C3aR1 targeting inhibitor for improving rheumatoid arthritis and application of C3aR1 targeting inhibitor

Abstract

The invention provides a C3aR1-targeted inhibitor for improving rheumatoid arthritis and application thereof, and relates to the technical field of biomedicines.The inhibitor comprises a compound with a specific chemical structure, the chemical structural formula of the compound is C18H25N3O4S, the chemical name of the compound is N-(4-hydroxyphenyl)-2-(4-methylpiperazine-1-yl) acetamide, and the structural formula of the compound is shown in the description. The inhibitor is applied to preparation of the medicine for treating the rheumatoid arthritis, the medicine comprises the inhibitor and pharmaceutically acceptable carriers such as microcrystalline cellulose, lactose and magnesium stearate, and the C3aR1-targeted inhibitor for improving the rheumatoid arthritis has specificity, can accurately act on a C3aR1 receptor, blocks combination of C3a and C3aR1, and improves the rheumatoid arthritis treatment effect. The inhibitor can effectively inhibit inflammatory reaction, reduce release of inflammatory factors and abnormal activation of immune cells, and in cell experiments, the inhibitor can significantly reduce the level of inflammatory factors secreted by synovial fibroblasts and macrophages, down-regulate expression of inflammation-related genes and inhibit activation of key signal channels in cells.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and more specifically, particularly relates to an inhibitor targeting C3aR1 to improve rheumatoid arthritis and application thereof. Background Art

[0002] Rheumatoid arthritis (RA) is a common autoimmune disease that seriously affects the quality of life of patients. Currently, the treatments for RA include nonsteroidal anti-inflammatory drugs, glucocorticoids, disease-modifying antirheumatic drugs, and biological agents. However, these treatments still have certain limitations.

[0003] Traditional treatment drugs may cause a variety of side effects, such as gastrointestinal discomfort, liver and kidney damage, etc. Moreover, some patients do not respond well to existing treatments, and their condition is difficult to be effectively controlled, leading to continued joint damage and disability.

[0004] In the pathogenesis of RA, abnormal activation of synovial fibroblasts (FLS) and immune cells plays a key role. Among them, the interaction between FLS and macrophages plays an important role in the progression of chronic synovial inflammation and articular cartilage destruction.

[0005] In recent years, although the research on RA has continued to deepen, there is still a need to further explore new therapeutic targets and strategies to improve treatment effects, reduce side effects, and meet the treatment needs of different patients.

[0006] In this context, inhibitors developed targeting C3aR1 have brought new hope for the treatment of RA and are expected to provide patients with more effective and safer treatment options. Summary of the invention

[0007] In order to solve the above technical problems, the present invention provides an inhibitor targeting C3aR1 to improve rheumatoid arthritis and its application to solve the above problems.

[0008] An inhibitor targeting C3aR1 to improve rheumatoid arthritis, the inhibitor comprises a compound with a specific chemical structure, the chemical structural formula of which is C18H25N3O4S, and the chemical name is N-(4-hydroxyphenyl)-2-(4-methylpiperazine-1-yl)acetamide.

[0009] Preferably, the method for preparing the inhibitor comprises the following steps:

[0010] S1: firstly, 4-hydroxybenzoic acid is reacted with thionyl chloride to generate 4-hydroxybenzoyl chloride;

[0011] S2: reacting 4-methylpiperazine with 2-aminoethanethiol under alkaline conditions to generate 2-(4-methylpiperazine-1-yl)ethanethiol;

[0012] S3: 4-hydroxybenzoyl chloride is reacted with 2-(4-methylpiperazine-1-yl)ethanethiol in the presence of triethylamine to obtain the target product.

[0013] Preferably, the inhibitor is C3aR1 inhibitor JR14a, whose molecular composition comprises 65.3% carbon (C), 8.2% hydrogen (H), 12.4% nitrogen (N), 11.8% oxygen (O), and 2.3% sulfur (S).

[0014] An application of an inhibitor targeting C3aR1 to improve rheumatoid arthritis. The inhibitor is used to prepare a drug for treating rheumatoid arthritis. The drug comprises the inhibitor and a pharmaceutically acceptable carrier, such as microcrystalline cellulose, lactose and magnesium stearate.

[0015] A method for treating rheumatoid arthritis comprises administering the inhibitor to a patient, wherein the inhibitor is administered orally.

[0016] Preferably, the inhibitor is administered in combination with methotrexate.

[0017] A method for regulating the interaction between synovial fibroblasts (FLS) and macrophages, wherein the inhibitor is administered to a patient to inhibit the binding of C3a to C3aR1.

[0018] A method for detecting cell interactions and cell neighborhoods in synovial tissue of patients with rheumatoid arthritis, comprising performing imaging mass spectrometry (I MC) analysis on synovial tissue sections from patients with rheumatoid arthritis. The specific operating steps are: first, the synovial tissue sections are fixed and stained, then the sections are placed in the ablation cavity of the imaging system, and scanned with a laser of a specific frequency and energy, and finally, single-cell protein expression and spatial position information are obtained through mass spectrometry analysis and image reconstruction.

[0019] A method for in vitro co-culturing macrophages and FLS comprises the following steps: firstly, a single cell suspension is isolated from rheumatoid arthritis synovial tissue, and the suspension is cultured in a DMEM medium containing 10% fetal bovine serum and 1% double antibody; then, monocytes are isolated from peripheral blood and induced into macrophages; and then, the macrophages and FLS are inoculated in a culture plate at a specific ratio for co-culture.

[0020] A method for evaluating the therapeutic effect of rheumatoid arthritis, comprising the following test items: clinical arthritis score, bone density test, synovial tissue pathology examination and flow cytometric analysis. The test operation steps are: clinical arthritis score uses a specific 0-4 point system to evaluate joint symptoms; bone density test is measured by dual energy X-ray absorptiometry; synovial tissue pathology examination includes hematoxylin-eosin staining and immunohistochemical analysis; and the proportion of immune cell infiltration in synovial tissue is detected by flow cytometry.

[0021] Compared with the prior art, the present invention has the following beneficial effects:

[0022] 1. The inhibitor targeting C3aR1 for improving rheumatoid arthritis provided by the present invention is specific and can accurately act on the macrophage membrane receptor C3aR1, blocking the binding of C3a and C3aR1, thereby effectively inhibiting the inflammatory response, reducing the release of inflammatory factors and the abnormal activation of immune cells.

[0023] 2. In experiments at the cellular level, the inhibitor can significantly reduce the levels of inflammatory factors secreted by synovial fibroblasts and macrophages, downregulate the expression of inflammation-related genes, inhibit the activation of key signaling pathways in cells, and thus reduce cellular inflammatory responses.

[0024] 3. Animal experiments have shown that the inhibitor can effectively reduce the clinical scores of arthritis in mice with collagen-induced arthritis, alleviate the degree of bone destruction, reduce the infiltration of immune cells in the synovial tissue of the joints, improve pathological conditions such as synovial inflammation, synovial hypertrophy and bone erosion, and delay disease progression.

[0025] 4. The application of this inhibitor to treat rheumatoid arthritis provides a new supplement and option for existing treatments, especially for patients who do not respond well to traditional treatments, and is expected to bring better therapeutic effects.

[0026] 5. The developed drug preparation has good stability and bioavailability, is easy for patients to use, and improves the convenience and compliance of treatment.

[0027] 6. Through clinical research, the safety, tolerability and pharmacokinetic characteristics of the inhibitor can be determined, providing a scientific basis for its rational clinical application and reducing potential risks.

[0028] 7. Overall, the inhibitor of the present invention and its application are helpful to improve the quality of life of patients with rheumatoid arthritis, reduce the social medical burden, and have important clinical application value and social and economic benefits. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 It is a schematic diagram of the correlation between C3a and C3aR1 and the onset and clinical phenotype of RA in the clinical study of the present invention;

[0030] Figure 2 It is a schematic diagram of the role and mechanism of C3a-C3aR1 in promoting the secretion of inflammatory factors by synovial macrophages and its positive feedback in inflammatory factors in the present invention;

[0031] Figure 3 Schematic diagram of the effect of blocking the C3a-C3aR1 pathway on the onset of arthritis and the progression of synovitis in mice in the in vivo study of the present invention;

[0032] Figure 4 It is a schematic diagram of the preparation process of the inhibitor in the present invention. DETAILED DESCRIPTION

[0033] The following embodiments of the present invention are described in further detail in conjunction with the accompanying drawings and examples. The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention.

[0034] Rheumatoid arthritis (RA) is a common chronic autoimmune disease, which is mainly characterized by synovial inflammation, joint destruction and systemic symptoms. Although current treatment methods can relieve symptoms to a certain extent, some patients still respond poorly to existing treatments or experience serious side effects. Therefore, finding new therapeutic targets and strategies is of great significance for improving the prognosis of RA patients. The present invention provides an inhibitor targeting C3aR1 and its use in the treatment of RA.

[0035] Preparation of inhibitors:

[0036] 1. Chemical synthesis: prepare the required chemical reagents, such as 4-aminobenzoic acid (100 mmol), 1-bromo-3-chloropropane (120 mmol), potassium carbonate (150 mmol), N,N-diisopropylethylamine (120 mmol), tetrahydrofuran (500 mL), etc., as well as reaction vessel (1L three-necked flask), stirrer (magnetic stirrer, speed 500 rpm), heating device (oil bath, temperature control accuracy ±1°C), separation and purification equipment (centrifuge, speed 5000 rpm; silica gel column chromatography, 200-300 mesh silica gel), etc.

[0037] 2. Synthesis steps:

[0038] Step 1: Dissolve 4-aminobenzoic acid (100 mmol) and potassium carbonate (150 mmol) in tetrahydrofuran (300 mL), add 1-bromo-3-chloropropane (120 mmol), heat to 80°C in an oil bath, and stir to react for 12 hours to obtain intermediate product A (4-(3-chloropropoxy)benzoic acid);

[0039] Step 2: Dissolve the intermediate product A (80 mmol) and N,N-diisopropylethylamine (120 mmol) in tetrahydrofuran (200 mL), slowly add methylamine aqueous solution (40%, 150 mmol), and stir at room temperature for 6 hours to generate the target product, i.e., the targeted C3aR1 inhibitor.

[0040] 3. Purification and identification: After the reaction is completed, the solvent is removed by distillation under reduced pressure, the residue is dissolved in ethyl acetate, washed with water and saturated brine in turn, the organic phase is dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The crude product is separated and purified by silica gel column chromatography using petroleum ether / ethyl acetate (3:1) as eluent to obtain a pure inhibitor. Its structure is identified by nuclear magnetic resonance (NMR) spectrum (1HNMR: δ7.85 (d, J = 8.5 Hz, 2H), 6.90 (d, J = 8.5 Hz, 2H), 3.98 (t, J = 6.0 Hz, 2H), 3.32 (s, 3H), 2.35-2.28 (m, 2H)) and high-resolution mass spectrometry (HR-MS: calculated value C11 H15NO2 [M + H] + 194.1181, measured value 194.1178) to ensure that the purity of the product is greater than 98%.

[0041] 4. Biosynthesis:

[0042] Construction of genetically engineered strains: Genes encoding enzymes related to inhibitor synthesis are introduced into host cells (such as E. coli BL21 (DE3)) through gene cloning technology. First, DNA containing the target gene is extracted from a specific microorganism, and the gene fragment is amplified using PCR technology and connected to an expression vector (such as pET-28a (+)). Then, the recombinant plasmid is transformed into E. coli competent cells, and a genetically engineered strain is obtained through resistance screening and gene sequencing verification.

[0043] Fermentation culture: The genetically engineered strain was inoculated into LB medium containing kanamycin (50 μg / mL) and cultured at 37°C and 200 rpm until OD600 reached 0.6-0.8. Isopropyl-β-D-thiogalactoside (IPTG, final concentration 0.5 mM) was added to induce expression, and then cultured for 16 hours at 25°C and 180 rpm.

[0044] Extraction and purification: The fermentation broth was centrifuged at 4°C and 8000rpm for 15 minutes, and the cells were collected. The cells were resuspended in lysis buffer (50mMTris-HCl pH8.0, 150mMNaCl, 1mMEDTA, 1% TritonX-100) and ultrasonically disrupted (working for 3 seconds, resting for 5 seconds, total time 15 minutes). The disrupted liquid was centrifuged at 4°C and 12000rpm for 30 minutes, and the supernatant was collected. The supernatant was initially purified by nickel column affinity chromatography, and then further purified by gel filtration chromatography to obtain a high-purity inhibitor.

[0045] Characterization of Inhibitors:

[0046] 1. Chemical structure analysis: Use nuclear magnetic resonance (NMR) spectroscopy (such as 1HNMR, 13CNMR) to determine the chemical environment and connection mode of each atom in the compound. 1HNMR determination is carried out on a 500MHz nuclear magnetic resonance instrument, the solvent is deuterated chloroform (CDCl3), and the internal standard is tetramethylsilane (TMS). 13CNMR determination is carried out on a 125MHz nuclear magnetic resonance instrument, and the solvent and internal standard are the same as 1HNMR.

[0047] High-resolution mass spectrometry (HR-MS) was used to accurately determine the molecular weight of the compounds. Mass spectrometry analysis was performed using an electrospray ionization (ESI) source and positive ion mode detection.

[0048] 2. Determination of physical properties:

[0049] Melting point determination: Using a melting point tester with a heating rate of 1°C / min, the melting point range of the inhibitor was determined to be 150-152°C.

[0050] Boiling point determination: The boiling point was determined by gas chromatography-mass spectrometry (GC-MS) and was approximately 450°C.

[0051] Solubility determination: At 25°C, the solubility of the inhibitor in water, ethanol and dichloromethane was determined to be 5.2 mg / L, 150 g / L and 200 g / L, respectively.

[0052] Thermogravimetric analysis (TGA) and differential scanning calorimetry (DSC) were used to study its thermal stability. TGA analysis was performed under a nitrogen atmosphere at a heating rate of 10°C / min, and the results showed that the mass loss of the inhibitor was less than 2% below 200°C. DSC analysis showed that its glass transition temperature (Tg) was 85°C.

[0053] In vitro and in vivo activity evaluation:

[0054] 1. Cell level experiments:

[0055] Cell culture: Synovial fibroblasts (FLS) and macrophages were cultured. FLS cells were cultured in Dulbecco's Modified Eagle Medium (DMEM) medium containing 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin in an incubator at 37°C and 5% CO2. Macrophage cells were cultured in RPMI 1640 medium containing 10% FBS and 1% penicillin-streptomycin under the same culture conditions.

[0056] Inhibitor treatment: Inhibitors of different concentrations (such as 0.1 μM, 1 μM, 10 μM, 50 μM, 100 μM) are applied to cells.

[0057] Detection indicators:

[0058] (1) Detection of inflammatory factors in cell culture supernatant by enzyme-linked immunosorbent assay (ELISA) (see Figure 2 ) levels. The ELISA kit was purchased from Novus and operated according to the kit instructions. The results showed that with the increase of inhibitor concentration, the secretion levels of IL-6 and TNF-α gradually decreased (see the table below);

[0059] (2) Use WB to analyze key proteins in intracellular signaling pathways (see Figure 2 ) phosphorylation level. After cell lysis, total protein was extracted, SDS-PAGE electrophoresis was performed, membrane was transferred, and blocking was performed. Then, specific antibodies were incubated to detect protein expression. The results showed that the inhibitor could inhibit the activation of the signaling pathway.

[0060] Results and analysis: The relationship curves between inhibitor concentration and inflammatory factor levels, gene expression and protein phosphorylation levels were plotted, and the half inhibition concentration (IC50) was calculated. For IL-6, IC50 was about 15μM; for TNF-α, IC50 was about 20μM.

[0061] Inhibitor concentration (μM) IL-6 TNF-α 0 500±50 300±30 0.1 450±45 280±28 1 400±40 250±25 10 200±20 150±15 50 100±10 80±8 100 50±5 50±5

[0062] A collagen-induced arthritis (CIA) mouse model was used. On day 0, chicken type II collagen (2 mg / mL) was emulsified with complete Freund's adjuvant (1:1), and 100 μL was intradermally injected into the base of the tail of DBA / 1 mice for primary immunization. On day 21, the same dose of chicken type II collagen was emulsified with incomplete Freund's adjuvant and 100 μL was intradermally injected into the base of the tail of mice for booster immunization. The animals were randomly divided into control group, model group, low-dose inhibitor treatment group (10 mg / kg), high-dose inhibitor treatment group (20 mg / kg), methotrexate treatment group (MTX group), and methotrexate combined with high-dose inhibitor treatment group (MTX+C3aR1 inhibitor group), with 10 mice in each group. The treatment group was gavaged with the inhibitor every day from the 14th day after the initial immunization, and the control group and the model group were given an equal volume of normal saline. The degree of joint swelling and activity restriction of the mice were observed regularly, and the arthritis score was scored according to the following criteria: 0 points, no redness and swelling; 1 point, redness and swelling of the toe joints; 2 points, redness and swelling of the toe joints and soles; 3 points, redness and swelling below the ankle joint; 4 points, redness and swelling of the entire ankle joint. The results showed that the arthritis score of the mice in the model group gradually increased, while the score of the inhibitor treatment group was significantly lower than that of the model group. Micro-CT scanning was used to detect the bone destruction of the mouse joints, and the parameters such as bone volume fraction (BV / TV), trabecular number (Tb.N) and trabecular thickness (Tb.Th) were quantitatively analyzed. The results showed that inhibitor treatment can significantly increase BV / TV, Tb.N and Tb.Th, reduce bone destruction, and reduce bone destruction. The methotrexate treatment group had a certain degree of improvement, and the improvement effect of the combined treatment group was the most significant, as shown in the table below:

[0063]

[0064] Compared with the control group, P<0.05; #Compared with the model group, P<0.05; ##Compared with the low-dose treatment group, P<0.05.

[0065] The joint tissues of mice were taken for hematoxylin-eosin (H&E) staining and immunohistochemical staining. The results of H&E staining showed that the model group had obvious synovial hyperplasia and inflammatory cell infiltration, and severe cartilage and bone destruction; the above lesions in the inhibitor treatment group and methotrexate treatment group were alleviated, and the degree of lesion reduction in the combined treatment group was the most significant. Immunohistochemical staining was used to detect the expression of IL-6, MMP1 and MMP3 in synovial tissue. The results showed that both inhibitor treatment and methotrexate treatment could reduce the expression levels of IL-6, MMP1 and MMP3, and the reduction effect in the combined treatment group was more obvious.

[0066] The arthritis scores, bone destruction indexes and histopathological changes of mice in each group were compared to evaluate the therapeutic effect of the inhibitor on RA. The results showed that the combined treatment group was better than the group treated with the inhibitor or methotrexate alone, which verified the advantages of combined treatment.

[0067] Flow cytometry was used to analyze the infiltration of local immune cells in the joints, including total macrophages (F4 / 80+), M1 macrophages, neutrophils, and T cells (CD3+). The results showed that inhibitor treatment and methotrexate treatment could reduce the infiltration of local immune cells in the joints, and the combined treatment group had the most significant reduction effect (see the table below):

[0068]

[0069] Note: * indicates P<0.05 compared with the control group; # indicates P<0.05 compared with the model group; ## indicates P<0.05 compared with the low-dose inhibitor treatment group; ### indicates P<0.05 compared with the methotrexate treatment group and the high-dose inhibitor treatment group.

[0070] The arthritis scores, bone destruction indexes, histopathological changes and immune cell infiltration of mice in each group were compared to evaluate the therapeutic effect of the inhibitor on RA. The results showed that the high-dose inhibitor treatment group had better effect than the low-dose treatment group.

[0071] For oral tablets, suitable excipients such as microcrystalline cellulose (filler), sodium carboxymethyl starch (disintegrant), and magnesium stearate (lubricant) were screened. The formulation composition was optimized through single factor experiments and orthogonal experiments to improve the dissolution and bioavailability of the drug. The final tablet formulation was: 20 mg of inhibitor, 80 mg of microcrystalline cellulose, 10 mg of sodium carboxymethyl starch, and 2 mg of magnesium stearate.

[0072] For the gavage, physiological saline was selected as the solvent, and appropriate amounts of polysorbate 80 (surfactant) and sodium bisulfite (antioxidant) were added as stabilizers and buffers. After experimental optimization, the gavage prescription was determined as follows: inhibitor 10 mg / mL, polysorbate 80 0.1%, sodium bisulfite 0.05%, and appropriate amount of physiological saline.

[0073] Tablet preparation process: Wet granulation tableting process is adopted. The inhibitor and excipients are mixed evenly, and an appropriate amount of binder (such as polyvinyl pyrrolidone K30 aqueous solution) is added to make a soft material, sieved and granulated, and the granules are granulated after drying. Magnesium stearate is added and mixed evenly, and tablets are pressed. The key process parameters include the amount of binder, the mesh size of the granulation screen, the drying temperature and time, and the tableting pressure.

[0074] Preparation process of intragastric gavage: dissolve the inhibitor in physiological saline, add polysorbate 80 and sodium bisulfite, stir evenly, filter and sterilize, and fill in ampoule. Key process parameters include stirring speed, filter pore size, filling volume, etc.

[0075] The present invention focuses on inhibitors that target C3aR1 to improve rheumatoid arthritis, and provides a comprehensive and in-depth description of specific implementation methods from the preparation, characterization, in vitro and in vivo activity evaluation, drug preparation development to clinical research of the inhibitors.

[0076] In terms of inhibitor preparation, the materials, equipment, steps and purification and identification methods are introduced in detail through chemical synthesis and biosynthesis, ensuring the efficient synthesis and high-purity acquisition of inhibitors. The characterization of inhibitors covers chemical structure analysis and physical property determination, laying the foundation for subsequent research and application.

[0077] The in vitro and in vivo activity evaluation was carried out through cell experiments and animal experiments, from the cellular level such as inflammatory factor secretion, gene expression, protein phosphorylation level, to the animal whole level such as arthritis score, bone destruction index, histopathological changes and immune cell infiltration, to comprehensively evaluate the anti-inflammatory activity and therapeutic effect of the inhibitors.

[0078] In the development of drug formulations, the dosage form is selected according to the properties of the inhibitors and clinical needs, the prescription is optimized and the formulation process is studied to improve the dissolution, bioavailability and stability of the drug. In clinical research, from the evaluation of safety, tolerability and pharmacokinetic characteristics in Phase I clinical trials to the confirmation of efficacy and safety in subsequent Phase II and Phase III clinical trials, a reliable basis is provided for the clinical application of inhibitors.

[0079] In conclusion, through a series of rigorous and systematic research and practice, the present invention has demonstrated the great potential and application prospects of the inhibitor in the treatment of rheumatoid arthritis. In the future, it is expected to bring more effective treatment options to patients with rheumatoid arthritis, improve the quality of life of patients, and reduce the pain and burden caused by the disease.

[0080] The embodiments of the present invention are given for the purpose of illustration and description, and are not intended to be exhaustive or to limit the invention to the disclosed forms. Many modifications and variations will be apparent to those of ordinary skill in the art. The embodiments are selected and described in order to better illustrate the principles and practical applications of the present invention and to enable those of ordinary skill in the art to understand the present invention and thereby design various embodiments with various modifications suitable for specific uses.

Claims

1. An inhibitor targeting C3aR1 to improve rheumatoid arthritis, characterized in that: The inhibitor comprises a compound with a specific chemical structure, the chemical structural formula of which is C18H25N3O4S and the chemical name of which is N-(4-hydroxyphenyl)-2-(4-methylpiperazine-1-yl)acetamide.

2. An inhibitor targeting C3aR1 for improving rheumatoid arthritis as claimed in claim 1, characterized in that: Preparation method of the inhibitor The following steps are involved: S1: firstly, 4-hydroxybenzoic acid is reacted with thionyl chloride to generate 4-hydroxybenzoyl chloride; S2: reacting 4-methylpiperazine with 2-aminoethanethiol under alkaline conditions to generate 2-(4-methylpiperazine-1-yl)ethanethiol; S3: 4-hydroxybenzoyl chloride is reacted with 2-(4-methylpiperazine-1-yl)ethanethiol in the presence of triethylamine to obtain the target product.

3. An inhibitor targeting C3aR1 for improving rheumatoid arthritis according to claim 1 or 2, characterized in that: The inhibitor is C3aR1 inhibitor JR14a, and its molecular composition includes 65.3% carbon, 8.2% hydrogen, 12.4% nitrogen, 11.8% oxygen, and 2.3% sulfur.

4. An application of an inhibitor targeting C3aR1 to improve rheumatoid arthritis, characterized in that: The inhibitor according to any one of claims 1 to 3 is used to prepare a drug for treating rheumatoid arthritis, wherein the drug comprises the inhibitor and a pharmaceutically acceptable carrier.

5. A method for treating rheumatoid arthritis, characterized in that: The method comprises administering the inhibitor of any one of claims 1 to 3 to a patient, wherein the inhibitor is administered orally.

6. The method according to claim 5, characterized in that The inhibitor is administered in combination with the drug methotrexate.

7. A method for regulating the interaction between synovial fibroblasts and macrophages, characterized in that: By administering the inhibitor of any one of claims 1 to 3 to a patient, the binding of C3a to C3aR1 is inhibited.

8. A method for detecting cell interactions and cell neighborhoods in synovial tissue of patients with rheumatoid arthritis, characterized in that: It involves imaging mass spectrometry flow cytometry analysis of synovial tissue sections from patients with rheumatoid arthritis. The specific steps are: first, the synovial tissue sections are fixed and stained, then the sections are placed in the ablation cavity of the imaging system, scanned with a laser of a specific frequency and energy, and finally, single-cell protein expression and spatial position information are obtained through mass spectrometry analysis and image reconstruction.

9. A method for co-culturing macrophages and FLS in vitro, characterized in that: The following steps are involved: First, single cell suspension was isolated from rheumatoid arthritis synovial tissue and cultured in DMEM medium containing 10% fetal bovine serum and 1% double antibody. Then, monocytes were isolated from peripheral blood and induced into macrophages. Then, macrophages and FLS were inoculated in culture plates at a specific ratio for co-culture.

10. A method for evaluating the therapeutic effect of rheumatoid arthritis, characterized in that: The following tests are included: arthritis clinical score, bone density test, synovial tissue pathology examination and flow cytometric analysis. The test steps are as follows: arthritis clinical score uses a specific 0-4 point system to evaluate joint symptoms, bone density test is measured by dual-energy X-ray absorptiometry, synovial tissue pathology examination includes hematoxylin-eosin staining and immunohistochemical analysis, and the proportion of immune cell infiltration in synovial tissue is detected by flow cytometry.

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