Application of isoprenylcysteine carboxymethyltransferase as a therapeutic target for rheumatoid arthritis in the preparation of drugs for treating rheumatoid arthritis

Abstract

The application discloses application of isoprenyl cysteine carboxymethyltransferase as a rheumatoid arthritis treatment target in preparation of a rheumatoid arthritis treatment drug. The application finds that the expression of ICMT is down-regulated after NSUN2 is knocked out, further research on the expression level of ICMT in RA FLS finds that the mRNA and protein expression of RA FLS ICMT is obviously up-regulated, the cell migration and invasion ability is reduced after ICMT is knocked down, and the expression of MMPs such as MMP1, MMP3 and MMP9 of RA FLS is obviously inhibited. The above results show that ICMT can regulate the abnormal migration and invasion of RA FLS, and targeting ICMT can become a new means to prevent RA joint destruction. Therefore, the application provides an effective treatment target for RA, and provides a new idea for clinical treatment of RA.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine, specifically relating to the application of isoprenylcysteine ​​carboxymethyltransferase as a therapeutic target for rheumatoid arthritis in the preparation of drugs for treating rheumatoid arthritis. Background Technology

[0002] Rheumatoid arthritis (RA) is an autoimmune disease characterized by chronic synovitis, pannus formation, and cartilage and bone destruction, with a global prevalence of approximately 0.5%–1.0%. Its core pathological mechanism involves the abnormal activation of fibroblast-like synoviocytes (FLS), manifested as excessive proliferation, apoptosis resistance, and excessive secretion of inflammatory factors (such as IL-6 and TNF-α) and matrix metalloproteinases (MMPs), ultimately leading to joint destruction and loss of function. Current clinical treatments primarily utilize traditional synthetic disease-modifying antirheumatic drugs (csDMARDs, such as methotrexate, leflunomide, hydroxychloroquine, and sulfasalazine), biologic DMARDs (bDMARDs, such as TNF-α inhibitors, IL-6R antagonists, and CTLA4-Ig fusion proteins), and targeted synthetic DMARDs (tsDMARDs, mainly JAK inhibitors). However, these therapies have significant limitations: firstly, approximately 30%–40% of patients do not respond to initial csDMARD treatment; secondly, safety risks further constrain clinical choices: methotrexate among csDMARDs may cause bone marrow suppression and hepatotoxicity, bDMARDs (especially TNF-α inhibitors) significantly increase the risk of infection, and tsDMARDs require strict patient screening due to JAK inhibitor-related thrombotic events and cardiovascular side effects. These issues collectively lead to a narrow therapeutic window, necessitating a new strategy that breaks through the existing framework in clinical practice. More importantly, existing drugs mostly focus on inhibiting downstream inflammatory mediators (such as TNF-α and IL-6), and cannot intervene in upstream epigenetic regulation or abnormal phenotypic transformation of FLS (such as excessive proliferation, apoptosis resistance, and enhanced migration and invasion), making it difficult to fundamentally block disease progression.

[0003] Salvianolic Acid A (SAA), one of the main water-soluble active ingredients of Danshen (Salvia miltiorrhiza), has had its chemical properties and pharmacological mechanisms preliminarily explored, but its mechanism of action needs to be strictly distinguished from the multi-component mixture of Danshen. The core mechanism of SAA focuses on anti-oxidative stress and bone metabolism regulation: preclinical studies have shown that SAA reduces oxidative damage by downregulating the FoxO3a signaling pathway, promotes osteogenic differentiation by activating the Wnt / β-catenin pathway, and improves bone microcirculation disorders by regulating the VEGF pathway. In summary, SAA regulates the balance of the bone and joint microenvironment and oxidative inflammation through multiple targets. For example, patent CN112315951A discloses that a composition of salvianolic A and chitosan can significantly improve osteoarthritis and can be used to prepare drugs for the prevention and treatment of osteoarthritis. Furthermore, the first human pharmacokinetic study of SAA confirmed its safety and nonlinear pharmacokinetic characteristics after intravenous administration. However, there are currently no reports on the therapeutic and regulatory mechanisms of SAA for RA.

[0004] In recent years, epigenetic regulation has become an emerging direction in the treatment of rheumatoid arthritis (RA). NSUN2 (NOP2 / Sun RNAmethyltransferase 2) is a major RNA m... 5 C-methyltransferases can catalyze the m-transferase of target genes. 5 C-modification. The article "Fat mass and obesity-associated protein inhibit the pathology of rheumatoid arthritis through the NSUN2 / SFRP1 / Wnt / β-cateninsignal axis" points out that compared with normal controls, NSUN2 expression was significantly increased in RA and collagen-induced arthritis (CIA) rats. Knockdown of NSUN2 blocked the Wnt / β-catenin signaling pathway and inhibited RA pathological factors such as MMP3, fibronectin, and interleukins. Therefore, NSUN2-mediated RNA methylation is also related to the pathological process of RA. However, existing NSUN2 inhibitors face significant challenges in clinical translation: for example, covalent inhibitors are prone to off-target toxicity at high doses, requiring strict control of dosage to avoid damage to normal tissues; furthermore, none of the current NSUN2 inhibitors have entered the clinical trial stage, and their human safety remains to be determined.

[0005] Isoprenylcysteine ​​carboxymethyltransferase (ICMT) is a key enzyme catalyzing the final step of isoprenylation modification of proteins. It regulates membrane localization and signal transduction by adding a methyl group to the carboxyl terminus of isoprenized proteins (such as the RAS superfamily). RAS proteins are well-established cancer-related molecules, and ICMT catalyzes the final isoprenylation step of various oncoproteins, including RAS, regulating their carcinogenic biological activity. For example, ICMT promotes liver cancer growth, migration, survival, and chemotherapy resistance; activates RAS / AKT signal-dependent cell migration in melanoma; and ICMT gene knockout inhibits KRAS-induced oncogenic transformation of mouse fibroblasts and reduces tumor development in KRAS-transformed mammary epithelial cell xenografts. However, its role in RA has not yet been reported in the literature.

[0006] Therefore, further exploring the relationship between ICMT and RA, and providing a new therapeutic target or drug for RA, is of great significance. Summary of the Invention

[0007] The purpose of this invention is to overcome the above-mentioned defects and deficiencies in the prior art and to provide the application of isoprenylcysteine ​​carboxymethyltransferase (ICMT) as a therapeutic target for rheumatoid arthritis in the preparation of drugs for treating rheumatoid arthritis.

[0008] A second objective of this invention is to provide the use of an agent that inhibits the expression of isoprenylcysteine ​​carboxymethyltransferase in the preparation of a medicament for treating rheumatoid arthritis.

[0009] A third objective of this invention is to provide the use of salvianolic acid A in the preparation of a medicament for treating rheumatoid arthritis.

[0010] A fourth objective of this invention is to provide the application of cerium-based nanomaterials loaded with salvianolic acid A in the preparation of medicaments for treating rheumatoid arthritis.

[0011] The above-mentioned objective of this invention is achieved through the following technical solution:

[0012] Rheumatoid arthritis (RA) is characterized by the abnormal proliferation, migration, and invasion of fibroblast-like synovial cells (FLS), as well as the excessive secretion of pro-inflammatory factors (such as IL-6 and TNF-α) and matrix metalloproteinases (MMPs). Existing treatments have limited efficacy and systemic side effects. This invention discovers that RA FLS mRNA 5-methylcytosine (5-methylcytosine, m... 5 C) Levels of FLS and RA FLS m were higher than in the normal control group. 5C-methyltransferase NSUN2 (NOP2 / Sun RNA methyltransferase 2) mRNA and protein expression were significantly upregulated in synovial cells or synovial tissue. Knockdown of NSUN2 resulted in decreased intracellular mRNA expression in RA FLS. 5 The C level was significantly reduced, resulting in a marked decrease in the number of cells migrating and invading to the contralateral side of the membrane. It also inhibited the expression of MMPs such as MMP1, MMP3, and MMP9, as well as ICMT (isopreneylcysteine ​​carboxymethyltransferase) in RA FLS. Further investigation into ICMT expression levels in RA FLS revealed that both ICMT mRNA and protein expression levels were significantly upregulated. Knockdown of ICMT decreased cell migration and invasion abilities and significantly inhibited the expression of MMPs such as MMP1, MMP3, and MMP9 in RA FLS. These results indicate that ICMT can regulate abnormal migration and invasion in RAFLS, suggesting that targeting ICMT could be a novel approach to prevent RA joint damage.

[0013] Therefore, this invention provides the application of ICMT as a therapeutic target for rheumatoid arthritis in the preparation of medicaments for treating rheumatoid arthritis.

[0014] The present invention also provides the use of an agent that inhibits ICMT expression in the preparation of a medicament for treating rheumatoid arthritis.

[0015] Furthermore, the preparation is a small interfering RNA, the nucleotide sequences of which are shown in SEQ ID No. 4-5, respectively.

[0016] Furthermore, the small interfering RNA also includes a small interfering RNA targeting NSUN2, the nucleotide sequences of which are shown in SEQ ID No. 1 to 2, respectively.

[0017] Furthermore, the drug achieves treatment by inhibiting the migration and invasion ability of fibroblast-like synovial cells in rheumatoid arthritis or by inhibiting the expression of matrix metalloproteinases MMP1, MMP3, and MMP9.

[0018] This invention also examined the ankle joints of RA rats after NSUN2 knockdown. The results showed that the ankle joint swelling of rats in the NSUN2 siRNA treatment group was significantly reduced, and the symptoms of arthritis in both lower limbs, ankle joint circumference and foot volume scores were significantly reduced. Imaging showed that joint destruction was significantly improved. Histologically, synovial inflammation, hyperplasia and cartilage and bone destruction of the joint were also significantly reduced. In addition, the expression of NSUN2 and ICMT in the synovium of rats in the siNsun2 treatment group was reduced.

[0019] Further research showed that salvianolic acid A (SAA) can directly bind to NSUN2 protein and inhibit m 5 C-methyltransferase activity, thereby reducing abnormally elevated RA FLS m5 C-level and ICMT expression levels; this action can block the expression of downstream MMP1, MMP3, and MMP9, and significantly inhibit the migration and invasion ability of RA FLS. The above mechanism indicates that ICMT, as a core effector molecule in this pathway, can directly intervene in the pathological process of RA by inhibiting its expression, that is, SAA can target m 5 C-NSUN2-ICMT axis for the treatment of rheumatoid arthritis.

[0020] Therefore, the present invention provides the use of salvianolic acid A in the preparation of a medicament for treating rheumatoid arthritis.

[0021] Nanomaterials (inorganic or organic particles with sizes ranging from 1 to 100 nm) have demonstrated potential for targeted delivery and synergistic effects in the treatment of cancer and infectious diseases. Their application in rheumatoid arthritis (RA) hinges on precise intervention of the joint microenvironment. Studies have shown that pathological vascular hyperpermeability, abnormal macrophage infiltration, and oxidative stress microenvironment in RA synovial tissue collectively constitute the biological basis for nano-targeting. Current strategies primarily utilize passive targeting (such as enhanced permeability and retention effect, EPR) to enrich highly permeable synovial vessels and active design (such as hyaluronic acid modification targeting CD44-overexpressing FLS) to enhance joint drug accumulation. Among these, cerium-based nanomaterials (CeO2), by mimicking superoxide dismutase activity, can efficiently scavenge reactive oxygen species in RA joints while simultaneously promoting macrophage transformation to the anti-inflammatory M2 phenotype, providing a new tool for regulating oxidative stress. Based on the unique acidic microenvironment within the joint cavity of rats with rheumatoid arthritis (RA) and the aforementioned mechanisms, this invention innovatively constructs a cerium-based SAA nanosystem. Through the EPR effect, it passively targets the joints of mice with collagen-induced arthritis (CIA), overcoming the deficiency of insufficient joint distribution of free SAA. This provides a delivery solution for SAA that enhances efficacy and reduces toxicity, offering a new approach to intervene in abnormal activation of fibrocystic spondylitis (FLS) and cartilage destruction. Animal experiments show that treatment with the SAA-cerium-based nanodelivery system significantly reduces joint inflammation scores, synovial hyperplasia, and bone erosion in CIA mice. Compared to traditional immunosuppressants, SAA targets m... 5 The C-NSUN2-ICMT axis RA FLS invasive function, the nanosystem further enhances the therapeutic window by synergistic effect and toxicity reduction, while the passive targeting properties avoid the off-target toxicity risk of traditional NSUN2 inhibitors.

[0022] Therefore, the present invention provides the application of cerium-based nanomaterials loaded with salvianolic acid A in the preparation of medicaments for treating rheumatoid arthritis.

[0023] Furthermore, the preparation method of the cerium-based nanomaterial loaded with salvianolic acid A is to mix salvianolic acid A with (NH4)2Ce(NO3)6 at a concentration ratio of 1:3 and react to obtain the nanomaterial.

[0024] Furthermore, the above-mentioned drugs target m 5 The C-NSUN2-ICMT axis enables the treatment of rheumatoid arthritis.

[0025] Furthermore, the concentration of the above-mentioned salvianolic acid A is 10 μM to 160 μM.

[0026] Furthermore, the drug is formulated as an oral or injectable form.

[0027] Compared with the prior art, the present invention has the following beneficial effects: This invention provides the application of isoprenylcysteine ​​carboxymethyltransferase (ICMT) as a therapeutic target for rheumatoid arthritis (RA) in the preparation of drugs for treating RA. This invention discovered that knocking out NSUN2 downregulates ICMT expression. Further research on ICMT expression levels in RA FLS revealed that ICMT mRNA and protein expression in RA FLS were significantly upregulated. Knockdown of ICMT decreased cell migration and invasion abilities and significantly inhibited the expression of MMPs such as MMP1, MMP3, and MMP9 in RA FLS. These results indicate that ICMT can regulate the abnormal migration and invasion of RA FLS, and targeting ICMT could be a new approach to prevent RA joint destruction. Further investigation revealed that SAA can target m... 5 The C-NSUN2-ICMT axis inhibits NSUN2 methyltransferase activity and reduces the mRNA levels of target genes such as ICMT. 5 This invention modifies the C-level, thereby reducing its mRNA and protein expression, and provides a new approach to intervening in abnormal FLS activation and cartilage destruction in RA. Therefore, this invention provides an effective therapeutic target for RA and offers new insights for clinical treatment of RA. Attached Figure Description

[0028] Figure 1 For m 5 C-methylation and NSUN2 expression in synovial tissue and FLS. Among them, Figure 1 A in m 5 C-dotblot assay for m in synovial cells (FLS) of RA patients and normal individuals 5 C level; B is the m level of FLS in RA patients and normal individuals as detected by LC-MS / MS. 5C represents the mRNA expression level of NSUN2 in RA patients and normal individuals with synovial fibrosis (FLS) detected by RT-qPCR; D represents the protein expression level of NSUN2 in RA patients and normal individuals with FLS detected by Western blotting; E represents the difference in NSUN2 expression in RA patients and normal individuals with FLS detected by cellular immunofluorescence; F represents the expression level of NSUN2 in synovial tissue of RA patients and normal individuals detected by tissue immunofluorescence. *: Represents comparison with the normal control group, *P<0.05, **P<0.01.

[0029] Figure 2 NSUN2 regulates the migration, invasion, and ICMT expression of RA FLS. Among these, Figure 2 A in m 5 C-dotblot assay showed the effect of RA FLS mRNA knockdown on NSUN2. 5 C represents the effect of NSUN2 knockdown on RA FLS migration as detected by scratch assay; B represents the effect of NSUN2 knockdown on RA FLS migration and invasion as detected by transwell assay; D represents the effect of NSUN2 knockdown on RA FLS MMP expression as detected by RT-qPCR; E represents the effect of NSUN2 knockdown on RA FLS ICMT mRNA expression level as detected by RT-qPCR; F represents the effect of NSUN2 knockdown on RA FLS ICMT protein expression level as detected by Western blot. *: Represents comparison with the siC group, *P<0.05, **P<0.01, ***P<0.001.

[0030] Figure 3 To investigate the therapeutic effect of intra-articular NSUN2 knockdown on a rat model of collagen-induced arthritis (CIA). Among other things, Figure 3 The top image (A) shows the appearance of the ankle joint in a rat CIA model after injection of PBS, NC, and siNsun2, respectively; the middle image shows the imaging changes of the joint (micro-CT 3D reconstruction image of the joint (3-matic software)); the bottom image shows H&E staining (×200x) and Safranin O-Fixed Green staining (×200x); B shows the symptoms of arthritis and the clinical scores of the ankle joints of both lower limbs: body weight, arthritis score, ankle joint circumference, and foot volume; C shows the pathological scores of the ankle joint: synovial inflammation and cartilage and bone destruction of the joint; D shows the expression of NSUN2 and ICMT in the synovial tissue of rats with CIA by immunohistochemistry (×100x). * represents comparison with PBS, **P<0.01, ***P<0.001; # represents comparison with NC, #P<0.05, ##P<0.01, ###P<0.001.

[0031] Figure 4 The expression of ICMT in synovial tissue and FLS. Figure 4 In this table, A represents the mRNA expression level of ICMT in RA patients and normal individuals with synovial fibrosis detected by RT-qPCR; B represents the protein expression level of ICMT in RA patients and normal individuals with synovial fibrosis detected by Western blotting; and C represents the expression level of ICMT in synovial tissue of RA patients and normal individuals detected by immunohistochemistry. * indicates comparison with the normal control group, *P<0.05, **P<0.01.

[0032] Figure 5 ICMT regulates the migration and invasion of RA FLS. Among them, Figure 5 In the table, A represents the effect of ICMT knockdown on RA FLS migration and invasion as detected by transwell assay; B represents the effect of ICMT knockdown on RA FLS migration as detected by scratch assay; and C represents the effect of ICMT knockdown on RA FLS MMP expression as detected by RT-qPCR. * indicates a comparison with the siC group, ***P<0.001.

[0033] Figure 6 The study investigated the binding of salvianolic acid A (SAA) to NSUN2 protein and its effect on ICMT. Figure 6 In the diagram, A represents the affinity of SAA for interaction with Human NSUN2 protein detected by SPR; B represents m 5 C-dot blot detection of SAA on RA FLS mRNA m 5 C represents the effect of methylation; C represents the effect of SAA on the mRNA expression level of RA FLS ICMT detected by RT-qPCR; D represents the effect of SAA on the protein expression level of RA FLS ICMT detected by Western blotting. *: represents the difference compared with the DMSO group, *P<0.05, **P<0.01, ***P<0.001.

[0034] Figure 7 This is to regulate the migration and invasion of RA FLS by SAA. Figure 7 In the table, A represents the effect of SAA on RAFLS migration and invasion detected by transwell assay; B represents the effect of SAA on RA FLS migration detected by scratch assay; and C represents the effect of SAA on RA FLS MMP expression detected by RT-qPCR. * indicates comparison with the DMSO group, *P<0.05, **P<0.01, ***P<0.001.

[0035] Figure 8 To establish a mouse CIA model, the therapeutic effect of SAA-cerium-based nanoparticles (Ce / SAA-NPs) on arthritis was investigated. Among other things, Figure 8Figure A shows the appearance of the ankle joint in a mouse CIA model after intraperitoneal injection of SAA, Ce / SAA-NPs, or Ce (top image), with H&E staining (×200x) and Safranin O-Fixed Green staining (×200x) (bottom image); Figure B shows the arthritis score in Figure A; Figure C shows the ankle joint pathology score: synovitis and cartilage and bone destruction. *: represents the comparison between Ce / SAA-NPs (20mg / kg) and DMSO groups, *P<0.05, **P<0.01; # represents the comparison between Ce / SAA-NPs (40mg / kg) and DMSO groups, ##P<0.01, ###P<0.001. Detailed Implementation

[0036] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, but the embodiments do not limit the present invention in any way. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in this technical field.

[0037] Unless otherwise specified, all reagents and materials used in the following examples are commercially available.

[0038] Salvianolic Acid A (SAA, CAS Registry No.: 96574-01-5) was purchased from MCE. It was dissolved in DMSO to prepare SAA solution, and then diluted with culture medium according to experimental requirements.

[0039] Example 1: NSUN2-mediated m 5 Expression of C-modification in RA synovium and its regulation of biological behavior of RA FLS I. Experimental Methods 1. Collect synovial membranes from rheumatoid arthritis (RA) patients and healthy controls (HC) to obtain primary fibroblast-like synovial cells (FLS), and perform cell culture, passage, and cryopreservation.

[0040] 2, m 5 C dot blot (1) Extract at least 20 μg of RNA according to the RNA extraction method, and isolate mRNA from the total RNA according to the mRNA purification method; (2) mRNA concentration was detected by serial dilution with nuclease-free water to 250 ng / μL, 125 ng / μL, and 62.5 ng / μL; (3) Denatured mRNA: 95°C for 3 min, and immediately transfer to ice after the process to prevent the formation of secondary structures of mRNA; (4) Drop 2 μL of mRNA directly onto the Hybond-N+ membrane; (5) Ultraviolet crosslinker 1200 microjoules (×100) 24-50s, crosslink twice; (6) Wash with PBST at room temperature on a shaker for 5 min to remove unbound mRNA; (7) 1% methylene blue / ddH2O, incubate at room temperature for 10 min, wash 2-3 times, 5 min each time, and take a photo in open field after cleaning the methylene blue background; (8) Wash away as much methylene blue as possible; (9) Please refer to WB for the subsequent blocking, incubation and antibody exposure steps.

[0041] 3. Liquid chromatography-tandem mass spectrometry (LC-MS / MS) (1) Extract total RNA; (2) Enrichment of mRNA; (3) 1 μg of sample was added to a buffer solution containing S1 nuclease, phosphodiesterase and alkaline phosphatase at 37°C for enzymatic digestion to convert it into nucleosides; (4) Chloroform extraction to remove the upper aqueous phase; (5) Sample loading and detection using liquid chromatography; (6) Mass spectrometry tandem analysis, for m 5 The target chromatographic peak (C) is integrated, and quantitative analysis is performed using a standard curve.

[0042] 4. Real-time quantitative PCR (RT-qPCR) Prepare the PCR reaction according to the AG instructions, add it to a 96-well qPCR plate, and amplify it using a PCR instrument.

[0043] Table 1 Preparation of PCR reaction solution

[0044] Table 2 PCR reaction conditions

[0045] The CT values ​​of the target gene and the internal reference gene (ACTIN) were compared, and the relative expression level of the target gene was represented by 2. -△△CT Calculation by method.

[0046] 5. Western blot (WB) assay (1) Gel preparation: Prepare a suitable concentration of polyacrylamide gel separating gel (60-200kD 6%, 40-100kD 8%, 20-70kD 10%, 20-60kD 12%, 10-40kD 15%) according to the size of the target molecules. Pour the separating gel and add ddH2O / isopropanol to isolate it from the air. After the separating gel solidifies, discard the ddH2O / isopropanol, pour the stacking gel, insert a comb to avoid air bubbles, and wait for the gel to solidify. (2) Electrophoresis: Assemble the vertical electrophoresis tank, fill the inner tank with electrophoresis buffer, remove the comb, load 20 μL of protein or 2-5 μL of marker into each well, and fill wells with less than 20 μL with 1× loading buffer. Electrophore at 100V constant voltage until the appropriate position is reached. (3) Transfer: Activate the PVDF membrane with methanol for 15s, remove the gel, assemble the "sandwich" of "black gel and white membrane" in the transfer solution, avoid air bubbles, and transfer the membrane under ice bath conditions. Generally, the constant voltage is 100V for 90min. In practice, the transfer conditions can be optimized according to the target molecule size. (4) After the transfer is completed, Ponceau S staining can be used to identify the transfer effect. The target band can be cut according to the staining effect and marker position. The Ponceau S should be rinsed clean with ddH2O. (5) 5% BSA / 5% skim milk in TBST room temperature shaker for 1 hour; (6) Dilute the primary antibody with the primary antibody dilution buffer according to the instructions (generally 1:1000), incubate overnight at 4°C, and TBST for 5 min 3 times; (7) Incubate the secondary antibody according to the source of the primary antibody (the concentration of the secondary antibody is generally 1:10000), incubate on a shaker at room temperature for 1 hour, and repeat TBST for 5 minutes 3 times; (8) Prepare the luminescent solution immediately and drop it evenly onto the membrane (200 μL / membrane). Expose the membrane in an exposure machine to obtain bands for subsequent analysis and statistics.

[0047] 6. Cell immunofluorescence (1) Wash cells twice with PBS; (2) Fixation: Fix with 4% paraformaldehyde at room temperature for 15-20 min, wash with PBS for 5 min × 3 times; (3) Punching: Punch with 0.3-0.5% Triton X-100 / PBS for 10 min, then wash with PBS for 5 min × 3 times; (4) Blocking: Block with 5% BSA / PBS at room temperature for 1 hour; (5) Incubate the primary antibody overnight in a humidified chamber at 4°C; (6) Discard the primary antibody and wash with PBS for 5 min × 3 times; (7) Incubate the secondary antibody at room temperature for 1 hour; (8) Discard the secondary antibody and wash with PBS for 5 min × 3 times; (9) Stain the nucleus with DAPI for 5 min, then wash with PBS for 5 min × 3 times; (10) Seal, dry, and photograph.

[0048] 7. Tissue immunofluorescence (1) Paraffin embedding and sectioning of synovial tissue were entrusted to Wuhan Saiweier Company; (2) Bake the slices at 60℃ for 1 hour; (3) Dewaxing and rehydration: xylene 10min×2 times, gradient alcohol (100% 5min×2 times, 95% 5min×2 times, 90% 5min×2 times, 75% 5min×1 time), ddH2O 5min×1 time; (4) Antigen retrieval: Boil 1× sodium citrate / Tris-EDTA antigen retrieval solution in a pressure cooker or microwave oven for 5 minutes, then cool to room temperature; PBS 5 minutes × 3 times; (5) Blocking endogenous peroxidase: circle the tissue block with a drawing pen, 3% H2O2 at room temperature for 10 min, PBS for 5 min 3 times; (6) Blocking: Add 1%-5% BSA / PBS and block in a humidified chamber at room temperature for half an hour; (7) Incubate primary antibody (1:50-1:200): Discard blocking solution, incubate primary antibody at 4 degrees Celsius overnight or at room temperature for 2 hours, PBS for 5 min × 3 times; (8) Incubation of secondary antibody: Incubate the histochemical secondary antibody at room temperature for 20 min, then PBS for 5 min three times; (9) Discard the secondary antibody and wash with PBS for 5 min × 3 times; (10) Stain the nucleus with DAPI for 5 min, then wash with PBS for 5 min × 3 times; (11) Seal, dry, and photograph.

[0049] 8. Cell transfection with siRNA (1) The siRNA was synthesized by Guangzhou Ruibo Biotechnology. Table 3 siRNA sequences

[0050] (2) Cell confluence before transfection was 70%–80%; (3) Taking a 6-well plate as an example, prepare 50 μL of solution A + 50 μL of solution B and incubate at room temperature for 5 min, then add 900 μL of 10% FBS medium. Add 1 mL of transfection solution to each well; Solution A: Dilute 3 μL of lipo3000 with 50 μL of Opti-MEM medium and incubate for 5 min; Solution B: Dilute 20 μM siRNA stock solution with 50 μL Opti-MEM medium (use final concentration 100 nM) and incubate for 5 min; (4) Change the medium 6-8 hours after transfection and continue culturing for 1-3 days for subsequent experiments.

[0051] 9. Scratch test (1) The cell density was about 80% before scratching, and the culture medium was replaced with serum-free medium overnight; (2) Draw straight lines on the culture plate, wash away floating cells with PBS, and then add 10% FBS cell culture medium for culture; (3) Take photos of the same location under a microscope at 0h and 24h.

[0052] 10. Detection of in vitro cell migration and invasion (1) Before migration and invasion, the cell confluence was about 80%, and the culture was changed to serum-free medium and cultured overnight; (2) For the Transwell chamber used in the invasion experiment, 50 μL of matrix adhesive diluted 1:20 should be evenly added to the upper chamber of the chamber before the experiment. The invasion chamber can only be used for the invasion experiment after the adhesive has solidified for more than 4 hours. (3) Add 600 μL and 700 μL of culture medium containing 15% FBS to the lower chamber of the Transwell chamber and the invasion chamber, respectively; (4) Prepare cells to 6.0 × 10⁶ cells according to the cell digestion steps. 4 / mL of cell suspension; (5) 200 μL and 400 μL of the above cell suspension were added to the upper chamber of the Transwell migration chamber and invasion chamber, respectively; (6) Remove the Transwell migration chamber after 6-8 hours and the invasion chamber after 12-16 hours; (7) Fix with 4% paraformaldehyde at room temperature for 15 min, then stain with crystal violet (0.3%~0.5% / PBS) for 15 min; (8) Wash the chamber once with PBS, wipe the cells in the upper chamber that have not migrated or invaded with cotton swabs, and take pictures and count them under a microscope.

[0053] 11. Collagen-induced arthritis (CIA) in rats and intra-articular injection of siRNA (1) Experimental animals: Eight-week-old male Wistar rats were selected and housed in the SPF-grade animal room of the Experimental Animal Center at the North Campus of Sun Yat-sen University. This study was approved by the Ethics Committee of the First Affiliated Hospital of Sun Yat-sen University. The animal experiment followed the "3R" principle and focused on ensuring animal welfare.

[0054] (2) CIA model construction: a: Emulsifier preparation method: Bovine type II collagen solution (2 mg / mL) and complete Freund's adjuvant (CFA) are mixed at a volume ratio of 1:1 and fully emulsified on ice; b: First immunization (D0): Inject 200 μL of type II collagen emulsion intradermally into the rat tail 2 cm from the base of the tail (the needle tip should be 0.5 cm from the base of the tail); c: Second booster immunization (D7): 7 days after the first immunization, inject 100μL of type II collagen emulsion subcutaneously at a point 3cm from the base of the tail (1.5cm from the needle tip to the base of the tail); The Wistar rat CIA model began to show symptoms such as joint swelling and pain about 15 days after the first immunization, peaking on day 30 and decreasing after day 40.

[0055] (3) Intra-articular injection of siRNA: a: Administration time: Day 8 (D8) and Day 15 (D15) after the first immunization. b: Administration method: PBS, NC, or siNsun2 siRNA were injected into the joint cavity of the left ankle of the rat using an insulin injector; the nucleotide sequence of siNsun2 siRNA is as follows: GAAGATGAAGGTCATTAAC (SEQ ID NO.3); c: Dosage: 5 nmol (siRNA dissolved in 0.9% physiological saline, 30 μL volume); (4) Observation indicators and detection methods: Starting from D8, the following observations and measurements were recorded every other day until 23 days after the first immunization (D23). a: General observation: rat activity level, coat color, weight, etc.; b: Use a micrometer to measure the left-right and anteroposterior diameters of the ankle joints of both hind limbs to calculate the ankle joint circumference; c: Volumetric method for measuring foot volume; d: The arthritis symptom scoring criteria are shown in Table 4 below: Table 4. Arthritis symptom scores (both lower extremities only):

[0056] (5) Animal euthanasia and tissue sampling: 21 days after the first immunization (D21), rats were anesthetized by intraperitoneal injection of sodium pentobarbital (50 mg / kg), and blood samples were collected by inferior vena cava sampling. The rats were internally fixed by instilling 4% paraformaldehyde into the left ventricle. The lower hind limbs and ankle joints were cut, the fur removed, and fixed in 4% paraformaldehyde. The ankle joint was examined by MicroCT to assess bone erosion and destruction. The ankle joint tissue was decalcified, dehydrated, embedded, sectioned, and then subjected to subsequent immunohistochemistry, HE staining, and Safranin-Fix-Green staining.

[0057] 12. Hematoxylin-eosin staining (H&E) Alkaline hematoxylin staining solution can stain basophilic structures in tissues (such as cell nuclei, ribosomes, and ribonucleic acid in the cytoplasm) purple-blue; acidic eosin stains eosinophilic structures in tissues (such as proteins and most of the cytoplasm) pink, clearly showing the morphology of the entire cell tissue.

[0058] Paraffin embedding, sectioning, and HE treatment were outsourced to Wuhan Saiweier Company.

[0059] (1) For baking, dewaxing, and rehydration, refer to the immunohistochemistry section in Chapter 1; (2) Stain the nucleus with hematoxylin for 2 min, then rinse with running water; (3) Decolorize with 1% hydrochloric acid alcohol for about 20 seconds, then wash with water; (4) Keep hot water on the surface until the water turns blue, then rinse. (5) Stain with eosin for 5 min, then wash with water; (6) Dehydration, clearing, mounting and photographing are described in Chapter 1, Immunohistochemistry section.

[0060] (7) Joint synovial pathology scoring: Select HE-stained slides containing the synovial lining and sublining layer as qualified. The joint synovial pathology is scored from four aspects: extra-articular infiltration / hyperplasia, synovitis / inflammation, cartilage destruction, and bone erosion, with each item scored from 0 to 3 points.

[0061] 13. Safranine O-fast green staining Safranin-Fix Green staining is highly favored in the morphological study of articular cartilage. Cartilage appears uniformly red, while bone appears green, clearly distinguishable. Cartilage damage causes the red color to fade or disappear. Fast Green binds firmly to loosely structured collagen fibers and is not easily faded.

[0062] Paraffin embedding, sectioning, and safranin-fast green staining were outsourced to Wuhan Saiweier Company.

[0063] (1) For baking, dewaxing, and rehydration, refer to the immunohistochemistry section in Chapter 1; (2) Stain with Fast Green solution for 1-5 minutes, wash away excess solution with water until the cartilage is colorless, soak briefly in hydrochloric acid alcohol differentiation solution for a few seconds, and rinse briefly with tap water. (3) Stain with safranin solution for 15-30 seconds, then dehydrate rapidly with anhydrous ethanol 3 times; (4) For clearing, mounting and photographing, refer to the immunohistochemistry section in Chapter 1.

[0064] 14. Micro-computed tomography (Micro CT) analysis The hind limbs and ankle joints of rats with CIA model were scanned and analyzed on MicroCT. The X-ray tube voltage was 65kV, the current was 380μA, the pixel size was 25μm, the filter AI was 1mm, the shooting interval was 0.4°, the scanning angle rotation was 180°, and each rotation averaged two frames.

[0065] 15. Immunohistochemistry (1) Paraffin embedding and sectioning of synovial tissue were entrusted to Wuhan Saiweier Company; (2) Bake the slices at 60℃ for 1 hour; (3) Dewaxing and rehydration: xylene 10min×2 times, gradient alcohol (100% 5min×2 times, 95% 5min×2 times, 90% 5min×2 times, 75% 5min×1 time), ddH2O 5min×1 time; (4) Antigen retrieval: Boil 1× sodium citrate / Tris-EDTA antigen retrieval solution in a pressure cooker or microwave oven for 5 minutes, then cool to room temperature; PBS 5 minutes × 3 times; (5) Blocking endogenous peroxidase: circle the tissue block with a drawing pen, 3% H2O2 at room temperature for 10 min, PBS for 5 min 3 times; (6) Blocking: Add 1%-5% BSA / PBS and block in a humidified chamber at room temperature for half an hour; (7) Incubate primary antibody (1:50-1:200): Discard blocking solution, incubate primary antibody at 4 degrees Celsius overnight or at room temperature for 2 hours, PBS for 5 min × 3 times; (8) Incubation of secondary antibody: Incubate the histochemical secondary antibody at room temperature for 20 min, then PBS for 5 min three times; (9) DAB color development: Prepare fresh and use immediately, follow the instructions, develop strictly under a microscope (15s-2min), rinse with tap water for 10min; (10) Counterstaining: stain with hematoxylin for 30s-1min, rinse with tap water for 15min; (11) Differentiation: If the cell nuclei are stained too darkly, differentiate them with hydrochloric acid alcohol for 1 min; (12) Dehydration and clearing: Gradient alcohol (75% 5min×1 time, 90% 5min×2 times, 95% 5min×2 times, 100% 5min×2 times), xylene 5min×2 times; (13) Mounting and photographing: Mount the slides with neutral resin, air dry, and photograph and analyze under a microscope.

[0066] 16. Data Statistics Data were processed and analyzed using GraphPad Prism 9.5.0 statistical software. Differences between groups were assessed using t-tests and one-way ANOVA. P < 0.05 was considered statistically significant.

[0067] II. Experimental Results 1. Using m 5 C-dot blot and LC-MS / MS detection of synovial cell m 5 C level, results as follows Figure 1 AB shows the mRNA m of RAFLS 5 C levels were higher than those in the normal control group (HC) FLS.

[0068] 2. Compared with HC FLS, RA FLS m 5 C-methyltransferase NSUN2 mRNA expression was significantly upregulated. Figure 1 C). Immunoplastic and immunofluorescence results further confirmed that NSUN2 protein expression was significantly upregulated in RA FLS ( Figure 1 D) and is mainly located in the cell nucleus ( Figure 1 E). Immunofluorescence staining of NSUN2 expression in synovial tissue of normal controls and RA patients showed that NSUN2 was highly expressed in RA synovial tissue. Figure 1 F).

[0069] 3. m 5 C dot blot analysis showed that knocking down NSUN2 had an impact on m in RA FLS. 5 The effect of C levels was found that knocking down NSUN2 reduced intracellular m in RAFLS. 5 C levels decreased significantly ( Figure 2 A).

[0070] 4. After knocking down NSUN2, RA FLS migrated to the scratched area. Figure 2 The number of cells in B) was also significantly reduced, and the number of RA FLS cells that migrated and invaded to the opposite side of the membrane was significantly reduced in vitro. Figure 2 C).

[0071] 5. Knocking down NSUN2 significantly inhibited the expression of MMPs such as MMP1, MMP3, and MMP9 in RA FLS. Figure 2 D).

[0072] 6. RT-qPCR and Western blot analysis revealed that knocking down NSUN2 decreased ICMT mRNA and protein expression. Figure 2 EF).

[0073] 7. Compared with the control siRNA treatment group, the siNsun2 treatment group showed significantly reduced ankle swelling, significantly decreased symptoms of arthritis in both lower limbs, and significantly reduced ankle circumference and foot volume scores; simultaneously, imaging showed significantly improved joint destruction; histologically, synovial inflammation, hyperplasia, and cartilage and bone destruction in the joints were also significantly reduced. Figure 3 AC). Furthermore, the expression of NSUN2 and ICMT was detected by immunohistochemical staining. The results showed that the expression of both NSUN2 and ICMT was decreased in the synovium of rats treated with siNsun2. Figure 3 D).

[0074] The above experiments confirm that NSUN2-mediated m 5 C-methylation can regulate the abnormal migration and invasion of RA FLS, suggesting that NSUN2-mediated m 5 C abnormality modification plays an important role in regulating synovial invasion in RA. The CIA rat model has confirmed that in vivo inhibition of NSUN2 has a therapeutic effect on arthritis, suggesting that targeting NSUN2 may be a new means to prevent joint destruction in RA.

[0075] Example 2: Expression of ICMT in RA synovium and its regulation of biological behavior of RA FLS I. Experimental Methods The experimental methods for RT-qPCR, Western blot, cell transfection with siRNA (siRNA sequences are shown in Table 5 below), scratch assay, in vitro cell migration, invasion detection, immunohistochemistry, and data statistics are as described in Example 1.

[0076] Table 5 siRNA sequences

[0077] II. Experimental Results 1. Compared with HC FLS, RA FLS showed a significant upregulation of ICMT mRNA expression. Figure 4 A). Immunoplasty results further confirmed that ICMT protein expression was significantly upregulated in RA FLS ( Figure 4B). Immunohistochemical comparison of ICMT protein expression in synovial tissues of normal controls and RA patients showed that ICMT was highly expressed in RA synovial tissues. Figure 4 C).

[0078] 2. Knockdown of ICMT significantly reduced the number of cells that migrated and invaded the opposite side of the membrane. Figure 5 A); The number of cells that migrated to the scratch region was significantly reduced in the scratch assay (A); Figure 5 B).

[0079] 3. Knockdown of ICMT significantly inhibited the expression of MMPs such as MMP1, MMP3, and MMP9 in RA FLS. Figure 5 C).

[0080] The above experiments confirm that ICMT can regulate the abnormal migration and invasion of RA FLS, suggesting that targeting ICMT may become a new means to prevent RA joint damage.

[0081] Example 3: Regulatory effect of salvianolic acid A (SAA) on the biological behavior of RA FLS I. Experimental Methods 1. SPR testing (1) Protein coupling: The amino coupling method is proposed to fix protein.

[0082] a: Place the running buffer (200 mL 1×PBS Buffer), water bottle, and waste bottle in the left and right trays respectively, and insert the corresponding inlet tubes.

[0083] b: Hold the CM5 chip with the printed side facing up. Gently push the chip into the slot following the direction of the arrow on the chip, and finally close the chip compartment door.

[0084] c: Activate chip channel 2 with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC, GE Healthcare) and N-hydroxysuccinimide (NHS, GE Healthcare) at a flow rate of 10 μL / min.

[0085] d: Dilute the ligand protein to 50 μg / mL with sodium acetate at pH 4.5, and immobilize the protein in channel 2 of the chip at a flow rate of 10 μL / min.

[0086] e: Seal the channel with ethanolamine at a flow rate of 10 μL / min.

[0087] f: Repeat step ce for channel 1 as a reference, except that step d uses a protein-free acetate buffer.

[0088] (2) Human NSUN2 protein-analyte interaction test.

[0089] a: Solvent correction: Prepare a 5% DMSO concentration correction curve by mixing 4.5% and 5.8% mother liquor according to the table below: Table 6 Solvent Correction Solution Preparation Table

[0090] b: Assay assay: Take a 96-well plate, dilute the small molecule used for single-concentration screening to an initial concentration of 100 μM with 5% DMSO + 1*PBS-P+ solution, and flow at a rate of 10 μL / min for 150 s in each run. At the end of each flow, regenerate the chip with 10 mM glycine hydrochloride (pH 2.0) solution for 5 minutes, and repeat this process until all compounds have been assayed.

[0091] c: By using Biacore Insight evaluation software (Cytiva, Marlborough, MA, USA), the data were globally fitted to a 1:1 Langmuir binding model to obtain the binding and dissociation constants.

[0092] 2, m 5 For experimental methods including C-dot blot, RT-qPCR, Western blot, scratch assay, in vitro cell migration, invasion detection, and data statistics, please refer to Example 1.

[0093] II. Experimental Results 1. SPR detection shows that SAA can bind to NSUN2 ( Figure 6 A).

[0094] 2. After treating RA FLS with different concentrations (10 μM, 20 μM, 40 μM, 80 μM, 160 μM), m 5 C-dot blot results showed that SAA could inhibit RA FLS m 5 C level ( Figure 6 B).

[0095] 3. Treatment of RA FLS with different concentrations (20 μM, 40 μM, 80 μM) of SAA significantly decreased the expression levels of ICMT mRNA and protein in a concentration-dependent manner. Figure 6 CD).

[0096] 4. Treatment of RA FLS with different concentrations (20μM, 40μM, 80μM) of SAA significantly reduced the number of cells migrating and invading to the opposite side of the membrane. Figure 7 A); In the scratch assay, the number of cells that migrated to the scratch area decreased significantly, and this decrease was concentration-dependent. Figure 7 B).

[0097] 5. Treatment of RA FLS with different concentrations (20 μM, 40 μM, 80 μM) of SAA significantly inhibited the expression of MMPs such as MMP1, MMP3, and MMP9 in RA FLS in a concentration-dependent manner. Figure 7 C).

[0098] The above in vitro experiments confirmed that SAA binds to the NSUN2 protein and inhibits the m-type of NSUN2. 5 C levels, thereby reducing ICMT expression and subsequently regulating abnormal biological behaviors of RA FLS migration and invasion. These results suggest that SAA may target m 5 The C-NSUN2-ICMT axis regulates RA FLS to exert its therapeutic effect on RA.

[0099] Example 4: Therapeutic effect of SAA-cerium-based nanoparticles (Ce / SAA-NPs) on arthritis I. Experimental Methods 1. Synthesis of SAA-cerium-based nanoparticles (Ce / SAA-NPs) Ce / SAA-NPs were synthesized via a self-assembly method. The process is briefly described below: 1 mM aqueous solution of salvianolic acid A (SAA) was mixed with 3 mM methanol solution of (NH4)2Ce(NO3)6 under stirring. The reaction was carried out at room temperature for 2 hours. The resulting Ce / SAA-NPs were collected by centrifugation and washed three times each with methanol and deionized water.

[0100] 2. Collagen-induced arthritis (CIA) in mice (1) Experimental animals: 8-week-old DBA / 1J mice; (2) Emulsifier preparation method: Bovine type II collagen solution (2 mg / mL) and complete Freund's adjuvant (CFA) are mixed at a volume ratio of 1:1 and fully emulsified on ice; (3) First immunization (D0): 100 μL of collagen emulsion was injected subcutaneously at multiple points in the tail of mice; (4) Secondary immunization (D21): 21 days after the first immunization, a booster immunization was given, with each mouse receiving an intraperitoneal injection of 100 μL of bovine type II collagen solution; (5) After secondary immunization, intraperitoneal injection of DMSO, different concentrations of SAA (20 mg / kg or 40 mg / kg), different concentrations of Ce / SAA-NPs (20 mg / kg or 40 mg / kg), and Ce; (6) Mice began to show symptoms 3-4 days after the second immunization, and the disease peaked two weeks after the second immunization. (7) Animal euthanasia and tissue collection: 37 days after the first immunization (D37), mice were anesthetized by intraperitoneal injection of sodium pentobarbital (50 mg / kg), and blood samples were collected by orbital blood collection method. After internal fixation by instilling 4% paraformaldehyde into the left ventricle, the lower limbs were cut off, and excess muscle tissue from the fur was removed and fixed in 4% paraformaldehyde for 2-3 days. The ankle joint tissue was decalcified, dehydrated, embedded, sectioned, and then subjected to subsequent immunohistochemistry.

[0101] The data statistical experiment method is as described in Example 1.

[0102] II. Experimental Results Compared with the control DMSO treatment group, SAA treatment group, and Ce treatment group, the Ce / SAA-NPs (20 mg / kg or 40 mg / kg) treatment group showed significantly reduced ankle swelling. Figure 8 AB); histologically, synovial inflammation, hyperplasia, and cartilage and bone destruction in the joint were also significantly reduced ( Figure 8 C).

[0103] The above in vivo experiments confirmed that the SAA-cerium-based nanosystem can treat arthritis in CIA mice, thus effectively overcoming the defects in free drug distribution.

Claims

1. The application of isoprenylcysteine ​​carboxymethyltransferase as a therapeutic target for rheumatoid arthritis in the preparation of drugs for treating rheumatoid arthritis.

2. Application of preparations that inhibit the expression of isoprenylcysteine ​​carboxymethyltransferase in the preparation of drugs for treating rheumatoid arthritis.

3. The application according to claim 2, characterized in that, The preparation is a small interfering RNA, and its nucleotide sequences are shown in SEQ ID No. 4 to 5, respectively.

4. The application according to claim 3, characterized in that, The small interfering RNA also includes a small interfering RNA targeting NSUN2, the nucleotide sequences of which are shown in SEQ ID No. 1 to 2, respectively.

5. Application of salvianolic acid A in the preparation of drugs for treating rheumatoid arthritis.

6. Application of cerium-based nanomaterials loaded with salvianolic acid A in the preparation of drugs for treating rheumatoid arthritis.

7. The application according to claim 5 or 6, characterized in that, The drug targets m 5 The C-NSUN2-ICMT axis enables the treatment of rheumatoid arthritis.

8. The application according to claim 5 or 6, characterized in that, The concentration of salvianolic acid A is 10 μM to 160 μM.

9. The application according to any one of claims 1, 2, 5 or 6, characterized in that, The drug achieves its therapeutic effect by inhibiting the migration and invasion of fibroblast-like synovial cells in rheumatoid arthritis or by inhibiting the expression of matrix metalloproteinases MMP1, MMP3, and MMP9.

10. The application according to any one of claims 1, 2, 5 or 6, characterized in that, The drug is available in oral or injectable form.

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