Use of a traditional Chinese medicine composition in the preparation of a medicine for treating rheumatoid arthritis

Abstract

The application discloses application of a traditional Chinese medicine composition in preparation of a medicine for treating rheumatoid arthritis, and belongs to the technical field of traditional Chinese medicines. The traditional Chinese medicine composition is prepared from the following raw medicinal materials in parts by weight: 20-60 parts of smilax glabra, 10-20 parts of achyranthes bidentata, 15-30 parts of radix paeoniae alba, 10-30 parts of giant knotweed, 6-20 parts of cortex fraxini, 3-10 parts of rhizoma coptidis, 6-20 parts of phellodendri chinensis, 10-30 parts of lily, and 6-20 parts of radix tinosporae. The traditional Chinese medicine composition can significantly improve the joint redness and swelling of a rheumatoid arthritis model animal, reduce an arthritis index, inhibit systemic and local joint inflammatory reactions, reduce synovial membrane proliferation, cartilage erosion and bone destruction, and is safe in use, thereby providing a safe and effective new scheme for clinical treatment of rheumatoid arthritis.

Description

Technical Field

[0001] This invention belongs to the field of traditional Chinese medicine technology and relates to the application of a traditional Chinese medicine composition in the preparation of a drug for treating rheumatoid arthritis. Background Technology

[0002] Rheumatoid arthritis (RA) is a common chronic erosive autoimmune joint disease in clinical practice. Its core pathological feature is symmetrical, erosive synovitis, accompanied by abnormal synovial proliferation, inflammatory cell infiltration, and progressive destruction of articular cartilage and bone tissue, which can eventually lead to joint deformity, ankylosis, and limb disability.

[0003] Currently, the core of Western medicine clinical treatment for rheumatoid arthritis (RA) is disease-modifying anti-rheumatic drugs (DMARDs), with methotrexate (MTX) being the first-line anchoring drug. Other drugs include leflunomide, biologics, and glucocorticoids. While these drugs can control inflammatory symptoms and slow disease progression to some extent, they still have significant clinical limitations: traditional synthetic DMARDs have adverse reactions such as liver and kidney toxicity, bone marrow suppression, and gastrointestinal reactions, and long-term tolerability is poor; biologics and targeted drugs are expensive and pose risks such as increased infection risk and inducing autoimmune reactions, with some patients experiencing poor or no response, failing to meet the clinical needs of RA patients for long-term, safe treatment.

[0004] Rheumatoid arthritis (RA) falls under the categories of "arthritis" and "arthralgia" in Traditional Chinese Medicine (TCM). TCM treatment of RA has garnered significant attention due to its advantages of being "universal, simple, inexpensive, and effective." Our research group has long adhered to the principle of "returning classics to clinical practice." Based on the pathogenesis of RA patients—characterized by "wind-dampness obstruction, tendon and bone damage"—we have proposed a treatment method of "dispelling wind and dampness, promoting blood circulation, unblocking meridians, strengthening the spleen and kidneys, and strengthening tendons and bones" (i.e., the "dampness-dispelling and meridian-unblocking method"), and have conducted a series of clinical and experimental studies on this approach. TCM treatment of RA has a long history and boasts advantages such as multi-target, multi-pathway, holistic regulation, and high safety. Currently, several TCM compound formulas for treating RA are publicly available. However, existing formulas often suffer from problems such as complex formulations, unclear targets, lack of standardized pharmacodynamic verification, and inability to simultaneously address anti-inflammatory and analgesic effects while protecting joint structure. Therefore, they still cannot fully meet the diverse clinical treatment needs of RA patients.

[0005] Therefore, whether in the fields of Western medicine or traditional Chinese medicine, existing RA treatment plans cannot fully meet the needs of long-term, safe and effective clinical treatment. There is an urgent need to develop a treatment drug with a simple and clear formulation that can effectively control systemic and local joint inflammation of RA, protect articular cartilage and bone tissue, delay disease progression, and has good safety. Summary of the Invention

[0006] In view of the problems of existing RA treatment drugs, the purpose of this invention is to provide an application of a traditional Chinese medicine composition in the preparation of RA treatment drugs, so as to solve the problems in the prior art.

[0007] To achieve the above objectives, the present invention adopts the following technical solution.

[0008] This invention discloses the application of a traditional Chinese medicine composition in the preparation of a drug for treating rheumatoid arthritis (RA). The traditional Chinese medicine composition includes Smilax glabra, Achyranthes bidentata, Astragalus membranaceus, Polygonum cuspidatum, Fraxinus chinensis, Coptis chinensis, Phellodendron chinense, Lilium brownii, and Liquidambar formosana.

[0009] Furthermore, the traditional Chinese medicine composition is made from the following raw materials in parts by weight: 20-60 parts of Smilax glabra, 10-20 parts of Achyranthes bidentata, 15-30 parts of Astragalus membranaceus, 10-30 parts of Polygonum cuspidatum, 6-20 parts of Fraxinus chinensis, 3-10 parts of Coptis chinensis, 6-20 parts of Phellodendron chinense, 10-30 parts of Lilium brownii, and 6-20 parts of Liquidambar formosana.

[0010] The formulation of the traditional Chinese medicine composition of this invention closely aligns with the core pathogenesis of RA, namely "rheumatism and blood stasis, tendon and bone injury." The formulation principle is based on the treatment approach of "dispelling wind and dampness, promoting blood circulation, clearing the meridians, strengthening the spleen and kidneys, and strengthening tendons and bones." The entire formula addresses both the root cause and symptoms, supporting the body's resistance without suppressing pathogenic factors, and eliminating pathogenic factors without harming the body's vital energy. The compatibility and synergistic effects of the various raw materials are as follows: This formula heavily utilizes Smilax glabra, a herb with a sweet and neutral flavor, as the principal ingredient. It leverages its properties of eliminating dampness and turbidity, strengthening the middle jiao (spleen and stomach), fortifying muscles and bones, and promoting joint mobility. Rheumatoid arthritis (RA) is primarily caused by dampness, which contributes to its recurrence. Smilax glabra, with its sweet and neutral properties, effectively eliminates deep-seated dampness and turbidity in the muscles and bones, cutting off the root cause of the persistent disease. Furthermore, it promotes joint mobility, directly addressing the core pathological aspects of RA, such as synovial hyperplasia and joint swelling. Simultaneously, it strengthens muscles and bones, addressing the underlying pathogenesis of "muscle and bone damage" while eliminating pathogenic factors. This aligns perfectly with the principle of "eliminating dampness and unblocking the meridians," achieving both symptomatic and root-cause treatment.

[0011] The auxiliary herbs selected are Achyranthes bidentata, Astragalus membranaceus, and Polygonum cuspidatum, which work precisely with the principal herb, Smilax glabra. Achyranthes bidentata is good at promoting blood circulation and unblocking meridians, and facilitating joint movement, addressing the pathological changes of local blood stasis in RA joints. It also nourishes the liver and kidneys, strengthens tendons and bones, directly targeting the pathogenesis of "tendon and bone damage" in RA, thus compensating for the deficiency of existing similar compound formulas that "emphasize dampness removal and meridian unblocking, but neglect the root cause of bone damage." When used together with the principal herb, Smilax glabra, one treats the symptoms by eliminating dampness and turbidity, while the other strengthens tendons and kidneys to consolidate the root cause, achieving the goal of eliminating pathogens without harming the body's vital energy and supporting the body's vital energy without retaining pathogens. Astragalus membranaceus is good at tonifying qi and strengthening the spleen, and promoting diuresis and reducing swelling. RA patients suffer from long-term chronic inflammation and continuous consumption of vital energy, resulting in a deficiency of vital energy. They are unable to expel pathogens and are prone to internal dampness and turbidity. At the same time, long-term use of existing Western medicines can easily damage the spleen and stomach's vital energy. Astragalus can warm and tonify the body's vital energy, assisting the principal herb in strengthening the middle jiao (spleen and stomach), eliminating dampness and turbidity, and improving the body's consumption and weight loss caused by chronic inflammation in rheumatoid arthritis (RA), reducing the risk of gastrointestinal damage from long-term medication, and meeting the safety requirements of long-term RA treatment. Polygonum cuspidatum (Hu Zhang) is good at promoting blood circulation, removing blood stasis, eliminating dampness and unblocking the meridians, and clearing heat and detoxifying. It can not only assist the principal herb in removing rheumatic and turbid pathogens from the joints, but also disperse meridian stagnation, improving redness, swelling, heat, pain, and difficulty in flexion and extension of RA joints. At the same time, its heat-clearing and detoxifying effects can clear local heat in the joints and inhibit the inflammatory cascade reaction. When used with Achyranthes bidentata (Niu Xi), its blood-activating and meridian-unblocking effects are doubled, directly targeting the core pathogenesis of RA, "rheumatic stagnation." The three auxiliary herbs together cover the three core pathogenesis of RA: "dampness and stagnation, deficiency of vital energy, and malnourishment of tendons and bones," synergistically achieving multiple effects of "eliminating dampness, activating blood, strengthening the body, and strengthening bones," solving the core clinical pain point that existing compound formulas cannot simultaneously address anti-inflammatory and analgesic effects while protecting joint structure.

[0012] The formula is supplemented with bitter and cold herbs such as Fraxinus bark, Coptis chinensis, and Phellodendron amurense to clear heat and dry dampness. Redness, swelling, heat, and pain in the joints are common during the active phase of rheumatoid arthritis (RA), indicating dampness transforming into heat and the accumulation of heat toxins. The combination of these three herbs can clear local heat toxins in the joints and reduce redness and swelling due to synovial inflammation, thus addressing the pathological state during acute RA exacerbations. Furthermore, the addition of lily bulb, which is sweet, cold, and moistening, can prevent the bitter, cold, and drying herbs from damaging the body's yin fluids. It also nourishes the heart and lungs, calms the mind, and stabilizes the spirit. This addresses both the pulmonary fibrosis caused by interstitial pneumonia, a common complication in RA patients, and the mental anxieties resulting from long-term pain in RA patients.

[0013] The formula uses *Luffa cylindrica* as its guiding herb, leveraging its ability to "unblock the twelve meridians," thus promoting blood circulation and directing the medicinal ingredients directly to the affected area. Respiratory ulcers (RA) can affect multiple joints throughout the body and even damage internal organs. Patients often experience symptoms such as joint stiffness, limb numbness, and weakness due to stagnation of qi and blood in the meridians. *Luffa cylindrica*, with its ability to move and circulate freely, can guide the medicinal power of the entire formula through the twelve meridians, ensuring that the effects reach all parts of the body, enhancing the overall efficacy of the formula, and better relieving the patient's discomfort such as joint stiffness, swelling, pain, limb numbness, and weakness.

[0014] The formula works together to expel the turbid evils of wind-dampness and blood stasis, clear obstructed blood vessels, strengthen weak muscles and bones, and relieve swelling and pain in the joints. Hence, it is named the Turbidity-Expelling and Pain-Relieving Formula.

[0015] The dosage ranges of each active pharmaceutical ingredient in this invention are all based on the core pathogenesis of RA, the pathological characteristics of the entire disease course, and the staged treatment needs. They are precisely set in combination with the medicinal properties and clinical safe medication window. The dosage design is highly compatible with the disease treatment goals of RA, as detailed below: 20-60 parts of Smilax glabra (all parts mentioned below refer to weight): This dosage range precisely matches the core pathogenesis of RA, namely "rheumatism and blood stasis, tendon and bone injury". 20 doses is the starting dose, which can invigorate the spleen and stomach, clear away dampness and turbidity, and cut off the root cause of RA's internal dampness and turbidity, leading to recurrent and prolonged disease. Approximately 40 doses is the commonly used effective dose, which can promote joint mobility, inhibit abnormal synovial proliferation, and significantly improve the core symptoms of joint redness, swelling, heat, pain, and difficulty in flexion and extension. 60 doses is the enhanced dose, which can penetrate deep into the muscles and bones, help protect articular cartilage and bone tissue, delay bone erosion and structural damage, and is within the safe medication window of being mild and non-toxic throughout the entire course, perfectly meeting the efficacy and safety requirements of long-term treatment of RA. If the dosage is less than 20 doses, the medicinal power will be insufficient to effectively clear the dampness, turbidity, and stasis deep in the muscles and bones, making it difficult to achieve a stable therapeutic effect. If the dosage is more than 60 doses, it is easy to excessively promote dampness, deplete the body's yin fluids and vital energy, violate the principle of the whole formula of supporting the body's resistance without suppressing pathogens and eliminating pathogens without harming the body's resistance, and may also increase the risk of gastrointestinal discomfort with long-term use.

[0016] Achyranthes bidentata (10-20 parts): Prolonged RA inevitably damages the liver and kidneys, leading to irreversible damage to tendons and bones. The dosage of 10-20 parts is designed to tonify the liver and kidneys and strengthen tendons and bones, aiming to intervene in the core pathological outcome of bone destruction in RA. A dosage below 10 parts would be insufficient to effectively slow the bone erosion process; a dosage above 20 parts would be too strong in promoting blood circulation and regulating menstruation, potentially affecting the menstrual cycle of female patients, leading to excessive menstrual flow and prolonged periods.

[0017] Astragalus membranaceus (Huang Qi) 15-30 parts: RA is a chronic wasting disease, and patients often experience qi deficiency, emaciation, and low immunity. A dosage of 15-30 parts utilizes its qi-tonifying, exterior-strengthening, diuretic, and swelling-reducing effects. This not only supports the body's vital energy to expel toxins but also improves the nutritional status and immune function of RA patients. A dosage below 15 parts will not be sufficient to tonify qi and will be difficult to correct the prevalent qi deficiency in RA patients; a dosage above 30 parts will be excessively tonifying, potentially promoting dampness and heat, which is detrimental to the elimination of dampness and turbidity.

[0018] Polygonum cuspidatum (10-30 parts): In the acute phase of rheumatoid arthritis (RA), joint redness, swelling, heat, and pain indicate a combination of dampness, blood stasis, and heat. A dosage of 10-30 parts allows for flexible adjustment of its blood-activating, heat-clearing, and dampness-removing effects. For patients with significant joint swelling and pain, up to 30 parts can be used to enhance blood circulation, clear heat, and detoxify, quickly controlling inflammation. For patients with stable conditions, 10-15 parts can be used to maintain the effect of clearing the meridians and preventing the recurrence of blood stasis. A dosage below 10 parts will be insufficient to effectively relieve joint swelling and pain; a dosage above 30 parts will have excessive bitter and cold properties, easily damaging the spleen and stomach's yang energy and affecting long-term drug tolerance.

[0019] Fraxinus bark 6-20 parts, Coptis chinensis 3-10 parts, Phellodendron chinense 6-20 parts: These three bitter and cold herbs are used as adjuvants to target the damp-heat toxins causing "redness, swelling, heat, and pain" in the RA joints. The dosage of the three herbs needs to be coordinated. If the joints are significantly red, swollen, hot, and painful during an attack, and inflammatory indicators such as C-reactive protein and erythrocyte sedimentation rate are significantly elevated, a high dose (15-20 parts) of Phellodendron chinense and Fraxinus bark and a medium-to-high dose (6-10 parts) of Coptis chinensis can be used to effectively clear heat, dry dampness, and directly counteract the inflammation. If the heat symptoms are not obvious after the condition is relieved, or if there is spleen and stomach deficiency and cold, a low dose (6-10 parts of Fraxinus bark and Phellodendron chinense, 3-5 parts of Coptis chinensis) should be used to take advantage of their drying effect and avoid their bitter and cold nature, reflecting the principle of "eliminating pathogens without harming the body's vital energy" in the formulation.

[0020] Lily bulb (10-30 parts): RA patients often suffer from insomnia due to prolonged pain and restlessness. Interstitial pneumonia is a common complication of RA. A dosage of 10-30 parts can nourish the heart and lungs while preventing the consumption of yin fluids by bitter, cold, and warming herbs, thus playing a dual role of "adjuvant" and "yin protection." A dosage below 10 parts will be insufficient in nourishing yin, calming the mind, and moistening the lungs, making it difficult to counteract the yin-damaging effects of the drying and dampness-inducing herbs in the formula; a dosage above 30 parts will be too rich and cloying, hindering the transformation and transportation of dampness and turbidity.

[0021] Lulutong (6-20 parts): RA lesions are located in the joints of the limbs, and only products that promote blood circulation and unblock the meridians can guide the medicine directly to the affected area. A dosage of 6-20 parts utilizes its property of "unblocking the twelve meridians," which can both assist the principal and assistant medicines in promoting blood circulation and unblocking the meridians, and guide the medicinal power of other medicines to circulate throughout the body's meridians. A dosage below 6 parts will not effectively unblock the meridians, making it difficult to exert the function of "guiding the medicine to the meridians"; a dosage above 20 parts will have an excessively strong dispersing and penetrating effect, easily depleting qi and blood, which is not conducive to the "strengthening the body's resistance and consolidating the foundation" treatment needs of the chronic phase of RA.

[0022] Furthermore, the traditional Chinese medicine composition is made from the following raw materials in parts by weight: 40-60 parts of Smilax glabra, 10-20 parts of Achyranthes bidentata, 20-30 parts of Astragalus membranaceus, 15-25 parts of Polygonum cuspidatum, 10-15 parts of Fraxinus chinensis, 5-10 parts of Coptis chinensis, 10-15 parts of Phellodendron chinense, 10-20 parts of Lilium brownii, and 10-15 parts of Liquidambar formosana.

[0023] In some specific embodiments, the traditional Chinese medicine composition is made from the following raw materials in parts by weight: 60 parts of Smilax glabra, 10 parts of Achyranthes bidentata, 20 parts of Astragalus membranaceus, 15 parts of Polygonum cuspidatum, 10 parts of Fraxinus chinensis, 5 parts of Coptis chinensis, 10 parts of Phellodendron chinense, 10 parts of Lilium brownii, and 10 parts of Liquidambar formosana.

[0024] In some specific embodiments, the traditional Chinese medicine composition is made from the following raw materials in parts by weight: 60 parts of Smilax glabra, 20 parts of Achyranthes bidentata, 30 parts of Astragalus membranaceus, 30 parts of Polygonum cuspidatum, 20 parts of Fraxinus chinensis, 10 parts of Coptis chinensis, 20 parts of Phellodendron chinense, 30 parts of Lilium brownii, and 20 parts of Liquidambar formosana.

[0025] In some specific embodiments, the traditional Chinese medicine composition is made from the following raw materials in parts by weight: 20 parts of Smilax glabra, 10 parts of Achyranthes bidentata, 15 parts of Astragalus membranaceus, 10 parts of Polygonum cuspidatum, 6 parts of Fraxinus chinensis, 3 parts of Coptis chinensis, 6 parts of Phellodendron chinense, 10 parts of Lilium brownii, and 6 parts of Liquidambar formosana.

[0026] Furthermore, the preparation method of the traditional Chinese medicine composition includes the following steps: weighing the raw materials according to the ratio, extracting with water, and collecting the extract to obtain the final product.

[0027] Furthermore, the drug is used to inhibit RA-mediated systemic and local joint inflammatory responses.

[0028] Furthermore, the drug is used to reduce RA-mediated inflammatory cell infiltration and synovial hyperplasia in the synovial tissue of joints.

[0029] Furthermore, the drug is used to reduce articular cartilage erosion and bone destruction caused by RA, increase joint bone density, improve joint bone microstructure, and delay joint structural damage and functional loss.

[0030] Furthermore, the dosage form of the drug is decoction, granules, pills, capsules, tablets, powders, mixtures, or oral liquids.

[0031] Furthermore, the drug also includes pharmaceutically acceptable excipients.

[0032] Furthermore, the content of the traditional Chinese medicine composition in the drug is 1wt% to 99wt%.

[0033] Furthermore, the pharmaceutically acceptable excipients include one or more of the following: flavoring agents, preservatives, diluents, binders, disintegrants, lubricants, fragrances, excipients, wetting agents, encapsulating materials, plasticizers, opacifiers, colorants, coating materials, antioxidants, solubilizers, or suspending agents.

[0034] Specifically, the preservative includes one or more of benzoic acid, sodium benzoate, sorbic acid, and potassium sorbate; the diluent includes one or more of lactose, sucrose, starch, dextrin, and mannitol; the binder includes one or more of povidone, hydroxypropyl methylcellulose, and starch paste; the disintegrant includes one or more of sodium carboxymethyl starch, low-substituted hydroxypropyl cellulose, crospovidone, sodium bicarbonate, and citric acid; the lubricant includes one or more of magnesium stearate, micronized silica gel, talc, and stearic acid; and the fragrance includes one or more of tangerine peel volatile oil, menthol, borneol, and flavoring. The excipients include one or more of honey, rice paste, flour paste, water, and medicinal juice; the capsule material includes gelatin or hydroxypropyl methylcellulose; the plasticizer includes glycerin or sorbitol; the opacifier includes one or more of titanium dioxide, iron oxides, zinc oxide, and barium sulfate; the coating material includes sugar coating or film coating; the antioxidant includes one or more of sodium sulfite, vitamin C, sodium thiosulfate, and propyl gallate; the solubilizer includes one or more of polysorbate-80, poloxamer, and lecithin; and the suspending agent includes one or more of sodium carboxymethyl cellulose, gum arabic, glycerin, and gelatin.

[0035] Compared with the prior art, the present invention has the following beneficial effects: The dosage range of the raw materials in the herbal composition of this invention is based on the core pathogenesis of RA (rheumatoid arthritis) of "wind-dampness obstruction, tendon and bone injury," combined with the medicinal properties of each herb and the synergistic effects of the principal, assistant, adjuvant, and guide herbs. This was determined through multiple rounds of pharmacodynamic screening and dose gradient control. The precise proportions of each herb are tailored to the treatment needs throughout the entire course of RA, achieving a balance between maximizing efficacy and medication safety. The application of this herbal composition in the preparation of drugs for treating RA has been validated in a mouse model of collagen-induced arthritis (CIA). The herbal composition significantly improves the core symptoms of RA model animals, such as joint redness and swelling, and limited mobility; effectively reduces the arthritis index and paw edema thickness; inhibits the progressive progression of arthritis; and significantly alleviates disease-mediated weight loss, improving the body's consumption caused by systemic inflammatory response. The traditional Chinese medicine composition of this invention can dose-dependently increase the level of interleukin-10 (IL-10), a key anti-inflammatory cytokine for rheumatoid arthritis (RA), in the serum of model animals, and decrease the levels of tumor necrosis factor-α (TNF-α), IL-6, and IL-1β, key pro-inflammatory cytokines for RA, in the serum of model animals. The high-dose group can restore TNF-α levels to near-normal levels, thus fundamentally blocking the inflammatory cascade response of the disease. Pathological histological examination results show that the traditional Chinese medicine composition of this invention can significantly reduce abnormal synovial hyperplasia and inflammatory cell infiltration in the knee and ankle joints of model animals, demonstrating a clear intervention effect on the pathological changes of autoimmune synovitis at the core of RA. The high-dose group showed a superior inhibitory effect on synovial inflammation compared to the first-line clinical drug MTX.

[0036] Targeting the characteristic cartilage erosion and bone destruction pathological process of rheumatoid arthritis (RA), the traditional Chinese medicine composition of this invention can effectively protect the integrity of articular cartilage, significantly reduce cartilage erosion and bone destruction in the knee and ankle joints, and the high-dose group shows a better protective effect on cartilage than the first-line clinical drug methotrexate (MTX). Microscopic CT scanning and bone metabolism index analysis confirm that the traditional Chinese medicine composition of this invention can significantly increase joint bone mineral density and bone volume fraction in model animals, improve the pathological state of sparse and disordered trabeculae, reduce bone erosion, bone loss and pathological bone hyperplasia, inhibit damage to joint bone microstructure, and fundamentally delay the joint structural destruction and functional loss caused by RA. Simultaneously, the traditional Chinese medicine composition of this invention can significantly improve inflammation-related abnormal platelet elevation in model animals, has no adverse effects on peripheral blood leukocyte and lymphocyte levels, and shows no significant toxic damage in liver and kidney tissue pathological examination. It has excellent drug safety, can meet the clinical needs of long-term treatment for RA patients, and has good clinical application prospects and industrial transformation value. Attached Figure Description

[0037] To more clearly illustrate the technical solutions of the embodiments of this application or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of this application. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0038] Figure 1 The figure shows the effect of different treatment groups on the degree of swelling of the ankle joint in CIA mice.

[0039] Figure 2 The graphs show the effects of different treatment groups on body weight, paw edema thickness, and arthritis index (AI) in CIA mice; among them, Figure 2 In the figure, A represents the effect curves of different treatment groups on AI at different time points in CIA mice; Figure 2 B in the figure represents the effect curves of different treatment groups on paw edema thickness at different time points in CIA mice; Figure 2 In the figure, C represents the effect curves of different treatment groups on the body weight of CIA mice at different time points.

[0040] Figure 3 Pathological images showing the effects of different treatment groups on H&E staining of the knee and ankle joints of CIA mice.

[0041] Figure 4 Pathological images showing the effects of different treatment groups on safranin-fast green staining of the knee and ankle joints of CIA mice.

[0042] Figure 5 Micro-CT reconstruction images of the knee and ankle joints of CIA mice in different treatment groups.

[0043] Figure 6 Statistical graph showing the effects of different treatment groups on bone mineral density and related indicators of the knee and ankle joints in CIA mice; among them... Figure 6 In the figure, A represents the statistical graph of the effects of different treatment groups on the bone volume fraction (BV / TV) of the knee joint in CIA mice; Figure 6 B in the figure represents the statistical graph of the effect of different treatment groups on the ratio of bone surface area to bone volume (BS / BV) of the knee joint in CIA mice; Figure 6 C in the figure represents the statistical graph of the effect of different treatment groups on the trabecular bone space (Tb.Sp) of the knee joint in CIA mice; Figure 6 D in the figure represents the statistical graph of the effects of different treatment groups on the bone mineral density (BMD) of the knee joint in CIA mice; Figure 6 E in the figure represents the statistical graph of the effect of different treatment groups on the bone volume fraction (BV / TV) of the ankle joint in CIA mice; Figure 6F in the figure represents the statistical graph of the effect of different treatment groups on the ratio of bone surface area to bone volume (BS / BV) of the ankle joint in CIA mice; Figure 6 G in the figure represents the statistical graph of the effect of different treatment groups on Tb.Sp in the ankle joint of CIA mice; Figure 6 H in the figure represents the statistical graph of the effects of different treatment groups on ankle bone mineral density (BMD) in CIA mice.

[0044] Figure 7 A statistical graph showing the effects of different treatment groups on the levels of inflammatory cytokines in the serum of CIA mice; among them, Figure 7 In the figure, A represents the statistical graph showing the effect of different treatment groups on the serum TNF-α level of CIA mice; Figure 7 B in the figure represents the statistical graph showing the effect of different treatment groups on the serum IL-6 level in CIA mice; Figure 7 C in the figure represents the statistical graph showing the effect of different treatment groups on the serum IL-10 level in CIA mice; Figure 7 D in the figure represents the statistical effect of different treatment groups on the serum IL-1β level of CIA mice.

[0045] Figure 8 A statistical chart showing the effects of different treatment groups on key indicators of peripheral blood routine tests in CIA mice; among them, Figure 8 In the figure, A represents the statistical graph showing the effect of different treatment groups on the white blood cell count (WBC) in CIA mice. Figure 8 B in the figure represents the statistical graph of the effects of different treatment groups on the peripheral blood lymphocyte count (Lym) of CIA mice; Figure 8 C in the figure represents the statistical graph of the effects of different treatment groups on peripheral blood hemoglobin (HGB) content in CIA mice; Figure 8 D in the figure represents the statistical graph showing the effect of different treatment groups on the platelet count (PLT) in the peripheral blood of CIA mice.

[0046] Figure 9 Pathological images showing the effects of different treatment groups on H&E staining of liver and kidney tissues in CIA mice. Detailed Implementation

[0047] The experimental methods described in the following embodiments of the present invention, unless otherwise specified, are generally performed under conventional conditions or as recommended by the manufacturer.

[0048] Unless otherwise defined, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The terminology used in this specification is for the purpose of describing particular embodiments only and is not intended to limit the invention.

[0049] The following description is based on specific embodiments.

[0050] Example 1: Preparation of Traditional Chinese Medicine Composition A method for preparing a traditional Chinese medicine composition (also known as Xiezhuo Qutong Fang, XZQTF) is as follows: Take 60g of Smilax glabra, 10g of Achyranthes bidentata, 20g of Astragalus membranaceus, 15g of Polygonum cuspidatum, 10g of Fraxinus chinensis, 5g of Coptis chinensis, 10g of Phellodendron chinense, 10g of Lilium brownii, and 10g of Liquidambar formosana. Prepare the decoction according to clinical methods. For the first decoction, soak the herbs thoroughly in 10 times their volume of deionized water, then bring to a boil over high heat and simmer over low heat for 1 hour. Collect the decoction and filter the residue through gauze and a 200-mesh sieve to obtain the first decoction. For the second decoction, decoct the herbs in 8 times their volume of deionized water for 45 minutes, filter again, and collect the filtrate. Mix the two decoctions thoroughly. Concentrate the decoction using a rotary evaporator under reduced pressure, maintaining the water bath temperature at 70℃, until the concentration of the raw herbs is 3.9g / mL, thus obtaining the decoction of the traditional Chinese medicine composition.

[0051] Example 2: Preparation of Traditional Chinese Medicine Composition A method for preparing a traditional Chinese medicine composition is as follows: Take 60g of Smilax glabra, 20g of Achyranthes bidentata, 30g of Astragalus membranaceus, 30g of Polygonum cuspidatum, 20g of Fraxinus chinensis, 10g of Coptis chinensis, 20g of Phellodendron chinense, 30g of Lilium brownii, and 20g of Liquidambar formosana, and decoct them according to the clinical decoction method. For the first decoction, soak the herbs thoroughly in 10 times their weight-to-volume ratio of deionized water, then bring to a boil over high heat and simmer over low heat for 1 hour. Collect the decoction, filter the residue through gauze and a 200-mesh sieve to obtain the first decoction. For the second decoction, decoct the herbs in 8 times their weight of deionized water for 45 minutes, filter again and collect the filtrate, and mix the two decoctions thoroughly. Use a rotary evaporator to concentrate the decoction under reduced pressure, controlling the water bath temperature at 70℃, and concentrate the decoction (to a crude drug concentration of 3.9g / mL) to obtain the decoction of the traditional Chinese medicine composition.

[0052] Example 3: Preparation of Traditional Chinese Medicine Composition A method for preparing a traditional Chinese medicine composition is as follows: Take 20g of Smilax glabra, 10g of Achyranthes bidentata, 15g of Astragalus membranaceus, 10g of Polygonum cuspidatum, 6g of Fraxinus chinensis, 3g of Coptis chinensis, 6g of Phellodendron chinense, 10g of Lilium brownii, and 6g of Liquidambar formosana. Prepare the decoction according to clinical methods. For the first decoction, soak the herbs thoroughly in 10 times their volume of deionized water, then bring to a boil over high heat and simmer over low heat for 1 hour. Collect the decoction and filter the residue through gauze and a 200-mesh sieve to obtain the first decoction. For the second decoction, decoct the herbs in 8 times their volume of deionized water for 45 minutes, filter again, and collect the filtrate. Mix the two decoctions thoroughly. Concentrate the decoction using a rotary evaporator under reduced pressure, maintaining the water bath temperature at 70℃, until the concentration of the raw herbs is 3.9g / mL, to obtain the decoction of the traditional Chinese medicine composition.

[0053] The traditional Chinese medicine compositions with the expected effects of the present invention were all prepared in Examples 1 to 3, and all of them can achieve the effect of treating RA. The following uses the traditional Chinese medicine composition provided in Example 1 as an example to illustrate the effect.

[0054] Example 4: Therapeutic effect of traditional Chinese medicine composition on CIA mouse model 1. Experimental Materials 1.1 Laboratory animals: A CIA mouse model was established using male, 6-8 week old DBA / 1 mice. The mice were housed in the SPF-grade animal experimental center of Guangzhou University of Chinese Medicine, with room temperature maintained at (23±2℃) and humidity maintained at (55%±5%), and subjected to alternating light and dark cycles for 7 days for acclimatization.

[0055] 1.2 Construction of CIA Animal Model The first immunization was performed on day 0 (0.1 mL of complete Freund's adjuvant (4 mg / mL) and bovine type II collagen solution (2 mg / mL) were emulsified in equal volumes and injected subcutaneously into the tail of the mouse); the second immunization was performed on day 21 (0.1 mL of incomplete Freund's adjuvant (4 mg / mL) and bovine type II collagen solution (2 mg / mL) were emulsified in equal volumes and injected subcutaneously into the back of the mouse).

[0056] 1.3 Grouping and Administration Methods Grouping: Mice were randomly divided into 5 groups: blank group (NC group), model group (CIA group), methotrexate group (MTX group), low-dose Xiezhuo Qutong Fang group (XZQTF-L group), and high-dose Xiezhuo Qutong Fang group (XZQTF-H group). Except for the blank group, all other groups were subjected to CIA modeling.

[0057] Administration method: Mice in each group were given the corresponding drug intervention starting on day 21 after the second immunization. The blank group and the model group were given an equal volume of physiological saline once a day; the MTX group was given 2 mg / kg according to the literature dose once a week, and an equal volume of physiological saline for the remaining 6 days; the XZQTF-L and XZQTF-H groups were given the traditional Chinese medicine composition prepared in Example 1 (low dose 14.97 g / kg crude drug and high dose 29.94 g / kg crude drug), respectively, once a day, all by gavage, for 4 weeks.

[0058] Twenty-eight days after administration, blood was drawn from the eye after enucleation under isoflurane inhalation anesthesia. The blood was left to stand at 4°C for 1 hour and then inhaled at 4°C and 1000 rpm. -1 Centrifuge for 20 minutes (centrifugation radius 18 cm), aliquot the supernatant into EP tubes and store at -80℃. The mice were then euthanized by dislocation, and their hind limbs, liver, and kidneys were fixed in 4% formalin at room temperature.

[0059] 2 Experimental Methods 2.1 Arthritis Score to Assess the Severity of Arthritis in Mice Joint conditions in mice were observed and recorded starting 21 days after the second immunization. Paw swelling was observed every 3 days. The degree of inflammation in the paws was scored according to the arthritis scoring criteria, recorded on a scale of 0 to 4. The cumulative score was the AI ​​score for each mouse. The specific scoring method is shown in Table 1. Table 1 Arthritis Scoring Criteria 2.2 Detection of serum inflammatory factor levels Enzyme-linked immunosorbent assay (ELISA) was used to detect the protein expression levels of inflammation-related cytokines IL-6, TNF-α, IL-10, and IL-1β in mouse serum.

[0060] According to the ELISA kit instructions (purchased from Shanghai Jianglai Biotechnology Co., Ltd.), CIA mouse plasma was centrifuged at 1000g for 20 min, the supernatant was collected, and the sample was diluted appropriately. 100 μL of the sample or different concentrations of standards were added to each well of the ELISA plate, and the plate was incubated at 37°C for 60 min. The liquid was discarded, and 100 μL of biotinylated antibody working solution was added to each well, and the plate was incubated at 37°C for 60 min. The liquid was discarded, and 300 μL of 1× washing buffer was added to each well. The plate was allowed to stand for 1 min, the washing buffer was discarded, and the plate was patted dry on absorbent paper. The plate was washed three times. 100 μL of enzyme conjugate working solution was added to each well, and the plate was incubated at 37°C for 30 min. The liquid was discarded, and the plate was washed. 90 μL of substrate was added to each well, and the plate was incubated at 37°C in the dark for 15 min. The plate was removed, and 50 μL of stop solution was added directly to each well. The OD value of each well was measured at 450 nm, and the concentration of the sample index was calculated based on the OD value.

[0061] 2.3 Micro-CT scan of knee and ankle joints The knee and ankle joints were fixed with 4% paraformaldehyde. The lower limbs of mice were scanned and reconstructed in three dimensions using the SkyScan-1276 micro-computed tomography (micro-CT) system. Scanning parameters: resolution 7 mm, voltage 50 kV, current 72 μA. A semi-quantitative scoring method was used to assess bone destruction in the micro-CT scans. CTAn software was used to analyze bone mineral density (BMD) and related parameters of the mouse knee and ankle joints. The knee joint analysis focused on the tibial plateau, and the ankle joint analysis focused on the talus.

[0062] 2.4 Bone tissue pathological examination Tissue was placed in 4% paraformaldehyde for 48 hours, rinsed with PBS, and decalcified with 13% ethylenediaminetetraacetic acid (EDTA) solution at 4°C. The decalcification solution was changed every 4-5 days until a pin could easily penetrate the bone. After rinsing with PBS, the tissue was dehydrated using a gradient alcohol dehydrator. The paraffin-soaked tissue was then embedded in an embedding machine, the paraffin blocks were trimmed, and sections were prepared, each approximately 8µm thick. The sections were placed on a slide and flattened at 4°C. The tissue was then lifted off the slide and placed in a 60°C oven to bake. After the paraffin wax melted, the tissue was removed and stored at room temperature.

[0063] 2.5 Pathological examination of synovial inflammation and joint structural damage H&E staining of knee and ankle joints: 1. Dewaxing paraffin sections to hydration: Immerse sections sequentially in xylene I for 10 min → xylene II for 10 min → anhydrous ethanol I for 2 min → anhydrous ethanol II for 2 min → 95% ethanol for 2 min → 90% ethanol for 2 min → 85% ethanol for 2 min → 75% ethanol for 2 min → wash with distilled water. 2. Mayer's hematoxylin staining of cell nuclei: Immerse sections in Harris hematoxylin staining for 2 min, rinse with warm water for 10 min, differentiate with 75% hydrochloric acid ethanol for 1 min, and rinse with tap water for 30 s. 3. Eosin staining of cytoplasm: Immerse sections in eosin staining solution for 2 min, and rinse with tap water for 30 s. 4. Dehydration and mounting: Immerse the sections sequentially in 85% ethanol for 2 min → 95% ethanol I for 2 min → 95% ethanol II for 2 min → anhydrous ethanol I for 2 min → anhydrous ethanol II for 2 min → xylene I for 10 min → xylene II for 10 min to dehydrate and clear. Remove the sections from the xylene and allow them to dry slightly before mounting with neutral resin. 5. Microscopic examination and image acquisition and analysis. 6. Staining results: Cell nuclei are blue, cytoplasm is red.

[0064] 2.6 Pathological examination of articular cartilage erosion and bone destruction Safranin-Fix Green Staining of Knee and Ankle Joints: 1. Dewaxing Paraffin Sections to Water: Immerse sections sequentially in xylene I for 5 min → xylene II for 5 min → xylene III for 5 min → anhydrous ethanol I for 10 min → anhydrous ethanol II for 5 min → anhydrous ethanol III for 5 min → 95% ethanol for 5 min → 90% ethanol for 5 min → 80% ethanol for 5 min → 70% ethanol for 5 min → rinse with running water for 5 min → rinse with distilled water for 5 min. 2. Safranin O Staining: Immerse sections in Safranin O staining solution for 10 min, then rinse with running water for 5 min. 3. Fast Green Staining: Immerse sections in Fast Green staining solution for 2 min, then rinse with running water for 30 s. 4. Dehydration and Mounting: Differentiate sections sequentially in 1% glacial acetic acid for 1 min, then dehydrate and clear them in 95% ethanol I for 2 min → anhydrous ethanol I for 2 min → anhydrous ethanol II for 2 min → xylene I for 10 min → xylene II for 10 min. Remove sections from xylene, allow them to dry slightly until clear, then mount with neutral resin. 5. Microscopic examination, image acquisition and analysis.

[0065] 2.7 Detection of core indicators in mouse peripheral blood routine tests CIA mouse whole blood was analyzed using a fully automated biochemical analyzer, and the levels of WBC, Lym, HGB, PLT, and other indicators in the blood of each group of mice were recorded.

[0066] 2.8 H&E staining of liver and kidney in CIA mice to observe pathological changes H&E staining was performed as described above: after dewaxing and hydration of paraffin sections, Mayer's hematoxylin and eosin were used to stain the cell nuclei and cytoplasm, respectively, followed by dehydration, mounting, and image acquisition and analysis.

[0067] 2.9 Statistical Methods Statistical analysis was performed using SPSS 27.0. The Shapiro-Wilk test was used to test the normality of quantitative data. For quantitative or ordinal data that did not conform to a normal distribution, the median (interquartile range) [M(P] was used]. 25 -P 75 This indicates that the nonparametric Kruskal-Wallis rank-sum test was used for comparisons among multiple groups, and the nonparametric Mann-Whitney U rank-sum test was used for comparisons between two groups; data conforming to a normal distribution were expressed as mean ± standard deviation. The expression indicates that a homogeneity of variance test was performed. When variances were homogeneous, Bonferroni analysis in One-way ANOVA was used for comparisons among multiple groups, and the two independent samples t-test was used for comparisons between two groups. When variances were unequal, Tamhane's T² analysis in One-way ANOVA was used for comparisons among multiple groups, and the corrected t-test was used for comparisons between two groups. P < 0.05 was considered statistically significant.

[0068] 3 Experimental Results 3.1XZQTF can alleviate arthritis symptoms in CIA mice. A CIA mouse model was established using DBA / 1 mice to simulate the pathological manifestations of RA. Compared with the NC group, the CIA group mice showed significant redness and swelling in the ankle and paw joints, accompanied by limited mobility. In contrast, the XZQTF-L, XZQTF-H, and MTX groups showed significant improvement in joint redness and swelling and mobility, suggesting that XZQTF can significantly improve joint symptoms in CIA mice. Figure 1 ).

[0069] 3.2XZQTF can improve weight loss, increased paw edema thickness, and AI score in CIA mice. like Figure 2 As shown in Table 2, the AI ​​score of mice in the NC group remained at 0 throughout the experiment. The AI ​​score of mice in the CIA group continued to rise after the initial immunization, showing a significant difference compared with the NC group on days 4 and 7-28 (P<0.01). The AI ​​scores of mice in the XZQTF-L group, XZQTF-H group, and MTX group were lower than those in the CIA group at all time points. Among them, the XZQTF-L group showed a significant difference compared with the CIA group on days 10 and 13, the XZQTF-H group on days 10, 13, 22, and 25, and the MTX group on days 4, 10, 13, 22, and 25 (P<0.05). Moreover, the AI ​​scores of each treatment group showed a decreasing trend in the later stage of the experiment (day 28), and the arthritis symptoms were significantly relieved.

[0070] like Figure 2As shown in Table 2, the paw edema thickness of mice in the NC group did not change significantly throughout the experiment; the paw edema thickness of mice in the CIA group increased significantly after the second immunization, showing significant differences compared with the NC group at days 10, 13, 16, and 28 (P<0.05); the paw edema thickness of mice in the XZQTF-L group, XZQTF-H group, and MTX group was reduced compared with the CIA group at all time points, and the peak paw edema thickness decreased. Among them, the XZQTF-L group showed significant differences compared with the CIA group at day 10, and the MTX group showed significant differences at days 7-10 (P<0.05).

[0071] like Figure 2 As shown in Table 2, the body weight of mice in the NC group steadily increased throughout the experimental period; the CIA group showed a significant difference from the NC group on days 1-28 (P<0.01); the decrease in body weight of mice in the XZQTF-L and XZQTF-H groups was less severe than that in the CIA group; and the body weight of mice in the MTX group rebounded significantly in the later stage of the experiment (days 16-28).

[0072] Table 2. Effects of XZQTF on AI score, paw edema thickness, and body weight in CIA mice ( / M(P 25 -P 75 ), n=6) Note: ① AI scores: The Shapiro-Wilk test showed that the AI ​​scores of mice in each group on days 1, 4, 10, and 13 did not all follow a normal distribution. The nonparametric Kruskal-Wallis rank-sum test was used for comparisons among multiple groups, and the nonparametric Mann-Whitney U rank-sum test was used for comparisons between two groups. The AI ​​scores of mice in each group on days 7, 16, 19, 22, 25, and 28 followed a normal distribution. Among them, the p-values ​​of the homogeneity of variance test for mice in each group on days 22 and 25 were >0.05, indicating homogeneity of variance. Bonferroni analysis was used for comparisons between groups. The p-values ​​of the homogeneity of variance test for mice in each group on days 7, 16, 19, and 28 were <0.05, indicating heterogeneity of variance. After correction for heterogeneity of variance, one-way ANOVA and Tamhane's T2 were used for comparisons between groups. Compared with the NC group, the P-values ​​in the CIA group on days 4, 10, 13, 16, 19, 22, 25, and 28 were <0.05, indicating statistically significant differences. Compared with the CIA group, the P-values ​​in the XZQTF-L group on days 10 and 13 were <0.05, indicating statistically significant differences. Compared with the CIA group, the P-values ​​in the XZQTF-H group on days 10, 13, 22, and 25 were <0.05, indicating statistically significant differences. Compared with the CIA group, the P-values ​​in the MTX group on days 4, 10, 13, 22, and 25 were <0.05, indicating statistically significant differences.

[0073] ② Foot swelling thickness: The Shapiro-Wilk test showed that the AI ​​scores of mice in each group on days 1, 4, 7, 10, 13, 16, and 25 did not all follow a normal distribution. The nonparametric Kruskal-Wallis rank-sum test was used for comparisons among multiple groups, and the nonparametric Mann-Whitney U rank-sum test was used for comparisons between two groups. The AI ​​scores of mice in each group on days 19, 22, and 28 followed a normal distribution. The mice on day 28 had a p-value > 0.05 for homogeneity of variance, and Bonferroni analysis was used for comparisons between groups. The mice on days 19 and 22 had p-values ​​< 0.05 for homogeneity of variance, and variance inequality was corrected. One-way ANOVA and Tamhane's T2 were used for comparisons between groups. Compared with the NC group, the P-values ​​of the CIA group on days 10, 13, 16, and 28 were <0.05, indicating statistical significance; compared with the CIA group, the P-value of the XZQTF-L group on day 10 was <0.05, indicating statistical significance; compared with the CIA group, the P-value of the MTX group on day 10 was <0.05, indicating statistical significance.

[0074] ③ Body weight: The Shapiro-Wilk test showed that the AI ​​scores of mice in each group on days 7, 10, 13, and 16 did not all follow a normal distribution. The nonparametric Kruskal-Wallis rank-sum test was used for comparisons among multiple groups, and the nonparametric Mann-Whitney U rank-sum test was used for comparisons between two groups. The AI ​​scores of mice in each group on days 1, 4, 19, 22, 25, and 28 followed a normal distribution. The p-value of the homogeneity of variance test among the groups was >0.05, indicating homogeneity of variance. Bonferroni analysis was used for comparisons between groups. Compared with the NC group, the P-values ​​of the CIA group on days 1, 4, 7, 10, 13, 16, 19, 22, 25, and 28 were <0.05, indicating statistically significant differences; compared with the CIA group, the P-values ​​of the XZQTF-L group on days 10 and 13 were <0.05, indicating statistically significant differences; compared with the CIA group, the P-values ​​of the MTX group on days 10, 13, and 16 were <0.05, indicating statistically significant differences.

[0075] P<0.05, P<0.01, P<0.001, indicating a statistically significant difference compared to the NC group. # P<0.05, ## P<0.01, ### P<0.001, indicating a statistically significant difference compared to the CIA group.

[0076] 3.3XZQTF can alleviate knee and ankle joint inflammation in CIA mice. like Figure 3 As shown, H&E staining was performed on the knee and ankle joints of mice in each group, and the staining results were analyzed for inflammatory infiltration and synovial hyperplasia. The results indicated that XZQTF could alleviate the joint inflammation of the knee and ankle in CIA mice, and XZQTF-H was more effective than MTX in inhibiting synovial inflammation.

[0077] 3.4XZQTF can reduce cartilage erosion and bone destruction in the knee and ankle joints of CIA mice. like Figure 4 As shown, safranin-fast green staining was performed on the knee and ankle joints of mice in each group, and the staining results were analyzed for cartilage erosion and bone destruction. The results indicated that XZQTF could reduce cartilage erosion and bone destruction in the knee and ankle joints of CIA mice, and XZQTF-H was more effective than MTX in inhibiting cartilage erosion and bone destruction.

[0078] 3.5XZQTF can improve bone mineral density and related indicators in the knee and ankle joints of CIA mice. Micro-CT scans were performed on the knee and ankle joints of mice in each group. The three-dimensional reconstruction and two-dimensional coronal plane images of the mouse joints are shown in the figure. Figure 5 The results showed that the knee and ankle joint structures of mice in the NC group were normal, the bone surfaces were smooth and flat, and the trabecular structure was normal. In contrast, the joint structure of mice in the CIA group was significantly damaged, with multiple areas of bone erosion, and the trabeculae were sparse and disordered. The bone erosion and trabecular reduction of CIA mice treated with XZQTF-L, XZQTF-H, or MTX were significantly reduced compared with the CIA group.

[0079] Based on the micro-CT scan results, bone mineral density and related indicators of the knee and ankle joints of mice in each group were analyzed. Figure 6(Tables 3 and 4). The results showed that compared with the NC group, the CIA group mice had significantly lower BV / TV in the knee joint (P<0.01), significantly higher BS / BV (P<0.001), significantly increased Tb.Sp (P<0.05), and significantly lower BMD (P<0.01). Compared with the CIA group, the XZQTF-L and XZQTF-H groups had significantly increased BV / TV (P<0.01), significantly lower BS / BV in all treatment groups (P<0.01), significantly higher BMD in the XZQTF-L and XZQTF-H groups (P<0.05), and although Tb.Sp showed a decreasing trend, there was no statistically significant difference. These results indicate that XZQTF can alleviate pathological bone hyperplasia and bone microstructural disorder in the knee joint of CIA mice and improve bone quality. Compared with the NC group, the CIA group mice showed a significantly decreased bone volume fraction (BV / TV) in the ankle joint (P<0.05), a significantly decreased bone mineral density (BMD) (P<0.01), and a significantly increased bone surface area to bone volume ratio (BS / BV) (P<0.01). While the trabecular bone spacing (Tb.Sp) showed an increasing trend, there was no statistically significant difference. Compared with the CIA group, the XZQTF-H and MTX groups showed significantly increased BV / TV (P<0.05), while the XZQTF-L and XZQTF-H groups showed significantly decreased BS / BV (P<0.05), and the XZQTF-L and XZQTF-H groups showed significantly increased BMD (P<0.01), indicating that XZQTF can improve the reduced bone mass and bone microstructure damage in the ankle joint of CIA mice.

[0080] Table 3. Effects of XZQTF on BV / TV and related indicators in the knee joint of CIA mice ( / M(P 25 -P 75 ), n=6) Note: ① The normality test p-values ​​for the NC, CIA, XZQTF-L, XZQTF-H, and MTX groups in BV / TV were 0.028, 0.596, 0.113, 0.359, and 0.588, respectively, indicating that they did not all follow a normal distribution. The nonparametric Kruskal-Wallis rank-sum test was used for comparisons among multiple groups, and the nonparametric Mann-Whitney U rank-sum test was used for comparisons between two groups. Compared with the NC group, the p-value for the CIA group was 0.004, indicating a statistically significant difference. Compared with the CIA group, the p-values ​​for the XZQTF-L and XZQTF-H groups were 0.016 and 0.004, respectively, indicating statistically significant differences. Compared with the CIA group, the p-value for the MTX group was 0.055, indicating no statistically significant difference. ② The Shapiro-Wilk test showed that the normality p-values ​​for the NC, CIA, XZQTF-L, XZQTF-H, and MTX groups in the BS / BV were 0.172, 0.404, 0.316, 0.169, and 0.223, respectively, all indicating a normal distribution. The homogeneity of variance p-value was 0.052, indicating homogeneity of variance. Bonferroni analysis was used for inter-group comparisons. Compared with the NC group, the p-value for the CIA group was 0.000, indicating a statistically significant difference. Compared with the CIA group, the p-values ​​for the XZQTF-L, XZQTF-H, and MTX groups were 0.003, 0.008, and 0.003, respectively, indicating statistically significant differences. ③ The Shapiro-Wilk test showed that the normality p-values ​​for the NC, CIA, XZQTF-L, XZQTF-H, and MTX groups in Tb.SP were 0.292, 0.278, 0.300, 0.330, and 0.354, respectively, all indicating a normal distribution. The homogeneity of variance p-value was 0.017, indicating unequal variances. After correcting for unequal variances, one-way ANOVA and Tamhane's T2 were used for inter-group comparisons. Compared to the NC group, the CIA group showed a statistically significant p-value of 0.046. Compared to the CIA group, the XZQTF-L, XZQTF-H, and MTX groups showed no statistically significant p-values ​​of 0.425, 0.347, and 1.000, respectively.④ The Shapiro-Wilk test showed that the p-values ​​for normality in the NC, CIA, XZQTF-L, XZQTF-H, and MTX groups in BMD were 0.737, 0.923, 0.856, 0.031, and 0.721, respectively, indicating that they did not all follow a normal distribution. The non-parametric Kruskal-Wallis rank-sum test was used for comparisons among multiple groups, and the non-parametric Mann-Whitney U rank-sum test was used for comparisons between two groups. Compared with the NC group, the p-value for the CIA group was 0.004, indicating a statistically significant difference. Compared with the CIA group, the p-values ​​for the XZQTF-L and XZQTF-H groups were 0.030 and 0.003, respectively, indicating statistically significant differences. Compared with the CIA group, the p-value for the MTX group was 0.091, indicating no statistically significant difference. P<0.05, P<0.01, P<0.001, indicating a statistically significant difference compared to the NC group. # P<0.05, ## P<0.01, ### P<0.001, indicating a statistically significant difference compared to the CIA group.

[0081] Table 4 Effects of XZQTF on ankle BV / TV and related indicators in CIA mice ( / M(P 25 -P 75 ), n=6) Note: ① The normality test p-values ​​for the NC, CIA, XZQTF-L, XZQTF-H, and MTX groups in BV / TV were 0.903, 0.366, 0.489, 0.145, and 0.613, respectively, all of which followed a normal distribution; the homogeneity of variance p-value was 0.114, indicating homogeneity of variance; Bonferroni analysis was used for inter-group comparisons; compared with the NC group, the p-value for the CIA group was 0.003, indicating a statistically significant difference; compared with the CIA group, the p-values ​​for the XZQTF-H and MTX groups were 0.002 and 0.011, respectively, indicating statistically significant differences; compared with the CIA group, the p-value for the XZQTF-L group was 0.132, indicating no statistically significant difference. ② The Shapiro-Wilk test showed that the p-values ​​for normality in the NC, CIA, XZQTF-L, XZQTF-H, and MTX groups in BS / BV were 0.009, 0.645, 0.528, 0.092, and 0.391, respectively, indicating that they did not all follow a normal distribution. The non-parametric Kruskal-Wallis rank-sum test was used for comparisons among multiple groups, and the non-parametric Mann-Whitney U rank-sum test was used for comparisons between two groups. Compared with the NC group, the p-value for the CIA group was 0.025, indicating a statistically significant difference. Compared with the CIA group, the p-values ​​for the XZQTF-L and XZQTF-H groups were 0.010 and 0.004, respectively, indicating statistically significant differences. Compared with the CIA group, the p-value for the MTX group was 0.150, indicating no statistically significant difference. ③ The Shapiro-Wilk test showed that the normality p-values ​​for the NC, CIA, XZQTF-L, XZQTF-H, and MTX groups in Tb.Sp were 0.287, 0.437, 0.575, 0.086, and 0.971, respectively, all indicating a normal distribution. The homogeneity of variance p-value was 0.361, indicating homogeneity of variance. Bonferroni analysis was used for inter-group comparisons. Compared with the NC group, the p-value for the CIA group was 0.268, indicating no statistically significant difference. Compared with the CIA group, the p-values ​​for the XZQTF-L, XZQTF-H, and MTX groups were 1.000, 0.192, and 0.308, respectively, indicating no statistically significant difference.④ The Shapiro-Wilk test showed that the p-values ​​for normality in the NC, CIA, XZQTF-L, XZQTF-H, and MTX groups in BMD were 0.030, 0.838, 0.843, 0.688, and 0.382, respectively, indicating that they did not all follow a normal distribution. The nonparametric Kruskal-Wallis rank-sum test was used for comparisons among multiple groups, and the nonparametric Mann-Whitney U rank-sum test was used for comparisons between two groups. Compared with the NC group, the p-value for the CIA group was 0.006, indicating a statistically significant difference. Compared with the CIA group, the p-values ​​for the XZQTF-L and XZQTF-H groups were 0.008 and 0.004, respectively, indicating statistically significant differences. Compared with the CIA group, the p-value for the MTX group was 0.092, indicating no statistically significant difference. P<0.05, P<0.01, P<0.001, indicating a statistically significant difference compared to the NC group. # P<0.05, ## P<0.01, ### P<0.001, indicating a statistically significant difference compared to the CIA group.

[0082] 3.6 XZQTF can reduce serum inflammatory cytokine levels in CIA mice. Serum inflammatory cytokine levels in mice were detected using ELISA. Figure 7 (Table 5). As shown in Table 5, compared with the NC group, the serum levels of pro-inflammatory cytokines TNF-α, IL-6, and IL-1β in the CIA group mice were significantly increased (P<0.01), while the level of the anti-inflammatory cytokine IL-10 was slightly decreased. Compared with the CIA group, the TNF-α levels in the XZQTF-L, XZQTF-H, and MTX groups were significantly decreased (P<0.01), the IL-6 level in the XZQTF-H group was significantly decreased (P<0.05), and the IL-1β levels in the XZQTF-L and XZQTF-H groups were decreased. Meanwhile, the IL-10 levels in all treatment groups were increased. The results indicate that XZQTF can dose-dependently reduce the serum levels of pro-inflammatory cytokines TNF-α, IL-6, and IL-1β in CIA mice and increase the level of the anti-inflammatory cytokine IL-10, thereby effectively alleviating systemic inflammatory responses.

[0083] Table 5. Effects of XZQTF on serum inflammatory cytokine protein levels in CIA mice ( / M(P 25 -P 75 ), n=6) Note: ① According to the Shapiro-Wilk test, the p-values ​​for normality in the NC, CIA, XZQTF-L, XZQTF-H, and MTX groups of TNF-α were 0.839, 0.038, 0.601, 0.059, and 0.968, respectively, indicating that they did not all follow a normal distribution. The non-parametric Kruskal-Wallis rank-sum test was used for comparisons among multiple groups, and the non-parametric Mann-Whitney U rank-sum test was used for comparisons between two groups. Compared with the NC group, the p-value for the CIA group was 0.004, indicating a statistically significant difference. Compared with the CIA group, the p-values ​​for the XZQTF-L, XZQTF-H, and MTX groups were 0.010, 0.004, and 0.006, respectively, indicating statistically significant differences. ② According to the Shapiro-Wilk test, IL-6... The p-values ​​for normality testing of the NC group, CIA group, XZQTF-L group, XZQTF-H group, and MTX group were 0.911, 0.079, 0.596, 0.635, and 0.597, respectively, all indicating a normal distribution. The p-value for homogeneity of variance was 0.035, indicating heterogeneity of variance. One-way ANOVA was used to compare the significant differences among the groups, with a p-value of 0.005, indicating significant differences between groups. The adjusted t-test in the two independent samples t-test was used to test the significance of differences between the two groups. Compared with the NC group, the p-value for the CIA group was 0.005 (t=-3.798), indicating a statistically significant difference. Compared with the CIA group, the p-values ​​for the XZQTF-L group, XZQTF-H group, and MTX group were 0.125 (t=1.764), 0.035 (t=2.691), and 0.102 (t=1.802), respectively. ③ The Shapiro-Wilk test showed that the normality p-values ​​for the NC, CIA, XZQTF-L, XZQTF-H, and MTX groups of IL-10 were 0.687, 0.229, 0.485, 0.307, and 0.830, respectively, all indicating a normal distribution. The homogeneity of variance p-value was 0.922, indicating homogeneity of variance. Bonferroni analysis was used for inter-group comparisons. Compared with the NC group, the p-value for the CIA group was 1.000, indicating no statistically significant difference. Compared with the CIA group, the p-value for the MTX group was 0.035, indicating a statistically significant difference. Compared with the CIA group, the p-values ​​for the XZQTF-L and XZQTF-H groups were 1.000 and 0.735, respectively, indicating no statistically significant difference.④ The Shapiro-Wilk test showed that the normality p-values ​​for the NC, CIA, XZQTF-L, XZQTF-H, and MTX groups of IL-1β were 0.440, 0.334, 0.984, 0.164, and 0.474, respectively, all of which followed a normal distribution. The homogeneity of variance p-value was 0.401, indicating homogeneity of variance. Bonferroni analysis was used for inter-group comparisons. Compared with the NC group, the p-value for the CIA group was 0.001, indicating a statistically significant difference. Compared with the CIA group, the p-values ​​for the XZQTF-L, XZQTF-H, and MTX groups were 1.000, 0.113, and 1.000, respectively, indicating no statistically significant difference.

[0084] P<0.05, P<0.01, P<0.001, compared with the NC group, the difference was statistically significant; #P<0.05, ##P<0.01, ###P<0.001, compared with the CIA group, the difference was statistically significant.

[0085] 3.7XZQTF Effects on Peripheral Blood Related Indicators in CIA Mice like Figure 8 Table 6 shows that compared with the NC group, the platelet count in the CIA group was significantly increased (P<0.001), indicating that the CIA model mice had significant inflammation-related thrombocytopenia. Compared with the CIA group, the platelet counts in the XZQTF-L and XZQTF-H groups were significantly decreased (P<0.01), while the MTX group showed a decreasing trend but no statistical difference, indicating that XZQTF can effectively inhibit the increase in platelets in CIA mice. Compared with the NC group, the hemoglobin level in the CIA group was decreased; compared with the CIA group, the hemoglobin level in the XZQTF-L and XZQTF-H groups was increased, indicating that XZQTF improved hemoglobin levels. There were no statistically significant differences in white blood cell count and lymphocyte count among the groups, indicating that XZQTF had no significant effect on the total number of peripheral blood white blood cells and lymphocytes in CIA mice. The results showed that XZQTF could significantly reduce the elevated platelet levels in CIA mice, improve inflammation-related thrombocytosis, improve the reduced hemoglobin levels in CIA mice, and had no significant adverse effects on leukocytes and lymphocytes.

[0086] Table 6. Effects of XZQTF on peripheral blood-related parameters in CIA mice ( / M(P 25 -P 75 ), n=6) Note: ① The normality test p-values ​​for the NC group, CIA group, XZQTF-L group, XZQTF-H group, and MTX group in WBC were 0.139, 0.003, 0.440, 0.530, and 0.952, respectively, according to the Shapiro-Wilk test, indicating that they did not all follow a normal distribution. The nonparametric Kruskal-Wallis rank-sum test was used for comparisons among multiple groups, and the nonparametric Mann-Whitney U rank-sum test was used for comparisons between two groups. Compared with the NC group, the p-value for the CIA group was 0.873, indicating no statistically significant difference. Compared with the CIA group, the p-values ​​for the XZQTF-L group, XZQTF-H group, and MTX group were 0.748, 0.520, and 0.262, respectively, indicating no statistically significant difference. ② The Shapiro-Wilk test showed that the p-values ​​for normality in the NC, CIA, XZQTF-L, XZQTF-H, and MTX groups in Lym were 0.588, 0.008, 0.989, 0.162, and 0.549, respectively, indicating that they did not all follow a normal distribution. The non-parametric Kruskal-Wallis rank-sum test was used for comparisons among multiple groups, and the non-parametric Mann-Whitney U rank-sum test was used for comparisons between two groups. Compared with the NC group, the p-value for the CIA group was 0.297, indicating no statistically significant difference. Compared with the CIA group, the p-values ​​for the XZQTF-L, XZQTF-H, and MTX groups were 0.749, 0.337, and 0.150, respectively, indicating no statistically significant difference. ③ The Shapiro-Wilk test showed that the p-values ​​for normality in the NC, CIA, XZQTF-L, XZQTF-H, and MTX groups in HGB were 0.007, 0.754, 0.405, 0.247, and 0.032, respectively, indicating that they did not all follow a normal distribution. The non-parametric Kruskal-Wallis rank-sum test was used for comparisons among multiple groups, and the non-parametric Mann-Whitney U rank-sum test was used for comparisons between two groups. Compared with the NC group, the p-value for the CIA group was 0.261, indicating no statistically significant difference. Compared with the CIA group, the p-values ​​for the XZQTF-L and MTX groups were 0.077 and 0.470, respectively, indicating no statistically significant difference. Compared with the CIA group, the p-value for the XZQTF-H group was 0.024, indicating a statistically significant difference.④ According to the Shapiro-Wilk test, the p-values ​​for normality in the NC, CIA, XZQTF-L, XZQTF-H, and MTX groups of PLT were 0.165, 0.791, 0.280, 0.702, and 0.710, respectively, all indicating a normal distribution; the p-value for homogeneity of variance was 0.013, indicating heterogeneity of variance; using One-way... ANOVA was used to compare the significance of differences among multiple groups, and the p-value was 0.000, indicating a significant difference between groups. The adjusted t-test in the two independent samples t-test was used to test the significance of differences between the two groups. Compared with the NC group, the p-value of the CIA group was 0.000 (t=-12.059), which was statistically significant. Compared with the CIA group, the p-values ​​of the XZQTF-L group, XZQTF-H group, and MTX group were 0.003 (t=3.921), 0.001 (t=5.142), and 0.259 (t=1.242), respectively.

[0087] P<0.05, P<0.01, P<0.001, indicating a statistically significant difference compared to the NC group. # P<0.05, ## P<0.01, ### P<0.001, indicating a statistically significant difference compared to the CIA group.

[0088] Effects of 3.8XZQTF on HE staining of liver and kidney in CIA mice To clarify the relevant effects of XZQTF on the pathological characteristics of the liver and kidneys in CIA mice, hematoxylin and eosin (HE) staining was performed on the liver and kidneys. Figure 9 As shown, the liver tissue of mice in each group was intact, the hepatic cords were neatly arranged, the outline of the liver lobule structure was clear, the hepatocytes were of uniform size, without degeneration or necrosis, and the cell nuclei were large and round, mostly located in the center of the cell; the renal tubules and Bowman's capsule structure was clear, the renal tubular epithelial cells were of normal size, neatly arranged, without obvious cell swelling, degeneration, or necrosis, and the structure was intact with clear outlines; the glomeruli were regular in shape, without hypertrophy or atrophy.

[0089] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these modifications and improvements all fall within the scope of protection of the present invention. Therefore, the scope of protection of this patent should be determined by the appended claims.

Claims

1. The application of a traditional Chinese medicine composition in the preparation of a drug for treating rheumatoid arthritis, characterized in that, The traditional Chinese medicine composition is made from the following raw materials: Smilax glabra, Achyranthes bidentata, Astragalus membranaceus, Polygonum cuspidatum, Fraxinus chinensis, Coptis chinensis, Phellodendron chinense, Lilium brownii, and Liquidambar formosana.

2. The application as described in claim 1, characterized in that, The traditional Chinese medicine composition is made from the following raw materials in parts by weight: 20-60 parts of Smilax glabra, 10-20 parts of Achyranthes bidentata, 15-30 parts of Astragalus membranaceus, 10-30 parts of Polygonum cuspidatum, 6-20 parts of Fraxinus chinensis, 3-10 parts of Coptis chinensis, 6-20 parts of Phellodendron chinense, 10-30 parts of Lilium brownii, and 6-20 parts of Liquidambar formosana.

3. The application as described in claim 2, characterized in that, The traditional Chinese medicine composition is made from the following raw materials in parts by weight: 40-60 parts of Smilax glabra, 10-20 parts of Achyranthes bidentata, 20-30 parts of Astragalus membranaceus, 15-25 parts of Polygonum cuspidatum, 10-15 parts of Fraxinus chinensis, 5-10 parts of Coptis chinensis, 10-15 parts of Phellodendron chinense, 10-20 parts of Lilium brownii, and 10-15 parts of Liquidambar formosana.

4. The application as described in claim 1, characterized in that, The drug is used to inhibit systemic and local joint inflammatory responses mediated by rheumatoid arthritis.

5. The application as described in claim 1, characterized in that, The drug is used to reduce inflammatory cell infiltration and synovial hyperplasia in the synovial tissue of joints mediated by rheumatoid arthritis.

6. The application as described in claim 1, characterized in that, The drug is used to improve articular cartilage erosion and bone destruction caused by rheumatoid arthritis, increase joint bone density, improve bone microstructure, and delay joint structural damage and functional loss.

7. The application as described in claim 1, characterized in that, The dosage forms of the drug include decoction, granules, pills, capsules, tablets, powders, mixtures, or oral liquids.

8. The application as described in claim 1, characterized in that, The drug also includes pharmaceutically acceptable excipients.

9. The application as described in claim 8, characterized in that, The content of the traditional Chinese medicine composition in the drug is 1wt% to 99wt%.

10. The application as described in claim 8, characterized in that, The pharmaceutically acceptable excipients include one or more of the following: flavoring agents, preservatives, diluents, binders, disintegrants, lubricants, flavoring agents, excipients, wetting agents, encapsulating materials, plasticizers, opacifiers, colorants, coating materials, antioxidants, solubilizers, or suspending agents.

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