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

Abstract

The present invention provides an application of ATP1B3-CA9 as a target in a drug for treating RA. By synergistically targeting the ATP1B3 protein through drugs, the phase separation of ATP1B3 is promoted to target and activate CA9, causing lysosomal dysfunction and inducing alkali death, thereby alleviating cartilage damage and the formation of nerve injury markers, and having a better analgesic effect and better therapeutic effect on RA.

Description

Technical Field

[0001] The present invention relates to the field of medical technology, and in particular to an application of ATP1B3-CA9 as a target in a drug for treating RA. Background Art

[0002] Rheumatoid arthritis (RA) is a systemic inflammatory disease that primarily causes irreversible damage to articular cartilage and subchondral bone, as well as severe, chronic bone pain. It has a significant societal impact in terms of treatment costs, physical disability, and lost productivity. The continued development of disease-modifying antirheumatic drugs (DMARDs) has achieved remarkable success in preventing and alleviating disease activity in RA patients.

[0003] However, some RA patients still have limited responses to DMARDs (slow-acting anti-rheumatic drugs), and their pain persists. More importantly, the treatment of RA bone and joint pain is highly challenging and represents a large unmet medical need. Existing therapeutic drugs (non-steroidal anti-inflammatory drugs, analgesics, and steroids) cannot provide sustained pain relief and may have serious adverse reactions. Although biological therapy and joint replacement surgery can effectively relieve RA pain, they may only be suitable for patients with the most severe or advanced disease. If the side effects of treatment are too great, it will have a more adverse impact on the patient's physical and mental health, which puts new demands on special targets and new therapies. Understanding the pathogenic role of various molecules in RA will help discover potential targets for the treatment and relief of bone and joint pain. Summary of the Invention

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

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

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

[0007] Furthermore, the drug synergistically targets the protein ATP1B3 to promote the phase separation of the protein ATP1B3 and target activation of CA9, causing lysosomal dysfunction and inducing alkali death, thereby alleviating cartilage damage and the formation of nerve injury markers.

[0008] The present invention uses ATP1B3-CA9 as a target in drugs for treating RA. By synergistically targeting the ATP1B3 protein through drugs, the phase separation of ATP1B3 is promoted to target and activate CA9, causing lysosomal dysfunction and inducing alkaline death, thereby alleviating cartilage damage and the formation of nerve injury markers, and having a better analgesic effect and better therapeutic effect on RA. BRIEF DESCRIPTION OF THE DRAWINGS

[0009] Figure 1 This is a distribution diagram of secondary hyperalgesia indices in the sham-operated group, the icariin ICA-treated group, and the rheumatoid arthritis (RA) model rats at different time points in the first embodiment of the present invention, wherein A is the 50% paw withdrawal threshold (50% PWT) of the rat's left hind paw, and B is the withdrawal threshold of the pain measurement (PAMWT) of the rat's left knee;

[0010] Figure 2 Graph showing quantitative analysis of left hind paw (LH) strength, LH area, and LH swing speed and right hind paw (RH) at week 8 after sham surgery or collagen-induced arthritis surgery in the first embodiment of the present invention;

[0011] Figure 3 This is a quantitative analysis diagram of MMP13 and ColX cells in articular cartilage in the first embodiment of the present invention;

[0012] Figure 4 This is an intersection analysis diagram of up-regulated genes in the first embodiment of the present invention;

[0013] Figure 5 This is a graph showing the inhibition of chondrocyte damage by overexpression of ATP1B3 in the first embodiment of the present invention;

[0014] Figure 6 : is a map of the intrinsic disordered region of ATP1B3 calculated by the IUPred2 website in the first embodiment of the present invention;

[0015] Figure 7 This is a quantitative analysis of the droplet formation of ATP1B3-GFP using different buffers in the first embodiment of the present invention, wherein A represents the buffer containing different concentrations of salt and 10% PEG-8000 to form droplets, and B represents the buffer containing different protein concentrations to form droplets;

[0016] Figure 8 Schematic diagram showing the in vivo phase separation condensation product of endogenous ATP1B3 protein in C28 / I2 and RKO cells by IF in the first embodiment of the present invention;

[0017] Figure 91. The first embodiment of the present invention shows the distribution of phase-separated aggregates of exogenous ATP1B3-GFP protein in C28 / I2 cells in vivo and the fluorescence photobleaching recovery experiment of ATP1B3-GFP aggregates in C28 / I2 cells in vivo;

[0018] Figure 10 Schematic diagram of the screening strategy for ATP1B3 phase separation specific targets in the first embodiment of the present invention;

[0019] Figure 11 This is a schematic diagram of the 15 genes identified in the first embodiment of the present invention as targets of ATP1B3 phase separation condensates;

[0020] Figure 12 This is a correlation analysis diagram between CA9 and ATP1B3 in the first embodiment of the present invention;

[0021] Figure 13 This is a graph showing the expression level of CA9 detected by qPCR and WB in the first embodiment of the present invention;

[0022] Figure 14 This is a graph showing the enrichment of endogenous ATP1B3 at the CA9 site in C28 / I2 cells detected by ChIP-qPCR in the first embodiment of the present invention;

[0023] Figure 15 This is a diagram showing the quantitative analysis of cartilage damage and noxious nerve marker expression levels in the first embodiment of the present invention;

[0024] Figure 16 This is a graph showing the effects of CA9 activation on cartilage damage and the concentration distribution of CGRP, a noxious nerve marker, in the first embodiment of the present invention;

[0025] Figure 17 This is a graph showing the intracellular pH distribution of C28 / I2 cells after treatment with ICA (3 μm) for 24 hours in the first embodiment of the present invention;

[0026] Figure 18 This is a diagram showing the effect of ATP1B3 on the NF-κB-dependent CA9 pathway in the first embodiment of the present invention;

[0027] Figure 19 This is a graph showing Western blot analysis of protein expression in C28 / I2 cells after treatment with ICA (3 μm) for 6-24 hours in the first embodiment of the present invention;

[0028] Figure 20 This is a graph showing the cell death measurement of C28 / I2 cells after treatment with ICA (3 μm) for 24 hours in the presence of bafilomycin A1 (BafA1, 100 nM) in the first embodiment of the present invention;

[0029] Figure 21 This is an analysis diagram of the IP analysis of the interaction between CA9 and ATP1B3 in C28 / I2 cells after treatment with ICA (3 μM) for 24 hours in the first example of the present invention;

[0030] Figure 22 This is an analysis graph of image analysis of lysosomal membrane permeability in C28 / I2 cells after treatment with ICA (3 μM) for 12 or 24 hours in the first embodiment of the present invention;

[0031] Figure 23 This is a graph showing the cell viability of C28 / I2 cells after treatment with ICA for 24 hours in the presence of CA9 in the first embodiment of the present invention;

[0032] Figure 24 This is a graph showing the expression of ATP1B3 in the C28 / I2 cell line after treatment with the proteasome inhibitor MG132 in the first embodiment of the present invention;

[0033] Figure 25 This is a graph showing the results of a cycloheximide chase test of ICA in the first embodiment of the present invention;

[0034] Figure 26 Schematic diagram of co-immunoprecipitation (Co-IP) detection of ubiquitination of ATP1B3 after ICA treatment in the first embodiment of the present invention;

[0035] Figure 27 This is a molecular docking analysis and identification diagram between ICA and ATP1B3 in the first embodiment of the present invention;

[0036] Figure 28 This is a molecular docking analysis and identification diagram of the two binding residues (ARG177 and TYR217) between JTC801 and ATP1B3 in the first embodiment of the present invention;

[0037] Figure 29 This is a graph showing the efficacy of JTC801 combined with ICA in the first embodiment of the present invention, wherein A represents a graph showing cell viability detected by CCK8, B represents a quantitative analysis of the expression levels of cartilage damage and noxious nerve markers, and C represents a graph showing the distribution of CGRP concentrations in cartilage damage and noxious nerve markers.

[0038] The following specific embodiments will further illustrate the present invention in conjunction with the above-mentioned drawings. DETAILED DESCRIPTION

[0039] To facilitate understanding of the present invention, the present invention will be described more fully below with reference to the accompanying drawings. The drawings illustrate several embodiments of the present invention. However, the present invention may be implemented in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided to provide a more thorough and comprehensive understanding of the present invention.

[0040] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one skilled in the art to which this invention pertains. The terms used in this specification of the present invention are for the purpose of describing specific embodiments only and are not intended to limit the present invention. The term "and / or" as used herein includes any and all combinations of one or more of the associated listed items.

[0041] Example 1

[0042] The present invention proposes the use of ATP1B3-CA9 as a target in drugs for treating RA, wherein the drugs for treating RA are icariin and JTC-801;

[0043] Among them, JTC-801 is an opioid analgesic drug and an alkali death activator.

[0044] Specifically, the above drugs synergistically target the protein ATP1B3 to promote the phase separation of the protein ATP1B3 and target activation of CA9, causing lysosomal dysfunction and inducing alkali death, thereby alleviating cartilage damage and the formation of neuronal markers.

[0045] Ica alleviates joint pain and cartilage damage in RA and is associated with upregulation of ATP1B3

[0046] 1. In vivo verification that icariin (ICA) can relieve RA joint pain and cartilage damage:

[0047] To establish a collagen-induced arthritis model, type II collagen (a protein isolated from the immune system and abundant in articular cartilage) was dissolved in 0.1 mol / L acetic acid and stirred thoroughly at 4°C to a concentration of 2 g / L. The mixture was then refrigerated at 4°C overnight. Inactivated BCG vaccine was then dissolved in liquid paraffin and prepared with 2 g of complete Freund's adjuvant. Equal volumes of the two were mixed and emulsified to create a type II collagen emulsion. 0.1 ml of this emulsion was injected intradermally at the base of the rat's tail to induce inflammation. On the 21st day, 0.1 ml of the emulsion was injected intraperitoneally as a challenge. Hindlimb temperature changes and photothermal images were recorded using an infrared thermal imaging system until the 30th day after adjuvant administration. Paw width changes were measured with a caliper.

[0048] 1) Pain indicators: secondary pain sensation (50% PWT), primary pain sensation (PAMWT), and gait deficits (paw swing speed, contact area, and intensity);

[0049] 2) Articular cartilage damage and aging phenotype: cartilage degradation (Saftanin O / SOFG staining and OARSI scoring); IHC and WB detection of degradation markers (COLX, ACAN, and MMP13); senescence detection using β-galactosidase staining, or WB / HC detection of senescence markers (p16INK4a, p21, and SASP);

[0050] 3) Gene expression profiling: RNA-seq analysis and pathway enrichment analysis;

[0051] 4) ICA drug target identification: mass spectrometry analysis and functional enrichment analysis; Based on gene expression profiling 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, and the intersection gene is ATP1B3.

[0052] 2. In vivo analysis of the effector mechanism of ICA

[0053] 1) Level of nociceptive nerve (pain perception) innervation in subchondral bone: detection of relevant markers (immunofluorescence staining of calcitonin gene-related peptide (CGRP), substance p (SP), PIEZ02, and P2X3); quantitative positive nociceptive nerve fiber density; coupling of angiogenesis and sensory innervation (colocalization of CGRP and EMUN); and osteophyte volume: microcomputed tomography (μCT) analysis of the medial compartment (coronal) of the tibial cartilage.

[0054] 2) Subchondral bone degeneration: bone microarchitecture (trabecular parameters, structural model index, total pore space, and subchondral bone plate thickness); bone pathological changes (tibial trichrome staining, calcein-alizarin double labeling for bone formation and localization); IHC detection of levels of osteoprogenitor cell markers (Nestin and Osterix).

[0055] 3) ATP1B3 and alkali death levels in subchondral bone: IHC / WB detection; pH level detection; IHC detection of alkali death markers (NF-κB, RELA, p65 and CA9).

[0056] 3. In vitro validation of the alleviating effect of ICA targeting ATP1B3 on RA bone pain and cartilage damage:

[0057] 1) Construct an organoid model of chondrocytes derived from RA patients, add ICA treatment, and divide the cells into control, RA, and RA+ICA groups. Detect cartilage degradation, noxious neural markers, ATP1B3 expression, and alkali death levels.

[0058] 2) Primary FLS from RA patients (RA-FLS) were isolated and co-cultured with human immortalized chondrocytes (C28 / I2), which overexpressed ATP1B3. The cells were divided into two groups: Con-FLS (C28 / I2+NC, C28 / I2+ATP1B3) and RA-FLS (C28 / I2+NC, C28 / I2+ATP1B3). Chondrocyte damage in the RA environment was detected: cartilage degradation and senescence. CCK-8 was used to detect cell viability, noxious neural markers, and alkali death levels.

[0059] Phase separation of ATP1B3 targets CA9 to promote cartilage damage and the formation of nociceptive neural markers

[0060] 1. Explore whether ATP1B3 undergoes phase separation in cartilage injury

[0061] C28 / I2 cells were co-cultured with RA-FLS to construct an acidic mutant of ATP1B3 (which lost phase separation ability) and a fusion plasmid of the ATP1B3 mutant and hnRNPA1-IDR (which restored phase separation ability). After transfection, the following phase separation indicators were measured:

[0062] 1) Phase separation potential detection of ATP1B3:

[0063] 2) In vitro droplet formation experiment: GFP fluorescence labeling, different concentrations of NaCl and protein treatment, IF detection of ATP1B3 expression and localization;

[0064] 3) IF detection of endogenous ATP1B3 expression and morphology;

[0065] 4) After exogenous transfection of GFP-ATP1B3 or ATP1B3-MUT plasmids, the expression localization of ATP1B3 was detected by IF after photobleaching treatment or without treatment.

[0066] 2. Analyze the regulatory effect of ATP1B3 phase separation on cartilage damage and bone pain

[0067] C28 / I2 cells were co-cultured with RA-FLS and transfected with different plasmids. The cells were divided into control, ATP1B3, ATP1B3-MUT, and ATP1B3-MUT-IDR groups. The following indicators were measured: ATP1B3 expression, nociceptive nerve (bone pain) markers, cartilage degradation, alkali death levels, and cell survival by TUNEL / flow cytometry.

[0068] 3. Explore the molecular mechanism and direct targets of ATP1B3 phase separation in alleviating cartilage damage

[0069] 1) Screening for direct targets of ATP1B3 phase separation: ATP1B3-WT and ATP1B3-MUT were transfected into C28 / I2 cells and analyzed by ChIP-seq; ATP1B3-KO cells were subjected to RNA-seq analysis; ATP1B3 binding proteins and intersection genes were excluded from ChIP-seq of ATP1B3-MUT, and the ATP1B3 phase separation-specific target gene CA9 was selected.

[0070] 2) Verify the regulatory effect of ATP1B3 on CA9: C28 / I2 and RA-FLS co-culture system, ChIP-qPCR detection of CA9 endogenous enrichment of ATP1B3, as well as exogenous enrichment of ATP1B3-WT, ATP1B3-MUT, and ATP1B3-MUT-IDR; after knocking down ATP1B3 or ATP1B3-MUT plasmid, WB detection of CA9, COLX, ACAN and MMP13, as well as nociceptive nerve markers; alkali death level.

[0071] 3) Interaction mechanism between ATP1B3 and CA9: Forward and reverse Co-IP were used to detect the endogenous and exogenous binding of ATP1B3 and CA9; point mutations were combined with Co-IP to analyze the binding sites of ATP1B3 and CA9; Flag-tagged ATP1B3-WT and ATP1B3-MUT were co-transfected with full-length CA9, and changes in binding ability were detected by Co-IP.

[0072] 4) Analyze the necessity of ATP1B3 phase separation in cartilage protection by suppressing alkali death caused by CA9 overexpression

[0073] The regulation of cartilage damage and nociceptive neural markers by CA9 and alkali death was analyzed in C28 / I2+RA-FLS (NC, ATP1B3, ATP1B3-MUT, shCA9, ATP1B3+shCA9, and

[0074] ATP1B3-MUT+shCA9), adjust the cell pH to acidic to inhibit alkali death, and perform the following index detection:

[0075] Expression of ATP1B3 and CA9, levels of alkali death, nociceptive nerve markers, and cartilage degradation markers.

[0076] 3. Exploring the mechanism by which ICA & JTC801 target the ATP1B3-CA9 axis to induce lysosomal dysfunction and alkali-induced death

[0077] 1. Analysis of the regulatory effect of ICA on the ATP1B3-CA9 axis

[0078] 1) Endogenous and exogenous verification: C28 / I2 and HEK293T cells were treated with the proteasome inhibitor MG132 or the autophagy inhibitor hydroxychloroquine (HCQ) (CHQ), and then treated with or without ICA. Protein half-life was analyzed by CHX tracking, ubiquitination analysis was performed by Co-IP, and the expression of ATP1B3 and CA9 was detected by Western blotting.

[0079] 2) Molecular docking analysis of the binding sites of ICA, ATP1B3 and CA9: Previous studies have identified two JTC-801 binding residues (ARG177 and TYR217) on ATP1B3.

[0080] 3) The ARG177 and TYR217 residue mutants ATP1B3ARG177R and ATP1B3TYR217R were constructed and transfected into HEK293T and C28 / I2 cells. After adding ICA, the expression and interaction of ATP1B3 and CA9 were detected.

[0081] 2. Explore the regulatory mechanism of ATP1B3-CA9 on alkali death

[0082] 1) ATP1B3 was knocked down in C28 / I2 cells. The cells were treated or not with the lysosomal inhibitor bafilomycin A1 (BafA1). The level of cell alkali death was detected by flow cytometry, and cell viability, intracellular pH, and expression of alkali death-related proteins NF-KB / CA9 were detected by CCK-8.

[0083] 2) Lysosomal dysfunction detection: C28 / 2 cells were knocked down for ATP1B3 and treated or not with JTC-801. Intracellular pH homeostasis was detected: the ysoSensor yellow / blue DND160 pH indicator displayed the level of lysosomal acidification; lysosomal membrane permeability: RITC-dextran leakage test; Western blot detection of the expression of lysosomal membrane proteases CTSB and CTSD.

[0084] 3. P1B3 regulates and restores CA9 function

[0085] 1) ATP1B3 was knocked down in C28 / I2 cells, and the expression of CA9 was detected by WB; the protein half-life of CA9 was tracked by CHX.

[0086] 2) C28 / I2 cells were knocked down for both ATP1B3 and CA9, and treated with or without ICA. Alkaline death levels, lysosomal dysfunction, cartilage damage markers, and noxious nerve markers were tested.

[0087] Combination therapy:

[0088] Groups: AVV-ATP1B3, AVV-ATP1B3+JTC-801, AVV-ATP1B3+icariin, AVV-ATP1B3+JTC-801+icariin, JTC-801+icariin;

[0089] After intra-articular injection of adenovirus-encapsulated ATP1B3, combined with ICA and JTC-801 treatment, RA rats were subjected to pain behavior testing (primary and secondary pain sensation), articular cartilage damage assessment (Safranin O / SOFG staining, OARSI scoring, and IHC and WB detection of COLX, ACAN, and MMP13 expression), analysis of the level of nociceptive innervation (pain sensation) in subchondral bone (nociceptive nerve marker detection: CGRP, SP, PIEZ02, and P2X3; IF detection of CGRP and EMUN; assessment of osteophyte volume), analysis of subchondral bone degeneration (assessment of osteoid formation and positioning and tibial structure, WB / IHC detection of osteoprogenitor cell markers Nestin and Osterix), and analysis of ATP1B3 and alkali-death levels in cartilage and subchondral bone: IHC / WB / IF detection of ATP1B3, NF-κB, RELA, p65, and CA9. level.

[0090] In this example, the analgesic effect of ICA on injured joints was verified in an RA rat model. It was found that after two weeks of ICA treatment, the secondary hyperalgesia index (50% paw break threshold: 50% PWT) of RA rats was significantly higher than that of the RA rat model group ( Figure 1 A in the figure), and the mechanical stimulation paw withdrawal threshold (PAMWT) was significantly higher than that of the RA rat model group ( Figure 1Figure B) Eight weeks after collagen-induced arthritis surgery (compared to the sham group), the ipsilateral hind paw showed significantly reduced strength, contact area, and swing speed relative to 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 inhibits chondrocyte degeneration in osteoarthritis. It was found that the expression of ColX and MMP13 in the RA group was significantly higher than that in the sham operation group. After ICA treatment, the expression of ColX and MMP13 decreased. Subsequently, the intersection analysis of 386 ICA drug targets, genes downregulated in rheumatoid arthritis and sham operation arthritis, and genes upregulated by icariin and rheumatoid arthritis was performed, and ATP1B3 ( Figure 4 Through cell experiments, it was confirmed that CCK8 detects chondrocyte viability and found that overexpression of ATP1B3 can effectively inhibit chondrocyte damage ( Figure 5 ).

[0091] For details, please refer to Figure 6 , a map of the intrinsically disordered region (IDR) of ATP1B3 calculated using the IUPred2 website. The IDR (amino acids 1-182) is shown above the disease score map. A strong intrinsically disordered region (IDR) was found at the N-terminus of ATP1B3. Quantitative analysis of droplet formation with 10 mM ATP1B3-GFP in droplet formation buffer containing varying salt concentrations and 10% PEG-8000 revealed that the number of droplets decreased with increasing salt concentration. In vitro droplet formation assays demonstrated that ATP1B3 can form round droplets in saline solutions, and the number of droplets gradually decreased with increasing salt concentration (see ). Figure 7 Quantitative analysis of ATP1B3-GFP droplet formation in droplet formation buffer at different protein concentrations revealed that the number of droplets increased with increasing protein concentration. In vitro droplet formation assays showed that droplets were smaller at low protein concentrations, grew larger as protein concentration increased, and the number of droplets increased at higher protein concentrations (see Figure 7 B in ). Also, see Figure 8 The status of ATP1B3 was examined in the poorly differentiated colon cancer cell line RKO and the immortalized human chondrocyte cell line C28 / I2. Immunofluorescence (IF) revealed the in vivo phase-separated condensation complex C28 / I2 of endogenous ATP1B3 protein in poorly differentiated colon cancer cells RKO and immortalized human chondrocyte C28 / I2.

[0092] See also Figure 9When exogenous ATP1B3-green fluorescent protein was transfected into the human immortalized chondrocyte C28 / I2 cell line with low endogenous ATP1B3 expression, ATP1B3 was observed to form larger condensates. The fluorescence photobleaching recovery experiment of in vivo ATP1B3-green fluorescent protein condensates in human immortalized chondrocyte C28 / I2 cells found that the green fluorescent protein signal can recover quickly after photobleaching.

[0093] See also Figure 10 , shown is a schematic diagram of the screening strategy for ATP1B3 phase separation-specific targets, in which RNA-seq and ATP1B3 ChIP-seq were performed using different ATP1B3 patterns.

[0094] Direct targets of ATP1B3 phase-separated condensates were identified following a standard sequential approach: (1) collection of significantly differentially expressed genes (DEGs) in response to ATP1B3 knockdown by RNA-seq, and (2) identification of effective targets of ATP1B3 by ChIP-seq, collection of overlaps between DEGs and WT-ATP1B3-targeted genes, and exclusion of mutant-ATP1B3-targeted genes from WT-ATP1B3-targeted genes.

[0095] Then, see Figure 11 , we collected 671 significant DEGs in response to ATP1B3 knockdown, 3580 WT-ATP1B3 target genes, and 995 mutant ATP1B3 target genes. Based on these criteria, we finally identified 15 genes as targets of ATP1B3 phase-separated condensates. Figure 12 , through the correlation analysis of ATP1B3 and 15 genes, the results showed that CA9 was significantly negatively correlated with ATP1B3. Figure 13 , qPCR and WB assays confirmed that overexpression of ATP1B3 attenuated the induction effect of CA9. Figure 14ChIP-qPCR assays showed that endogenous ATP1B3 was enriched at the CA9 site in human immortalized cartilage C28 / I2 cells. ChIP-qPCR assays showed that exogenous wild-type ATP1B3 and mutant-ATP1B3-containing strongly intrinsically disordered regions were enriched at the CA9 site in human immortalized C28 / I2 cells. According to ChIP-qPCR results, the CA9 gene site was occupied by WT-ATP1B3, but not mutant-ATP1B3. Mut-IDR rescued the disabled function of the ATP1B3 mutant on CA9 binding. Collectively, these results indicate that ATP1B3 phase-separated condensates directly target CA9 to induce its expression. Also, please refer to Figures 15 and 16 In the rheumatoid arthritis group and the carbonic anhydrase group, the expression of ColX and MMP13 was significantly higher than that in the negative control group. In the rheumatoid arthritis group and the carbonic anhydrase group, the expression of CGRP (calcitonin gene-related peptide) was significantly higher than that in the negative control group. It can be seen that the activation of carbonic anhydrase (CA9) can promote the formation of cartilage damage and noxious nerve markers ( Figure 15 and Figure 16 ).

[0096] See also Figure 17 After treatment with ICA (3 μM) for 24 hours, the intracellular pH of human immortalized cartilage C28 / I2 cells was detected, and the pH value was suppressed in ATP1B3 knockdown C28 / I2 cells. However, after treatment with ICA (3 μM) for 6-24 hours, Western blot analysis of protein expression in human immortalized cartilage C28 / I2 cells revealed that ATP1B3 had no significant effect on the NF-κB-dependent CA9 pathway ( Figure 18 and Figure 19 ).

[0097] See also Figure 20 , after treatment with ICA (3µM) for 24 hours in the absence or presence of bafilomycin A1 (BafA1, 100 nM), human immortalized chondrogenic C28 / I2 cells were subjected to a cell death assay. Quantitative analysis was performed using Image J software. It was found that, similar to knockout of ATP1B3, bafilomycin A1 (BafA1), a specific inhibitor of lysosomal V-ATPase, effectively abolished the anticancer activity of JTC801; see [see ]. Figure 21 After treatment with ICA (3 μM) for 24 hours, IP analysis of the interaction between carbonic anhydrase (CA9) and ATP1B3 in human immortalized cartilage C28 / I2 cells was performed. IP experiments showed that ICA increased the formation of CA9-ATP1B3 complex in C28 / I2 cells.

[0098] See also Figure 22 Image analysis of lysosomal membrane permeability in human immortalized chondrogenic C28 / I2 cells after treatment with ICA (3 µM) for 12 or 24 hours. Acridine orange (AO) staining showed that ICA treatment for 24 hours upregulated lysosomal membrane permeability, and this process was inhibited by ATP1B3 knockout. Figure 23 The cell viability of human immortalized chondrogenic C28 / I2 cells was measured after treatment with ICA for 24 hours in the absence or presence of carbonic anhydrase (CA9). 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 C28 / I2 cells with knockdown of ATP1B3.

[0099] See also Figure 24 , ATP1B3 expression in the human immortalized cartilage C28 / I2 cell line after treatment with the proteasome inhibitor MG132 was found to be upregulated in the C28 / I2 cell line in a dose- and time-dependent manner, similar to ICA. However, the autophagy inhibitor hydroxychloroquine (HCQ) reduced ATP1B3 expression. Figure 25 The stability of ATP1B3 protein in the indicated immortalized human cartilage 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. Figure 26 , co-immunoprecipitation (Co-IP) was used to detect the ubiquitination of ATP1B3 after ICA treatment, and it was found that the ubiquitination of ATP1B3 increased after ICA treatment.

[0100] See also Figure 27 , the binding energy between ICA and ATP1B3 was identified to be −7.6 using molecular docking analysis. Figure 28 Two binding residues (ARG177 and TYR217) between JTC801 and ATP1B3 were identified.

[0101] After establishing an RA rat model, the efficacy of JTC801 combined with ICA was tested. Figure 29 The CCK8 assay in Figure A showed that the viability of human immortalized cartilage C28 / I2 cells in the JTC801 combined with ICA treatment group was significantly higher than that in the single treatment group, indicating that the synergistic treatment showed better efficacy. Figure 29Figures B and C tested the efficacy of JTC801 combined with ICA in an RA rat model. The results showed that the JTC801 combined with ICA treatment group had significantly lower levels of cartilage damage markers and nociceptive nerve markers ColX and calcitonin gene-related peptide (CGRP) than the JTC801 or icariin alone treatment groups.

[0102] In summary, the application of ATP1B3-CA9 as a target in the treatment of RA in the above embodiments of the present invention, through the synergistic drug targeting ATP1B3 protein, promotes the phase separation of ATP1B3 and targeted activation of CA9, causing lysosomal dysfunction and inducing alkaline death, thereby alleviating cartilage damage and the formation of nerve injury markers, and has better analgesic effect and better efficacy for RA.

[0103] The technical features of the above-mentioned embodiments can be combined arbitrarily. In order to make the description concise, not all possible combinations of the technical features in the above-mentioned embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0104] The above-described embodiments merely represent several implementation methods of the present application. While the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that a person skilled in the art could make various modifications and improvements without departing from the spirit of the present application, all of which fall within the scope of protection of the present application. Therefore, the scope of protection of the present patent application shall be determined by the appended claims.

Claims

1. Application of icariin and JTC-801 in combination in the preparation of a drug for the treatment of rheumatoid arthritis.

2. 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 and target activation of CA9, causing lysosomal dysfunction and inducing alkaline death, thereby alleviating cartilage damage and the formation of nerve injury markers.

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