Application of ATP1B3-CA9 as target spot in medicine for treating RA

Abstract

The invention provides an application of ATP1B3-CA9 as a target spot in a medicine for treating RA (rheumatoid arthritis), which is characterized in that ATP1B3 protein is synergistically targeted through the medicine, phase separation of ATP1B3 is promoted to target and activate CA9, lysosome dysfunction is caused to induce alkali death, cartilage injury and formation of injury nerve markers are relieved, and the analgesic effect and the curative effect on RA are better.

Description

Technical Field

[0001] The present invention relates to the field of pharmaceutical technologies, and particularly to the application of ATP1B3-CA9 as a target in drugs for treating RA. Background Art

[0002] Rheumatoid arthritis (RA) is a systemic inflammatory disease that mainly causes irreversible damage to articular cartilage and subchondral bone, as well as severe chronic bone pain. It has a significant social impact in terms of treatment costs, physical disability, and productivity loss. With the continuous development of disease-modifying anti-rheumatic drugs (DMARDs), remarkable success has been achieved in preventing and alleviating the disease activity of RA patients.

[0003] However, the response of some RA patients to DMARDs (slow-acting anti-rheumatic drugs) is still limited, and the pain of the patients still persists. More importantly, treating the bone and joint pain of RA is highly challenging, representing a large number of unmet medical needs. Existing therapeutic drugs (non-steroidal anti-inflammatory drugs, analgesics, and steroids) cannot continuously relieve pain and may produce serious adverse reactions. Although biotherapy and joint replacement surgery can effectively relieve RA pain, they may only be applicable to patients with the most severe or advanced diseases. If the treatment side effects are too large, it will have a more adverse impact on the physical and mental health of the patients, which poses new requirements for special targets and new therapies. Understanding the pathogenic role of various molecules in RA helps to discover potential targets for treating and alleviating bone and joint pain. Summary of the Invention

[0004] Based on this, the purpose of the present invention is to provide the application of ATP1B3-CA9 as a target in drugs for treating RA to at least solve the deficiencies in the above technologies.

[0005] The present invention proposes the application of ATP1B3-CA9 as a target in drugs for treating RA.

[0006] Further, the drugs for treating RA are icariin and JTC-801.

[0007] Further, the drugs synergistically target the protein ATP1B3 to promote the phase separation of the protein ATP1B3 to target and activate CA9, causing lysosomal dysfunction to induce alkali death, thereby alleviating cartilage damage and the formation of nociceptive nerve markers.

[0008] The application of ATP1B3-CA9 as a target in the present invention in drugs for treating RA. By synergistically targeting the ATP1B3 protein with drugs, it promotes the phase separation of ATP1B3 to target and activate CA9, causes lysosomal dysfunction to induce alkali death, thereby alleviating cartilage damage and the formation of nociceptive nerve markers, and has a better analgesic effect and better curative effect on RA. BRIEF DESCRIPTION OF THE DRAWINGS

[0009] Figure 1 It is a distribution diagram of secondary hyperalgesia indexes of sham operation group, icariin ICA treatment group, and rats with rheumatoid arthritis RA model at different time points in the first embodiment of the present invention. Among them, A is the 50% paw withdrawal threshold (50%PWT) of the left hind paw of the rat, and B is the withdrawal threshold of pain measurement (PAMWT) applied to the left knee of the rat; Figure 2 It is a quantitative analysis diagram of the intensity, area, and swing speed of the left hind paw (LH) and the right hind paw (RH) at the 8th week after sham operation or collagen-induced arthritis surgery in the first embodiment of the present invention; Figure 3 It is a quantitative analysis diagram of MMP13 and ColX cells in articular cartilage in the first embodiment of the present invention; Figure 4 It is an intersection analysis diagram of up-regulated genes in the first embodiment of the present invention; Figure 5 It is an inhibition analysis diagram of overexpressing ATP1B3 on chondrocyte damage in the first embodiment of the present invention; Figure 6 It is a diagram of the intrinsically disordered region of ATP1B3 calculated by the IUPred2 website in the first embodiment of the present invention; Figure 7 It is a quantitative analysis diagram of the droplet formation of ATP1B3-GFP with different buffers in the first embodiment of the present invention. Among them, A represents the buffer for droplet formation containing different concentrations of salt and 10% PEG-8000, and B represents the buffer for droplet formation with different protein concentrations; Figure 8 It is a schematic diagram of the in vivo phase separation condensate of endogenous ATP1B3 protein in C28 / I2 and RKO cells shown by IF in the first embodiment of the present invention; Figure 9 It is a distribution diagram of the in vivo phase separation condensate of exogenous ATP1B3-GFP protein and a fluorescence recovery after photobleaching experiment diagram of the in vivo ATP1B3-GFP condensate in C28 / I2 cells shown in the first embodiment of the present invention; Figure 10 It is a schematic diagram of the screening strategy for specific targets of ATP1B3 phase separation in the first embodiment of the present invention; Figure 11 Schematic diagram of 15 genes identified in the first embodiment of the present invention as targets of the ATP1B3 phase separation condensate; Figure 12 Correlation analysis chart between CA9 and ATP1B3 in the first embodiment of the present invention; Figure 13 Chart showing the expression levels of CA9 detected by qPCR and WB in the first embodiment of the present invention; Figure 14 ChIP-qPCR detection shows the enrichment map of endogenous ATP1B3 at the CA9 locus in C28 / I2 cells in the first embodiment of the present invention; Figure 15 Chart for quantitative analysis of the expression levels of cartilage injury and nociceptive nerve markers in the first embodiment of the present invention; Figure 16 Chart of the CGRP concentration distribution in cartilage injury and nociceptive nerve markers after activating CA9 in the first embodiment of the present invention; Figure 17 Chart of the intracellular pH value distribution of C28 / I2 after treatment with ICA (3μm) for 24 hours in the first embodiment of the present invention; Figure 18 Chart of the effect of ATP1B3 on the NF-κB-dependent CA9 pathway in the first embodiment of the present invention; Figure 19 Chart of the Western blot analysis of protein expression in C28 / I2 cells after treatment with ICA (3μm) for 6 - 24 hours in the first embodiment of the present invention; Figure 20 Chart of the cell death assay of C28 / I2 cells after treatment with ICA (3μm) for 24 hours in the presence of bafilomycin A1 (BafA1, 100nM) in the first embodiment of the present invention; Figure 21 Chart of the IP analysis of the interaction between CA9 and ATP1B3 in C28 / I2 cells after treatment with ICA (3μM) for 24 hours in the first embodiment of the present invention; Figure 22 Chart of the image analysis of lysosomal membrane permeability in C28 / I2 cells after treatment with ICA (3μM) for 12 or 24 hours in the first embodiment of the present invention; Figure 23 Chart of the cell viability assay of C28 / I2 cells after treatment with ICA for 24 hours in the presence of CA9 in the first embodiment of the present invention; Figure 24 Chart of the ATP1B3 expression in the C28 / I2 cell line after treatment with the proteasome inhibitor MG132 in the first embodiment of the present invention; Figure 25 It is the cycloheximide chase assay result graph of ICA in the first embodiment of the present invention; Figure 26 It is the schematic diagram of detecting the ubiquitination of ATP1B3 after ICA treatment by co-immunoprecipitation (Co-IP) in the first embodiment of the present invention; Figure 27 It is the molecular docking analysis and identification graph between ICA and ATP1B3 in the first embodiment of the present invention; Figure 28 It is the molecular docking analysis and identification graph of two binding residues (ARG177 and TYR217) between JTC801 and ATP1B3 in the first embodiment of the present invention; Figure 29 It is the efficacy detection graph of JTC801 combined with ICA in the first embodiment of the present invention. Among them, A represents the cell viability detection graph by CCK8, B represents the quantitative analysis of the expression levels of cartilage injury and nociceptive nerve markers, and C represents the concentration distribution graph of CGRP in cartilage injury and nociceptive nerve markers.

[0010] The following specific embodiments will further illustrate the present invention in combination with the above-mentioned drawings. Specific Embodiments

[0011] For the convenience of understanding the present invention, the present invention will be described more comprehensively below with reference to the relevant drawings. Several embodiments of the present invention are given in the drawings. However, the present invention can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, the purpose of providing these embodiments is to make the disclosure of the present invention more thorough and comprehensive.

[0012] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the technical field to which the present invention belongs. The terms used in the specification of the present invention herein are only for the purpose of describing specific embodiments and are not intended to limit the present invention. The term "and / or" used herein includes any and all combinations of one or more of the related listed items.

[0013] Example 1 The present invention proposes the application of ATP1B3-CA9 as a target in drugs for treating RA, wherein the drug for treating RA is icariin and JTC-801; Among them, JTC-801 is an opioid analgesic drug and belongs to an alkali death activator.

[0014] Specifically, the above drugs synergistically target the protein ATP1B3 to promote the phase separation of the protein ATP1B3 to target and activate CA9, causing lysosomal dysfunction to induce alkali death, thereby alleviating cartilage injury and the formation of nociceptive nerve markers 1. Icariin (ICA) alleviates RA joint pain and cartilage damage by upregulating ATP1B3 1. In vivo verification of the ability of icariin (ICA) to relieve RA joint pain and cartilage damage: Establish a collagen-induced arthritis model: Dissolve type II collagen (a protein isolated from the immune system and abundantly present in articular cartilage) in 0.1 mol / L acetic acid, stir well at 4°C until fully dissolved, with a concentration of 2 g / L, place it in a 4°C refrigerator overnight. Then, place inactivated BCG in liquid paraffin to prepare 2 g of complete Freund's adjuvant. Mix and emulsify the two in equal volumes to make a type II collagen emulsion. Intradermally inject 0.1 ml of this emulsion into the tail root of rats to induce inflammation, and intraperitoneally inject 0.1 ml of the emulsion on the 21st day as a booster injection. Use an infrared thermal imaging system to record the temperature changes and photothermal images of the hind limbs until the 30th day after adjuvant administration. Measure the change in paw width with a caliper.

[0015] 1). Pain indicators: secondary hyperalgesia (50% PWT), primary hyperalgesia (PAMWT), and gait defects (paw swing speed, contact area, and intensity); 2). Articular cartilage damage and senescence phenotypes: cartilage degradation (Saftanin O / SOFG staining and OARSI scoring); detect the expression of degradation markers (COLX, ACAN, and MMP13) by IHC and WB; detect senescence by β-galactosidase staining, or detect the expression of senescence markers (p16INK4a, p21, and SASP) by WB / HC; 3). Gene expression profile analysis: RNA-seq analysis and pathway enrichment analysis; 4). Identification of ICA drug targets: mass spectrometry analysis and functional enrichment analysis; based on gene expression profile analysis and ICA drug target identification, there are 386 ICA drug targets, genes downregulated in the RA group, and genes upregulated in the ICA treatment group. Take the intersection gene: ATP1B3.

[0016] 2. In vivo analysis of the mechanism of action of ICA 1). The level of nociceptive nerve (pain sensation) innervation in subchondral bone: detection of related markers (immunofluorescence staining of calcitonin gene-related peptide CGRP, substance P SP, PIEZ02, and P2X3); quantification of the density of positive nociceptive nerve fibers; coupling of angiogenesis and sensory innervation (co-localization of CGRP and endomucin EMUN); osteophyte volume: micro-computed tomography (μCT) analysis of the medial compartment (coronal) of the tibial chondral bone.

[0017] 2), Subchondral bone degeneration: Bone microstructure (trabecular bone parameters, structure model index, total pore space, and subchondral bone plate thickness); pathological changes of bone (tibial trichrome staining, calcein-alizarin double labeling for bone formation and localization); IHC detection of the levels of bone progenitor cell markers (Nestin and Osterix).

[0018] 3), ATP1B3 and alkali death levels in subchondral bone: IHC / WB detection; pH level detection; IHC detection of the levels of alkali death markers (NF-κB, RELA, p65, and CA9).

[0019] 3. In vitro verification of the alleviating effect of ICA targeting ATP1B3 on RA bone pain and cartilage damage: 1), Construct an organoid model of chondrocytes derived from RA patients, add ICA treatment, and group: Control, RA, and RA + ICA. Detect cartilage degradation, nociceptive nerve markers, the expression of ATP1B3, and alkali death levels.

[0020] 2), Isolate primary FLS from RA patients (RA-FLS) and co-culture with human immortalized chondrocytes (C28 / I2). Overexpress ATP1B3, and group: Con-FLS (C28 / I2 + NC, C28 / I2 + ATP1B3) and RA-FLS (C28 / I2 + NC, C28 / I2 + ATP1B3). Detect the damage of chondrocytes in the RA environment: cartilage degradation, senescence, CCK-8 assay for cell viability, nociceptive nerve markers, and alkali death levels.

[0021] II. Phase separation targeting activation of CA9 by ATP1B3 promotes cartilage damage and the formation of nociceptive nerve markers 1. Explore whether ATP1B3 undergoes phase separation in cartilage damage Co-culture C28 / I2 with RA-FLS, construct an acidic mutant of ATP1B3 (losing the ability of phase separation), and a fusion plasmid of ATP1B3 mutant and hnRNPA1-IDR (restoring the ability of phase separation). After transfection of cells respectively, detect the phase separation indicators: 1), Detection of the phase separation potential of ATP1B3: 2), In vitro droplet formation experiment: GFP fluorescence labeling, treatment with different concentrations of Nacl and proteins, and IF detection of the expression and localization of ATP1B3; 3), IF detection of the expression and morphology of endogenous ATP1B3; 4), Exogenously transfect GFP-ATP1B3 or ATP1B3-MUT plasmids, and after photobleaching treatment or without treatment, detect the expression and localization of ATP1B3 by IF.

[0022] 2. Analyze the regulatory effect of ATP1B3 phase separation on cartilage injury and bone pain C28 / I2 cells were co-cultured with RA-FLS, transfected with different plasmids, and grouped as Control, ATP1B3, ATP1B3-MUT, and ATP1B3-MUT-IDR. The following indicators were detected: the expression of ATP1B3, nociceptive nerve (bone pain) markers, cartilage degradation, alkali death level, and cell viability detected by TUNEL / flow cytometry.

[0023] 3. Explore the molecular mechanism and direct targets of ATP1B3 phase separation in alleviating cartilage injury 1). Screen the direct targets of ATP1B3 phase separation: Transfect C28 / I2 cells with ATP1B3-WT and ATP1B3-MUT, and perform ChIP-seq analysis; perform RNA-seq analysis on ATP1B3-KO cells; for the binding proteins of ATP1B3, exclude the genes in the ChIP-seq of ATP1B3-MUT from the intersection genes, and select the ATP1B3 phase separation-specific target gene: CA9.

[0024] 2). Verify the regulatory effect of ATP1B3 on CA9: In the C28 / I2 and RA-FLS co-culture system, detect the endogenous enrichment of CA9 on ATP1B3 by ChIP-qPCR, as well as the exogenous enrichment of ATP1B3-WT, ATP1B3-MUT, and ATP1B3-MUT-IDR; after knocking down ATP1B3 or ATP1B3-MUT plasmids, detect CA9, COLX, ACAN, MMP13, and nociceptive nerve markers by WB; alkali death level.

[0025] 3). Interaction mechanism between ATP1B3 and CA9: Detect the endogenous and exogenous binding of ATP1B3 and CA9 by forward and reverse Co-IP; analyze the binding site between ATP1B3 and CA9 by point mutation combined with Co-IP; co-transfect Flag-labeled ATP1B3-WT and ATP1B3-MUT with full-length CA9, and detect the change in binding ability by Co-IP.

[0026] 4). Analyze the necessity of ATP1B3 phase separation in participating in cartilage protection by the alkali death inhibited by overexpression of CA9 Regulation of CA9 and alkali death on cartilage injury and nociceptive nerve markers, grouping: C28 / I2+RA-FLS (NC, ATP1B3, ATP1B3-MUT, shCA9, ATP1B3+shCA9 and ATP1B3-MUT+shCA9). Adjust the cell pH value to acidic to inhibit alkali death, and detect the following indicators: Expressions of ATP1B3 and CA9, levels of alkali death, nociceptive nerve markers and cartilage degradation markers.

[0027] III. Explore the mechanism by which ICA&JTC801 target the ATP1B3-CA9 axis to induce lysosomal dysfunction and alkali death 1. Analyze the regulatory effect of ICA on the ATP1B3-CA9 axis 1). Exogenous and endogenous verification: For C28 / I2 and HEK293T cells, after treatment with the proteasome inhibitor MG132 or the autophagy inhibitor hydroxychloroquine (HCQ) (CHQ), add ICA with or without treatment, and use CHX to trace and analyze the protein half-life, Co-IP for ubiquitination analysis, and WB to detect the expressions of ATP1B3 and CA9.

[0028] 2). Molecular docking analysis of the binding sites of ICA with ATP1B3 and CA9: Two JTC-801 binding residues (ARG177 and TYR217) on ATP1B3 have been identified in previous studies.

[0029] 3). Construct mutants ATP1B3ARG177R and ATP1B3TYR217R of the two residues ARG177 and TYR217, transfect them into HEK293T and C28 / I2 cells, and after adding ICA, detect the expressions and interactions of ATP1B3 and CA9.

[0030] 2. Explore the regulatory mechanism of ATP1B3-CA9 on alkali death 1). Knock down ATP1B3 in C28 / I2 cells, and use the lysosomal inhibitor bafilomycin A1 (BafA1) with or without treatment to detect the level of cell alkali death: Flow cytometry to detect cell apoptosis, CCK-8 to detect cell viability, intracellular pH value, and the expressions of alkali death-related proteins NF-KB / CA9.

[0031] 2), Detection of lysosomal dysfunction: Knockdown of ATP1B3 in C28 / 2 cells, with or without treatment with JTC-801, to detect intracellular pH homeostasis: The ysoSensor yellow / blue DND160 pH indicator shows the level of lysosomal acidification; lysosomal membrane permeability: RITC-dextran leakage assay; WB is used to detect the expression of lysosomal membrane proteases CTSB and CTSD.

[0032] 3. Regulatory and restorative effects of P1B3 on CA9 function 1), Knockdown of ATP1B3 in C28 / I2 cells, and WB is used to detect the expression of CA9; CHX is used to track the protein half-life of CA9.

[0033] 2), Knockdown of both ATP1B3 and CA9 in C28 / I2 cells, with or without treatment with ICA, to detect the levels of alkali death, lysosomal dysfunction, cartilage damage markers, and nociceptive nerve markers.

[0034] Combined treatment: Grouping: AVV-ATP1B3, AVV-ATP1B3+JTC-801, AVV-ATP1B3+icariin, AVV-ATP1B3+JTC-801+icariin, JTC-801+icariin; After intra-articular injection of adenovirus-packaged ATP1B3 and combined treatment of RA rats with ICA and JTC-801, pain behavior detection (primary and secondary pain sensation), assessment of articular cartilage damage (Safranin O / SOFG staining, OARSI score, and IHC and WB are used to detect the expression of COLX, ACAN, and MMP13), analysis of the level of nociceptive nerve innervation (pain sensation) in subchondral bone (detection of nociceptive nerve markers: CGRP, SP, PIEZ02, and P2X3; IF is used to detect CGRP and EMUN; assessment of the volume of osteophytes), analysis of subchondral bone degeneration (assessment of osteoid formation and localization and tibial structure, WB / IHC is used to detect the markers Nestin and Osterix of osteoprogenitor cells), and analysis of the levels of ATP1B3 and alkali death in cartilage and subchondral bone: IHC / WB / IF is used to detect the levels of ATP1B3, NF-κB, RELA, p65, and CA9.

[0035] In this example, the analgesic effect of ICA on damaged joints was verified in a RA rat model. It was found that in RA rats treated with ICA for two weeks, the secondary hyperalgesia index (50% paw withdrawal threshold: 50%PWT) was significantly higher than that of the RA rat model group (Figure 1 in A), and the paw withdrawal mechanical threshold (PAMWT) was significantly higher than that of the RA rat model group ( Figure 1 in B), at the 8th week after the collagen-induced arthritis model surgery (compared with the sham operation group), the strength, contact area and swing speed of the affected hind paw were significantly reduced compared with the contralateral hind paw; however, these characteristics were significantly restored after ICA treatment ( Figure 2 ). According to Figure 3 it can be seen that ICA inhibited chondrocyte degeneration in osteoarthritis, and it was found that the expressions of ColX and MMP13 in the RA group were significantly higher than those in the sham operation group, and the expressions of ColX and MMP13 decreased after ICA treatment. Subsequently, intersection analysis was performed on 386 ICA drug targets, rheumatoid arthritis and sham operation arthritis down-regulated genes, and icariin and rheumatoid arthritis up-regulated genes, and finally ATP1B3 ( Figure 4 was obtained). It was confirmed by cell experiments that CCK8 was used to detect chondrocyte viability, and it was found that overexpression of ATP1B3 could effectively inhibit chondrocyte damage ( Figure 5 ).

[0036] Specifically, please refer to Figure 6 the intrinsically disordered region (IDR) map of ATP1B3 calculated by the IUPred2 website. The IDR (1 - 182 amino acids) is shown above the disease score map. It was found that the N-terminus of ATP1B3 has a strong intrinsically disordered region (IDR). Quantitative analysis of the droplet formation of 10 mM ATP1B3-GFP in a droplet formation buffer containing different concentrations of salt and 10% PEG-8000 was performed, and it was found that the number of droplets decreased with the increase in salt concentration. In vitro droplet formation experiments showed that ATP1B3 could form round droplets in a salt solution, and the number of droplets gradually decreased with the increase in salt concentration (please refer to Figure 7 in A). Quantitative analysis of the droplet formation of ATP1B3-GFP in a droplet formation buffer with different protein concentrations was performed, and it was found that the number of droplets increased with the increase in protein concentration. In vitro droplet formation experiments showed that the droplets became smaller at low protein concentrations, became larger as the protein concentration increased, and the number of droplets increased at higher protein concentrations (please refer to Figure 7 in B). In addition, please refer to Figure 8 to detect the status of ATP1B3 in the poorly differentiated colon cancer cell line RKO and the immortalized human chondrocyte cell line C28 / I2. Immunofluorescence (IF) showed the in vivo phase separation condensates of endogenous ATP1B3 protein in the poorly differentiated colon cancer cells RKO and immortalized human chondrocytes C28 / I2C28 / I2.

[0037] Please refer to Figure 9When exogenous ATP1B3-green fluorescent protein was transfected into the immortalized human chondrocyte C28 / I2 cell line with low endogenous ATP1B3 expression, it was observed that ATP1B3 formed larger condensates. In the fluorescence recovery after photobleaching experiment of ATP1B3-green fluorescent protein condensates in immortalized human chondrocyte C28 / I2 cells, it was found that the green fluorescent protein signal could recover rapidly after photobleaching.

[0038] Please refer to Figure 10 as shown in the schematic diagram of the screening strategy for specific targets of ATP1B3 phase separation. Among them, RNA-seq and ATP1B3 ChIP-seq were performed using different ATP1B3 patterns.

[0039] The direct targets of ATP1B3 phase separation condensates were obtained following the standard order: (1) Collect significantly differentially expressed genes (DEGs) responsive to ATP1B3 knockdown by RNA-seq, (2) Identify effective targets of ATP1B3 by ChIP-seq, collect the overlap between DEGs and WT-ATP1B3 target genes, and exclude mutant ATP1B3 target genes from WT-ATP1B3 target genes.

[0040] Subsequently, please refer to Figure 11 671 significantly DEGs responsive to ATP1B3 knockdown, 3580 WT-ATP1B3 target genes, and 995 mutant ATP1B3 target genes were collected respectively. According to these criteria, 15 genes were finally determined as the targets of ATP1B3 phase separation condensates. Please refer to Figure 12 By performing correlation analysis on ATP1B3 and 15 genes, the results showed that CA9 was significantly negatively correlated with ATP1B3. Please refer to Figure 13 Verified by qPCR and WB detection, overexpression of ATP1B3 weakened the induction effect of CA9. Please refer to Figure 14, ChIP-qPCR assays showed the enrichment of endogenous ATP1B3 at the CA9 locus in immortalized human chondrocyte C28 / I2 cells. ChIP-qPCR assays showed the enrichment of exogenous wild-type ATP1B3 and mutant -ATP1B3-containing a strong intrinsically disordered region, rather than mutant -ATP1B3, at the CA9 locus in immortalized human C28 / I2 cells. According to the ChIP-qPCR results, the CA9 gene locus was occupied by WT-ATP1B3 rather than mutant ATP1B3. Mut-IDR rescued the disabling function of the ATP1B3 mutant on CA9 binding. Collectively, these results suggest that the ATP1B3 phase-separated condensates directly target CA9 to induce its expression. Also see Figures 15 to 16 , the expressions of ColX and MMP13 in the rheumatoid arthritis group and carbonic anhydrase group were significantly higher than those in the negative control group. The expression of CGRP (calcitonin gene-related peptide) in the rheumatoid arthritis group and carbonic anhydrase group was significantly higher than that in the negative control group. It can be seen that activating carbonic anhydrase (CA9) can promote the formation of cartilage damage and nociceptive nerve markers ( Figure 15 and Figure 16 ).

[0041] Also see Figure 17 , after treating with ICA (3 μM) for 24 hours, the intracellular pH value of immortalized human chondrocyte C28 / I2 cells was detected, and the pH value was inhibited in ATP1B3-knockdown C28 / I2 cells. However, after treating with ICA (3 µM) for 6 - 24 hours, western blot analysis of protein expression in immortalized human chondrocyte C28 / I2 cells found that ATP1B3 had no significant effect on the NF-κB-dependent CA9 pathway ( Figure 18 and Figure 19 ).

[0042] Also see Figure 20 , in the absence or presence of bafilomycin A1 (BafA1, 100 nM), after treating with ICA (3µM) for 24 hours, cell death assays were performed on immortalized human chondrocyte C28 / I2 cells, and quantitative analysis was performed using Image J software. It was found that, like knocking out ATP1B3, the specific inhibitor of V-ATPase in lysosomes, bafilomycin A1 (BafA1), effectively eliminated the anti-cancer activity of JTC801; also see Figure 21 , after treating with ICA (3 μM) for 24 hours, IP analysis of the interaction between carbonic anhydrase (CA9) and ATP1B3 in immortalized human chondrocyte C28 / I2 cells showed that IP experiments demonstrated that ICA increased the formation of the CA9-ATP1B3 complex in C28 / I2 cells.

[0043] Please refer to Figure 22 After treatment with ICA (3 μM) for 12 or 24 hours, image analysis of lysosomal membrane permeability in immortalized human chondrocyte C28 / I2 cells was performed. Acridine orange (AO) staining showed that ICA treatment for 24 hours upregulated lysosomal membrane permeability, and this process was inhibited by ATP1B3 knockout. Please refer to Figure 23 After treatment with ICA for 24 hours in the absence or presence of carbonic anhydrase (CA9), the cell viability of immortalized human chondrocyte C28 / I2 cells was measured. It was found that knockdown of CA9 alone induced cell death and increased cell viability in C28 / I2 cells, but did not induce cell viability in ATP1B3-knockdown C28 / I2 cells.

[0044] Please refer to Figure 24 After treatment with the proteasome inhibitor MG132 in the immortalized human chondrocyte C28 / I2 cell line, the expression of ATP1B3 was detected. It was found that, like ICA, treatment with the proteasome inhibitor MG132 also induced upregulation of ATP1B3 in the C28 / I2 cell line in a dose- or time-dependent manner. However, the autophagy inhibitor hydroxychloroquine (HCQ) decreased ATP1B3 expression. Please refer to Figure 25 The stability of ATP1B3 protein in the indicated immortalized human chondrocyte C28 / I2 cells with or without ICA (3 μM) treatment was analyzed by cycloheximide (CHX) (30 μg / mL) chase assay. The cycloheximide chase assay confirmed that ICA increased the half-life of ATP1B3 protein. Please refer to Figure 26 Ubiquitination of ATP1B3 after ICA treatment was detected by co-immunoprecipitation (Co-IP). It was found that ubiquitination of ATP1B3 increased after ICA treatment.

[0045] Please refer to Figure 27 Using molecular docking analysis, the binding energy between ICA and ATP1B3 was identified as -7.6. Figure 28 Two binding residues (ARG177 and TYR217) between JTC801 and ATP1B3 were identified in

[0046] After establishing a rat model of RA, the efficacy of JTC801 combined with ICA was detected. Figure 29 CCK8 in A of was used to detect cell viability and found that the cell viability of immortalized human chondrocyte C28 / I2 cells in the JTC801 combined with ICA treatment group was significantly higher than that in the single treatment group, and the combination treatment showed better efficacy. Please refer to Figure 29In B and C of the established RA rat model, the efficacy of JTC801 combined with ICA was detected. The results showed that the cartilage injury markers and nociceptive nerve markers ColX and calcitonin gene-related peptide (CGRP) in the JTC801 combined with ICA treatment group were significantly lower than those in the JTC801 or icariin alone treatment group.

[0047] In summary, the application of ATP1B3-CA9 as a target in the above embodiments of the present invention in drugs for treating RA, through the drug synergistically targeting the ATP1B3 protein, promotes the phase separation of ATP1B3 to target and activate CA9, induces lysosomal dysfunction to cause alkali death, thereby alleviating cartilage injury and the formation of nociceptive nerve markers, and has a better analgesic effect and better curative effect on RA.

[0048] The technical features of the above embodiments can be combined arbitrarily. For the sake of brevity of description, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, it should be considered as the scope described in this specification.

[0049] The above embodiments only represent several implementation manners of the present application, and their descriptions are relatively specific and detailed, but they should not be construed as limiting the scope of the invention patent. It should be noted that for those of ordinary skill in the art, without departing from the concept of the present application, several modifications and improvements can still be made, and these all belong to the protection scope of the present application. Therefore, the protection scope of the patent of the present application shall be subject to the appended claims.

Claims

1. Application of ATP1B3-CA9 as a target in the treatment of RA.

2. The use according to claim 1, characterized in that: The RA treatment drugs are icariin and JTC-801.

3. The use according to claim 1, characterized in that: The drug synergistically targets the protein ATP1B3 to promote the phase separation of the protein ATP1B3 to target and activate CA9, causing lysosomal dysfunction to induce alkali death, thereby alleviating cartilage damage and the formation of nerve injury markers.

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