group is added with 20% kidney marrow Kangrongyin drug-containing serum.

[0158] 3) The above method is cultured for 7 days, washed once with 1x PBS for 3 minutes, fixed overnight, washed 3 times with 1x PBS for 3 minutes each time, added with Alcian blue for 30 minutes, washed 3 times with 1x PBS for 3 minutes each time. Observe and complete the sampling under a Zeiss upright microscope, and the results are shown in Figure 8 .

[0159] Figure 8The results showed that compared with the control group, 10 ng·ml -1 of IL-1 β induced model group of chondrocytes were unevenly stained and partially stained lighter, which visually reflected the pathological changes of chondrocytes in the model group; compared with the model group, the staining of chondrocytes treated with kidney marrow Kang drink containing serum was uniform, and the color was deepened, indicating that kidney marrow Kang drink containing serum could reverse the changes of chondrocytes in extracellular matrix composition and structure.

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

[0161] 6.1 Extraction of mRNA from primary chondrocytes

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

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

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

[0165] 6.2 Determination of mRNA concentration and reverse transcription

[0166] Take 1.5 μL of mRNA from each group of samples, and use ultramicro UV spectrophotometer to determine the RNA concentration; take 500 ng of mRNA from each group of samples, and prepare the reaction system (10 μL) according to the operation instruction in the reverse transcription kit to synthesize cDNA, and the remaining mRNA is stored in the refrigerator at -80°C.

[0167] The reverse transcription reaction system is as follows: mRNA 500 ng, 5x Mix 2 μL, and RNAase-free water to 10 μL.

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

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

[0170] The cDNA obtained by reverse transcription was diluted 5 times with 40 μL RNase-free water and used as the PCR reaction template. The reaction system (10 μL) was prepared according to the SYBR kit operation manual using 500 ng mRNA of each sample, and the remaining mRNA was stored in a refrigerator at -80℃. In this experiment, Mmp13 and Col2a1 were selected as the target genes, and Actb was used as the internal reference gene (see Table 2 for primer sequences). The target gene copy number / Actb ratio was used as the statistical value, and the real-time fluorescence PCR data were analyzed by the 2 -ΔΔCT method. The expression levels of the target genes in the primary chondrocytes of each group were analyzed by real-time PCR, and the results are shown in Figure 9

[0171] Real-time PCR reaction system:

[0172] cDNA template 2 μL, upstream primer (10 μM) 0.5 μL, downstream primer (10 μM) 0.5 μL, 2x Sybrgreen mix 5 μL, RNase-free water to 10 μL.

[0173] Real-time PCR reaction program:

[0174] Initial denaturation at 95℃ for 30 seconds, denaturation at 95℃ for 10 seconds, annealing and extension at 60℃ for 60 seconds (denaturation, annealing and extension cycle number 40), melting curve 60-95℃ for 5 seconds hold / 1℃.

[0175] Table 2 Primer sequences

[0176]

[0177] The results showed that, compared with the control group, the Mmp13 mRNA level in the chondrocytes of the model group induced by 10 ng·ml -1 of IL-1 β was significantly increased ( p <0.001 ) ( Figure 9 middle B), and the Col2a1 level was significantly decreased, and the difference was statistically significant ( p <0.01 ) ( Figure 9 middle A). Compared with the model group, the Mmp13 mRNA level decreased in a dose-dependent manner after treatment with Shen Su An Kang Yin drug-containing serum ( p <0.01 ) ( Figure 9 ​The Col2a1 mRNA level was increased in a dose-dependent manner, and the difference was statistically significant (P<0.01) p <0.01 ) ( Figure 9 The results of the experiment of Example 2 show that the Shenshen'an Kang Yin drug-containing serum can delay the IL-1 β induced KOA chondrocyte degeneration.

[0178] Obviously, the above examples of the present application are only examples for clearly illustrating the present application, and are not intended to limit the embodiments of the present application. Based on the above description, other different forms of changes or variations can be made by those skilled in the art. Here, it is not necessary and also impossible to enumerate all the embodiments. Any modification, equivalent replacement and improvement, etc. made within the spirit and principle of the present application shall be included in the protection scope of the claims of the present application.

Claims

1. A traditional Chinese medicine composition for the prevention and / or treatment of knee osteoarthritis, characterized in that, It is made from the following components in parts by weight: 6-10 parts salt-processed Eucommia ulmoides, 9-15 parts raw Rehmannia glutinosa, 15-20 parts Astragalus membranaceus, 9-12 parts Angelica sinensis, 9-12 parts Achyranthes bidentata, 6-9 parts Ligusticum chuanxiong, 9-12 parts steamed Cornus officinalis, 6-10 parts wheat bran Citrus aurantium, 9-15 parts wheat bran Paeonia lactiflora, and 3-5 parts prepared Glycyrrhiza uralensis. The preparation method is as follows: after weighing the medicinal materials according to the weight proportions, soak them in distilled water and decoct them using the reflux method. The concentrated medicinal liquid is the Chinese medicine composition.

2. The traditional Chinese medicine composition according to claim 1, characterized in that, It is made from the following components in parts by weight: 10 parts salted Eucommia ulmoides, 15 parts raw Rehmannia glutinosa, 20 parts Astragalus membranaceus, 12 parts Angelica sinensis, 12 parts Achyranthes bidentata, 9 parts Ligusticum chuanxiong, 12 parts steamed Cornus officinalis, 10 parts wheat bran Citrus aurantium, 15 parts wheat bran Paeonia lactiflora, and 5 parts roasted Glycyrrhiza uralensis.

3. The traditional Chinese medicine composition according to claim 1, characterized in that, The soaking time is 1 to 2 hours.

4. The traditional Chinese medicine composition according to claim 1, characterized in that, The decoction method is as follows: decoct twice, each time for 1 to 2 hours.

5. The traditional Chinese medicine composition according to claim 1, characterized in that, The method for concentrating the medicinal solution is as follows: concentrate to a crude drug content of 1.6 g·mL. -1 .

6. The use of the traditional Chinese medicine composition as described in claim 1 or 2 in the preparation of a medicament for the prevention and / or treatment of knee osteoarthritis.

Images