A traditional Chinese medicine composition for improving rheumatoid arthritis and bone metabolism abnormality-sarcopenia bone and muscle metabolism

Abstract

The present application belongs to the technical field of traditional Chinese medicine, and particularly relates to a traditional Chinese medicine composition for improving rheumatoid arthritis and bone metabolism abnormality-sarcopenia bone and muscle metabolism. The traditional Chinese medicine composition of the present application comprises eucommia ulmoides, rhizoma dispersae, semen euryales, astragalus, schisandra chinensis, poria cocos, angelica sinensis, huai niu xi, cornu cervi, persicae semen, drynaria, radix rehmanniae preparata, and can be used for improving rheumatoid arthritis and bone metabolism abnormality-sarcopenia bone and muscle metabolism, specifically, can effectively improve muscle mass and muscle strength, improve joint pain symptoms, effectively improve patient inflammatory chemotactic factor, limb skeletal muscle mass and bone metabolism index, reduce the risk of fracture, prevent falling, reduce the risk of fracture, effectively improve the metabolic network of rheumatoid arthritis and bone metabolism abnormality patients, help osteoblast growth, improve bone metabolism, and prevent and treat osteoporosis. The present application provides a new way for developing effective traditional Chinese medicine compound for treating rheumatoid arthritis and bone metabolism abnormality-sarcopenia.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of traditional Chinese medicine, and particularly relates to a traditional Chinese medicine composition for improving the bone and muscle metabolism of rheumatoid arthritis and bone metabolism abnormality-sarcopenia. BACKGROUND

[0002] Rheumatoid arthritis (RA) is an immune disease characterized by chronic synovitis, which is manifested as morning stiffness, joint pain and destruction caused by chronic, progressive and erosive polyarthritis. Sarcopenia is a condition characterized by decreased muscle strength and / or reduced muscle mass or physiological activity of the human body. Osteoporosis (OP) is a systemic metabolic bone disease characterized by decreased bone mass, damaged bone tissue microstructure, increased bone fragility and easy fracture. Bone and muscle are adjacent to each other, and have common pathogenesis (such as mechanical factors, chemical factors, genetic factors, endocrine factors, nutritional factors, reduction of individual exercise amount and weakening of neural function factors) and closely related signal pathways. Therefore, the concept of osteosarcopenia is proposed (Fielding RA, Vellas B, Evans WJ, et al. Sarcopenia: an undiagnosed condition in older adults. Current consensus definition: prevalence, etiology, and consequences. International working group on sarcopenia [J]. J Am Med Dir Assoc, 2011, 12: 249-256.). Among them, RA patients are prone to develop osteosarcopenia, thereby increasing the risk of falls in patients, leading to adverse consequences such as fractures, disabilities and even death (Salari N, Ghasemi H, Mohammadi L, et al. The global prevalence of osteoporosis in the world: a comprehensive systematic review and meta-analysis [J]. J Orthop Surg Res, 2021, 16(1): 609.), which brings great pressure to the life and economy of patients, and also increases the burden of public health and social medical care.

[0003] RA patients are prone to falls, which can lead to fractures, disability, and even death, causing great pressure on patients' lives and economy, and a great burden on public health and social medical care. Among the various causes of falls in RA patients, osteoporosis and sarcopenia account for a large proportion. A prospective cohort study of 559 RA patients followed up for 1 year found that the incidence of falls in RA patients was as high as 1.11 times per person per year, more than half of the falls caused moderate injury (including bruises, stiffness, joint pain, sprains and head impact, etc.), 7.6% caused serious injury (such as fractures, etc.), 15% needed general practitioners or other health professionals, and 8.8% needed emergency services. Further investigation of the causes of falls in this study found that osteoporosis and muscle weakness were the most common causes of falls in RA patients, and falls often caused fractures. According to the results of domestic and foreign research, the incidence of RA combined with OP is high, about 30% (about 2 times that of normal population), which means that 1 in every 3 RA patients has OP, the incidence of hip OP is 7.8%-15.8%, and the incidence of lumbar OP is 16.8%-19.3%. Once a fracture occurs, it needs to be in bed for a long time, which can induce more internal diseases, aggravate the condition, not only greatly increase the medical expenses, but also increase the pain of patients and the burden of society and family. Osteoporosis has also become a great burden in other countries. In the United States, the annual economic loss due to osteoporosis is tens of billions of dollars. In 2000, in Europe, about 2.7 million people had osteoporotic fractures, and 20% of patients with hip fractures would die within 1 year, costing about 36 billion euros. Traditional Chinese medicine is a unique health resource with the advantages of broad-spectrum, low toxicity, multiple targets and multiple pathways, and has opened up an effective and safe treatment path for RA in recent years. If we take advantage of the safety, non-toxicity, non-side effects, effectiveness and economy of traditional Chinese medicine, and through early diagnosis and early blocking of RA and sarcopenia-osteoporosis-induced fractures and other diseases, it will bring great blessings to society and patients, save a huge amount of medical expenses, and improve the quality of life of patients. Therefore, traditional Chinese medicine has great advantages in the treatment of diabetic osteoporosis. At the same time, traditional Chinese medicine treatment is safe, effective and economical, and easy to popularize, and is easy to accept by primary hospitals and the public. Therefore, the development of effective traditional Chinese medicine compound treatment of this disease has a good prospect. SUMMARY

[0004] To overcome the shortcomings of the existing technology, this invention proposes a traditional Chinese medicine composition for strengthening the spleen, tonifying the kidneys, and invigorating bones. This composition is achieved by rationally combining Eucommia ulmoides, Dipsacus asper, Cuscuta chinensis, Astragalus membranaceus, Schisandra chinensis, Poria cocos, Angelica sinensis, Achyranthes bidentata, deer antler glue, peach kernel, Drynaria fortunei, and Rehmannia glutinosa. This composition is used to improve bone and muscle metabolism in patients with rheumatoid arthritis and sarcopenia, providing a new approach for developing effective traditional Chinese medicine compound treatments for rheumatoid arthritis and sarcopenia.

[0005] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0006] This invention provides a traditional Chinese medicine composition for strengthening the spleen, tonifying the kidneys, and invigorating bones. By weight, the raw materials of the traditional Chinese medicine composition include: 10-20 parts of Eucommia ulmoides, 10-20 parts of Dipsacus asper, 10-20 parts of Cuscuta chinensis, 15-30 parts of Astragalus membranaceus, 5-15 parts of Schisandra chinensis, 10-15 parts of Poria cocos, 10-20 parts of Angelica sinensis, 10-20 parts of Achyranthes bidentata, 5-10 parts of deer antler glue, 10-15 parts of Prunus persica, 10-15 parts of Drynaria fortunei, and 10-20 parts of Rehmannia glutinosa.

[0007] Preferably, the raw materials of the traditional Chinese medicine composition, by weight, include: 15 parts of Eucommia ulmoides, 15 parts of Dipsacus asper, 15 parts of Cuscuta chinensis, 15 parts of Astragalus membranaceus, 6 parts of Schisandra chinensis, 12 parts of Poria cocos, 12 parts of Angelica sinensis, 12 parts of Achyranthes bidentata, 10 parts of deer antler powder, 10 parts of peach kernel, 10 parts of Drynaria fortunei, and 10 parts of Rehmannia glutinosa.

[0008] The second aspect of the present invention also provides the use of the spleen-tonifying, kidney-tonifying, and bone-strengthening traditional Chinese medicine composition described in the first aspect in the preparation of a medicament for improving bone and muscle metabolism in rheumatoid arthritis and bone metabolism abnormalities-sarcopenia.

[0009] Preferably, the drug further includes alendronate sodium.

[0010] The spleen-tonifying, kidney-nourishing, and bone-strengthening traditional Chinese medicine composition of this invention can improve bone and muscle metabolism in rheumatoid arthritis with bone metabolism abnormalities—sarcopenia—by promoting osteoblast proliferation and osteogenic processes. In particular, it can be used in synergistically with the Western medicine alendronate sodium to improve bone and muscle metabolism in rheumatoid arthritis with bone metabolism abnormalities—sarcopenia. Specifically, it can effectively improve muscle mass and strength, alleviate joint pain symptoms; effectively improve inflammatory chemokines, limb skeletal muscle mass, and bone metabolism indicators, reducing fracture risk and preventing falls; effectively improve the metabolic network in patients with rheumatoid arthritis with bone metabolism abnormalities; promote osteoblast growth, improve bone metabolism, and prevent osteoporosis. This invention provides experimental evidence and new ideas for developing effective traditional Chinese medicine compound treatments for rheumatoid arthritis with bone metabolism abnormalities—sarcopenia, and has significant application prospects.

[0011] Preferably, the drug further includes pharmaceutically acceptable excipients.

[0012] Preferably, the dosage form of the drug includes (but is not limited to) decoction, granules, ointment, pills, powder, tablets, and capsules.

[0013] More preferably, the dosage form of the drug is granules, and the specific preparation method includes the following steps:

[0014] S1. Decoction: Chop Eucommia ulmoides, Dipsacus asper, Cuscuta chinensis, Astragalus membranaceus, Schisandra chinensis, Poria cocos, Angelica sinensis, Achyranthes bidentata, deer antler glue, peach kernel, Drynaria fortunei, and Rehmannia glutinosa. After washing with water, soak for 1-2 hours. Then add water and decoct three times. For the first decoction, add water to slightly cover the herbs and decoct for 1-2 hours. For the second and third decoctions, gradually reduce the amount of water and decoct for 1-1.5 hours each time. Combine the three decoctions and filter through a 50-70 mesh sieve. Let it stand and settle for more than 12 hours until the supernatant is clear. Set aside.

[0015] S2, Concentration: Concentrate the supernatant of the medicine solution in S1 to a relative density of 1.35-1.38 to obtain the clear extract;

[0016] S3. Granulation: Mix twice the amount of sucrose powder and twice the amount of dextrin with the clear paste of S2, and spray in 75% ethanol to make a suitable soft material. Finally, according to the requirement of "forming a ball when squeezed, and dispersing when touched", use a swing-type No. 1 filter screen to granulate the soft material.

[0017] More preferably, the concentration is carried out in a vacuum-reduced concentration tank at a temperature of 70-85°C, a steam pressure of 0.14-0.16 MPa, and a vacuum degree of -0.06-0.08 MPa.

[0018] More preferably, the dosage form of the drug is a decoction, and the specific preparation method is as follows: add water to Eucommia ulmoides, Dipsacus asper, Cuscuta chinensis, Astragalus membranaceus, Schisandra chinensis, Poria cocos, Angelica sinensis, Achyranthes bidentata, Prunus persica, Drynaria fortunei and Rehmannia glutinosa, decoct over medium heat for 30-50 minutes, remove the dregs after decoction, decoct over low heat until one-third of the original volume, add deer antler glue, and melt to obtain the decoction;

[0019] Alternatively: First, add deer antler powder to water and simmer over high heat for 5-15 minutes. Then add eucommia bark, dipsacus root, dodder seed, astragalus root, schisandra fruit, poria cocos, angelica root, achyranthes root, peach kernel, drynaria rhizome, and prepared rehmannia root. Simmer over medium heat for 30-50 minutes. After simmering, remove the dregs and then simmer over low heat until the volume is reduced to one-third of the original volume to obtain a traditional Chinese medicine decoction.

[0020] Compared with the prior art, the beneficial effects of the present invention are:

[0021] This invention discloses a traditional Chinese medicine composition for strengthening the spleen, tonifying the kidneys, and invigorating bones. The composition includes Eucommia ulmoides, Dipsacus asper, Cuscuta chinensis, Astragalus membranaceus, Schisandra chinensis, Poria cocos, Angelica sinensis, Achyranthes bidentata, deer antler glue, peach kernel, Drynaria fortunei, and Rehmannia glutinosa. Among them, Eucommia ulmoides has the functions of tonifying the liver and kidneys, strengthening tendons and bones, and calming the fetus; Dipsacus asper has the functions of tonifying the liver and kidneys, strengthening tendons and bones, healing fractures, and stopping metrorrhagia; Cuscuta chinensis has the functions of tonifying the liver and kidneys, consolidating essence and reducing urination, calming the fetus, improving eyesight, and stopping diarrhea; Astragalus membranaceus has the functions of tonifying qi and raising yang, and strengthening the body's defenses; Schisandra chinensis has the functions of astringing and consolidating, tonifying qi and generating fluids, and tonifying the kidneys and calming the mind; Poria cocos has the functions of promoting diuresis and eliminating dampness, strengthening the spleen, and calming the mind and soothing the nerves; Angelica sinensis has the functions of tonifying blood and promoting blood circulation, regulating menstruation and relieving pain, and moistening the intestines and promoting bowel movements; Achyranthes bidentata has the functions of removing blood stasis and promoting menstruation, promoting diuresis and relieving strangury, tonifying the liver and kidneys, and strengthening tendons and bones; Cervi cornu powder has the functions of tonifying kidney yang, strengthening tendons and bones, promoting blood circulation and unblocking collaterals, stopping bleeding, and warming and tonifying kidney yang; Prunus persica has the functions of promoting blood circulation and removing blood stasis, and moistening the intestines and promoting bowel movements; Drynaria fortunei has the functions of healing wounds and relieving pain, and tonifying the kidneys and strengthening bones; Rehmannia glutinosa has the functions of tonifying blood and nourishing yin, and replenishing essence and marrow. Through the rational combination and scientific formulation of traditional Chinese medicine (TCM) formulas, and in conjunction with relevant clinical studies, it has been found that the TCM composition for strengthening the spleen, tonifying the kidneys, and invigorating bones, in combination with the Western medicine alendronate sodium, can effectively improve muscle mass and strength, and alleviate joint pain symptoms; it can effectively improve patients' inflammatory chemokines, limb skeletal muscle mass, and bone metabolism indicators, reducing fracture risk and preventing falls; it can effectively improve the metabolic network in patients with rheumatoid arthritis and bone metabolism disorders; it can promote osteoblast growth, improve bone metabolism, and prevent osteoporosis. This indicates that the TCM composition of this invention can be used to improve bone and muscle metabolism in patients with rheumatoid arthritis and bone metabolism disorders—sarcopenia, providing a new approach for developing effective TCM formulas to treat rheumatoid arthritis and bone metabolism disorders—sarcopenia. Attached Figure Description

[0022] Figure 1 Serum NMR spectra before and after treatment with traditional Chinese medicine (green represents before treatment, red represents after treatment);

[0023] Figure 2 PCA analysis before and after traditional Chinese medicine treatment (A represents before treatment, C represents after treatment);

[0024] Figure 3 PLS-DA analysis graphs before (green ●) and after (blue ●) traditional Chinese medicine treatment;

[0025] Figure 4 OPLS-DA analysis graphs before (green ●) and after (blue ●) traditional Chinese medicine treatment;

[0026] Figure 5 Volcano plot showing the differences in serum metabolites before and after traditional Chinese medicine treatment;

[0027] Figure 6Metabolic network analysis diagrams before and after treatment with traditional Chinese medicine (1. Glyoxylic acid and dicarboxylic acid metabolism; 2. Alanine, aspartic acid, and glutamate metabolism; 3. Valine, leucine, and isoleucine biosynthesis; 4. Citrate cycle (TCA cycle); 5. Pyruvate metabolism; 6. Arginine biosynthesis; 7. Phenylalanine, tyrosine, and tryptophan biosynthesis; 8. Butyrate metabolism; 9. Glycine, serine, and threonine metabolism; 10. Phenylalanine metabolism);

[0028] Figure 7 Survival rate of RAW264.7 cells under different intervention times with traditional Chinese medicine;

[0029] Figure 8 Survival rates of RAW264.7 cells under different ALE intervention times;

[0030] Figure 9 The survival rate of BMSC cells under different intervention times of traditional Chinese medicine. Detailed Implementation

[0031] The specific embodiments of the present invention will be further described below. It should be noted that these descriptions are for the purpose of aiding understanding the present invention, but do not constitute a limitation thereof. Furthermore, the technical features involved in the various embodiments of the present invention described below can be combined with each other as long as they do not conflict with each other.

[0032] Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods, and the experimental materials used in the following embodiments are all available through conventional commercial channels.

[0033] Example: A traditional Chinese medicine decoction or granule for strengthening the spleen, tonifying the kidneys, and invigorating bones, and its preparation method.

[0034] 1. Traditional Chinese medicine composition:

[0035] By weight, one dose of the traditional Chinese medicine composition includes: 15g of Eucommia ulmoides, 15g of Dipsacus asper, 15g of Cuscuta chinensis, 15g of Astragalus membranaceus, 6g of Schisandra chinensis, 12g of Poria cocos, 12g of Angelica sinensis, 12g of Achyranthes bidentata, 10g of deer antler powder, 10g of peach kernel, 10g of Drynaria fortunei, and 10g of Rehmannia glutinosa.

[0036] 2. Preparation of Traditional Chinese Medicine Decoctions:

[0037] Chinese herbal decoction 1: (1) According to the prescription of the above Chinese herbal combination, add 400mL of water to Eucommia ulmoides, Dipsacus asper, Cuscuta chinensis, Astragalus membranaceus, Schisandra chinensis, Poria cocos, Angelica sinensis, Achyranthes bidentata, Prunus persica, Drynaria fortunei and Rehmannia glutinosa, and decoct over medium heat for 40 minutes.

[0038] (2) Remove the dregs from the decoction after step (1), then simmer it over low heat until it reaches 300 mL. Add the deer antler glue to the decoction and melt it to obtain a traditional Chinese medicine decoction for strengthening the spleen, benefiting the kidneys and strengthening bones.

[0039] Chinese herbal decoction 2: (1) Add deer antler powder to 800mL of water and decoct over high heat for 10 minutes. Then add Eucommia ulmoides, Dipsacus asper, Cuscuta chinensis, Astragalus membranaceus, Schisandra chinensis, Poria cocos, Angelica sinensis, Achyranthes bidentata, Prunus persica, Drynaria fortunei and Rehmannia glutinosa. Decoction over medium heat for 40 minutes.

[0040] (2) Remove the dregs from the decoction after step (1), and then simmer over low heat until concentrated to 300 mL to obtain a traditional Chinese medicine decoction for strengthening the spleen, benefiting the kidneys and strengthening bones.

[0041] 3. Preparation of Traditional Chinese Medicine Granules:

[0042] Based on the above prescription of traditional Chinese medicine composition, it is prepared into traditional Chinese medicine granules by decoction according to the requirements for granule preparation, specifically including the following steps:

[0043] (1) Preparation of medicinal materials: Eucommia ulmoides, Dipsacus asper, Cuscuta chinensis, Astragalus membranaceus, Schisandra chinensis, Poria cocos, Angelica sinensis, Achyranthes bidentata, deer antler glue, peach kernel, Drynaria fortunei, Rehmannia glutinosa.

[0044] (2) Decoction: Put the chopped medicinal materials from step (1) into a multi-functional extraction tank (steam pressure 0.2MPa; manufacturer: Dongguan Minghang Environmental Protection Machinery Co., Ltd., model: MH-500), rinse with water until there are no impurities on the surface, soak for one hour, then turn on the steam valve to decoct. Add water and decoct three times. The first time, add water to slightly cover the medicinal materials and decoct for 1.5 hours. The amount of water added in the second and third times is gradually reduced (the amount of water added is 80% of the previous amount), and decoct for 1 hour each time. After filtering with a 60-mesh sieve, put the decoction into the designated storage tank and record the amount of decoction added through the tank level gauge. After combining the three decoctions, let them stand for sedimentation for more than 12 hours until the supernatant is clear and ready for use.

[0045] (3) Concentration: Close the vent valve, open the vacuum valve and the condensate valve, and draw the supernatant in the drug storage tank into the vacuum depressurization concentrator (temperature is 80℃, steam pressure is 0.15MPa, vacuum degree is 0.01MPa; manufacturer: CNNC Mechanical Engineering Co., Ltd., model: 300l). Open the steam valve on the heater and concentrate the drug solution to a relative density of 1.36 (55℃) and then stop the concentration to obtain the clear paste.

[0046] (4) Granulation: Add 300g of sucrose powder and 300g of dextrin into a trough mixer (manufacturer: Changzhou Debang Drying Engineering Co., Ltd., model: CH) and mix. After mixing, slowly add about 750g of the clear paste obtained in step (3) and spray 75% ethanol as a binder while adding the clear paste until the clear paste adheres and forms a suitable soft material. Finally, according to the requirement of "forming a ball when squeezed and dispersing when touched", use a swing-type small pulverizer (manufacturer: Shenzhen Leitong Industrial Co., Ltd., model: DFT-1000) to granulate the soft material.

[0047] Experimental Example: Study on the effects and mechanisms of traditional Chinese medicine for strengthening the spleen, tonifying the kidneys, and invigorating bones in improving bone and muscle metabolism in rheumatoid arthritis and bone metabolism disorders - sarcopenia.

[0048] 1. Experimental Methods

[0049] 1.1 Case selection criteria:

[0050] (1) Diagnostic criteria for TCM syndrome differentiation: Referring to the "2019 Guidelines for the Diagnosis and Treatment of Osteoporosis with Integrated Traditional Chinese and Western Medicine" and the "2020 Guidelines for the Diagnosis and Treatment of Rheumatoid Arthritis with Traditional Chinese Medicine," patients whose syndrome differentiation meets the criteria for Spleen and Kidney Yang Deficiency and Blood Stasis are:

[0051] 1) Main symptoms: joint pain, cold pain in the lower back and knees, poor appetite and loose stools, weakness in the limbs;

[0052] 2) Secondary symptoms: aversion to cold and preference for warmth, abdominal distension, and sallow complexion;

[0053] 3) Tongue and pulse appearance: The tongue is dark red with ecchymosis, and the coating is thin white or thick and greasy; the pulse is deep and wiry or deep, wiry and thin.

[0054] (2) Basis of Western medicine diagnostic criteria:

[0055] 1) Diagnostic criteria for rheumatoid arthritis: meeting the RA classification criteria of the American College of Rheumatology (ACR) in 1987 or the 2018 Chinese Guidelines for the Diagnosis and Treatment of Rheumatoid Arthritis (Chinese Rheumatology Society. 2018 Chinese Guidelines for the Diagnosis and Treatment of Rheumatoid Arthritis [J]. Chinese Journal of Internal Medicine, 2018, 57(2): 242-251.);

[0056] 2) Diagnostic criteria for osteoporosis: Refer to the diagnostic criteria for osteoporosis recommended for Chinese patients (Chinese Society of Osteoporosis and Bone Mineral Diseases. Guidelines for the diagnosis and treatment of primary osteoporosis (2017) [J]. Chinese Journal of Osteoporosis, 2019, 25(3): 281-309.). If the BMD of one or more sites is lower than the normal peak bone mass of the same sex by 1.0 standard deviation, it is diagnosed as osteopenia; if it is lower than the normal peak bone mass of the same sex by 2.5 standard deviations, it is diagnosed as OP.

[0057] 3) Sarcopenia: Chinese expert consensus (Wang Lili, Liang Jie. Research progress on screening tools and treatment of sarcopenia [J]. World's Latest Medical Information, 2019, 19(99):108-110.): The ratio of limb skeletal muscle mass (kg) to the square of height (m) (i.e., SMI) is less than 2 standard deviations below the average skeletal muscle mass of young adults of the same race, or less than 7 kg / m² for men using the BIA method. 2 Women below 5.7 kg / m 2 .

[0058] 1.2 Inclusion criteria for patients with RA and sarcopenia-osteoporosis:

[0059] (1) Meets the above diagnostic criteria of both traditional Chinese and Western medicine; (2) Age 20-70 years; (3) Good compliance, able to receive treatment and various tests in accordance with the guidelines.

[0060] 1.3 Exclusion criteria: pregnant or lactating women and patients with mental illness; patients with other rheumatic diseases; patients with other secondary osteoporosis; patients with severe malnutrition or with severe damage to the heart, brain, kidneys, or hematopoietic system.

[0061] 1.4 Case Grouping: All patients were from the outpatient and inpatient departments of the Rheumatology Department of Dongguan Hospital of Guangzhou University of Chinese Medicine between October 2022 and October 2023. Sixty-six consecutive random numbers were selected from a random number table and randomly divided into two groups according to the order of patient visits: a traditional Chinese medicine treatment group (compound traditional Chinese medicine + alendronate sodium; also known as the traditional Chinese medicine group) and a Western medicine control group (alendronate sodium alone; also known as the Western medicine group). The treatment group consisted of 33 patients (4 males, 12%; 29 females, 88%, mean age 65.33±7.54), and the control group consisted of 33 patients (3 males, 9%; 30 females, 91%, mean age 63.61±7.412). There were no statistically significant differences in gender or age between the two groups (P = 0.692 > 0.05).

[0062] 1.5 Drug treatment: The treatment group took decoction 1 of traditional Chinese medicine, sourced from Dongguan Traditional Chinese Medicine Hospital, one dose per day, for a total of 6 months; the control group took alendronate sodium, one tablet per week, for 6 months.

[0063] 1.6 Observation Indicators and Methods

[0064] (1) Clinical and laboratory data (treatment group and control group)

[0065] This study collected data on insulin-like factors, vitamin D, bone metabolism indicators, bone mineral density, inflammatory factors (erythrocyte sedimentation rate, CRP, TNF-α, IL-6), syndrome scores (blood stasis index, spleen deficiency index, liver and kidney deficiency index), physical indicators (VAS score, simplified physical function assessment, 6m walk test, 5 chair sit-stand tests, 3m turnaround time, hand grip strength, calf circumference), disease activity assessment, and limb skeletal muscle mass measured by bioelectrical impedance analysis in patients with rheumatoid arthritis and sarcopenia-osteoporosis before and after treatment. Among them, insulin-like factors, vitamin D, and inflammatory factors (erythrocyte sedimentation rate, CRP, TNF-α, IL-6) were measured by immunofluorescence assay (tool: BS-220 fully automated biochemical analyzer, company: Mindray); bone mineral density was measured by X-ray bone densitometer (GE Healthcare Systems Ultrasound and Basic Medical Diagnostics Co., Ltd.); bone metabolism indicators mainly examined CTX index (CTX indicates osteoclast activity, the lower the better); syndrome scores (blood stasis index score, spleen deficiency index score, liver and kidney deficiency index score) and physical test indicators (VAS score, simplified physical function test) were also assessed. The methods for measuring the following parameters (estimated weight, 6m walking test, 5 chair sitting tests, 3m turnaround time, hand grip strength, and calf circumference) were as follows: “Liu Yanhui, Chen Shuchun. Interpretation and implications of the 2022 European Society for Clinical Nutrition and Metabolism and European Society for the Study of Obesity’s Consensus on the Definition and Diagnostic Criteria of Sarcopenic Obesity [J]. Chinese General Practice, 2023, 26(12):1422-1428.”; the assessment of disease activity was as follows: “2018 Chinese Guidelines for the Diagnosis and Treatment of Rheumatoid Arthritis, Chinese Rheumatology Branch [J]. Clinical Medical Research and Practice, 2018, 3(12):201.”.

[0066] (2) Metabolomics experiment (treatment group)

[0067] 1) Pretreatment of subject blood samples

[0068] In the traditional Chinese medicine (TCM) treatment group, 20 individuals were randomly selected for metabolomics testing. Specifically, 20 patients with rheumatoid arthritis (RA) and sarcopenia-osteoporosis before treatment (Group A) were selected, and after six months of TCM treatment, they became the TCM group (Group C). Fasting blood samples were collected from the elbow vein before and after treatment for RA and sarcopenia-osteoporosis, as well as from the healthy group. Immediately after collection, the samples were centrifuged at 3000 rpm for 15 minutes, and serum was collected. 300 μL of serum was placed in an NMR tube, and then 200 μL of 0.2 mol / L phosphate buffer (pH = 7.4) and 50 μL of heavy water were added, followed by shaking to mix thoroughly.

[0069] 2) NMR experiment

[0070] A superconducting nuclear magnetic resonance spectrometer (Varian, Inc., USA) was used. The room temperature was set to 25°C, the pulse sequence was Carr-Purcell-Mei boom-Gill, the echo time was set to 1 ms, the spectral width to 10000 Hz, the number of sampling points to 64 kJ, the number of cycles to 64, and the number of NMR scans to 128. The acquired NMR signals were converted into spectra using Fourier transform.

[0071] 3) Spectrum processing

[0072] The spectrum was analyzed using the professional software MestReNova (version 8.0.1). The spectrum was processed by phase adjustment, baseline adjustment, and automatic integration normalization. The lactic acid peak (δ1.33) was used as the chemical shift reference peak. The total region was defined as δ0.5-9.0, with an integration interval of 0.005 ppm. The water peak in the δ4.7-5.1 region was eliminated (the integration value was set to zero). The data was then saved in Excel format for further multivariate statistical analysis.

[0073] 1.7 In vitro experiments

[0074] (1) Experimental materials

[0075] Reagents: Fetal bovine serum (Gibco BR); 0.25% trypsin (Sigma-Aldrich, USA); penicillin-streptomycin antibiotics (Beyotime, China); DMEM medium (FBS, Gibco BR); PBS buffer (Shanghai Dalsey, China); Rat bone marrow mesenchymal stem cell osteogenic differentiation induction kit (Cyagen, China); 4% paraformaldehyde solution (Beyotime, China); TRAP / ALP double staining kit (Fujiflim, Japan); Alizarin Red staining solution (pH 5.1-5.3, Cyagen, China); Red blood cell lysis buffer (Boster Biologics, China).

[0076] Animal source: Two male SD rats aged 4–6 weeks were purchased from the Laboratory Animal Center of Guangdong Provincial Hospital of Traditional Chinese Medicine. All animal experiments were conducted in accordance with the 2013 ARRIVE and AVMA guidelines on euthanasia. The procedures met the requirements of the Medical Ethics Committee.

[0077] Cell source: RAW264.7 cells were purchased from Pronoss Biotechnology Co., Ltd. in China.

[0078] (2) Cell Culture

[0079] Culture of bone marrow mesenchymal stem cells (BMSCs): Two 4-6 week old SD rats were selected and sacrificed by cervical dislocation. Cells were isolated from the bone marrow cavity of the femur and tibia under aseptic conditions. The collected cell suspension was repeatedly mixed by blow-dip mixing with a sterile Pasteur pipette. Red blood cells were lysed by adding erythrocyte lysis buffer at a volume ratio of 1:4. After passing through a cell filter, the cells were transferred to a centrifuge. Erythrocyte lysis buffer was then added to the erythrocyte lysis buffer at a volume ratio of 1:4 to lyse the red blood cells. The cells were then centrifuged at 100×g at 20℃ for 5 min. The resuspended cells were transferred to a culture dish containing 10% FBS in low-glucose DMEN medium and incubated at 37℃ in a 5% CO2 incubator. The medium was changed every 2-3 days. After 3-10 passages, the cells were used for subsequent experiments.

[0080] Macrophage (RAW264.7) culture: The cells were seeded in culture dishes containing 10% FBS and 1% penicillin antibiotics in high-glucose DMEN medium and placed in an incubator at 37°C and 5% CO2. The medium was changed every 2-3 days. After 3-10 generations, the cells were used for subsequent experiments.

[0081] (3) Drug preparation

[0082] The traditional Chinese medicine decoction 1 was prepared according to Example 1, and then concentrated to a concentration of 2g of raw medicinal material per mL using a rotary evaporator (Shanghai Shensheng). After freezing at -80℃ for 72 hours, it was placed in a vacuum freeze dryer (LABCONCO) and dried. The powder was collected into 50mL centrifuge tubes, with a net weight of 63.788g, and stored at 4℃ for later use. The lyophilized powder was dissolved in DMEN containing 10% FBS and 1% double antibiotics to prepare a 10mg / mL solution. The solution was sterilized using a 0.22μm microporous membrane, dispensed, and frozen at -20℃ for later use.

[0083] (4) Experimental methods

[0084] CCK-8 assay for BMSC proliferation: The effects of different concentrations of lyophilized traditional Chinese medicine powder and osteogenic induction differentiation medium on BMSC proliferation were observed using CCK-8 assay to determine the maximum safe concentration. BMSCs were seeded into 96-well plates, with three replicates per group and a density of 1×10⁶ wells. 4Each well contained one sample of lyophilized Chinese medicine (TCM) and was cultured in DMEM medium containing 10% FBS. After 1 day, the samples were incubated with different concentrations of TCM lyophilized powder (final concentrations in the medium were 0 μg / L, 50 μg / L, 100 μg / L, 200 μg / L, 400 μg / L, 600 μg / L, 800 μg / L, and 1000 μg / L) and osteogenic induction differentiation medium (replacing the DMEM medium with an equal volume of osteogenic induction differentiation medium) for 24 h, 48 h, and 96 h. Then, 10 μL of CCK-8 buffer was added to each well and the samples were incubated at 37 °C for 70 min. The optical density was measured at 450 nm using a microplate reader.

[0085] CCK-8 assay for RAW264.7 proliferation: The effects of different concentrations of lyophilized traditional Chinese medicine powder and different concentrations of alendronate sodium on RAW264.7 proliferation were observed using CCK-8 assay to determine the maximum safe concentration. RAW264.7 was seeded into 96-well plates, with three replicates per group at a density of 0.5 × 10⁻⁶ wells. 4 Cells / well were cultured in DMEM medium containing 10% FBS / 1% double antibiotics. One day later, cells were treated with different concentrations of lyophilized traditional Chinese medicine powder (final concentrations in the culture medium were 0 μg / L, 25 μg / L, 50 μg / L, 100 μg / L, 200 μg / L, 400 μg / L, 600 μg / L, 800 μg / L, and 1000 μg / L) and different concentrations of alendronate sodium (final concentrations in the culture medium were 0 μmol / L, 50 μmol / L, 100 μmol / L, 200 μmol / L, 400 μmol / L, 600 μmol / L, 800 μmol / L, and 1000 μmol / L) for 24 h, 48 h, and 96 h. Then, 10 μL of CCK-8 buffer was added to each well and the cells were incubated at 37 °C for 70 min. The absorbance (optical density, OD) was measured at 450 nm using a microplate reader, and the cell viability of each group was calculated. [Cell viability = (OD) / (Cell viability = ...] 实验孔 -OD 空白孔 ) / (OD 阴性对照孔 -OD 空白孔 )】.

[0086] (3) Statistical methods

[0087] Statistical analysis was performed using Grappad Pism 9.5 statistical software. Quantitative data were expressed as mean ± standard deviation. The results indicate that the t-test was used for comparisons between two groups, one-way ANOVA was used for comparisons among multiple groups, and LSD-t test was used for pairwise comparisons between groups. A p-value < 0.05 was considered statistically significant.

[0088] 2. Experimental Results

[0089] 2.1 General Information of the Traditional Chinese Medicine Group and the Western Medicine Group

[0090] As shown in Table 1, the ages of both the traditional Chinese medicine group and the Western medicine group conformed to a normal distribution (Shapiro-Wilk = 0.942, 0.975, P = 0.078, 0.639 > 0.05); the homogeneity of variance test showed that the variances were homogeneous (F = 0.002, P = 0.963 > 0.05); the independent samples t' test showed no significant difference in age between the traditional Chinese medicine group and the Western medicine group (t = 0.938, P = 0.352 > 0.05), which was not statistically significant. Meanwhile, the normality of the sexes in the traditional Chinese medicine group and the Western medicine group was tested (Shapiro-Wilk = 0.384, 0.328, P = 0.000, 0.000 < 0.05). Compared with the Western medicine group, there was no significant difference in sex in the traditional Chinese medicine group (Mann-Whitney U = 528.000, P = 0.692 > 0.05), which was not statistically significant and therefore comparable.

[0091] Table 1 Comparison of ages between the traditional Chinese medicine treatment group and the Western medicine control group

[0092]

[0093] 2.2 Comparison of TCM syndrome efficacy indicators before and after treatment between the TCM group and the Western medicine group

[0094] As shown in Table 2, the blood stasis scores of patients in the traditional Chinese medicine (TCM) group all followed a normal distribution (Shapiro-Wilk = 0.974, 0.957, P = 0.603, 0.216 > 0.05); however, the variances were not homogeneous (P = 0.014 < 0.05). Welch's test showed a significant difference in blood stasis scores between patients before and after TCM treatment (F = 79.022, P = 0.000 < 0.05).

[0095] As shown in Table 3, the spleen deficiency score followed a normal distribution before treatment (Shapiro-Wilk = 0.947, P = 0.108 > 0.05); after treatment, it did not follow a normal distribution (Shapiro-Wilk = 0.929, P = 0.034 < 0.05); the test showed a significant difference in spleen deficiency score before and after Western medicine treatment (Mann-Whitney U = 73.5, P = 0.000 < 0.05).

[0096] As shown in Table 4, the liver and kidney deficiency scores all followed a normal distribution (Shapiro-Wilk = 0.956, 0.951, P = 0.195, 0.138 > 0.05); the variances were homogeneous (F = 0.274, P = 0.603 > 0.05). A t-test was used for comparison, and there was a significant difference in liver and kidney deficiency scores before and after traditional Chinese medicine treatment (t = 10.342, P = 0.000 < 0.05).

[0097] As shown in Table 5, the blood stasis scores of patients in the Western medicine group all followed a normal distribution (Shapiro-Wilk = 0.969, 0.952, P = 0.464, 0.148 > 0.05), and the variances were homogeneous (F = 0.899, P = 0.347 > 0.05). A t-test was used for comparison, and compared with the scores before Western medicine treatment, there was a significant difference in blood stasis scores (t = 5.104, P = 0.000 < 0.05).

[0098] As shown in Table 6, the spleen deficiency scores in the Western medicine group did not follow a normal distribution before treatment (Shapiro-Wilk = 0.907, P = 0.015 < 0.05); after treatment, they followed a normal distribution (Shapiro-Wilk = 0.947, P = 0.108 > 0.05). The difference in spleen deficiency scores before and after Western medicine treatment was statistically significant (Mann-Whitney U = 98.0, P = 0.000 < 0.05).

[0099] As shown in Table 7, the liver and kidney deficiency scores in the control group all followed a normal distribution (Shapiro-Wilk = 0.943, 0.962, P = 0.085, 0.287 > 0.05); the variances were homogeneous (F = 0.090, P = 0.765 > 0.05). A t-test showed a significant difference in liver and kidney deficiency scores before and after Western medicine treatment (t = 4.945, P = 0.000 < 0.05).

[0100] After treatment, the TCM syndrome scores of both the traditional Chinese medicine (TCM) group and the Western medicine group decreased significantly, with the TCM group showing a higher efficacy than the Western medicine group. As shown in Table 8, in the TCM group, there were 0 cases of clinical cure, 0 cases of significant improvement, 32 cases of improvement, and 1 case of ineffectiveness; in the Western medicine group, there were 0 cases of clinical cure, 0 cases of significant improvement, 22 cases of improvement, and 11 cases of ineffectiveness. The clinical efficacy of TCM syndromes between the two groups was statistically different (Mann-Whitney U = 191.50, P = 0.000 < 0.05). The total effective rate of the TCM group was 97%, significantly higher than that of the Western medicine group (66.7%). Fisher's chi-square test showed that the total effective rate of the TCM group was significantly better than that of the Western medicine group (P = 0.003 < 0.05).

[0101] Table 2 Comparison of blood stasis scores before and after treatment with traditional Chinese medicine and Western medicine.

[0102]

[0103] Note: Tests showed that, compared to treatment with traditional Chinese medicine, [the results were satisfactory]. # P < 0.05.

[0104] Table 3 Comparison of spleen deficiency scores before and after treatment with traditional Chinese medicine and Western medicine [M(M 25 M75)]

[0105]

[0106] Note: Tests showed that, compared to treatment with traditional Chinese medicine, [the results were satisfactory]. # P < 0.05.

[0107] Table 4 Comparison of Liver and Kidney Deficiency Scores Before and After Treatment with Traditional Chinese Medicine and Western Medicine Table 4 Comparison of Liver and Kidney Deficiency Scores Before and After Treatment with Traditional Chinese Medicine and Western Medicine

[0108]

[0109] Note: Based on the t-test, compared with traditional Chinese medicine treatment, # P < 0.05.

[0110] Table 5 Comparison of blood stasis scores before and after Western medicine treatment.

[0111]

[0112] Note: Tests showed that, compared to treatment with Western medicine, # P > 0.05.

[0113] Table 6 Comparison of Spleen Deficiency Scores Before and After Treatment with Western Medicine [M(M)] 25 M 75 )]

[0114]

[0115] Note: Based on the t-test, compared with Western medicine treatment, # P > 0.05.

[0116] Table 7 Comparison of liver and kidney insufficiency scores before and after Western medicine treatment.

[0117]

[0118] Note: Based on the t-test, compared with Western medicine treatment, # P > 0.05.

[0119] Table 8 Comparison of overall effective rates of Western medicine and Western medicine treatment (n=33)

[0120]

[0121] Note: # Compared with the control group, P < 0.05.

[0122] 2.3 Comparison of clinical observation indicators before and after treatment between the traditional Chinese medicine group and the Western medicine group

[0123] The VAS scores before and after treatment are shown in Table 9. Intergroup comparisons: There was no statistically significant difference between the traditional Chinese medicine group and the Western medicine group before treatment (Z = -1.785, P = 0.074 > 0.05), but a statistically significant difference was found after treatment (Z = -2.896, P = 0.004 < 0.05). Intragroup comparisons: Before treatment, the VAS score in the traditional Chinese medicine group followed a normal distribution (Shapiro-Wilk = 0.941, P = 0.075 > 0.05); after treatment, it did not follow a normal distribution (Shapiro-Wilk = 0.873, P = 0.001 < 0.05); the VAS score before and after traditional Chinese medicine treatment showed a significant difference (Mann-Whitney U = 315.000, P = 0.003 < 0.05). The VAS scores in the Western medicine group before and after treatment did not conform to a normal distribution (Shapiro-Wilk = 0.908, 0.881, P = 0.009, 0.002 < 0.05); the VAS scores before and after Western medicine treatment showed a significant difference (Mann-Whitney U = 186.000, P = 0.000 < 0.05). This indicates that the VAS scores of both the traditional Chinese medicine group and the Western medicine group decreased significantly, with the treatment effect of the traditional Chinese medicine group being higher than that of the Western medicine group.

[0124] Table 9 Comparison of VAS scores before and after treatment [M(M)] 25 M 75 )]

[0125]

[0126]

[0127] Note: # Compared with the Western medicine group, P < 0.05.

[0128] The balance ability tests before and after treatment are shown in Table 10. Intergroup comparisons: There were no statistically significant differences between the traditional Chinese medicine group and the Western medicine group before treatment (Z = -0.842, P = 0.400 > 0.05), and no significant differences after treatment either (Z = -0.870, P = 0.384 > 0.05). Intragroup comparisons: The balance ability tests before and after treatment in the traditional Chinese medicine group did not follow a normal distribution (Shapiro-Wilk = 0.766, 0.711, P = 0.000, 0.000 < 0.05); the test showed no significant difference in balance ability before and after traditional Chinese medicine treatment (Mann-Whitney U = 477.000, P = 0.334 > 0.05). Balance ability tests in the Western medicine group before and after treatment did not conform to a normal distribution (Shapiro-Wilk = 0.718, 0.763, P = 0.000, 0.000 < 0.05); the test showed no significant difference in balance ability tests before and after Western medicine treatment (Mann-Whitney U = 492.500, P = 0.456 > 0.05). These results indicate that there was no significant increase in balance ability tests in either the traditional Chinese medicine group or the Western medicine group.

[0129] Table 10 Comparison of balance ability before and after treatment [M(M)] 25 M 75 )]

[0130]

[0131] Note: # Compared with the Western medicine group, P>0.05.

[0132] The 4m walking speed tests before and after treatment are shown in Table 11. Intergroup comparisons: There were no statistically significant differences between the traditional Chinese medicine group and the Western medicine group before treatment (Z = -0.842, P = 0.400 > 0.05), but significant statistical differences were observed after treatment (Z = -4.757, P = 0.000 < 0.05). Intragroup comparisons: The 4m walking speed tests before and after treatment in the traditional Chinese medicine group did not follow a normal distribution (Shapiro-Wilk = 0.874, 0.766, P = 0.001, 0.000 < 0.05); the 4m walking speed test before and after traditional Chinese medicine treatment showed a significant difference (Mann-Whitney U = 258.000, P = 0.000 < 0.05). The 4m walking speed test results in the Western medicine group did not conform to a normal distribution before and after treatment (Shapiro-Wilk = 0.882, 0.855, P = 0.002, 0.000 < 0.05); the test showed no significant difference in 4m walking speed test results before and after Western medicine treatment (Mann-Whitney U = 413.50, P = 0.456 > 0.05). These results indicate that the 4m walking speed test result was significantly increased in the traditional Chinese medicine group, while no significant difference was observed in the Western medicine group.

[0133] Table 11 Comparison of 4m walking speed test before and after treatment [M(M)] 25 M 75 )]

[0134]

[0135]

[0136] Note: # Compared with the Western medicine group, P < 0.05.

[0137] Table 12 shows the results of five chair sitting tests before and after treatment. Intergroup comparisons: There were no statistically significant differences between the traditional Chinese medicine group and the Western medicine group before treatment (Z = -0.053, P = 0.957 > 0.05), but significant statistical differences were observed after treatment (Z = -3.740, P = 0.000 < 0.05). Intragroup comparisons: The five chair sitting tests before and after treatment in the traditional Chinese medicine group did not follow a normal distribution (Shapiro-Wilk = 0.881, 0.735, P = 0.002, 0.000 < 0.05); however, the results showed a significant difference in the five chair sitting tests before and after traditional Chinese medicine treatment (Mann-Whitney U = 382.000, P = 0.028 < 0.05). In the Western medicine group, the five chair-sitting tests before and after treatment did not follow a normal distribution (Shapiro-Wilk = 0.886, 0.898, P = 0.002, 0.005 < 0.05); the test showed no significant difference in the five chair-sitting tests before and after Western medicine treatment (Mann-Whitney U = 424.50, P = 0.109 > 0.05). These results indicate that the five chair-sitting tests in the traditional Chinese medicine group showed a significant increase, while the Western medicine group showed no significant difference.

[0138] Table 12 Comparison of 5 chair sitting tests before and after treatment [M(M)] 25 M 75 )]

[0139]

[0140] Note: # Compared with the Western medicine group, P < 0.05.

[0141] Table 13 shows the SARC-F scores before and after treatment. Intergroup comparisons: There were no statistically significant differences between the traditional Chinese medicine group and the Western medicine group before treatment (Z = -0.396, P = 0.692 > 0.05), but significant statistical differences were observed after treatment (Z = -2.150, P = 0.032 < 0.05). Intragroup comparisons: The SARC-F scores before and after treatment in the traditional Chinese medicine group did not follow a normal distribution (Shapiro-Wilk = 0.930, 0.900, P = 0.034, 0.005 < 0.05); the SARC-F scores before and after treatment with traditional Chinese medicine showed a significant difference (Mann-Whitney U = 381.000, P = 0.033 < 0.05). In the Western medicine group, the SARC-F scale scores before and after treatment both followed a normal distribution (Shapiro-Wilk = 0.942, 0.940, P = 0.077, 0.069 > 0.05); however, there was no significant difference in the SARC-F scale scores before and after Western medicine treatment (Z = -0.364, P = 0.716 > 0.05). This indicates that the SARC-F level was significantly increased in the traditional Chinese medicine group, while no significant difference was observed in the Western medicine group.

[0142] Table 13 Comparison of SARC-F scale before and after treatment [M(M 25 M 75 )]

[0143]

[0144]

[0145] Note: # Compared with the Western medicine group, P < 0.05.

[0146] Table 14 shows the limb skeletal muscle mass measured by bioelectrical impedance analysis before and after treatment. Intergroup comparisons: There was no statistically significant difference between the traditional Chinese medicine group and the Western medicine group before treatment (t = 0.081, P = 0.935 > 0.05), but a significant statistical difference was observed after treatment (t = 2.907, P = 0.005 < 0.05). Intragroup comparisons: The limb skeletal muscle mass measured by bioelectrical impedance analysis before and after treatment in the traditional Chinese medicine group followed a normal distribution (Shapiro-Wilk = 0.970, 0.958, P = 0.472, 0.226 > 0.05); the variances were homogeneous (F = 0.005, P = 0.946 > 0.05); the t-test showed a significant difference in limb skeletal muscle mass measured by bioelectrical impedance analysis before and after traditional Chinese medicine treatment (t = -2.524, P = 0.014 < 0.05). The bioelectrical impedance analysis of limb skeletal muscle mass before and after treatment with the Western medicine group showed a normal distribution (Shapiro-Wilk = 0.972, 0.960, P = 0.524, 0.259 > 0.05); the homogeneity of variance was tested and found to be homogeneous (F = 0.002, P = 0.956 > 0.05); the t-test showed no significant difference in limb skeletal muscle mass before and after treatment with the Western medicine group (t = 0.081, P = 0.936 > 0.05).

[0147] Table 14 Comparison of limb skeletal muscle mass measured by bioelectrical impedance method before and after treatment.

[0148]

[0149] Note: # Compared with the Western medicine group, P < 0.05.

[0150] Bone mineral density (lumbar spine) before and after treatment is shown in Table 15. Intergroup comparisons: There were no statistically significant differences between the traditional Chinese medicine group and the Western medicine group before treatment (t = -0.197, P = 0.844 > 0.05), and no significant differences after treatment either (t = -0.207, P = 0.836 > 0.05). Intragroup comparisons: Bone mineral density before and after treatment in the traditional Chinese medicine group followed a normal distribution (Shapiro-Wilk = 0.976, 0.971, P = 0.656, 0.496 > 0.05); however, the bone mineral density before and after treatment with traditional Chinese medicine was statistically significant (t = -3.400, P = 0.002 < 0.05). Bone mineral density (BMD) in the Western medicine group before and after treatment followed a normal distribution (Shapiro-Wilk = 0.942, 0.940, P = 0.077, 0.069 > 0.05); however, the difference in BMD before and after Western medicine treatment was statistically significant (t = -2.254, P = 0.031 > 0.05). These results indicate that there were significant differences in BMD between the traditional Chinese medicine group and the Western medicine group after treatment.

[0151] Table 15 Comparison of bone mineral density (lumbar spine) before and after treatment

[0152]

[0153]

[0154] Note: # Compared with the Western medicine group, P>0.05.

[0155] 2.4 Comparison of inflammatory markers and cell chemokines before and after treatment in the traditional Chinese medicine group and the Western medicine group

[0156] Table 16 shows the CRP levels before and after treatment. Intergroup comparisons: There were no statistically significant differences between the traditional Chinese medicine (TCM) group and the Western medicine group before treatment (Z = -0.372, P = 0.710 > 0.05), and no significant differences after treatment either (Z = -0.385, P = 0.700 > 0.05). Intragroup comparisons: The CRP levels in the TCM group before and after treatment did not follow a normal distribution (Shapiro-Wilk = 0.833, 0.865, P = 0.000, 0.001 < 0.05); the test showed no significant difference in CRP levels before and after TCM treatment (Mann-Whitney U = 523.500, P = 0.788 > 0.05). CRP levels in the Western medicine group did not follow a normal distribution before and after treatment (Shapiro-Wilk = 0.873, 0.887, P = 0.001, 0.003 < 0.05); however, there was no significant difference in CRP levels before and after Western medicine treatment (Mann-Whitney U = 508.000, P = 0.640 > 0.05).

[0157] Table 16 Comparison of CRP levels before and after treatment [M(M25, M75)]

[0158]

[0159] Note: * Compared with before treatment, P>0.05; # Compared with the Western medicine group, P>0.05.

[0160] The ESR values ​​before and after treatment are shown in Table 17. Intergroup comparisons: There were no statistically significant differences between the traditional Chinese medicine (TCM) group and the Western medicine group before treatment (Z = -0.462, P = 0.644 > 0.05), and no significant differences after treatment either (Z = -0.308, P = 0.758 > 0.05). Intragroup comparisons: The ESR values ​​before and after treatment in the TCM group did not follow a normal distribution (Shapiro-Wilk = 0.800, 0.920, P = 0.000, 0.018 < 0.05); the test showed no significant difference in ESR before and after TCM treatment (Mann-Whitney U = 506.000, P = 0.621 > 0.05). The ESR values ​​before and after treatment in the Western medicine group did not follow a normal distribution (Shapiro-Wilk = 0.895, 0.925, P = 0.004, 0.026 < 0.05); the test showed no significant difference in ESR before and after Western medicine treatment (Mann-Whitney U = 480.000, P = 0.408 > 0.05).

[0161] Table 17 Comparison of ESR before and after treatment [M(M)] 25 M 75 )]

[0162]

[0163]

[0164] Note: * Compared with before treatment, P > 0.05; # Compared with the Western medicine group, P>0.05.

[0165] Table 18 shows the TNF-α levels before and after treatment. Intergroup comparisons: There was no statistically significant difference in TNF-α levels between the traditional Chinese medicine (TCM) group and the Western medicine group before treatment (t = 0.767, P = 0.446 > 0.05), but a significant statistical difference was observed after treatment (Z = -2.154, P = 0.035 < 0.05). Intragroup comparisons: TNF-α levels before and after treatment in the TCM group followed a normal distribution (Shapiro-Wilk = 0.964, 0.962, P = 0.342, 0.294 > 0.05); however, the variances were not homogeneous (P = 0.042 < 0.05). Welch's test showed a significant difference in TNF-α levels after TCM treatment compared to before treatment (F = 7.730, P = 0.007 < 0.05). Both TNF-α levels before and after treatment with the Western medicine group followed a normal distribution (Shapiro-Wilk = 0.960, 0.950, P = 0.265, 0.133 > 0.05); the variances were homogeneous (F = 0.000, P = 0.995 > 0.05); and there was no significant difference in TNF-α levels before and after treatment with the Western medicine group (t = 0.024, P = 0.981 > 0.05).

[0166] Table 18 Comparison of TNF-α before and after traditional Chinese medicine treatment

[0167]

[0168] Note: * Compared with before treatment, P < 0.05; # Compared with the Western medicine group, P < 0.05.

[0169] Table 19 shows the IL-6 levels before and after treatment. Intergroup comparisons: There were no statistically significant differences in IL-6 levels between the traditional Chinese medicine (TCM) group and the Western medicine group before treatment (Z = -0.508, P = 0.612 > 0.05), and no significant differences were observed after treatment (Z = -1.566, P = 0.117 > 0.05). Intragroup comparisons: IL-6 levels in the TCM group before and after treatment did not follow a normal distribution (Shapiro-Wilk = 0.617, 0.644, P = 0.000, 0.000 < 0.05); however, the difference in IL-6 levels before and after TCM treatment was statistically significant (Mann-Whitney U = 388.000, P = 0.044 < 0.05). In the Western medicine group, IL-6 levels before and after treatment did not follow a normal distribution (Shapiro-Wilk = 0.584, 0.700, P = 0.000, 0.000 < 0.05); however, there was no significant difference in IL-6 levels before and after Western medicine treatment (Mann-Whitney U = 524.000, P = 0.792 > 0.05).

[0170] Table 19 Comparison of IL-6 levels before and after treatment [M(M)]25 M 75 )]

[0171]

[0172]

[0173] Note: * Compared with before treatment, P < 0.05; # Compared with the Western medicine group, P>0.05.

[0174] 2.5 Comparison of bone metabolism indicators before and after treatment in the traditional Chinese medicine group and the Western medicine group

[0175] Table 20 shows the IGF-1 levels before and after treatment. Intergroup comparisons: There were no statistically significant differences between the traditional Chinese medicine (TCM) group and the Western medicine group before treatment (Z = -0.366, P = 0.715 > 0.05), and no significant differences after treatment either (Z = -0.988, P = 0.323 > 0.05). Intragroup comparisons: IGF-1 levels in the TCM group before and after treatment did not follow a normal distribution (Shapiro-Wilk = 0.927, 0.931, P = 0.028, 0.038 < 0.05); however, the difference in IGF-1 levels before and after TCM treatment was statistically significant (Mann-Whitney U = 234.000, P = 0.000 < 0.05). In the Western medicine group, IL-6 levels before and after treatment did not follow a normal distribution (Shapiro-Wilk = 0.930, 0.921, P = 0.036, 0.019 < 0.05); the difference in IL-6 levels before and after Western medicine treatment was statistically significant (Mann-Whitney U = 336.000, P = 0.007 < 0.05).

[0176] Table 20 Comparison of IGF-1 levels before and after treatment [M(M)] 25 M 75 )]

[0177]

[0178] Note: * Compared with before treatment, P < 0.05; # Compared with the Western medicine group, P>0.05.

[0179] Table 21 shows the CTX levels before and after treatment. Intergroup comparison: There was no statistically significant difference in CTX between the traditional Chinese medicine group and the Western medicine group before treatment (t = 1.385, P = 0.171 > 0.05). There was also no significant difference after treatment (t = 1.353, P = 0.181 > 0.05). Intragroup comparison: CTX levels in the traditional Chinese medicine group before and after treatment followed a normal distribution (Shapiro-Wilk = 0.971, 0.938, P = 0.514, 0.058 > 0.05); the variances were homogeneous (F = 3.541, P = 0.064 > 0.05); the t-test showed a significant difference in CTX levels before and after traditional Chinese medicine treatment (t = 3.160, P = 0.002 < 0.05). CTX levels in the Western medicine group followed a normal distribution before and after treatment (Shapiro-Wilk = 0.946, 0.940, P = 0.105, 0.069 > 0.05); the variances were homogeneous after the homogeneity test (F = 1.832, P = 0.181 > 0.05); the t-test showed no significant difference in CTX levels before and after Western medicine treatment (t = 1.282, P = 0.209 > 0.05).

[0180] Table 21 Comparison of CTX before and after treatment

[0181]

[0182]

[0183] Note: *P<0.05 compared with pre-treatment levels; #P>0.05 compared with the Western medicine group.

[0184] 2.6 Metabolomics analysis of traditional Chinese medicine group

[0185] (1) Before and after treatment with traditional Chinese medicine 1 H-NMR analysis

[0186] By comparing serum metabolites before and after treatment with traditional Chinese medicine 1 H-NMR spectrum ( Figure 1 Differences were identified by referring to relevant literature (Hang, H.; Robinson, PN; Wang, K. Phenolyzer: Phenotype-based prioritization of candidate genes for human diseases. Nat. Methods 2015, 12, 841-843.) and the HMDB metabolite database (http: / / www.hmdb.ca) for this experiment. 1 Identification was performed using H-NMR spectra.

[0187] Figure 1The serum NMR spectra of the Chinese medicine group before and after treatment are shown (green represents before treatment, red represents after treatment). The differences between the groups are as follows: the two groups are basically distinguishable, but there is still some overlap, indicating a good pattern, which can be further analyzed by PLS-DA.

[0188] (2) Unsupervised PCA analysis before and after treatment in the traditional Chinese medicine group

[0189] Unsupervised PCA analysis was performed on patients in the traditional Chinese medicine group before and after treatment. Figure 2 The figure shows the PCA analysis before and after treatment with traditional Chinese medicine (A represents before treatment, and C represents after treatment). The distribution of the traditional Chinese medicine group before and after treatment was observed in the figure (PC1 vs. PC2, R2 = 95.4%, Q2 = 71.8%). R2x and R2Y, i.e. Q2, are both greater than 50%, indicating that the model is good and has high reliability. The two groups are basically separated and can be used for OPLS-DA analysis.

[0190] (3) Analysis of PLS-DA and OPLS-DA before and after treatment in the traditional Chinese medicine group

[0191] Further PLS-DA analysis was performed on the above PCA statistical results, and the results are as follows: Figure 3 (R²x = 94.8%, R²Y = 98.3%, Q²(cum) = 94.3%). Based on this, OPLS-DA analysis was further performed to improve the judgment ability of the pattern recognition method, and the results are as follows. Figure 4 As shown in (R2x=91.4%, R2Y=98.3%, Q2(cum)=94.3%), using a weighting factor (VIP) > 1 and combined with the metabolic profile, a total of 41 metabolites were screened out (see Table 22).

[0192] Meanwhile, the differences in serum metabolites before and after traditional Chinese medicine treatment were analyzed using volcano plots. Figure 5 As shown in Table 22, compared with before treatment, the levels of HDL, α-ketoglutarate, phenylalanine, alanine, acetate, butyric acid, dimethylamine, glutamate, glutamine, citrulline, succinic acid, xanthine, methionine, methionine, tyrosine, leucine, citric acid, taurine, proline, prostaglandin D2, hydroxybutyrate, tryptophan, aspartic acid, homocysteine, adenine, isoleucine, and histidine increased after traditional Chinese medicine treatment, while the levels of 3-hydroxybutyric acid, VLDL / LDL, glycerol, glycine, creatinine, lysine, glucose, threonine, acetate, lactic acid, and betaine all showed a decreasing trend.

[0193] Table 22 Differences in serum metabolites before and after traditional Chinese medicine treatment

[0194]

[0195] (4) Analysis of potential metabolic pathways before and after treatment with traditional Chinese medicine

[0196] Changes in metabolites can reflect the overall metabolic information network and help identify abnormal metabolic pathways. Abnormal metabolites before and after traditional Chinese medicine treatment were analyzed using MetaboAnalyst 5.0, revealing 30 important metabolic pathways. 14 pathways with a P-value less than 0.05 or an impact greater than 0.1 were selected as potential target pathways. Figure 6 Table 23 shows the metabolism of glyoxylate and dicarboxylic acid, alanine, aspartic acid and glutamic acid, valine, leucine and isoleucine biosynthesis, citric acid cycle (TCA cycle), pyruvate metabolism, arginine biosynthesis, phenylalanine, tyrosine and tryptophan biosynthesis, butyrate metabolism, glycine, serine and threonine metabolism, β-alanine metabolism, phenylalanine metabolism, glycolysis / gluconeogenesis, cysteine ​​and methionine metabolism, and histidine metabolism. Combined with the KEGG database, the metabolic pathways involved include energy metabolism, glucose metabolism, amino acid metabolism, lipid metabolism, and immune disorders.

[0197] Table 23 Changes in metabolic pathways before and after traditional Chinese medicine treatment

[0198]

[0199] (5) Results of in vitro experiments

[0200] In cell experiments, RAW264.7 was induced to undergo osteoclastogenesis using RANKL and M-SCF, and after intervention with different concentrations of traditional Chinese medicine and alendronate sodium, the results showed ( Figure 7 , 8 The OD values ​​of all concentrations of traditional Chinese medicine (TCM) and Western medicine were higher than those of the control group, and the OD value of the TCM group after 72 hours was significantly higher than that of the Western medicine group, indicating that TCM and alendronate sodium (ALE) had no toxic effects on cells, while the cell survival rate was higher after TCM intervention. In rat bone marrow mesenchymal stem cells, osteoblasts were induced using osteogenic induction culture medium and different concentrations of TCM, and the results showed... Figure 9 The OD values ​​of all concentrations of traditional Chinese medicine (TCM) and osteogenic induction culture medium were higher than those of the control group, and the OD value of the TCM group was significantly higher than that of the osteogenic induction culture medium group. This indicates that TCM and osteogenic induction culture medium have no toxic effect on cells. The cell survival rate after TCM intervention was higher, suggesting that TCM helps osteoblast growth. This is consistent with the conclusion in the clinical study that the TCM group was more effective in treating osteoporosis than the Western medicine group.

[0201] In summary, the spleen-tonifying, kidney-tonifying, and bone-strengthening traditional Chinese medicine composition of this invention, in combination with the Western medicine alendronate sodium, can effectively improve muscle mass and strength, and alleviate joint pain symptoms. It can effectively improve patients' inflammatory chemokines, limb skeletal muscle mass, and bone metabolism indicators, reducing fracture risk and preventing falls, which is superior to alendronate sodium treatment alone. The traditional Chinese medicine compound effectively improves the metabolic network in patients with rheumatoid arthritis and bone metabolism disorders. The traditional Chinese medicine compound promotes osteoblast growth, improves bone metabolism, and prevents osteoporosis. This demonstrates that it can effectively improve bone and muscle metabolism in patients with rheumatoid arthritis and bone metabolism disorders (sarcopenia), providing experimental evidence and new ideas for developing effective traditional Chinese medicine compound treatments for rheumatoid arthritis and bone metabolism disorders (sarcopenia).

[0202] The embodiments of the present invention have been described in detail above, but the present invention is not limited to the described embodiments. For those skilled in the art, various changes, modifications, substitutions, and variations can be made to these embodiments without departing from the principles and spirit of the present invention, and these variations still fall within the protection scope of the present invention.

Claims

1. The application of a traditional Chinese medicine composition for invigorating the spleen, tonifying the kidneys, and strengthening bones in the preparation of a drug for improving bone and muscle metabolism in rheumatoid arthritis and bone metabolism disorders—sarcopenia, characterized in that, The drug comprises a traditional Chinese medicine composition for strengthening the spleen, tonifying the kidneys, and invigorating bones, and sodium alendronate; by weight, the raw materials of the traditional Chinese medicine composition for strengthening the spleen, tonifying the kidneys, and invigorating bones are: 10-20 parts of Eucommia ulmoides, 10-20 parts of Dipsacus asper, 10-20 parts of Cuscuta chinensis, 15-30 parts of Astragalus membranaceus, 5-15 parts of Schisandra chinensis, 10-15 parts of Poria cocos, 10-20 parts of Angelica sinensis, 10-20 parts of Achyranthes bidentata, 5-10 parts of deer antler glue, 10-15 parts of peach kernel, 10-15 parts of Drynaria fortunei, and 10-20 parts of Rehmannia glutinosa.

2. The application according to claim 1, characterized in that, The raw materials of the spleen-strengthening, kidney-tonifying, and bone-strengthening traditional Chinese medicine composition, by weight, are: 15 parts Eucommia ulmoides, 15 parts Dipsacus asper, 15 parts Cuscuta chinensis, 15 parts Astragalus membranaceus, 6 parts Schisandra chinensis, 12 parts Poria cocos, 12 parts Angelica sinensis, 12 parts Achyranthes bidentata, 10 parts deer antler powder, 10 parts peach kernel, 10 parts Drynaria fortunei, and 10 parts Rehmannia glutinosa.

3. The application according to claim 1, characterized in that, The traditional Chinese medicine composition also includes pharmaceutically acceptable excipients.

4. The application according to claim 3, characterized in that, The dosage form of the traditional Chinese medicine composition is selected from decoctions, granules, ointments, pills, powders, tablets, and capsules.

5. The application according to claim 4, characterized in that, The dosage form of the traditional Chinese medicine composition is granules, and the specific preparation method includes the following steps: S1. Decoction: Chop Eucommia ulmoides, Dipsacus asper, Cuscuta chinensis, Astragalus membranaceus, Schisandra chinensis, Poria cocos, Angelica sinensis, Achyranthes bidentata, deer antler glue, peach kernel, Drynaria fortunei, and Rehmannia glutinosa. After washing with water, soak for 1-2 hours. Then add water and decoct three times. For the first decoction, add water to slightly cover the herbs and decoct for 1-2 hours. For the second and third decoctions, gradually reduce the amount of water and decoct for 1-1.5 hours each time. Combine the three decoctions and filter through a 50-70 mesh sieve. Let it stand and settle for more than 12 hours until the supernatant is clear. Set aside for later use. S2. Concentration: Concentrate the supernatant of the drug solution in S1 to a relative density of 1.35-1.38 to obtain the clear extract; S3. Granulation: Mix twice the amount of sucrose powder and twice the amount of dextrin with the clear paste of S2, and spray in 75% ethanol to make a suitable soft material. Finally, according to the requirement of "forming a ball when squeezed, and dispersing when touched", use a swing-type No. 1 filter screen to granulate the soft material.

6. The application according to claim 5, characterized in that, The concentration is carried out in a vacuum-reduced concentration tank at a temperature of 70-85℃, a steam pressure of 0.14-0.16 MPa, and a vacuum degree of -0.06-0.08 MPa.

7. The application according to claim 4, characterized in that, The dosage form of the traditional Chinese medicine composition is a decoction. The specific preparation method is as follows: add water to Eucommia ulmoides, Dipsacus asper, Cuscuta chinensis, Astragalus membranaceus, Schisandra chinensis, Poria cocos, Angelica sinensis, Achyranthes bidentata, Prunus persica, Drynaria fortunei, and Rehmannia glutinosa, decoct over medium heat for 30-50 minutes, remove the dregs after decoction, and then decoct over low heat until it is reduced to three-quarters of the original volume. Then add deer antler glue, melt it to obtain the decoction. Alternatively: First, add deer antler powder to water and simmer over high heat for 5-15 minutes. Then add eucommia bark, dipsacus root, dodder seed, astragalus root, schisandra fruit, poria cocos, angelica root, achyranthes root, peach kernel, drynaria rhizome, and prepared rehmannia root. Simmer over medium heat for 30-50 minutes. After simmering, remove the dregs and then simmer over low heat until the volume is reduced to three-eighths of the original volume to obtain a traditional Chinese medicine decoction.

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