Traditional Chinese medicine composition for treating rheumatoid arthritis, its preparation method and application

Abstract

This invention relates to the field of traditional Chinese medicine preparation technology, specifically to a traditional Chinese medicine composition for treating rheumatoid arthritis, its preparation method, and its application. The traditional Chinese medicine composition consists of *Sargentodoxa cuneata*, *Acanthopanax senticosus*, *Ligusticum striatum*, *Drynaria fortunei*, and *Clematis chinensis*. Its preparation method includes the following steps: extraction; filtration; and concentration. Animal experiments and clinical trials have confirmed that the traditional Chinese medicine composition provided by this invention has a significant therapeutic effect on rheumatoid arthritis, with good safety and no obvious adverse reactions, and has great clinical application value.

Description

Technical Field

[0001] This invention relates to the field of traditional Chinese medicine preparation technology, specifically to a traditional Chinese medicine composition for treating rheumatoid arthritis, its preparation method, and its application. Background Technology

[0002] Rheumatoid arthritis (RA) is a chronic autoimmune disease caused by the combined effects of environmental and genetic factors. Its pathological changes include persistent inflammation of the synovial membrane, pannus formation, and subsequent destruction of cartilage and bone. RA is characterized by high incidence, high recurrence rate, and high disability rate, affecting 0.3% to 1% of the global population. Its high medical costs and loss of work due to joint disability impose a heavy economic burden on society and severely impact patients' quality of life.

[0003] Currently, drugs used clinically to treat rheumatoid arthritis (RA) include: nonsteroidal anti-inflammatory drugs (NSAIDs) such as ibuprofen and diclofenac sodium; glucocorticoids such as prednisolone acetate and dexamethasone acetate; antirheumatic drugs such as methotrexate tablets and leflunomide tablets; and biologics such as adalimumab injection and infliximab injection. While these drugs achieve some therapeutic effect, they still have drawbacks such as significant side effects and high treatment costs. Therefore, there is an urgent need to develop new RA treatment drugs with significant therapeutic effects and fewer side effects.

[0004] To address the aforementioned problems, this invention provides a traditional Chinese medicine composition derived from a combination of Sargentodoxa cuneata, Acanthopanax senticosus, Scutellaria baicalensis, Drynaria fortunei, and Clematis chinensis, aiming to offer a new drug option for the clinical treatment of rheumatoid arthritis and improve the quality of life for patients. Summary of the Invention

[0005] The purpose of this invention is to provide a traditional Chinese medicine composition for treating rheumatoid arthritis and its preparation method.

[0006] Another object of the present invention is to provide the use of the above-mentioned traditional Chinese medicine composition in the preparation of a medicament for treating rheumatoid arthritis.

[0007] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0008] The traditional Chinese medicine composition for treating rheumatoid arthritis described in this invention consists of the following components in parts by weight: 20-40 parts of *Sargentodoxa cuneata*, 10-20 parts of *Acanthopanax senticosus*, 20-40 parts of *Drynaria fortunei*, and 10-30 parts of *Clematis chinensis*.

[0009] Preferably, the traditional Chinese medicine composition for treating rheumatoid arthritis according to the present invention is composed of the following components in parts by weight: 25-35 parts of *Sargentodoxa cuneata*, 12-18 parts of *Acanthopanax senticosus*, 25-35 parts of *Drynaria fortunei*, and 15-25 parts of *Clematis chinensis*.

[0010] More preferably, the traditional Chinese medicine composition for treating rheumatoid arthritis according to the present invention is composed of the following components in parts by weight: 30 parts of *Sargentodoxa cuneata*, 15 parts of *Acanthopanax senticosus*, 30 parts of *Smilax china*, 30 parts of *Drynaria fortunei*, and 20 parts of *Clematis chinensis*.

[0011] The preparation method of the traditional Chinese medicine composition for treating rheumatoid arthritis according to the present invention includes the following steps:

[0012] S1. Extraction:

[0013] Weigh out the following five herbs according to the formula: large blood vine, five-capsule bark, blood-sucking herb, bone-drying herb, and clematis root. Add 10-15 times the total weight of the herbs to water, heat to boiling, then simmer over low heat for 0.5-1.5 hours.

[0014] S2. Filtration and Concentration:

[0015] Filter the decoction containing Chinese medicinal herbs obtained in step S1 to remove the dregs, take the filtrate, and concentrate it over a low heat for 0.5-1.5 hours to obtain the decoction.

[0016] S3, Preparation:

[0017] The decoction obtained in step S2 is further heated and boiled to concentrate it into a clear paste with a relative density of 1.05-1.15 at 60°C. Pharmaceutically acceptable excipients are added to prepare a pharmaceutically acceptable formulation.

[0018] Preferably, in the preparation method of the traditional Chinese medicine composition for treating rheumatoid arthritis according to the present invention, the amount of water added in step S1 is 12 times the total mass of the medicinal materials.

[0019] Preferably, in the preparation method of the traditional Chinese medicine composition for treating rheumatoid arthritis according to the present invention, the simmering time in step S1 is 1 hour.

[0020] Preferably, in the preparation method of the traditional Chinese medicine composition for treating rheumatoid arthritis according to the present invention, the simmering time in step S2 is 1 hour.

[0021] Preferably, in the preparation method of the traditional Chinese medicine composition for treating rheumatoid arthritis according to the present invention, the preparation in step S3 includes a solid preparation or a liquid preparation.

[0022] In a further preferred embodiment, in the preparation method of the traditional Chinese medicine composition for treating rheumatoid arthritis according to the present invention, the solid preparation includes tablets, capsules, granules, and pills, and the liquid preparation includes oral liquids and injections.

[0023] The application of the traditional Chinese medicine composition described in this invention in the preparation of a medicament for treating rheumatoid arthritis.

[0024] The beneficial effects of this invention are:

[0025] This invention provides a traditional Chinese medicine composition for treating rheumatoid arthritis (RA) and its preparation method, derived from five medicinal herbs: *Sargentodoxa cuneata*, *Acanthopanax senticosus*, *Ligusticum striatum*, *Drynaria fortunei*, and *Clematis chinensis*. Animal experiments have confirmed that the traditional Chinese medicine composition provided by this invention can improve arthritis in a rat model of RA (CIA), reduce joint swelling, lower AI scores, reduce the secretion of inflammatory factors TNF-α, IL-1β, and IL-6, promote IL-10 secretion, regulate the balance of M1 / M2 macrophages, and improve the expression of HIF-1α, GPX4, SLC7A11, and GSH proteins, thus treating RA. Furthermore, clinical trials have demonstrated that the traditional Chinese medicine composition provided by this invention, combined with conventional Western medicine, can improve the achievement rate of ACR20 / 50 / 70 and the overall effective rate (75.76% in the treatment group and 66.67% in the control group) compared to conventional Western medicine treatment for RA with cold-dampness obstruction syndrome, significantly improving cold-dampness obstruction syndrome, improving patients' quality of life, and demonstrating good safety with no significant adverse reactions, thus possessing significant clinical application value. Attached Figure Description

[0026] Figure 1 Photos of joint swelling in rats from each group;

[0027] Figure 2 HE staining results of rat joints in each group;

[0028] Figure 3 Flow cytometry plots of M1 and M2 macrophage expression in each group;

[0029] Figure 4 The expression bands of HIF-1α, GPX4, SLC7A11, GSH, and β-ACTIN in each group were displayed. Detailed Implementation

[0030] The technical solution of the present invention will be described in detail below with reference to specific embodiments. The following embodiments are for explanation and illustration only, and do not constitute a limitation on the technical solution of the present invention.

[0031] Example 1

[0032] The traditional Chinese medicine composition for treating rheumatoid arthritis consists of the following ingredients: 30 g of *Sargentodoxa cuneata*, 15 g of *Acanthopanax senticosus*, 30 g of *Sargentodoxa cuneata*, 30 g of *Drynaria fortunei*, and 20 g of *Clematis chinensis*.

[0033] Example 2

[0034] The traditional Chinese medicine composition for treating rheumatoid arthritis consists of the following ingredients: 20 g of *Sargentodoxa cuneata*, 10 g of *Acanthopanax senticosus*, 20 g of *Sargentodoxa cuneata*, 20 g of *Drynaria fortunei*, and 13 g of *Clematis chinensis*.

[0035] Example 3

[0036] The traditional Chinese medicine composition for treating rheumatoid arthritis consists of the following ingredients: 25 g of *Sargentodoxa cuneata*, 12 g of *Acanthopanax senticosus*, 25 g of *Sargentodoxa cuneata*, 25 g of *Drynaria fortunei*, and 17 g of *Clematis chinensis*.

[0037] Example 4

[0038] The traditional Chinese medicine composition for treating rheumatoid arthritis consists of the following ingredients: 35 g of *Sargentodoxa cuneata*, 17 g of *Acanthopanax senticosus*, 35 g of *Sargentodoxa cuneata*, 35 g of *Drynaria fortunei*, and 23 g of *Clematis chinensis*.

[0039] Example 5

[0040] The traditional Chinese medicine composition for treating rheumatoid arthritis consists of the following ingredients: 40 g of *Sargentodoxa cuneata*, 20 g of *Acanthopanax senticosus*, 40 g of *Sargentodoxa cuneata*, 40 g of *Drynaria fortunei*, and 27 g of *Clematis chinensis*.

[0041] The traditional Chinese medicine compositions of Examples 1-5 were prepared using any of the preparation methods of Examples 6-9.

[0042] Example 6

[0043] The preparation method of the traditional Chinese medicine composition for treating rheumatoid arthritis includes the following steps:

[0044] (1) Extraction:

[0045] According to the formula in Example 1, weigh out five medicinal materials: large-leaved vine, five-leaved bark, bloodwort, bone-drying herb, and clematis root. Add 1500 mL of water, heat to boiling, then simmer for 1 hour until the volume of the decoction is 800 mL.

[0046] (2) Filtration and concentration

[0047] Filter the decoction containing Chinese medicinal materials obtained in step (1), remove the dregs, take the filtrate, and concentrate it over a low heat for 1 hour until the volume of the decoction is 300 mL to obtain the decoction.

[0048] Example 7

[0049] The preparation method of the traditional Chinese medicine composition for treating rheumatoid arthritis includes the following steps:

[0050] (1) Extraction:

[0051] According to the formula in Example 1, weigh out five medicinal materials: large-leaved vine, five-leaved bark, blood-sucking herb, bone-drying herb, and clematis root. Add 1250 mL of water, heat to boiling, then simmer over low heat for 0.5 h until the volume of the decoction is 900 mL.

[0052] (2) Filtration and concentration

[0053] Filter the decoction containing Chinese medicinal materials obtained in step (1), remove the dregs, take the filtrate, concentrate it over a low heat for 1.2 h, and heat it to concentrate the decoction to a volume of 300 mL to obtain the decoction.

[0054] Example 8

[0055] The preparation method of the traditional Chinese medicine composition for treating rheumatoid arthritis includes the following steps:

[0056] (1) Extraction:

[0057] According to the formula in Example 1, weigh out five medicinal materials: large-leaved vine, five-leaved bark, bloodwort, bone-drying herb, and clematis root. Add 1800 mL of water, heat to boiling, then simmer for 1.5 hours until the volume of the decoction is 800 mL.

[0058] (2) Filtration and concentration

[0059] Filter the decoction containing Chinese medicinal materials obtained in step (1), remove the dregs, take the filtrate, concentrate it over a low flame for 0.8 h, and heat it to concentrate the decoction to a volume of 400 mL to obtain the decoction.

[0060] Example 9

[0061] The preparation method of the traditional Chinese medicine composition for treating rheumatoid arthritis includes the following steps:

[0062] (1) Extraction:

[0063] According to the formula in Example 1, weigh out five medicinal materials: large-leaved vine, five-leaved bark, bloodwort, bone-drying herb, and clematis root. Add 1500 mL of water, heat to boiling, then simmer for 1 hour until the volume of the decoction is 800 mL.

[0064] (2) Filtration and concentration

[0065] Filter the decoction containing Chinese medicinal materials obtained in step (1), remove the dregs, take the filtrate, and concentrate it over a low heat for 1 hour until the volume of the decoction is 300 mL to obtain the decoction.

[0066] (3) Formulation

[0067] The decoction obtained in step S2 is further heated and boiled to concentrate it into a clear paste with a relative density of 1.05-1.15 at 60°C. Pharmaceutically acceptable excipients are added to prepare a pharmaceutically acceptable formulation, including tablets, capsules, granules, oral liquids, and decoctions.

[0068] Example 10

[0069] The traditional Chinese medicine composition prepared by the preparation method of Example 6 was used to treat rheumatoid arthritis, and the usage and dosage were as follows: after cooling at room temperature, take 100 mL each after breakfast, lunch, and dinner.

[0070] In order to further verify the reliability of the present invention, the inventors conducted a series of experiments, which are specifically as follows:

[0071] I. Analysis of the formula of Xueteng Qubi Decoction

[0072] The prescription of Xueteng Qubi Decoction consists of 30 g of Sargentodoxa cuneata, 15 g of Acanthopanax gracilistylus, 30 g of Aristolochia acuminata, 30 g of Drynaria fortunei, and 20 g of Speranskia tuberculata. This prescription was formed under the guidance of Dong medicine theory. The "Five-in-One" theory is the core of Dong medical thought, which refers to the dialectical relationship that heaven, earth, qi, water, and man are an organic whole, emphasizing the interaction and integration between nature composed of heaven, earth, qi, and water and man. There are 12 categories of disease classification and naming in Dong medicine, such as wind, syndrome, fright, dysentery, sore, sha, pain, etc., and there are 72 types regarding "wind" and "wind diseases". Wind diseases are those that are caused by feeling wind pathogens or being weak and generating wind, with manifestations of wind movement. Professor Long Yunguang, a national famous veteran TCM doctor and Dong medicine scholar, summarized: "Most of the diseases called 'wind' and 'wind diseases' in Dong medicine are related to the pathogenicity of wind pathogens, and only a few names have no substantial meaning." Based on the theory of wind diseases and combined with the experience of clinically treating RA, Professor Long Yunguang believes that RA belongs to one type of "wind disease". Dong medicine believes that "when qi is excessive or insufficient, people get sick, and when people die, qi stops and essence turns into water", and when qi and water are out of balance, people will get sick; the quantitative and qualitative changes of qi and water are the pathological processes of diseases. Therefore, the etiology of RA is the invasion of abnormal winds in the four seasons due to insufficient qi and blood in the body, resulting in the pathogenesis of obstruction of qi movement in meridians and viscera. Combining the Dong medicine principle of "identifying symptoms, seeking causes, and exploring essence", the treatment principle of "dispelling pathogenic wind, regulating viscera, and assisting in regulating emotions" was proposed. According to the theory of the six natures and six flavors of Dong medicine, the medication principle for treating RA was proposed, that is, bitter taste can reduce fever, spicy (pungent) taste can dispel cold, promote blood circulation, and remove stasis, fragrant taste can reduce swelling and relieve pain, and sweet taste can enrich blood and boost qi.

[0073] The following is the analysis of the formula of Xueteng Qubi Decoction according to the main drug, assisting drug, and adjunct drug:

[0074] (I) Main drug

[0075] Sargentodoxa cuneata has a bitter and flat nature and flavor, and mainly has the effects of detoxifying and dissipating carbuncles, promoting blood circulation to stop pain, dispelling wind and dampness, and killing insects. Its bitter taste can reduce fever, and its cold nature can clear heat pathogens in the body and help detoxify. Sargentodoxa cuneata has the effects of promoting blood circulation to remove stasis and dredging collaterals, and can reduce inflammation and relieve pain, especially suitable for joint swelling and pain caused by rheumatoid arthritis (RA).

[0076] Drynaria fortunei, bitter in taste and warm in nature, primarily tonifies the kidneys and strengthens bones, promotes healing of injuries and relieves pain. This herb helps enhance joint repair function, reduces damage from rheumatoid arthritis, and warms and tonifies cold and dampness, making it suitable for use in joint pain and dysfunction caused by rheumatism and cold-dampness. Its bitter taste has heat-clearing and anti-inflammatory effects.

[0077] (ii) Assisting with medicine

[0078] Acanthopanax bark is pungent and bitter in taste, and warm in nature. It has the effects of dispelling wind and dampness, tonifying the liver and kidneys, and strengthening tendons and bones. Acanthopanax bark can warm and unblock the joints, and relieve joint pain caused by cold and dampness. Especially when wind, cold and dampness obstruct the joints, it can warm and unblock the meridians, dispel cold and dampness, and help the large blood vine and bone drynaria to exert their effects.

[0079] Jianxuefei (literally "blood-dispelling herb") is pungent and warm in nature, possessing the effects of dispelling wind and cold, promoting blood circulation, relaxing muscles and tendons, and relieving pain. Jianxuefei helps promote blood circulation, eliminate pain and swelling caused by blood stasis, enhance the overall effect of the prescription on promoting blood circulation and removing blood stasis in the joints, and further alleviate the symptoms of rheumatic arthritis.

[0080] (III) Dispensing medicine

[0081] Clematis chinensis, with its pungent and warm properties, possesses the functions of dispelling wind, eliminating dampness, relaxing muscles and tendons, promoting blood circulation, and relieving pain. Clematis chinensis can help improve blood supply to joints by accelerating blood circulation and metabolism, reducing swelling and pain caused by inflammation, especially in the chronic stage of rheumatoid arthritis. Its pungent flavor can warm and unblock the meridians, promoting smooth blood flow.

[0082] The Xue Teng Qu Bi Tang formula works synergistically to dispel wind and dampness, invigorate blood circulation, and unblock the meridians. Based on the theory of six natures and six flavors, its formula has a clear therapeutic purpose: the main herbs (Da Xue Teng and Gu Sui Bu) focus on reducing fever, invigorating blood circulation, removing blood stasis, and clearing the meridians to help relieve inflammation and pain in rheumatoid arthritis; the auxiliary herbs (Wu Jia Pi and Jian Xue Fei) further enhance the effect of invigorating blood circulation and removing blood stasis, eliminating swelling and pain caused by blood stasis; the accompanying herb (Tou Gu Cao) supplements the functions of clearing the meridians, detoxifying, and reducing swelling, promoting the overall efficacy of the formula. The Xue Teng Qu Bi Tang formula dispels wind and dampness, invigorates blood circulation, and unblocks the meridians. When treating the symptoms of rheumatoid arthritis (such as joint swelling and pain, and limited mobility), this formula can alleviate joint discomfort by comprehensively regulating the systemic rheumatic pathology.

[0083] II. Preparation method of Xue Teng Qu Bi Decoction

[0084] (a) Extraction

[0085] Weigh out 30 g of *Sargentodoxa cuneata*, 15 g of *Acanthopanax senticosus*, 30 g of *Sargentodoxa cuneata*, 30 g of *Drynaria fortunei*, and 20 g of *Clematis chinensis*. Add 1500 mL of water, heat to boiling, then simmer over low heat for 1 hour until the volume of the herbal decoction is 800-850 mL.

[0086] (ii) Filtering

[0087] The decoction containing Chinese medicinal herbs is filtered through a strainer to remove the dregs and retain the liquid.

[0088] (III) Concentration

[0089] Transfer the filtered decoction to a decoction pot and simmer over low heat for 1 hour until the volume is 300 mL.

[0090] Dosage and administration of Xue Teng Qu Bi Tang: Cool to room temperature and take 100 mL after breakfast, lunch and dinner.

[0091] III. Animal Experiments to Verify the Efficacy of Xue Teng Qu Bi Decoction

[0092] (I) Experimental Methods

[0093] (1) Laboratory animals

[0094] Seventy-two Lewis female rats (weighing 200 ± 20 g, clean grade, provided by the Animal Experiment Center of Guizhou University of Traditional Chinese Medicine) were used. Based on statistical estimation, the required number of animals was 12 per group. The rats were randomly divided into 6 groups: blank control group; model group; low, medium, and high dose groups of Xue Teng Qu Bi Tang; and HIF-1α inhibitor PX-478 group.

[0095] (2) Animal model preparation

[0096] The CIA rat model exhibits clinical, histopathological, and immunological changes similar to those in humans and is currently the internationally recognized animal model for bone destruction. Type II collagen was dissolved in 0.1 mol / L acetic acid to prepare a 2 mg / mL solution. After stirring and dissolving, the solution was incubated overnight at 4°C. An equal volume of this solution was then mixed with incomplete Freund's adjuvant and emulsified to prepare a type II collagen emulsion. 0.1 mL of this emulsion was injected intradermally at multiple points at the base of the rat's tail to induce inflammation and establish a stable CIA model. A booster immunization was administered on day 8 using the same method. Successful model establishment was evaluated using the arthritis scoring method; successful modeling was indicated by redness and swelling from the toes to the ankle joint, with an arthritis score greater than 4.

[0097] (3) Administration method

[0098] Rats with successfully established models were administered drugs by gavage. The treatment groups received low, medium, and high doses of Xue Teng Qu Bi Tang and PX-478, respectively, while the control group and model group received an equal volume of physiological saline. Starting from day 1 of drug administration, the time of onset of bilateral hind paw swelling in each group and the time when swelling appeared in all model groups (100% swelling time) were recorded. On days 7, 14, 21, and 28, the volume of the rats' bilateral hind paws was measured using a water volume analyzer. Two hours after gavage on day 28, the rats were sacrificed, and blood was collected to separate serum. The skin of the knee and ankle joints of both hind limbs was removed to collect synovial tissue and joint tissue for various parameter tests.

[0099] (4) Drug dosage

[0100] The preparation of Xue Teng Qu Bi Decoction involved soaking the medicinal materials in 7 times their volume of water for 30 minutes, then decocting for 1 hour. The decoction was filtered through gauze, and then decocted again in 7 times their volume of water for 0.5 hours. The decoction was filtered through gauze, and the two decoctions were combined and concentrated to a concentration of 5 g / mL. The solution was stored at 4°C for later use. The rat dosage was approximately 6.17 times the clinical dosage for a 60 kg adult. The dosage was calculated using a 1:2:4 ratio for low, medium, and high doses. The administration volume was 4 mL, with the low-dose group having a concentration of 0.64 g / mL, the medium-dose group 1.29 g / mL, and the high-dose group 2.57 g / mL. PX-478 was administered at a dose of 10 mg / kg, three times a week (Monday, Wednesday, and Friday).

[0101] (5) Indicator detection

[0102] 1. Swelling rate and histopathological morphology of synovial membrane in rats

[0103] Mark the ankle joint of the right hind limb of rats. Measure the volume (mL) of the marked area using the isohydrophilic method. Measurements were taken before modeling, before drug administration, and on days 7, 14, 21, and 28 after drug administration, and the joint swelling rate was calculated. Joint swelling rate = (volume of water displacement measured each time - volume of water displacement measured on day 1) / volume of water displacement measured on day 1. Three rats were randomly selected from each experimental group. Synovial membrane and cartilage tissue were fixed in 10% neutral formalin, decalcified, dehydrated, embedded in paraffin, sectioned, and stained with hematoxylin and eosin (HE). Pathological changes in the synovial membrane and cartilage tissue were observed under a light microscope.

[0104] 2. Arthritis AI score in rats

[0105] The experiment used a 5-point scoring system to evaluate the degree of joint involvement in rats. The sum of the joint lesion scores for each rat was the AI ​​(Arthritis Arthritis Index) for each rat. 0 points: no arthritis; 1 point: mild swelling of the little toe joint; 2 points: swelling of the little toe joint and metatarsal; 3 points: swelling of the paw below the ankle joint; 4 points: swelling of all joints, including the ankle joint. The AI ​​score was calculated based on the degree of joint swelling and deformity. A higher score indicated more severe joint inflammation, with a maximum AI score of 16 points per rat.

[0106] 3. ELISA detection of serum cytokines TNF-α, IL-1β, IL-6, and IL-10

[0107] Follow the ELISA kit instructions: Add 50 μL of different concentrations of standards and 50 μL of the sample to be tested to the enzyme-labeled plate; add 50 μL of antibody mixture to each well; shake at 400 rpm for 2 h at room temperature. Wash the plate three times with 300 μL of 1X washing buffer. Add 100 μL of TMB solution to each well and incubate at 400 rpm for 10 min in the dark. Add 100 μL of stop solution to each well and react for 1 min. Measure the OD value at 450 nm and calculate the concentration of each sample.

[0108] 4. FCM was used to detect the expression of CD86 and CD206 in tissue cells, and the M1 / M2 ratio was calculated.

[0109] Synovial membrane was digested with 0.5 g / L collagenase IV for 1 h, followed by digestion with 0.25% trypsin for 30 min to obtain single cells. Cells were then blocked with 10% rabbit serum for 30 min, centrifuged, and the supernatant discarded. Cells were resuspended in 50 µL of PBS, and 10 µL each of PE-CD86 and APC-CD206 antibodies were added. The cells were incubated at 4°C in the dark for 30 min. After washing once with PBS, the cells were resuspended in 100 µL of PBS and analyzed by flow cytometry.

[0110] 5. Western blot analysis of HIF-1α, SLC7A11, GSH, and GPX4 protein expression in synovial tissue.

[0111] Tissue was homogenized at low temperature, and protein lysis buffer was added and lysed on ice for 40 min. The mixture was then centrifuged at 12000 r / min for 20 min at 4°C, and the supernatant was collected. Protein concentration was determined using the BCA method: 20 μg of protein sample was subjected to 10% SDS-PAGE electrophoresis, and the protein was transferred to a PVDF membrane. The membrane was then blocked in blocking buffer at 37°C for 1 h. Primary antibody was incubated overnight at 4°C. The membrane was washed, and secondary antibody was incubated on a shaker at room temperature for 1.5 h. The membrane was washed again, developing buffer was added, and the membrane was exposed and developed using a chemiluminescence imaging system. Images were saved. The relative expression levels of each protein were calculated using ImageJ software.

[0112] (II) Results and Analysis

[0113] (1) Changes in joint swelling

[0114] Photos of joint swelling in rats in each group are shown below. Figure 1 As shown in the table. The results showed that rats in the CIA model group gradually developed the disease after modeling, exhibiting obvious redness and swelling of the paws. Compared with the CIA model group, the redness and swelling of the joints in the Xue Teng Qu Bi Tang group and the PX-478 group were significantly reduced. Compared with the model group, after 1, 2, 3, and 4 weeks of treatment, the degree of swelling of the right hind limb ankle joint in each treatment group was significantly improved (P < 0.05), and the drainage volume of the right hind limb ankle joint in each group is shown in Table 1.

[0115] ;

[0116] (2) Joint synovial tissue HE

[0117] HE staining results of rat joints in each group are as follows: Figure 2 As shown in the figure. Pathological results of the synovium showed that in the blank control group, the synovial tissue of rats showed normal nuclei and cytoplasmic structures within the joint cavity, with no obvious inflammatory changes or pannus formation; in the CIA model group, a large number of inflammatory cells infiltrated, the synovial tissue showed significant hyperplasia and thickening, exhibiting granulomatous changes, neovascularization, pannus formation, and significant destruction of bone structure; in the Xue Teng Qu Bi Tang group and the PX-478 group, the inflammatory cell infiltration and hyperplasia of the synovial tissue were significantly reduced compared to the model group, with very little neovascularization, and no significant destruction of joint bone and cartilage.

[0118] (3) Joint AI Index Score

[0119] The results of the joint AI index scores are shown in Table 2. The results showed that Xue Teng Qu Bi Tang effectively reduced the AI ​​scores of CIA rats. Compared with the model group, after 4 weeks of treatment, the AI ​​scores of each treatment group decreased significantly (P < 0.05).

[0120] ;

[0121] (4) Expression of inflammatory factors

[0122] The expression of inflammatory factors is shown in Table 3. The results showed that after 4 weeks of treatment with Xue Teng Qu Bi Tang, compared with the CIA model group, TNF-α, IL-1β, and IL-6 were significantly decreased (P<0.05), while IL-10 was significantly increased (P<0.05).

[0123] ;

[0124] (5) Expression of M1 / M2 macrophages

[0125] Flow cytometry plots of M1 and M2 macrophage expression in each group are shown below. Figure 3 As shown in Table 4, the expression of M1 / M2 macrophages in each group was analyzed. The results showed that after treatment with Xue Teng Qu Bi Tang, the M1 / M2 ratio was significantly lower than that in the CIA model group (P < 0.05).

[0126] ;

[0127] (6) Expression levels of HIF-1α, GPX4, SLC7A11, and GSH proteins

[0128] The expression bands of HIF-1α, GPX4, SLC7A11, GSH, and β-ACTIN in each group are as follows: Figure 4As shown in the figure. The relative expressions of HIF-1α, GPX4, SLC7A11, and GSH are shown in Table 5. The results showed that after treatment with Jixueteng Qubi Decoction, compared with the CIA model group, the relative expressions of HIF-1α, GPX4, SLC7A11, and GSH were significantly decreased (P < 0.05).

[0129] ;

[0130] (III) Summary

[0131] Jixueteng Qubi Decoction can improve arthritis in the CIA rat model of rheumatoid arthritis, relieve joint swelling, reduce the AI score, reduce the secretion of inflammatory factors TNF-α, IL-1β, and IL-6, promote the secretion of IL-10, regulate the balance of M1 / M2 macrophages, improve the expression of HIF-1α, GPX4, SLC7A11, and GSH proteins, and treat RA. In summary, the Dong medicine Jixueteng Qubi Decoction can treat the RA inflammatory model, providing a theoretical basis for its further clinical promotion and application.

[0132] IV. Clinical efficacy verification

[0133] (I) Inclusion / exclusion criteria

[0134] (1) Inclusion criteria:

[0135] 1. Age 18 - 75 years old, gender not limited;

[0136] 2. Patients with initial treatment;

[0137] 3. Patients who meet the 2010 ACR / EULAR RA diagnostic criteria;

[0138] 4. No contraindications to the use of methotrexate or hydroxychloroquine;

[0139] 5. Obtained informed consent;

[0140] 6. Belong to the syndrome of cold-dampness obstruction of collaterals.

[0141] (2) Exclusion criteria:

[0142] 1. Patients with other autoimmune diseases;

[0143] 2. Pregnant or lactating women;

[0144] 3. Those with a history of cardiovascular and cerebrovascular diseases such as stroke, TIA, MI, HF (NYHA class II-IV), coronary artery surgery (such as angioplasty, stent implantation, bypass grafting, etc.);

[0145] 4. Those with a history of peptic ulcer or gastrointestinal bleeding;

[0146] 5. Individuals with active liver disease or abnormal liver function, whose AST, ALT, or GGT levels are 1.2 times higher than the upper limit of normal;

[0147] 6. Individuals with abnormal renal function and serum creatinine (sCr) concentration exceeding 1.2 times the upper limit of normal;

[0148] 7. Abnormal laboratory tests 7 days prior to medication, with a white blood cell count <4.0×10⁻⁶. 9 / L, or platelet count <100×10 9 / L;

[0149] 8. Individuals with concurrent malignant tumors or mental illness;

[0150] 9. Those with a history of alcohol or drug dependence, or those who need to use painkillers daily for any reason;

[0151] 10. Individuals who participated in other clinical studies within 3 months prior to enrollment;

[0152] 11. Other circumstances that researchers deem unsuitable for participation in the clinical study.

[0153] (II) Observation Indicators

[0154] (1) Characteristics of joint symptoms

[0155] The number of swollen joints, the number of tender joints, and the duration of morning stiffness were recorded before and after treatment in both groups.

[0156] (2) Traditional Chinese Medicine Symptom Scores

[0157] Referring to the "Guiding Principles for Clinical Research of New Traditional Chinese Medicine Drugs (Trial Implementation)," three primary symptoms were included: joint swelling, joint pain, and morning stiffness; seven secondary symptoms were included: joint tenderness, limited joint flexion and extension, migratory joint pain, joint pain aggravated by cold, heaviness in the limbs, aversion to cold and wind, and fatigue. A four-level scoring method was used for evaluation, with 0 points for primary symptoms (none), 2 points for mild, 4 points for moderate, and 6 points for severe; and 0 points for secondary symptoms (none), 1 point for mild, 2 points for moderate, and 3 points for severe. The total score ranged from 0 to 39 points, with higher scores indicating more severe TCM syndromes.

[0158] (3) Inflammatory markers

[0159] Fasting blood samples were collected from the elbow vein in the morning before and after treatment in each group to measure erythrocyte sedimentation rate (ESR) and C-reactive protein (CRP) levels.

[0160] (4) Improvement in disease activity

[0161] Disease activity is determined based on the Disease Activity Score (DAS-28) of 28 joints. DAS28-ESR = [0.56×sqrt(T28) + 0.28×sqrt(SW28) + 0.70×Ln(ESR)]×1.08+0.16. Note: T28 is the number of tender joints: bilateral proximal interphalangeal joints (10), metacarpophalangeal joints (10), wrist joints (2), elbow joints (2), shoulder joints (2) and knee joints (2), totaling 28 joints with tenderness or tenderness during passive movement; SW28 is the number of swollen joints: whether the above 28 joints are swollen, the number of swollen joints SW28 is obtained.

[0162] The proportion of patients meeting the ACR classification criteria of 20%, 50%, and 70% improvement in disease activity was recorded (ACR20 / 50 / 70). The ACR remission criteria were based on the following seven points: ① improvement in the number of tender joints; ② improvement in the number of swollen joints; ③ patient evaluation of pain; ④ patient's overall evaluation of disease activity; ⑤ physician's overall evaluation of disease activity; ⑥ HAO-DI score; ⑦ improvement in acute reactants (ESR, CRP). The percentage improvement for each indicator = (previous value - postvious value) / previous value × 100%. ACR20: improvement rate >20% for all seven criteria; ACR50: improvement rate >50% for all seven criteria; ACR70: improvement rate >70% for all seven criteria.

[0163] (5) Standards for TCM syndrome differentiation and treatment

[0164] Refer to the "Guiding Principles for Clinical Research of New Traditional Chinese Medicines (Trial Implementation)". Clinical cure: Significant or near-complete disappearance of TCM clinical symptoms and signs, with a syndrome score reduction >95%; Marked effect: Significant improvement in TCM clinical symptoms and signs, with a syndrome score reduction ≥70% but <95%; Effective: Some improvement in TCM clinical symptoms and signs, with a syndrome score reduction ≥30% but <70%; Ineffective: No significant improvement in TCM clinical symptoms and signs, or even worsening, with a syndrome score reduction <30%. Total effective rate = (Pre-treatment score - Post-treatment score) / Pre-treatment score × 100%.

[0165] (6) Adverse reactions

[0166] Blood routine tests and liver and kidney function tests were performed at weeks 1, 4, 8, and 12 of the start of treatment. Adverse reactions during treatment were observed and recorded.

[0167] (III) Treatment methods

[0168] Control group: Methotrexate tablets (specification: 2.5 mg, Shanghai Shangyao Xinyi Pharmaceutical Co., Ltd., approval number: National Drug Approval Number H31020644; 10 mg / time, once a week, orally) + folic acid (specification: 2.5 mg, Shanghai Shangyao Xinyi Pharmaceutical Co., Ltd., approval number: National Drug Approval Number H31020644; 10 mg / time, once a week, orally) + hydroxychloroquine tablets (specification: 0.1 g, Shanghai Shangyao Zhongxi Pharmaceutical Co., Ltd., approval number: National Drug Approval Number H19990263; 0.2 g / time, once a day, orally) were used for treatment.

[0169] The observation group was given a combination of methotrexate tablets (10 mg once a week, orally) + folic acid (specification: 2.5 mg, Shanghai Shangyao Xinyi Pharmaceutical Co., Ltd., approval number: National Drug Approval Number H31020644; 10 mg once a week, orally) + hydroxychloroquine tablets (0.2 g once a day, orally) plus Xue Teng Qu Bi Tang.

[0170] The Xue Teng Qu Bi Decoction consists of 30 g of Da Xue Teng (Spatholobus suberectus), 15 g of Wu Jia Pi (Acanthopanax senticosus), 30 g of Jian Xue Fei (Spatholobus suberectus), 30 g of Gu Sui Bu (Drynaria fortunei), and 20 g of Tou Gu Cao (Clematis chinensis). It is prepared by the Chinese Pharmacy Department of the Second Affiliated Hospital of Guizhou University of Traditional Chinese Medicine. The dosage is 100 mL each time, 3 times a day. One month is one course of treatment, and the treatment lasts for 3 months.

[0171] (iv) Clinical Enrollment

[0172] Patients with rheumatoid arthritis (RA) treated at the outpatient and inpatient departments of the Department of Rheumatology and Immunology, Second Affiliated Hospital of Guizhou University of Traditional Chinese Medicine, were selected and randomly divided into a treatment group and a control group. The treatment group consisted of 33 patients (2 males and 31 females), aged 22–72 years (mean 51.45 ± 10.21 years), with a disease duration of 2–18 months (mean 9.27 ± 3.89 months). The control group consisted of 31 patients (3 males and 28 females), aged 33–68 years (mean 51.10 ± 8.29 years), with a disease duration of 3–15 months (mean 8.26 ± 3.71 months). There were no statistically significant differences in general characteristics between the two groups (P > 0.05), indicating comparability. This study protocol was approved by the Medical Ethics Committee of the Second Affiliated Hospital of Guizhou University of Traditional Chinese Medicine (KY202401212).

[0173] (V) Results and Analysis

[0174] (1) Comparison of the number of swollen joints, the number of tender joints, and the duration of morning stiffness before and after treatment

[0175] The results of comparing the number of swollen joints, the number of tender joints, and the duration of morning stiffness before and after treatment in each group are shown in Table 6. The results show that the number of swollen joints, the number of tender joints, and the duration of morning stiffness in the treatment group were all lower after treatment than before treatment (P < 0.05); the number of swollen joints, the number of tender joints, and the duration of morning stiffness in the control group were all lower after treatment (P < 0.05); and the number of tender joints and the duration of morning stiffness in the treatment group were lower than those in the control group after treatment.

[0176] ;

[0177] (2) Comparison of TCM symptom scores, ESR, and CRP before and after treatment

[0178] The results of comparing TCM symptom scores, ESR, and CRP before and after treatment in each group are shown in Table 7. The results show that the TCM symptom scores, ESR, and CRP in the treatment group decreased after treatment compared to before treatment (P<0.05); the TCM symptom scores, ESR, and CRP in the control group also decreased after treatment (P<0.05); and after treatment, the TCM symptom scores, ESR, and CRP in the treatment group were all lower than those in the control group (P<0.05).

[0179] ;

[0180] (3) Comparison of DAS28, ACR20, ACR50 and ACR70 before and after treatment

[0181] Table 8 shows the comparison results of DAS28, ACR20, ACR50, and ACR70 before and after treatment in each group. The results indicate that: DAS28 in the treatment group decreased after treatment compared to before treatment (P<0.05); DAS28 in the control group decreased after treatment compared to before treatment (P<0.05); after treatment, DAS28 in the treatment group was lower than that in the control group (P<0.05); after treatment, ACR20, ACR50, and ACR70 in the treatment group were all higher than those in the control group (P<0.05).

[0182] ;

[0183] (4) Comparison of TCM syndromes and their therapeutic effects

[0184] The results of the comparison of TCM syndrome efficacy are shown in Table 9. The results show that the total effective rate in the treatment group was 75.76%, while the total effective rate in the control group was 66.67%. The TCM syndrome efficacy in the treatment group was better than that in the control group (P<0.05).

[0185] ;

[0186] (5) Adverse reactions

[0187] During the treatment process, 1 case of nausea and 1 case of mild abdominal distension occurred in the treatment group, and the incidence of adverse reactions was 6.06%. In the control group, 2 cases of nausea due to upper respiratory tract infection occurred, and the incidence of adverse reactions was 6.45%. There was no significant difference in the incidence of adverse reactions between the two groups (P>0.05).

[0188] (VI) Summary

[0189] The treatment of RA with cold-dampness obstruction syndrome by Xuefeng Qubi Decoction combined with conventional western medicine can improve the attainment rates of ACR20 / 50 / 70 after treatment, improve the syndrome of cold-dampness obstruction, generally improve the quality of life of patients, and has good safety, no obvious adverse reactions, and has great clinical application value.

[0190] Although the present invention has been described in detail above with general descriptions, specific embodiments and experiments, some modifications or improvements can be made based on the present invention, which are obvious to those skilled in the art. Therefore, these modifications or improvements made without departing from the spirit of the present invention all fall within the scope of protection of the present invention.

Claims

1. A traditional Chinese medicine composition for treating rheumatoid arthritis, characterized in that, The traditional Chinese medicine composition is made of components with the following mass parts: Sargentodoxa 20-40 parts, Acanthopanax 10-20 parts, Saurauia 20-40 parts, Drynaria 20-40 parts, and Asarum 13-27 parts.

2. The traditional Chinese medicine composition for treating rheumatoid arthritis according to claim 1, characterized in that, The traditional Chinese medicine composition is made of components with the following mass parts: Sargentodoxa 25-35 parts, Acanthopanax 12-18 parts, Saurauia 25-35 parts, Drynaria 25-35 parts, and Asarum 17-23 parts. 3.The traditional Chinese medicine composition for treating rheumatoid arthritis according to claim 2, characterized in that, The traditional Chinese medicine composition is made of components with the following mass parts: Sargentodoxa 30 parts, Acanthopanax 15 parts, Saurauia 30 parts, Drynaria 30 parts, and Asarum 20 parts.

4. The method for preparing the traditional Chinese medicine composition for treating rheumatoid arthritis as described in any one of claims 1-3, characterized in that, The preparation method comprises the following steps: S1, extraction: Sargentodoxa, Acanthopanax, Saurauia, Drynaria, and Asarum are weighed according to the prescription composition, and water with a total mass of 10-15 times that of the medicine is added, and then heated to boiling, followed by gentle heat for 0.5-1.5 h; S2, filtration and concentration: The soup liquid mixed with the traditional Chinese medicine prepared in step S1 is filtered to remove the dregs, and the filtrate is obtained, and then concentrated for 0.5-1.5 h on a low heat to obtain a decoction; S3, preparation: The decoction obtained in step S2 is further heated to boiling, and concentrated into a clear paste with a relative density of 1.05-1.15 at 60℃, and then pharmaceutically acceptable adjuvants are added to prepare a pharmaceutically acceptable preparation.

5. The preparation method according to claim 4, characterized in that, The amount of water added in step S1 is 12 times the total mass of the medicine.

6. The preparation method according to claim 4, characterized in that, The time for gentle heat in step S1 is 1 h.

7. The preparation method according to claim 4, characterized in that, The time for concentration on a low heat in step S2 is 1 h.

8. The preparation method according to claim 4, characterized in that, The preparation in step S3 comprises a solid preparation or a liquid preparation.

9. The production method according to claim 8, characterized by, The solid preparation comprises tablets, capsules, granules, and pills, and the liquid preparation comprises oral liquid and injection.

10. Use of the traditional Chinese medicine composition according to any one of claims 1-3 in the preparation of a medicine for treating rheumatoid arthritis.

Images