Application of paeonol in preparation of medicine for treating rheumatoid arthritis

Abstract

The invention discloses application of paeonol in preparation of a medicine for treating rheumatoid arthritis, and belongs to the technical field of biological medicine. Experimental research verifies that the paeonol monomer has excellent improvement effects in reducing joint swelling degree, improving ankle joint bone destruction, improving bone loss of knee joints and inhibiting osteoclast differentiation. The invention relates to a medicine for treating rheumatoid arthritis, which takes paeonol as an active component, and has a certain relieving effect on joint swelling and bone loss of the rheumatoid arthritis, so that the joint is protected, and the aim of treating the rheumatoid arthritis is fulfilled.

Description

Technical Field

[0001] The present invention belongs to the field of biomedical technologies, and particularly relates to the application of paeonol in the preparation of drugs for treating rheumatoid arthritis. Background Art

[0002] Rheumatoid arthritis (RA) is a systemic autoimmune disease characterized by irreversible damage to articular cartilage and secondary bone erosion. It is characterized by polyarticular, symmetrical, and invasive joint inflammation of small joints in the hands and feet, often accompanied by involvement of extra-articular organs. The cause of rheumatoid arthritis is not yet clear. Modern medicine believes that rheumatoid arthritis is a disease closely related to factors such as genetics, environment, and cells.

[0003] The main purpose of treating rheumatoid arthritis is to reduce joint inflammatory reactions, inhibit the development of the disease and irreversible bone destruction, protect the functions of joints and muscles as much as possible, and ultimately achieve the goal of complete remission of the disease or low disease activity. At present, the treatment of rheumatoid arthritis mostly aims at inflammation and reducing the progression of structural damage. Non-steroidal anti-inflammatory drugs (NSAIDs) or other painkillers can relieve joint pain, but can only be used in combination with DMARD treatment.

[0004] The prior art discloses that oroxylin A can effectively inhibit paw swelling in a rheumatoid arthritis model mouse, inhibit ankle bone mass loss, inhibit the formation of RA synovitis, and inhibit cartilage destruction; the results of gene analysis show that oroxylin A can effectively reduce the expression levels of Th17 cell characteristic genes FOXP3, IL-17A, RORA, and CCR7; the application of total iridoid glycosides of Morinda officinalis How in the preparation of drugs for preventing and treating rheumatoid arthritis or osteoporosis, etc., have therapeutic effects on rheumatoid arthritis with monomeric chemical components as active ingredients.

[0005] There are also some compound formulas or those that can only have therapeutic effects after drug combination in the prior art. For example, a drug for treating rheumatoid arthritis specifically includes a JAK3 inhibitor and also includes a compound synergist; the compound synergist is a mixture of resveratrol, puerarin, and paeonol; the JAK3 inhibitor is tofacitinib, and the JAK3 inhibitor and the compound synergist are mixed in a mass ratio of 5:1, wherein the compound synergist is a mixture of resveratrol, puerarin, and paeonol in a mass ratio of 1:2:4, and experiments have proved that none of the above raw materials can be missing.

[0006] The above technologies are mostly compound formulas, compositions, or compound products, and have relatively high and complicated requirements for the processing and preparation processes of raw materials. Summary of the Invention

[0007] The object of the present invention is to provide an application of paeonol in the preparation of drugs for treating rheumatoid arthritis, specifically the application of paeonol in reducing the degree of joint swelling, improving ankle joint bone destruction, improving bone loss in the knee joint, and inhibiting osteoclast differentiation.

[0008] The second object of the present invention is to provide a drug for treating rheumatoid arthritis. The drug uses paeonol as the active ingredient, which has a certain degree of alleviating effect on joint swelling and bone loss in rheumatoid arthritis, thereby protecting joints and achieving the purpose of treating rheumatoid arthritis.

[0009] The present invention is achieved through the following technical solutions:

[0010] Application of paeonol in the preparation of drugs for treating rheumatoid arthritis.

[0011] Preferably, the rheumatoid arthritis symptoms include joint swelling, ankle joint bone destruction, knee joint bone loss, and osteoclast differentiation.

[0012] Preferably, the application of the drug in reducing the degree of joint swelling, improving ankle joint bone destruction, improving knee joint bone loss, and inhibiting osteoclast differentiation.

[0013] Preferably, the application in improving ankle joint bone destruction is specifically to reduce ankle joint bone volume, reduce bone surface area, and reduce bone surface area / bone volume.

[0014] Preferably, the application in improving bone loss in the knee joint is specifically to increase bone volume fraction, increase trabecular bone thickness, reduce trabecular bone pattern factor, reduce structure model index, and reduce trabecular bone separation.

[0015] A drug for treating rheumatoid arthritis, which is formed by mixing paeonol with pharmaceutically acceptable drug excipients.

[0016] Preferably, the mass concentration of paeonol is 6.25 - 200 μg / ml.

[0017] Preferably, the pharmaceutically acceptable excipients include carriers or excipients, disintegrants or binders.

[0018] Preferably, the drug dosage form includes powders, ointments, powders, injections, aqueous solutions, enteric-coated sustained-release preparations, or injections.

[0019] Compared with the prior art, the present invention has at least the following technical effects:

[0020] The present invention provides an application of paeonol in the preparation of drugs for treating rheumatoid arthritis.

[0021] After experimental research and verification, this paeonol monomer has excellent effects in reducing the degree of joint swelling, improving ankle joint bone destruction, improving bone loss in the knee joint, and inhibiting osteoclast differentiation.

[0022] A drug for treating rheumatoid arthritis, which uses paeonol as the active ingredient and has a certain degree of alleviating effect on joint swelling and bone loss in rheumatoid arthritis, thereby protecting joints and achieving the purpose of treating rheumatoid arthritis. Brief Description of the Drawings

[0023] Figure 1 It is a schematic diagram showing that paeonol can improve the joint swelling degree of rats with collagen-induced arthritis (CIA) model in Experiment 1, where (A) is the joint swelling diagram of experimental comparison rats in each group; (B) is the line graph of administration time and arthritis swelling index.

[0024] Figure 2 It is a schematic diagram showing that paeonol can improve the bone destruction of the ankle joint of rats with CIA model in Experiment 2, where (A) is the bone destruction diagram of the ankle joint of experimental comparison rats in each group; (B) is the schematic diagram for different degrees of bone destruction in each group.

[0025] Figure 3 It is a schematic diagram showing that paeonol can improve the bone loss of the knee joint of rats with CIA model in Experiment 3, where (A) is the bone loss diagram of the knee joint of experimental comparison rats in each group; (B) is the schematic diagram for different degrees of bone loss in the knee joint in each group.

[0026] Figure 4 It is a schematic diagram of TRAP staining showing that paeonol can inhibit osteoclast differentiation in Experiment 3. Detailed Embodiments

[0027] The following will describe the implementation schemes of the present invention in detail in combination with embodiments. However, those skilled in the art will understand that the following embodiments are only used to illustrate the present invention and should not be regarded as limiting the scope of the present invention. For the specific conditions not specified in the embodiments, they are carried out according to conventional conditions or the conditions recommended by the manufacturer. For the reagents or instruments not specified for the manufacturer, they are all conventional products that can be obtained through commercial purchase.

[0028] The technical solution of a specific implementation manner of the present invention is:

[0029] The application of paeonol in the preparation of a drug for treating rheumatoid arthritis.

[0030] The symptoms of rheumatoid arthritis include joint swelling, ankle joint bone destruction, knee joint bone loss, and osteoclast differentiation.

[0031] The application of the drug in reducing the degree of joint swelling, improving ankle joint bone destruction, improving knee joint bone loss, and inhibiting osteoclast differentiation.

[0032] The application in improving ankle bone destruction is specifically to reduce the ankle bone volume, reduce the bone surface area, and reduce the bone surface area / bone volume.

[0033] The application in improving the bone loss of the knee joint is specifically to increase the bone volume fraction, increase the trabecular thickness, reduce the trabecular pattern factor, reduce the structural model index, and reduce the trabecular separation.

[0034] The chemical structure of paeonol is as follows:

[0035]

[0036] 1. Experimental verification method:

[0037] 1. Preparation of CIA Rat Model

[0038] Take an appropriate amount of bovine type II collagen solution (concentration of 2 mg / mL) (Chondrex, item number 20022) and add it dropwise to an equal volume of incomplete Freund's adjuvant (Chondrex, item number 7002). Use a homogenizer in an ice bath to fully emulsify until it does not diffuse when dropped into water. Take 0.1 mL of the emulsified mixture and inject it intradermally at the right side of the tail base. After 7 days, use the same method and dosage to boost immunization once at the left side of the tail base of rats.

[0039] 2. Animal Grouping and Dosing

[0040] Thirty-four male SD rats aged 6 to 8 weeks were randomly selected as the normal control group after one week of adaptive feeding, and the remaining 26 rats were used for CIA modeling. The rats with successful modeling were randomly divided into three groups: CIA group, CIA+Pae group (40 mg / kg / d) (China Food and Drug Administration Researcher, Product No. 110708-201407), and methotrexate group (1.5 mg / kg, twice a week) (Solarbio, Product No. SM9600), with 8 rats in each group. The drug was administered by gavage, with a volume of 1 mL / 100 g rat body weight. The model group was given an equal amount of distilled water, and the intervention lasted for 28 days.

[0041] 3. Micro-CT scanning and analysis

[0042] The fixed rat femurs and hind paws with 4% paraformaldehyde were scanned using a Skyscan 1276 Micro-CT, and the three-dimensional images of trabecular bone were reconstructed with N-Recon software to obtain a three-dimensional model. CT-AN software was used for three-dimensional analysis to detect the following indicators: bone volume fraction (BV / TV), bone surface area (BS), bone volume (BV), trabecular thickness (Tb.Th), trabecular pattern factor (Tb.Pf), trabecular number (Tb.N), trabecular separation (Tb.Sp), and structure model index (SMI).

[0043] 4. Cell culture

[0044] RAW264.7 cells were cultured in DMEM complete medium (Gibco, catalog number C11995500BT) containing 10% fetal bovine serum (Gibco, catalog number 10099-141C) in a 37°C, 5% CO2 incubator. The cells could be passaged when they grew to 85% density in the culture flask, and were passaged once every 2 - 3 days.

[0045] 5. Osteoclast (OCs) induction and drug intervention

[0046] RAW264.7 cells were seeded in 96-well plates at a density of 2×103 cells / well, and the total volume of the medium in each well was 200 μL. After the cells were completely adherent, the old medium was discarded, and complete medium containing 50 ng / mL RANKL (R&D, catalog number 462-TEC-010) was added for induction for 48 h. Subsequently, paeonol at 6.25 μg / ml, 12.5 μg / ml, 25 μg / ml, 50 μg / ml, 100 μg / ml, and 200 μg / ml (National Institutes for Food and Drug Control, catalog number 110708-201407) was added for intervention for 48 h, and TRAP staining was performed to detect the differentiation of OCs.

[0047] 6. TRAP staining

[0048] TRAP staining was performed according to the method of the TRAP staining kit (Sigma, catalog number 378A-1KT). The specific steps of TRAP staining are as follows. First, prepare the citrate-acetone fixative: 25 ml of citrate + 65 ml of acetone + 8 ml of 37% formaldehyde, mix well, store at 4°C in a glass bottle, and let it stand at room temperature before use for fixing cells.

[0049] (1) Prepare deionized water, preheat it to 37°C for standby, and detect it with a thermometer before use.

[0050] (2) Mix 0.5 ml of Fast Gamet GBC and 0.5 ml of Sodium Nitrite Solution with a pipette for 30 s, and let it stand for 2 min.

[0051] (3) Mix the following reagents in the following proportions to obtain the TRAP staining solution: 45 ml of deionized water + 1 ml of the liquid obtained in step (2) + 0.5 ml of Naphthol AS-BI + 2 ml of acetate Solution + 1 ml of tartrate solution.

[0052] (4) Preheat the TRAP staining solution to 37 °C under light protection and detect it with a thermometer before use.

[0053] (5) Place the prepared citrate-acetone fixing solution at room temperature in advance. Take out the cells from the CO2 incubator, drop sufficient fixing solution into each well of the 96-well plate to completely cover the cells, and wash them 3 times with deionized water after 10 min (do not allow the cells to dry during the staining process).

[0054] (6) After cell fixation, take the preheated TRAP staining solution at 37 °C and drop sufficient staining solution into each well of the 96-well plate to completely cover the cells, and incubate at 37 °C in the dark for 40 min.

[0055] (7) Wash 3 times with deionized water, observe and take pictures under a microscope. The cytoplasm of osteoclasts successfully induced is purple-red, and there are multiple nuclei, which are osteoclast-like cells.

[0056] II. Experimental conclusions:

[0057] Experiment 1: Paeonol can improve the joint swelling degree of rats in the collagen-induced arthritis (CIA) model

[0058] As Figure 1 shown, it is a schematic diagram of paeonol having the effect of improving the joint swelling degree of rats in the CIA model. Among them, (A) is the joint swelling diagram of experimental comparison rats in each group; (B) is the line graph of the administration time and the arthritis swelling index. Note: Compared with the normal group, ##p < 0.01; compared with the model group, *p < 0.015, **p < 0.01.

[0059] As Figure 1 shown in (A) therein, compared with the normal group, the hind paw joint of the model group rats was severely swollen, and the arthritis index increased significantly. Compared with the model group, paeonol can significantly improve the joint swelling of CIA rats.

[0060] As Figure 1 shown in (B) therein, paeonol can significantly reduce the arthritis index.

[0061] Experiment 2: Paeonol can improve the bone destruction of the ankle joint of rats in the CIA model

[0062] As Figure 2As shown in the figure, it is a schematic diagram of paeonol improving bone destruction in the ankle joints of CIA model rats. Among them, (A) is the Micro-CT scan of the ankle joints of experimental rats in each group; (B) is the comparison of three-dimensional analysis data of BV, BS, and BS / BV of the ankle joints of rats in each group. Note: Compared with the normal group, ##p < 0.01; compared with the model group, *p < 0.015, **p < 0.01, ***p < 0.001.

[0063] Combined with Figure 2 (A), it can be seen that compared with the normal group, the bone destruction in the ankle joints of rats in the model group is severe; compared with the model group, paeonol can significantly improve the bone destruction in CIA rats.

[0064] Combined with Figure 2 (B), it can be seen that compared with the normal group, the bone volume (BV), bone surface area (BS), and bone surface area / bone volume (BS / BV) in the ankle joints of rats in the model group are all significantly increased. Compared with the model group, paeonol can significantly reduce BV, BS, and BS / BV.

[0065] Experiment 3: Paeonol can improve bone loss in the knee joints of CIA model rats

[0066] As Figure 3 shown in the figure, it is a schematic diagram of paeonol improving bone loss in the knee joints of CIA model rats. Among them, (A) is the Micro-CT scan of the knee joints of experimental rats in each group; (B) is the comparison of three-dimensional analysis data of BV / TV, Tb.Th, Tb.N, Tb.Pf, SMI, and Tb.Sp of the knee joints of rats in each group. Note: Compared with the normal group, ##p < 0.01, p < 0.001; compared with the model group, *p < 0.05, ***p < 0.001; compared with the paeonol group, $p < 0.05, $$p < 0.01.

[0067] Combined with Figure 3 (A), it can be seen that compared with the normal group, the bone loss in the knee joints of rats in the model group is severe; compared with the model group, paeonol can significantly improve the bone loss in the knee joints of CIA rats.

[0068] Combined with Figure 3 (B), it can be seen that compared with the normal group, the bone volume fraction (BV / TV) and trabecular bone thickness (Tb.Th) of rats in the model group are significantly reduced, and the trabecular bone number (Tb.N) is significantly decreased; compared with the model group, paeonol can significantly increase BV / TV and significantly increase Tb.N.

[0069] Compared with the normal group, the trabecular bone pattern factor (Tb.Pf) and structure model index (SMI) of rats in the model group are significantly increased. Compared with the model group, paeonol can significantly reduce Tb.Pf and SMI in CIA rats.

[0070] Compared with the normal group, the trabecular bone separation (Tb.Sp) of rats in the model group increased. Compared with the model group, paeonol could reduce the Tb.Sp of CIA rats, but there was no statistical difference.

[0071] Experiment 4: Paeonol can inhibit osteoclast differentiation

[0072] As Figure 4 shown, it is a schematic diagram of TRAP staining showing that paeonol has the effect of inhibiting osteoclast differentiation. The scale bar is 200 μm and the magnification is 100 times. Note: Compared with the 0 μg / ml group, *p < 0.05, **p < 0.01. Among them, (A) is the result graph of osteoclast TRAP staining; (B) is the statistical graph of the number of TRAP-positive osteoclasts.

[0073] Combined Figure 4 it can be seen that the results of TRAP staining showed that paeonol (6.25 μg / ml, 12.5 μg / ml, 25 μg / ml, 50 μg / ml, 100 μg / ml, 200 μg / ml) could significantly inhibit the differentiation of osteoclasts.

[0074] Finally, it should be noted that the above are only the preferred embodiments of the present invention and are not used to limit the protection scope of the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. Application of paeonol in the preparation of drugs for the treatment of rheumatoid arthritis.

2. The use according to claim 1, characterized in that: The symptoms of rheumatoid arthritis include joint swelling, ankle bone destruction, knee bone loss and osteoclast differentiation.

3. The use according to claim 1, characterized in that: The drug is used in reducing the degree of joint swelling, improving ankle joint bone destruction, improving knee joint bone loss and inhibiting osteoclast differentiation.

4. The use according to claim 3, characterized in that: The application in improving ankle bone destruction is specifically to reduce the ankle bone volume, reduce the bone surface area, and reduce the bone surface area / bone volume.

5. The use according to claim 3, characterized in that: The application in improving the bone loss of the knee joint is specifically to increase the bone volume fraction, increase the trabecular thickness, reduce the trabecular pattern factor, reduce the structural model index, and reduce the trabecular separation.

6. A drug for treating rheumatoid arthritis, characterized in that: The medicine comprises the paeonol as claimed in claim 1 mixed with pharmaceutically acceptable excipients.

7. A drug for treating rheumatoid arthritis according to claim 6, characterized in that: The mass concentration of the paeonol is 6.25-200 μg / ml.

8. The drug for treating rheumatoid arthritis according to claim 6, characterized in that: The pharmaceutically acceptable excipients include carriers or excipients, disintegrants or binders.

9. The drug for treating rheumatoid arthritis according to claim 6, characterized in that: The drug dosage forms include powders, ointments, powders, injections, aqueous solutions, enteric-coated sustained-release preparations or injections.

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