Application of cimicifugin in preparation of medicine for promoting chondrogenic differentiation and medicine for treating cartilage injury diseases

Abstract

The invention discloses application of cimicifugin in preparation of a medicine for promoting chondrogenic differentiation of bone marrow mesenchymal stem cells. The invention further discloses application of the cimicifugin in preparation of drugs for treating cartilage injury related diseases. The cimicifugin can promote proliferation of the bone marrow mesenchymal stem cells and promote chondrogenic differentiation of the bone marrow mesenchymal stem cells; in addition, the cimicifugin can reduce chondrocyte inflammation and generation of oxidative stress related product active oxygen; more importantly, the cimicifugin can inhibit the generation of chondrocyte extracellular matrix catabolic proteins (MMP3 and MMP13), promote the generation of chondrocyte extracellular matrix synthetic proteins (ACAN and SOX9) and protect the integrity of ECM (extracellular matrix). Cimicifugin is injected into an in-vivo articular cavity, so that cartilage repair can be promoted, cartilage fibrosis can be inhibited, articular surface cartilage abrasion can be relieved, cartilage matrix synthesis can be increased, and articular cartilage can be protected. In addition, the cimicifugin has the advantages of small toxic and side effects, high safety and the like.

Description

Technical Field

[0001] The invention belongs to the field of natural medicine compounds, and relates to the application of cimicifuga glycosides in the preparation of drugs for promoting cartilage differentiation and in the preparation of drugs for treating cartilage damage diseases. Technical Background

[0002] The destruction and loss of cartilage are important pathological changes in many orthopedic diseases, including osteoarthritis. Due to the special anatomical and tissue characteristics of cartilage, such as the lack of blood supply, nerve tissue and lymphatic distribution, its self-repair ability is abnormally insufficient, so it is of great clinical significance to repair cartilage damage by artificial methods. Mesenchymal stem cells have the characteristics of wide sources, good proliferation ability, multi-differentiation potential, and good biocompatibility with various 3D scaffold materials, which makes them have promising potential in cartilage repair applications. Bone marrow mesenchymal stem cells are an important member of the stem cell family and can differentiate into osteoblasts, chondrocytes, adipocytes and other cells. In the current field of bone regenerative medicine and cell therapy, bone marrow mesenchymal stem cells are the most studied ideal seed cells. Therefore, it is particularly important to discover natural compounds that can promote the chondrogenic differentiation of bone marrow mesenchymal stem cells and reshape the microenvironment of articular cartilage.

[0003] Rheumatoid arthritis (RA) and osteoarthritis (OA) are both systemic diseases of chronic joint diseases. Although they are essentially different, they are closely related to the degeneration of articular cartilage and the degradation of extracellular matrix. The cartilage extracellular matrix (ECM) is mainly composed of collagen and aggrecan, of which collagen is mainly Collagen II and aggrecan is mainly composed of ACAN (aggregable proteoglycan). Chondrocytes are single-cell components in ECM and mainly maintain cartilage homeostasis through the balance between catabolism and anabolism. The key enzymes for cartilage destruction are mainly matrix-degrading enzymes, including matrix metalloproteinases (MMPs) and integrins and metalloproteinases with thrombospondin-like motifs (ADAMTS). In addition, the increased expression of intracellular reactive oxygen species (ROS) and inflammation-related genes in the pathological joint environment will further cause a vicious cycle of oxidative stress, inflammation and chondrocyte apoptosis, ultimately leading to ECM degradation and accompanied by inflammatory reactions. Therefore, the integrity of the extracellular matrix (ECM) plays an important role in the growth and repair of cartilage.

[0004] Prim-O-glucosylcimifugin (POG) is a chromone, mainly derived from plants such as Saposhnikovia divaricata. Its structural formula is as follows:

[0005]

[0006] Studies have shown that cimicifuga glycosides have a variety of pharmacological properties, including antibacterial, anticancer, antioxidant, antipyretic, anti-inflammatory and analgesic effects, and are used to treat diseases such as fever, rheumatism and cancer.

[0007] Currently, there are no reports on the effects of cimicifuga glycosides on the chondrogenic differentiation of bone marrow mesenchymal stem cells and cartilage damage-related diseases. Summary of the invention

[0008] The inventor creatively discovered through experiments that: Cimicifuga glycosides can promote the proliferation of bone marrow mesenchymal stem cells and promote their chondrogenic differentiation; in addition, Cimicifuga glycosides can reduce the production of reactive oxygen species related to chondrocyte inflammation and oxidative stress; more importantly, Cimicifuga glycosides can inhibit the production of cartilage cell extracellular matrix decomposition proteins (MMP3, MMP13), promote the production of cartilage cell extracellular matrix synthesis proteins (ACAN, SOX9), and protect the integrity of ECM. Injection of Cimicifuga glycosides into the joint cavity in vivo can promote cartilage repair and inhibit cartilage fibrosis, reduce articular surface cartilage wear, increase the synthesis of cartilage matrix, and have a protective effect on articular cartilage. In addition, Cimicifuga glycosides have the advantages of small toxic and side effects and high safety. The present invention provides a new idea and strategy for the treatment of cartilage injury-related diseases, and has broad application prospects.

[0009] One object of the present invention is to provide the use of cimicifuga glycosides in the preparation of a drug for promoting the proliferation and chondrogenic differentiation of bone marrow mesenchymal stem cells.

[0010] The inventors have found that cimicifuga glycosides can promote the chondrogenic differentiation of bone marrow mesenchymal stem cells. When the concentration of cimicifuga glycosides in the culture medium is 0.1, 1, 2.5, 5, 10, and 20 μM, the effect of chondrogenic differentiation of bone marrow mesenchymal stem cells gradually increases, promoting the expression of cartilage synthesis proteins and genes ACAN, Collagen II2, and SOX9 to play its role in chondrogenic differentiation.

[0011] Furthermore, the application is the use of cimicifuga glycosides in the preparation of drugs for promoting the chondrogenic differentiation of bone marrow mesenchymal stem cells by promoting the levels of ACAN, Collagen II, and SOX9 proteins.

[0012] Furthermore, the application is the application of cimicifuga glycoside in the preparation of drugs for promoting the expression of ACAN and Collagen II genes and promoting the differentiation of bone marrow mesenchymal stem cells into cartilage.

[0013] Another object of the present invention is to provide the use of cimicifuga glycosides in the preparation of drugs for treating diseases related to cartilage damage.

[0014] The cartilage damage-related diseases are osteoarthritis and rheumatoid arthritis.

[0015] Furthermore, the application is the use of cimicifuga glycosides in the preparation of drugs for treating cartilage damage diseases by inhibiting the expression of proteins related to cartilage extracellular matrix degradation (MMP13, MMP3).

[0016] Furthermore, the application is the application of cimicifuga glycoside in the preparation of drugs for treating cartilage damage-related diseases by increasing the expression of proteins related to cartilage extracellular matrix synthesis (ACAN, SOX9).

[0017] Furthermore, the application is the application of cimicifuga glycoside in the preparation of drugs for treating cartilage damage-related diseases by inhibiting chondrocyte inflammation and oxidative stress.

[0018] Furthermore, the application is the application of cimicifuga glycoside in the preparation of a drug for treating cartilage damage-related diseases by promoting cartilage repair and inhibiting cartilage fibrosis.

[0019] Furthermore, the drug uses cimicifuga glycoside as an effective ingredient or a main effective ingredient and is prepared into a pharmaceutically acceptable dosage form with a pharmaceutically acceptable carrier.

[0020] Furthermore, the pharmaceutically acceptable carrier includes one or more of a lubricant, a binder, a filler, a disintegrant, and a lubricant. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 This is a bar graph showing the effect of cimicifuga glycosides on the proliferation of bone marrow mesenchymal stem cells.

[0022] Figure 2 This is the effect of cimicifuga glycosides on the expression levels of ACAN, Collagen II and SOX9 proteins in bone marrow mesenchymal stem cells.

[0023] Figure 3 The results show the effect of cimicifuga glycosides on the gene expression of ACAN (A) and Collagen II (B) in bone marrow mesenchymal stem cells; compared with the control group, ns means no significant difference, ** means P < 0.01, *** and **** means P < 0.001.

[0024] Figure 4 The effect of cimicifuga glycosides on the cytotoxicity of rat chondrocytes.

[0025] Figure 5 To investigate the effect of cimicifuga glycosides on the expression of proteins related to extracellular matrix degradation and synthesis (ACAN, SOX9, MMP-13, MMP-3) in rat chondrocytes induced by IL-1β.

[0026] Figure 6The results show that cimicifuga glycosides have an effect on the expression of inflammation-related genes in rat chondrocytes induced by IL-1β; A shows the effect of cimicifuga glycosides on the expression of INOS, a gene related to inflammation in rat chondrocytes induced by IL-1β; B shows the effect of cimicifuga glycosides on the expression of COX2, a gene related to inflammation in rat chondrocytes induced by IL-1β; compared with the control group, ns represents no significant difference, ** represents P<0.01, *** and **** represent P<0.001.

[0027] Figure 7 The effect of cimicifuga glycosides on the production of reactive oxygen species in rat chondrocytes induced by IL-1β; A is the ROS fluorescence image, and B is the ROS fluorescence quantitative image; compared with the control group, ns represents no significant difference, ** represents P<0.01, *** and **** represent P<0.001.

[0028] Figure 8 Safranin fast green staining was used to evaluate the effects of cimicifuga glycosides on the structural changes of cartilage and subchondral bone in rats with cartilage injury.

[0029] Fig. 9 Collagen II immunohistochemistry was used to evaluate the effect of cimicifuga glycosides on cartilage matrix synthesis in rats with cartilage injury. DETAILED DESCRIPTION

[0030] The technical scheme of the present invention is further described below in conjunction with specific examples and the accompanying drawings, but the examples do not limit the present invention in any form. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art.

[0031] Preparation of the working solution of cimicifuga glycoside: Weigh 1 mg of cimicifuga glycoside, dissolve it with DMSO to prepare a cimicifuga glycoside mother solution with a final concentration of 100 mM, and then dilute it with α-MEM complete medium (Example 1-Example 3, α-MEM medium + 10% FBS + 1% double antibody) or DMEM complete medium (Example 4-Example 7, DMEM medium + 10% fetal bovine serum + 1% penicillin-streptomycin solution) to obtain cimicifuga glycoside working solutions of different concentrations.

[0032] Example 1

[0033] Effects of cimicifuga glycosides on cytotoxicity and proliferation of rat bone marrow mesenchymal stem cells

[0034] The MTT method was used to determine the effects of cimicifuga glycosides on the cytotoxicity and proliferation ability of bone marrow mesenchymal stem cells.

[0035] Rat bone marrow mesenchymal stem cells were extracted from 2-week-old SD rats. The whole process required aseptic operation. The extraction steps were as follows: the rats were killed by cervical dislocation, and then completely immersed in 75% ethanol for thorough disinfection for 10 minutes. The long bones and femurs of the legs on both sides were carefully cut, and the skin and muscle tissue were removed to expose the bone marrow cavity. A 1mL syringe was used to absorb α-MEM complete medium (α-MEM medium + 10% fetal bovine serum + 1% penicillin-streptomycin solution) to gently flush the bone marrow cavity to flush out the cells. The bone marrow cavity was repeatedly flushed until the bone marrow cavity was light pink. The cells were collected and inoculated into a cell culture bottle, placed in a cell culture incubator at 37°C and 5% CO2, and the medium was changed semi-quantitatively after 24 hours, and the medium was completely changed after 48 hours. During the liquid change process, PBS was used to gently rinse the cell residues.

[0036] Rat bone marrow mesenchymal stem cells in the logarithmic growth phase were taken, the culture medium was discarded, washed twice with PBS, digested with 0.25% trypsin for 5 minutes, counted with a cell counter, and the cells were inoculated in a 96-well plate, with 5000 cells per well. After 24 hours of attachment, the culture medium was discarded and replaced with different concentrations of cimicifuga glycoside working solution (the final concentrations of cimicifuga glycoside were 0, 0.625, 1.25, 2.5, 5, 10, 20, 40, 80, and 160 μM, respectively, and cultured in a cell culture incubator at 37°C and 5% CO2 for 24h / 48h. After the culture was completed, 20 μL of MTT solution was added to each well, and the well was placed in an incubator for incubation at 37°C for 4h, the culture medium was gently discarded, 150 μL of DMSO was added to each well, and the well was placed in a shaker and shaken at room temperature for 10min, and then the well was measured using a Molecular Devices Spectra Max Plus384 microplate reader was used to measure the OD value, and the detection wavelength was set to 570nm and the reference wavelength was set to 630nm. The average value was calculated by measuring three times.

[0037] The results are as follows Figure 1 The results showed that when rat bone marrow mesenchymal cells were intervened with cimicifuga glycosides within 160 μM for 24 and 48 hours, there was no obvious growth inhibition of the cells, indicating that cimicifuga glycosides had no cytotoxicity to rat bone marrow mesenchymal stem cells; and when rat bone marrow mesenchymal cells were intervened with cimicifuga glycosides within 20 μM for 48 hours, the proliferation of bone marrow mesenchymal stem cells was promoted.

[0038] Example 2

[0039] Effects of cimicifuga glycosides on the expression of ACAN, Collagen II and SOX9 proteins in bone marrow mesenchymal stem cells

[0040] Western blot was used to detect the expression of matrix synthesis and degradation-related proteins (ACAN, SOX9, and Collagen II) in chondrocytes.

[0041] The rat bone marrow mesenchymal stem cells in the logarithmic growth phase of Example 1 were inoculated into a culture dish. After the cells adhered to the wall, the final concentrations of 0 μM, 0.1 μM, 1 μM, 2.5 μM, 5 μM, 10 μM, and 20 μM of the cimicifuga glycoside working solution were added for 48 hours. The cells were lysed on ice with RIPA lysis solution, and the cells were scraped and transferred to a centrifuge tube. Centrifugation was performed and the supernatant was taken. The protein concentration was determined by the BCA method. 10% SDS-PAGE was used to separate equal amounts of protein and transferred to a PVDF membrane, which was then blocked with 5% skim milk powder for 2 hours. After washing the membrane 3 times with TBST, the membrane was incubated with the primary antibody at 4 ° C overnight. After washing the membrane 3 times with TBST, the membrane was incubated with the corresponding secondary antibody at room temperature for 2 hours. The color was developed with a high-sensitivity ECL chemiluminescence detection kit. ImageLab was used to calculate the gray value of the target protein band relative to the gray value of the internal reference, and the result was the relative expression of the target protein of a certain sample.

[0042] The results are as follows Figure 2 As shown in the results, different concentrations of cimicifuga glycosides can increase the levels of ACAN, Collagen II and SOX9 proteins in bone marrow mesenchymal stem cells, indicating that cimicifuga glycosides can promote the chondrogenic differentiation of bone marrow mesenchymal stem cells.

[0043] Example 3

[0044] Effects of cimicifuga glycosides on the expression of ACAN and Collagen II genes in bone marrow mesenchymal stem cells

[0045] The expression of matrix synthesis and degradation-related genes ACAN and Collagen II in chondrocytes was detected by PCR. Rat bone marrow mesenchymal stem cells in the logarithmic growth phase of Example 1 were inoculated into a culture dish. After the cells adhered to the wall, the working solution of cimicifuga glycosides (the final concentration of cimicifuga glycosides was 0μM, 0.01μM, 0.1μM, 1μM, 2.5μM, 5μM, 10μM, and 20μM) was added for 48 hours, and the total cell RNA was extracted using the RNA-Quick Purification Kit. RNA was reversed into cDNA under the action of HiScript IIQRTSuperMix. The target genes ACANACAN and Collagen II were detected by real-time fluorescence quantitative PCR using the ChamQ SYBR qPCR Master Mix kit. The fold change of the target gene was based on 2 -△△Ct GAPDH mRNA was used as an internal control.

[0046] Table 1. Primer sequences of Rat-derived GAPDH, ACAN, and Collagen II genes

[0047]

[0048] The results are as follows Figure 3 As shown, when the concentration of cimicifuga glycosides was ≥0.1 μM, different concentrations of cimicifuga glycosides could increase the ACAN and Collagen II gene levels of bone marrow mesenchymal stem cells in a dose-dependent manner, further indicating that cimicifuga glycosides could promote the chondrogenic differentiation of bone marrow mesenchymal stem cells.

[0049] Example 4

[0050] Effects of cimicifuga glycosides on cytotoxicity and proliferation of rat chondrocytes

[0051] The MTT method was used to determine the effects of cimicifuga glycosides on the cytotoxicity and proliferation of rat chondrocytes.

[0052] Chondrocytes were extracted from the knee cartilage of 5-7 day old SD rats. The extraction steps were as follows: the rats were killed by cervical dislocation, placed in a 50 mL EP tube containing 75% alcohol and irradiated under ultraviolet light in a clean bench for 10 minutes, the rats were taken out and placed in a clean culture dish, the knee joints were cut off, the cartilage tissue of the knee joints was carefully peeled off, and it was washed once in PBS containing 2% double antibody, and then washed once in PBS without double antibody. The separated cartilage was placed in a 5 mL EP tube and chopped, and an appropriate amount of 0.25% trypsin was added, and digested in a cell culture incubator at 37°C and 5% CO2 for 30 minutes, centrifuged at 1000 rpm for 5 minutes, the supernatant was discarded, and an appropriate amount of 0.2% collagenase II was added to digest in a cell culture incubator at 37°C and 5% CO2 for 8-12 hours, centrifuged at 1000 rpm for 5 minutes, and the digested cells were inoculated in a cell culture dish (10 cm 2 ) and cultured in DMEM complete medium (DMEM medium + 10% fetal bovine serum + 1% penicillin-streptomycin solution). After the cells adhered for 24 hours, the old medium was discarded, PBS was added to wash twice, and fresh complete medium was added. To avoid loss of chondrocyte phenotype, chondrocytes of passages 1-3 were used.

[0053] Take rat chondrocytes in logarithmic growth period, discard the culture medium, wash twice with PBS, digest with 0.25% trypsin containing EDTA for 5min, count with cell counter, inoculate cells in 96-well plate, 5000 cells per well, adhere to the wall for 24h, discard the culture medium, add different concentrations of cimicifuga glycoside working solution (0, 0.625, 1.25, 2.5, 5, 10, 20, 40, 80, 160μM), culture in cell culture incubator at 37℃, 5% CO2 for 24h or 48h, after the culture, add 20μL MTT solution to each well, put in incubator at 37℃ for 4h, discard the culture medium gently, add 150μL DMSO to each well, put in shaker, shake at room temperature for 10min, and then measure OD value with Molecular Devices Spectra Max Plus384 microplate reader, set the detection wavelength to 570nm, and the reference wavelength to 630nm. Measure three times to get the average value.

[0054] The results are as follows Figure 4 The results showed that when chondrocytes were intervened with cimicifuga glycosides within 160 μM for 24 and 48 hours, there was no obvious growth inhibition of the cells, indicating that cimicifuga glycosides had no cytotoxicity to chondrocytes; and that intervention of chondrocytes with cimicifuga glycosides within 160 μM for 48 hours could promote chondrocyte proliferation.

[0055] Example 5

[0056] Effects of cimicifuga glycosides on the expression of proteins related to extracellular matrix degradation and synthesis in rat chondrocytes induced by IL-1β

[0057] Take rat chondrocytes in the logarithmic growth phase of Example 4, discard the culture medium, wash twice with PBS, digest with 0.25% trypsin for 5min, count with a cell counter, inoculate the cells in a culture dish, 700,000 cells per dish, adhere to the wall for 24h, discard the culture medium, and pretreat the cimicifuga glycosides group with cimicifuga glycosides working solution (the final concentration of cimicifuga glycosides was 2.5, 5, 10, and 20 μM, respectively) for 2h, and then co-treat with 10ng / mL of IL-1β for 48h, the model group was treated with 10ng / mL of IL-1β for 48h, and the blank group only needed to replace fresh culture medium, all of which were carried out in a cell culture incubator at 37°C and 5% CO2. Extract chondrocyte proteins, and detect the expression of matrix synthesis and degradation-related proteins (ACAN, SOX9, MMP13, MMP3) in chondrocytes by Western blot. Rat chondrocytes were lysed on ice with RIPA lysis buffer, and the cells were scraped and transferred to a centrifuge tube. Centrifugation was performed, and the supernatant was taken. The protein concentration was determined by the BCA method. Equal amounts of protein were separated by 10% SDS-PAGE and transferred to a PVDF membrane, which was then blocked with 5% skim milk powder for 2 hours. After washing the membrane 3 times with TBST, the membrane was incubated with the primary antibody at 4°C overnight. After washing the membrane 3 times with TBST, the membrane was incubated with the corresponding secondary antibody at room temperature for 2 hours. The color was developed using a high-sensitivity ECL chemiluminescence detection kit. ImageLab was used to calculate the gray value of the target protein band relative to the gray value of the internal reference, and the result was the relative expression of the target protein in a certain sample.

[0058] The results are as follows Figure 5 , showing that compared with the blank group, the expression of ACAN and SOX9 in the model group was decreased, and the expression of MMP-13 and MMP-3 was increased, indicating that IL-1β-induced chondrocytes can cause the expression of extracellular matrix anabolism-related proteins to decrease, and cause the expression of extracellular matrix catabolism-related proteins to increase, and this phenomenon was reversed after administration, indicating that cimicifuga glycosides can effectively inhibit the degradation of extracellular matrix.

[0059] Example 6

[0060] Effects of cimicifuga glycosides on the expression of inflammation-related genes in rat chondrocytes induced by IL-1β

[0061] The rat chondrocytes in the logarithmic growth phase of Example 4 were inoculated in a 6-well plate with 200,000 cells per dish. After 24 hours of adhesion, the cimicifuga administration group, model group, and blank group were set up respectively for administration treatment. The administration method was the same as that of Example 5. The cimicifuga administration group was first pretreated with cimicifuga working solution (the final concentration of cimicifuga was 2.5, 5, 10, and 20 μM, respectively) for 2 hours, and then co-treated with 10 ng / mL of IL-1β for 48 hours. The model group was treated with 10 ng / mL of IL-1β for 48 hours. The blank group only needed to replace the fresh culture medium. All of the above were carried out in a cell culture incubator at 37°C and 5% CO2. The total RNA of the cells was extracted using the RNA-QuickPurification Kit. The RNA was reversed into cDNA under the action of HiScript IIQRT SuperMix. ChamQ SYBR qPCR Master Mix Kit was used to detect the target genes INOS (inducible nitric oxide synthase) and COX2 (Cyclooxygenase2) by real-time fluorescence quantitative PCR. The fold change of the target gene was calculated based on 2 -△△Ct GAPDH mRNA was used as an internal control.

[0062] The results are as follows Figure 6 , showing that compared with the blank group, the expressions of INOS and COX2 in the model group were increased, indicating that IL-1β stimulation of chondrocytes can cause inflammation, and the administration of cimicifuga glycoside reversed this phenomenon in a dose-dependent manner, especially when the concentration of cimicifuga glycoside was 20μM, the reversal effect was more obvious, indicating that cimicifuga glycoside can effectively inhibit chondrocyte inflammation.

[0063] Table 2. Primer sequences of Rat-derived GAPDH, INOS, and COX2 genes

[0064]

[0065] Example 7

[0066] Effects of cimicifuga glycosides on the production of reactive oxygen species in rat chondrocytes induced by IL-1β

[0067] Take rat chondrocytes in the logarithmic growth phase of Example 4, discard the culture medium, wash twice with PBS, digest with 0.25% trypsin for 5 minutes, count with a cell counter, inoculate the cells in a culture dish, 700,000 cells per dish, adhere to the wall for 24 hours, discard the culture medium, the cimicifuga glycoside administration group is first pretreated with cimicifuga glycoside working solution (the final concentration of cimicifuga glycoside is 2.5, 5, 10, and 20 μM, respectively) for 2 hours, and then co-treated with 10 ng / mL IL-1β for 48 hours, the model group is treated with 10 ng / mL IL-1β for 48 hours, and the blank group only needs to replace the fresh culture medium, all of which are carried out in a cell culture incubator at 37°C and 5% CO2. Discard the drug-containing culture medium, add the DCFH-DA probe and incubate for 30 minutes, then add 150 μL PBS to each well, and use Confocal microscopy was used to take pictures.

[0068] The results are as follows Figure 7 , showing that the fluorescence intensity of the model group was higher than that of the blank group, indicating that the ROS of chondrocytes increased after IL-1β intervention, and the fluorescence area decreased after administration, indicating that cimicifuga glycosides inhibited the production of ROS in chondrocytes and had an antioxidant effect.

[0069] Example 8

[0070] Histological analysis to evaluate the effects of cimicifuga glycosides on the structural changes of cartilage and subchondral bone in rats with cartilage injury

[0071] 1. Establishment of animal model and administration method

[0072] All animal experimental procedures were approved by the Animal Ethics Committee of China Pharmaceutical University.

[0073] 24 SPF grade SD male rats (180-200g, purchased from Shanghai Bikai) were fed with free diet adaptability for one week, and then 18 rats were subjected to DMM (medial meniscus instability) modeling. The specific scheme was as follows: the rats were fixed after anesthesia, the hair near the knee joint of the right leg of the rats was removed, and the rats were wiped with 75% alcohol and iodine tincture. A longitudinal incision was made on the medial side of the patella at the knee joint of each rat's right leg, and then the medial meniscus was removed. Finally, the joint capsule and the skin were sutured. The same incision was used for 6 normal group mice, but the meniscus was not removed. After modeling, penicillin 100,000 U / time was given once a day for three consecutive days. After modeling for one week, 18 rats were randomly divided into: DMM model group, low-dose administration group of cimicifuga glycoside (1mg / kg), high-dose administration group of cimicifuga glycoside (2mg / kg), 6 in each group. Cimicifuga glycoside was dissolved in 40% PEG400 aqueous solution to obtain cimicifuga glycoside solution. Each rat in the high-dose and low-dose groups of cimicifuga glycosides received an intra-articular injection of 60 μL of cimicifuga glycoside solution, and the normal group and DMM model group were injected with an equal volume of 40% PEG400 aqueous solution. The injections were continued for 8 weeks, twice a week. After the end of the administration, the right knee joint was removed, the fat tissue was removed, and fixed in 4% paraformaldehyde for subsequent histological analysis.

[0074] 2. Safranin Fast Green Dyeing

[0075] The tissue fixed with 4% paraformaldehyde was decalcified in 10% EDTA for 4 weeks; after decalcification, it was dehydrated step by step with different concentrations of ethanol, then embedded in paraffin, and sampled by continuous longitudinal sections. The thickness of the sections was about 5μm, and each tissue section was 6 sheets. The obtained sections were dewaxed with xylene, and then hydrated with gradient ethanol to remove the residual xylene. After staining with safranin fast green, the sections were dehydrated with gradient ethanol, then permeabilized with xylene, and finally sealed with neutral gum. A panoramic scan was performed using a slice scanner. Safranin can bind to proteoglycans to appear red, and safranin fast green staining can intuitively reflect the changes in the structure of articular cartilage and subchondral bone. The results are shown in Figure 8 .

[0076] 3. Collagen II immunohistochemical analysis

[0077] CollagenⅡ is the main component of cartilage matrix (ECM). The tissue fixed with 4% paraformaldehyde was decalcified in 10% EDTA for 4 weeks; after decalcification, it was dehydrated step by step with different concentrations of ethanol, then embedded in paraffin, and serial longitudinal sections were taken. The thickness of the sections was about 5μm, and each tissue section was 6. Immunohistochemical staining was then performed, first incubated with 3% hydrogen peroxide for 5 minutes, then soaked in PBS, and then incubated with 10% goat serum at room temperature for 10 minutes, and collagenⅡ (Affinity, #AF0135, 1:70) primary antibody was added and incubated overnight at 4°C. The next day, it was washed three times with PBS, and then biotin-labeled secondary antibody was added and incubated at room temperature for 30 minutes. After washing with PBS, it was labeled with horseradish peroxidase for 30 minutes, then washed with PBS, DAB was used for color development, and then washed, counterstained with hematoxylin, and sealed. Results are shown in Fig. 9 .

[0078] Depend on Figure 8 It can be seen that in the normal group, the surface of the subchondral bone was flat, the boundary between cartilage and subchondral bone was clear, the tide line was complete, and the joint space was large. In the DMM model group, the color of safranin became lighter, the joint surface became rough and uneven, the cartilage was severely eroded, proteoglycans were lost, and the joint space was significantly narrowed. Compared with the DMM model group, cimicifuga glycosides can significantly improve these changes. Fig. 9 It can be seen that compared with the DMM model group, the expression level of CollagenⅡ in the high-dose administration group (2 mg / kg) of cimicifuga glycosides was significantly increased, indicating that intra-articular injection of cimicifuga glycosides can delay the degradation of ECM in DMM model rats, indicating that cimicifuga glycosides can promote cartilage repair and inhibit cartilage fibrosis to alleviate cartilage damage.

[0079] In summary, cimicifuga glycosides can promote the differentiation of bone marrow mesenchymal stem cells into cartilage. In addition, cimicifuga glycosides can not only reduce the production of reactive oxygen species related to inflammation and oxidative stress in chondrocytes, but more importantly, they can inhibit the production of cartilage extracellular matrix decomposition proteins (MMP3, MMP13), promote the production of cartilage extracellular matrix synthesis proteins (ACAN, SOX9), and protect the integrity of ECM. Intraarticular injection of cimicifuga glycosides can promote cartilage repair and inhibit cartilage fibrosis, reduce articular surface cartilage wear, increase cartilage matrix synthesis, and have a protective effect on articular cartilage. In addition, cimicifuga glycosides have the advantages of low toxicity and side effects and high safety, and have very good development and application prospects.

[0080] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various changes and modifications. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. Application of cimicifuga glycosides in the preparation of drugs for promoting the proliferation and chondrogenic differentiation of bone marrow mesenchymal stem cells.

2. The use according to claim 1, characterized in that: The application is the application of cimicifuga glycoside in the preparation of a drug for promoting the cartilage differentiation of bone marrow mesenchymal stem cells by promoting the levels of ACAN, Collagen II and SOX9 proteins.

3. The use according to claim 1, characterized in that: The application is the application of cimicifuga glycoside in the preparation of drugs for promoting the expression of ACAN and Collagen II genes and promoting the differentiation of bone marrow mesenchymal stem cells into cartilage.

4. The application of cimicifuga glycosides in the preparation of drugs for treating cartilage damage related diseases.

5. The use according to claim 4, characterized in that: The cartilage damage-related diseases are osteoarthritis and rheumatoid arthritis.

6. The use according to claim 4, characterized in that: The application is the application of cimicifuga glycoside in the preparation of a drug for treating cartilage damage diseases by inhibiting the expression of cartilage cell extracellular matrix degradation-related proteins MMP13 and MMP3.

7. The use according to claim 4, characterized in that: The application is the application of cimicifuga glycoside in the preparation of a drug for treating cartilage damage diseases by promoting the expression of cartilage cell extracellular matrix synthesis-related proteins ACAN and SOX9.

8. The use according to claim 4, characterized in that: The application is the application of cimicifuga glycoside in the preparation of medicines for treating cartilage damage diseases by inhibiting chondrocyte inflammation and oxidative stress.

9. The use according to claim 4, characterized in that: The application is the application of cimicifuga glycoside in the preparation of medicines for treating cartilage damage diseases by promoting cartilage repair and inhibiting cartilage fibrosis.

10. The use according to claim 1 or 4, characterized in that: The drug uses cimicifuga glycoside as the effective ingredient or main effective ingredient and is prepared into a pharmaceutically acceptable dosage form with a pharmaceutically acceptable carrier; the pharmaceutically acceptable carrier includes one or more of a lubricant, a binder, a filler, a disintegrant, and a lubricant.

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