Composition for preventing or treating osteoarthritis targeting glycosylation

Abstract

The present specification relates to a composition for preventing or treating osteoarthritis, wherein the composition targets glycosylation. More specifically, the composition can inhibit abnormal accumulation of UDP-GlcNAc in the cytoplasm of chondrocytes and restore normal UDP-GlcNAc distribution to the endoplasmic reticulum and / or Golgi body, and thereby inhibit excessive O-GlcNAcization caused by cytoplasmic UDP-GlcNAc accumulation in chondrocytes, and can ultimately prevent or treat osteoarthritis.

Description

Composition for the prevention or treatment of osteoarthritis targeting glycosylation

[0001] Cross-reference regarding related applications

[0002] This application claims priority to Korean Patent Application No. 10-2024-0155038, filed November 5, 2024, the entire contents of which are incorporated by reference herein. This application claims priority to Korean Patent Application No. 10-2025-0160920, filed October 30, 2025, the entire contents of which are incorporated by reference herein.

[0003] This specification relates to a composition for the prevention or treatment of osteoarthritis targeting glycosylation, and a composition for the diagnosis or prognosis prediction of osteoarthritis.

[0004] [National R&D projects that supported this invention]

[0005] [Project ID] 2710076477

[0006] [Project No.] 0409-20250080

[0007] [Ministry Name] Ministry of Science and ICT

[0008] [Name of Project Management (Specialized) Agency] National Research Foundation of Korea

[0009] [Research Project Name] Individual Basic Research (Ministry of Science and ICT)

[0010] [Project Title] Study on the Liver-Joint Transport Pathway of Trace Element Selenium and the Mechanism of Cellular Aging and Degenerative Arthritis Development Caused by Abnormal Selenium Metabolism in Chondrocytes

[0011] [Name of Project Performing Organization] Seoul National University

[0012] [Research Period] 2025.03.01 ~ 2026.02.28

[0013]

[0014] Osteoarthritis (OA) is a major cause of disability, with the number of patients increasing by 48% worldwide between 1990 and 2019. Various etiological risk factors, such as aging, obesity, and mechanical overload, contribute to the development and progression of osteoarthritis. The progression of osteoarthritis is characterized by pathological changes such as cartilage degeneration, subchondral bone sclerosis, osteophyte formation, and synovial inflammation.

[0015] Osteoarthritis is characterized by the loss of proteoglycans (PGs) in articular cartilage and chronic inflammation. The disease is exacerbated by senescent chondrocytes accumulating in the cartilage, including in the superficial zone, which secrete SASPs containing inflammatory cytokines, chemokines, and cartilage matrix degrading enzymes. Uridine diphosphate N-acetylglucosamine (UDP-GlcNAc) is a sugar precursor constituting the glucosaminoglycan (GAG) chain and serves as a substrate for O-linked N-acetylglucosamine (O-GlcNAcylation). O-GlcNAcylation is a reversible post-translational modification catalyzed by O-GlcNAc transferase (OGT) and removed by O-GlcNAcase (OGA), and is known as an important mechanism integrating nutritional and metabolic states with the regulation of cellular responses. Dysregulated O-GlcNAcylation is associated with various human diseases, including cancer, type 2 diabetes, and cardiovascular disease.

[0016] Meanwhile, glucosamine is used as a material to efficiently generate UDP-GlcNAc and is widely supplemented with the expectation that it will maintain proteoglycan levels in patients with osteoarthritis and the elderly, but its clinical effects are controversial.

[0017] Despite the importance of cellular senescence and SASP in the pathogenesis of osteoarthritis, there is currently a lack of effective treatments capable of selectively regulating SASP in senescent chondrocytes.

[0018]

[0019] [Prior Art Literature]

[0020] [Patent Literature]

[0021] Japanese Published Patent No. 2021-004745 A

[0022]

[0023] One aspect of the present specification aims to provide a composition for the prevention or treatment of osteoarthritis.

[0024] One aspect of the present specification aims to provide a composition for diagnosing or predicting the prognosis of osteoarthritis.

[0025] One exemplary aspect of this specification aims to elucidate the pathogenic redistribution phenomenon of UDP-GlcNAc observed in osteoarthritis and to control the induction / maintenance of SASP due to increased O-GlcNAcylation. Another exemplary aspect of this specification aims to block the transition to a chronic inflammatory secretory pathway by inhibiting the selective autophagic degradation of GATA4 (GATA binding protein 4) mediated by p62 adapter proteins through the control of O-GlcNAcylation. Yet another exemplary aspect of this specification aims to provide a therapeutic strategy that inhibits the structural / symptomatic progression of osteoarthritis by targeting upstream (SLC35 transporter family) and downstream (OGT and GATA4) alone or in combination.

[0026]

[0027] A composition for the prevention or treatment of osteoarthritis according to one aspect of the present specification comprises: a preparation that increases the expression or activity of a gene belonging to solute carrier family 35 (SLC35); a protein encoded by said gene; or an inhibitor that inhibits the accumulation of uridine diphosphate N-acetylglucosamine (UDP-GlcNAc) in the cytoplasm of chondrocytes.

[0028] A composition for diagnosing or predicting the prognosis of osteoarthritis according to one aspect of the present specification comprises: a preparation for measuring the expression level of a gene belonging to the solute transporter family 35 (SLC35); a preparation for measuring the level of a protein encoded by said gene; or a preparation for measuring the level of uridine diphosphate N-acetylglucosamine.

[0029] A composition for the prevention or treatment of osteoarthritis according to one aspect of the present specification comprises at least one GATA4 inhibitor selected from the group consisting of NSC140905 (a compound represented by [Chemical Formula 1] below, 2-(1,3-Benzodioxol-5-ylmethyl)butanedioic acid); or GATA4 (GATA binding protein 4) siRNA consisting of the nucleotide sequences of SEQ ID NO. 1 (5′-GAGCAAACCAGAGCCUAGAdTdT-3′) and SEQ ID NO. 2 (5′-UCUAGGCUCUGGUUUGCUCdTdT-3').

[0030] [Chemical Formula 1]

[0031] .

[0032]

[0033] A composition for the prevention or treatment of osteoarthritis according to one aspect of the present specification can inhibit the abnormal accumulation of UDP-GlcNAc in the cytoplasm (e.g., cytoplasm of chondrocytes) and restore the normal distribution of UDP-GlcNAc to the endoplasmic reticulum and / or Golgi apparatus. By doing so, excessive O-GlcNAc formation caused by cytoplasmic UDP-GlcNAc accumulation in chondrocytes can be inhibited, and ultimately, osteoarthritis can be prevented or treated. For example, a composition for the prevention or treatment of osteoarthritis according to one aspect can reactivate the delivery of UDP-GlcNAc to the endoplasmic reticulum or Golgi apparatus and resolve cytoplasmic UDP-GlcNAc accumulation by restoring / enhancing the function of the SLC35 transporter family (e.g., inducing expression, activating, or acting as a carrier), and can increase PG secretion and synthesis and reduce SASP. For example, a composition for the prevention or treatment of osteoarthritis according to one aspect can inhibit SASP by reducing O-GlcNAcylation of GATA4 through the inhibition of O-GlcNAc transferase (OGT), which mediates O-GlcNAcylation, thereby restoring binding with the p62 adapter and increasing GATA4 degradation by selective autophagy. For example, a composition for the prevention or treatment of osteoarthritis according to one aspect can mitigate the onset and progression of osteoarthritis through a significant synergistic effect compared to targeting each of these individually by co-targeting upstream (SLC35 transporter family) and downstream (OGT and GATA4).

[0034] A composition for preventing or treating osteoarthritis according to one aspect of the present specification can prevent or treat osteoarthritis by inhibiting the level of GATA4 in chondrocytes or a cartilage layer. For example, a composition for preventing or treating osteoarthritis according to one aspect can block the SASP transcription program with a GATA4 inhibitor (e.g., small molecule NSC140905, siRNA sequence for Gata4, etc.).

[0035] A composition according to one aspect of the present invention is applicable to various types of osteoarthritis, such as post-traumatic osteoarthritis, inflammatory osteoarthritis, and degenerative osteoarthritis, and is manufactured as an injectable formulation to provide a local and effective therapeutic effect through direct administration into the joint.

[0036] A composition for diagnosis or prognosis prediction according to one aspect of the present specification can diagnose the onset or progression of osteoarthritis early or predict the prognosis by measuring the expression level of the SLC35 gene, the level of the protein encoded by it, or the level of UDP-GlcNAc in the cytoplasm. This can be usefully utilized in establishing individualized treatment strategies for patients with osteoarthritis.

[0037] One aspect of this specification identifies that excessive glucosamine supplementation can increase cytoplasmic UDP-GlcNAc accumulation and O-GlcNAc conversion of GATA4, thereby stabilizing GATA4 and exacerbating post-traumatic osteoarthritis, and thus provides a guideline that excessive glucosamine intake should be carefully considered in individuals experiencing joint instability or trauma.

[0038] Compositions, methods, etc. according to one aspect of the present invention alleviate total articular OA phenotypes such as cartilage destruction, subluxatory osteosclerosis, osteophytes, and synovitis, reduce mechanical pain (incapacitance, von Frey), and GATA4, SASP, and aging indicators (p16 INK4a It can reduce the expression of HMGB1 and induce a decrease in the production of ECM damage markers (CTX-II, NITEGE).

[0039] The effects disclosed in this specification are demonstrated by improvements in histological / pain indicators, reductions in collagen / PG damage indicators, reductions in SASP / aging indicators, and preservation of the cartilage matrix in individual, tissue, and cell models of human and mouse cartilage.

[0040]

[0041] Figure 1a shows a heatmap of the fold change in expression levels of UDP-GlcNAc and UDP-GalNAc transporters in human osteoarthritis cartilage. The publicly available transcriptome datasets GSE16464, GSE43923, GSE64394, GSE113825, GSE117999, GSE178557, and GSE186220 were analyzed.

[0042] Figure 1b shows a heatmap of the fold change in expression levels of UDP-GlcNAc and UDP-GalNAc transporters in chondrocytes treated with IL-1β. The publicly available transcriptome datasets GSE6119, GSE104793, and GSE163080 were analyzed.

[0043] Figure 1c shows a heatmap of the fold change in expression levels of UDP-GlcNAc and UDP-GalNAc transporters in the cartilage of osteoarthritis mouse and rat models. Mouse datasets GSE26475, GSE53857, GSE101573, GSE110268, and GSE143447 and rat dataset GSE28958 were analyzed.

[0044] Figure 1d shows SA-β-Gal staining and quantification of positive rate of C28 / I2 chondrocytes treated with 100 nM doxorubicin (left), representative immunofluorescence images and BrdU integrated quantification (middle), and relative mRNA expression levels of CDK inhibitor or Lmnb1 (right).

[0045] Figure 1e shows SA-β-Gal staining and positive rate quantification of mouse chondrocytes treated with 50 μg / mL bleomycin (left), representative immunofluorescence images and BrdU integrated quantification (middle), and relative mRNA expression levels of CDK inhibitor or Lmnb1 (right).

[0046] Figure 1f shows the relative mRNA expression levels of UDP-GlcNAc and UDP-GalNAc carriers in C28 / I2 chondrocytes treated with doxorubicin.

[0047] Figure 1g shows the relative mRNA expression levels of UDP-GlcNAc and UDP-GalNAc carriers in mouse chondrocytes treated with bleomycin.

[0048] Figure 1h shows the results of quantifying UDP-GlcNAc levels in the whole cell (left), endoplasmic reticulum, Golgi apparatus, and cytoplasmic compartment (right) of C28 / I2 chondrocytes treated with vehicle, DON, or doxorubicin.

[0049] Figure 1i shows the results of quantifying UDP-GlcNAc levels in the whole cells (left) and cytoplasm (right) of mouse chondrocytes treated with vehicle, DON, or bleomycin.

[0050] Figure 1j shows the results of measuring the secretion of sulfated glycoprotein (sGAG) of C28 / I2 chondrocytes treated with vehicle or doxorubicin using the sGAG assay.

[0051] Figure 1k shows the secretion of sulfated glycoprotein (sGAG) from mouse chondrocytes treated with bleomycin.

[0052] Scale bar: Fig. 1d (left), Fig. 1e (left), 50 μm, Fig. 1d (center), Fig. 1e (center), 25 μm. Fig. 1d-k, data represent mean ± standard error. P values ​​were obtained from two-sided t-tests (Fig. 1d-Fig. 1g, Fig. 1j, Fig. 1k) or one-way ANOVA with Dunnett post-hoc test (Fig. 1h, Fig. 1i).

[0053] Figure 2a shows intact and damaged sections of human osteoarthritis cartilage stained with Alcian blue and O-GlcNAc and p16 INK4a O-GlcNAc and p16 immunohistochemically stained for INK4a Shows a representative image of the co-immunofluorescence.

[0054] Figure 2b shows the immunofluorescence staining of O-GlcNAc or p16 in intact and damaged areas of human osteoarthritis cartilage. INK4aShows a pie chart quantifying positive cells.

[0055] Figures 2c, 2d, and 2e show Western blot analysis of SA-β-Gal positivity and global O-GlcNAcification of primary cultured chondrocytes isolated from humans (Figure 2c) or mice (Figures 2d, 2e). Figure 2c was treated with vehicle or 100 nM doxorubicin, Figure 2d with vehicle or 50 μg / mL bleomycin, and Figure 2e with vehicle or 100 μM H2O2.

[0056] Figures 2f and 2g show the results of γ-H2AX immunofluorescence and SA-β-Gal staining of primary cultured chondrocytes treated with bleomycin (50 μg / mL) for 24 hours followed by treatment with vehicle (DMSO) or the OGT inhibitor ST045849 (20 μM) for 72 hours. Figure 2f is a representative image, and Figure 2g is the quantification of the γ-H2AX and SA-β-Gal positive rates.

[0057] Figure 2h shows the relative mRNA expression levels of aging markers in chondrocytes treated with bleomycin for 24 hours followed by treatment with vehicle or ST045849 (20 μM) for 72 hours.

[0058] Figure 2i shows the p16 of primary cultured human chondrocytes treated with the indicated dose of doxorubicin for 14 days. INK4a These are the results of Western blot analysis for , GATA4, and SASPs. Asterisks indicate the active form of the MMP.

[0059] Figure 2j shows the immunohistochemistry for GATA4 and SASP factors in intact and damaged areas of human osteoarthritis cartilage.

[0060] Figure 2k shows representative co-immunofluorescence images of O-GlcNAc and GATA4 and a pie chart quantifying O-GlcNAc or GATA4 positive cells in intact and damaged areas of human osteoarthritis cartilage.

[0061] Figure 2l shows the relative mRNA expression levels of SASP factor in chondrocytes treated with bleomycin for 24 hours followed by treatment with vehicle or ST045849 (20 μM) for 5 days.

[0062] Figure 2m shows GSEA using the "extracellular matrix degradation" gene set on RNA-seq data of chondrocytes treated with vehicle or ST045849 for 5 days after 24 hours of treatment with bleomycin.

[0063] Scale bar: Fig. 2a (top), Fig. 2f (bottom), Fig. 2j, 50 μm, Fig. 2a (bottom), Fig. 2f (top), Fig. 2k, 25 μm. Fig. 2c, Fig. 2d, Fig. 2e, Fig. 2g, Fig. 2h, Fig. 2l, data represent mean ± standard error. P values ​​are obtained from a two-sided t-test (Fig. 2c, Fig. 2d, Fig. 2e) or a two-way ANOVA with Tukey post-hoc test (Fig. 2g, Fig. 2h, Fig. 2l).

[0064] Figure 3a shows the co-immunoprecipitation of FLAG-tagged GATA4 and HA-tagged OGT in HEK293T cells.

[0065] Figure 3b shows a pull-down immunoblot using sWGA-conjugated agarose beads with and without thiamet-G treatment in HEK293T cells.

[0066] Figure 3c shows the O-GlcNAc immunoblot of HA-tagged GATA4 in HEK293T cells treated with vehicle or thiamet-G. The HA-tagged protein was pulled down with an anti-HA-tagged antibody. MG132 (10 μM) was treated for 6 hours prior to lysis.

[0067] Figure 3d shows GATA4 immunoblots in HEK293T cells transfected with Tet-HA-GATA4, depending on doxycycline (Doxy) treatment and the presence or absence of thiamet-G.

[0068] Figure 3e shows that GATA reporter gene analysis was performed on HEK293T cells transfected with a TK promoter-driven empty vector (EV) or HA-GATA4, followed by treatment with a control solvent, Tiamat-G, or ST045849 for 36 hours. The reporter gene construct contains a GATA binding site in the promoter region of the firefly luciferase coding sequence.

[0069] Figure 3f shows the CHX trace analysis of HEK293T-FLAG-GATA4 treated with vehicle or thiamet-G (20 μM).

[0070] Figure 3g shows GSEA using the "GATA4 target gene" gene set for RNA-seq data of chondrocytes treated with vehicle or ST045849 for 5 days after 24 hours of treatment with bleomycin.

[0071] Figures 3h and 3i show the relative mRNA expression levels of Mmp3 (Figure 3h) and Il6 (Figure 3i) in chondrocytes transfected with negative control siRNA (si-NC) or siRNA targeting Gata4 after 24 hours of treatment with bleomycin, and treated with vehicle or ST045849 (20 μM) for 5 days.

[0072] Figure 3j shows the Western blot analysis of HEK293T cells transfected with FLAG-tagged GATA4 wild-type (WT) or variants (S212A or S406A). Bafilomycin A1 (BafA1) treatment conditions were used as a positive control to neutralize the autophagy-mediated degradation of GATA4.

[0073] Figure 3k shows the co-immunoprecipitation of FLAG-tagged GATA4 wild-type or S406A variant and HA-tagged p62 in HEK293T cells. Cells were treated with vehicle or thiamet-G (20 μM, 48 hours) and BafA1 (50 nM, 24 hours). Asterisks indicate immunoglobulin G (IgG) heavy chains.

[0074] The data represent the mean ± standard error. P values ​​are obtained from a two-sided t-test (Fig. 3e), a one-sided permutation test corrected for the family-wise error rate (FWER) for multiple comparisons (Fig. 3g), or a two-way ANOVA with Tukey post-hoc test (Figs. 3h, 3i).

[0075] Figure 4a shows the results of immunohistochemical staining of O-GlcNAc and GATA4 proteins in the superficial layer of articular cartilage of normal mice aged 20 weeks.

[0076] Fig. 4b is Gata4 fl / fl ;Col2a1-CreER T2 This shows tissue images of articular cartilage stained with Safranin-O, Fast Green, and Hematoxylin after sham or DMM surgery was performed on mice administered vehicle (corn oil) or tamoxifen (TMX).

[0077] Figure 4c shows the results of scoring and quantifying cartilage destruction, subchondral bone sclerosis, osteophyte formation, and synovitis in the mouse of Figure 4b.

[0078] Figure 4d shows the results of measuring the weight-bearing ratio between the surgical side (DMM) leg and the contralateral leg in the same mouse using a static weight-bearing test.

[0079] Fig. 4e is Gata4 fl / fl ;Prg4-CreER T2 This shows the Safranin-O / Fast Green staining results of cartilage tissue that underwent DMM surgery in mice following TMX treatment.

[0080] Figure 4f shows the results of scoring the degree of cartilage destruction, subchondral bone changes, osteophytes, and synovitis in the model of Figure 4e.

[0081] Figure 4g shows Gata4 after sham or DMM surgery. fl / fl ;Prg4-CreER T2This is a cross-sectional image of a mouse knee joint taken with μCT.

[0082] Figure 4h shows the results of immunohistochemical staining of MMP3, MMP13, and IL-6 in the cartilage tissue of cartilage surface-specific Gata4-defective mice.

[0083] Fig. 4i is Gata4 fl / fl ;Prg4-CreER T2 This shows the results of measuring the weight-bearing ratio of the forelimbs on the surgical side and the opposite side in mice.

[0084] Figure 4j shows the results of mechanical allodynia evaluated by von Frey analysis on the leg on the side where surgery was performed in the same mouse.

[0085] Scale bar: Fig. 4a, Fig. 4h, 25 μm, Fig. 4b, 500 μm, Fig. 4e, 200 μm. Figs. 4c, Fig. 4d, Fig. 4f, Fig. 4i, Fig. 4j, data represent mean ± standard error. P values ​​are obtained from the Kruskal-Wallis test with a two-sided Mann-Whitney U test (Fig. 4c), a two-sided t-test (Fig. 4d, Fig. 4i, Fig. 4j), or the Mann-Whitney U test (Fig. 4f).

[0086] Figure 5a shows that after performing DMM surgery on 12-week-old C57BL / 6J wild-type mice, vehicle or thiamet-G was injected into the intra-articular (IA) once a week. Knee joint tissues were stained with safranin O, fast green, and hematoxylin to observe the degree of cartilage damage, and magnified images are presented at the bottom and right.

[0087] Figure 5b quantified the degree of cartilage destruction and synovitis by scoring.

[0088] Figure 5c shows that weight bearing reduction due to joint pain was evaluated using a static weight bearing test, and pain was assessed by calculating the weight bearing ratio between the operated leg (DMM-operated limb) and the opposite leg.

[0089] Figure 5d shows transcriptome analysis performed after treating bleomycin-induced chondrocytes with an OGT inhibitor (ST045849) or a vehicle, and GSEA confirmed that the expression of a gene set ("Upregulated genes in OA") increased in osteoarthritis patients was inhibited.

[0090] Figure 5e shows that ST045849 was loaded onto an ascorbyl palmitate-based hydrogel formulation and administered intra-articularly once a week to 12-week-old wild-type mice after DMM surgery. The joints were stained with safranin O, fast green, and hematoxylin to analyze cartilage lesions.

[0091] Figures 5f, 5g, and 5h show the results of immunohistochemical staining performed on mouse cartilage administered with a vehicle or ST045849 after DMM surgery; Figure 5f shows GATA4 and cartilage matrix degradation products (Type II collagen C-terminal telopeptide (CTX-II), aggrecan NITEGE), Figure 5g shows MMP3, MMP13, IL-6, and Figure 5h shows aging markers (p16 INK4a The expression of HMGB1) and HGF, an inflammation-independent SASP factor, was confirmed.

[0092] Figure 5i quantitatively analyzed cartilage destruction, subchondral bone sclerosis, osteophyte formation, and synovitis by scoring.

[0093] Figure 5j evaluated osteoarthritis pain sensitivity as the weight-bearing ratio of the surgical leg versus the non-surgical leg in a static weight distribution test.

[0094] Figure 5k shows that mechanical hypersensitivity to pain was measured using an electronic von Frey test.

[0095] Scale bar: Fig. 5a, Fig. 5e, 200 μm, Fig. 5f, Fig. 5g, Fig. 5h, 25 μm. Fig. 5b, Fig. 5c, Fig. 5i, Fig. 5j, Fig. 5k, data represent mean ± standard error. P values ​​are obtained from a two-sided Mann-Whitney U test (Fig. 5b, Fig. 5i), a two-sided t-test (Fig. 5c, Fig. 5j, Fig. 5k), or a one-sided permutation test corrected for the family-wise error rate (FWER) for multiple comparisons (Fig. 5d).

[0096] Figure 6a shows the results of Western blot analysis (left) and quantification (right) of the total O-GlcNAcylation level of proteins when cellular senescence was induced in mouse primary cultured chondrocytes by treating them with bleomycin for 24 hours, followed by additional treatment with glucosamine sulfate at various concentrations for 72 hours. It shows that O-GlcNAcylation significantly increases with increasing glucosamine treatment concentration.

[0097] Figure 6b shows the results of confirming the stability of the GATA4 protein by performing a CHX trace assay after expressing FLAG-tagged GATA4 in HEK293T cells and treating them with either glucosamine sulfate (2 mM) or a vehicle. It shows that the degradation of the GATA4 protein is delayed and stability is increased under the glucosamine treatment condition.

[0098] Figure 6c shows the experimental design in which glucosamine sulfate (5 mg / mL) was administered to 12-week-old C57BL / 6J mice starting 7 days before DMM surgery to induce traumatic osteoarthritis.

[0099] Figure 6d compares the change in body weight before and after the administration of glucosamine sulfate in DMM-operated mice, and no significant difference in the change in body weight was observed depending on whether glucosamine was administered.

[0100] Figure 6e shows the results of observing knee joint tissues stained with safranin O, fast green, and hematoxylin after administering or not administering glucosamine sulfate in the control group and the DMM surgery group.

[0101] Figure 6f shows μCT images of changes in subchondral bone depending on oral administration of glucosamine sulfate in mice that underwent sham or DMM surgery.

[0102] Figure 6g shows the results of immunohistochemical analysis of the expression of GATA4, IL-6, MMP13, and ADAMTS5 in cartilage tissue of mice administered glucosamine sulfate after DMM surgery. The expression of these degrading enzymes and inflammatory proteins increased in the glucosamine administration group.

[0103] Figure 6h shows the results of quantifying cartilage destruction and subchondral bone sclerosis by scoring. It shows that glucosamine administration exacerbates DMM-induced osteoarthritis.

[0104] Figure 6i shows the results of mechanical hyperalgesia evaluated by the electronic von Frey test.

[0105] Figure 6j shows the results of measuring the weight-bearing ratio of the operated leg relative to the contralateral leg using a static weight distribution test. Worsening of the pain response was confirmed in the glucosamine administration group.

[0106] Scale bar: Fig. 6e, 200 μm, Fig. 6g, 25 μm. Figs. 6a, 6d, 6h, 6i, and 6j, data represent mean ± standard error. P values ​​are obtained from one-way ANOVA with Dunnett post-hoc test (Fig. 6a), two-way ANOVA with Bonferroni post-hoc test (Fig. 6d), two-way Mann-Whitney U test (Fig. 6h), or two-way t-test (Figs. 6i, 6j).

[0107] Figure 7a shows the results of confirming whether Slc35d1 mRNA was overexpressed 48 hours after transfecting mouse chondrocytes with an adenovirus (Ad-Slc35d1) overexpressing SLC35D1 at 400 MOI (Multiplicity of infection).

[0108] Figure 7b shows images of mouse chondrocytes introduced with various aging models, including doxorubicin, bleomycin, hydrogen peroxide, and oxidative stress long-term culture models (passage 2-3), transfected with an adenovirus overexpressing SLC35D1 (Ad-Slc35d1) or an empty vector adenovirus (Ad-Null) at 400 MOI and stained immunofluorescence using an anti-calreticulin antibody, an endoplasmic reticulum marker. The fragmentation of endoplasmic reticulum in aging chondrocytes was reduced through the overexpression of SLC35D1.

[0109] Figure 7c shows images of mouse chondrocytes introduced with various aging models, including doxorubicin, bleomycin, hydrogen peroxide, and oxidative stress long-term culture models (passage 2-3), transfected with an adenovirus overexpressing SLC35D1 (Ad-Slc35d1) or an empty vector adenovirus (Ad-Null) at 400 MOI and stained immunofluorescence using the anti-GM130 antibody, a Golgi apparatus marker. The fragmentation and irregular distribution of the Golgi apparatus in aging chondrocytes are reduced through the overexpression of SLC35D1, causing the Golgi apparatus to condense and be located close to the nucleus.

[0110] Figure 7d shows the results of a quantitative analysis of mRNA expression levels of major SASP factors (Mmp3, Mmp13, Il6) and Adamts5 when mouse chondrocytes were treated with bleomycin for 24 hours to induce cellular senescence, then transfected with Ad-Slc35d1 at 400 MOI for overexpression and analyzed after 5 days. When SLC35d1 was overexpressed, the expression of various SASPs was statistically significantly reduced.

[0111] Figures 7a and 7d show the mean ± standard error of the data. P values ​​are obtained from a two-sided t-test (Figure 7a) and a one-way analysis of variance with Tukey's post-hoc test (Figure 7d).

[0112] Figure 8 shows the results of comparing the mRNA expression levels of major SASP factors (Mmp3, Mmp13, Il6) and Adamts5 in mouse chondrocytes after inducing cellular senescence by treating them with bleomycin for 24 hours, followed by transfection with Ad-Null or Ad-Slc35d1 at 400 MOI or treatment with the vehicle or GATA4 inhibitor NSC140905 (50 μM) for 5 days. Overexpression of SLC35D1 and treatment with NSC140905 (or NSC) inhibited the expression of SASP and ADAMTS5 genes, suggesting that a synergistic effect occurs in which SASP expression decreases when SLC35D1 is overexpressed together with GATA4 inhibition. These results can be seen from the fact that gene expression was statistically significantly reduced (P < 0.05) when comparing the Bleo+NSC group and the Bleo+Ad-Slc35d1+NSC group.

[0113] Figure 8 shows the data as mean ± standard error. The P-value is obtained from the one-way analysis of variance with Tukey's post-hoc test (Figure 8).

[0114] Figure 9 shows the results of comparing the mRNA expression levels of major SASP factors (Mmp3, Mmp13, Il6) and Adamts5 in mouse chondrocytes after inducing cellular senescence by treating them with bleomycin for 24 hours, followed by transfection with Ad-Null or Ad-Slc35d1 at 400 MOI or treatment with the vehicle or OGT inhibitor ST045849 (10 μM) for 5 days. Overexpression of SLC35D1 and treatment with ST045849 (or ST) inhibited the expression of SASP and ADAMTS5 genes, suggesting a synergistic effect in which SASP expression decreases when SLC35D1 is overexpressed in addition to inhibiting OGT. These results are supported by the statistically significant decrease in gene expression (P < 0.05) when comparing the Bleo+ST group with the Bleo+Ad-Slc35d1+ST group.

[0115] Figure 9 shows the data as mean ± standard error. The P-value is obtained from the one-way analysis of variance with Tukey's post-hoc test (Figure 9).

[0116] Figure 10 shows the results of SA-β-Gal staining performed on chondrocytes irradiated with 5 Gy of ionic radiation (IR) and the results of a quantitative analysis of the proportion of SA-β-Gal-positive cells (left photo and graph). In addition, Figure 10 shows representative images of BrdU immunofluorescence staining performed under the same conditions and the results of quantifying the proportion of BrdU-positive cells (middle photo and graph). Through this, the inhibition of cell proliferation was confirmed. Figure 10 shows the results of quantifying the mRNA expression levels of aging marker genes (CDK inhibitor gene and Lmnb1) in chondrocytes after IR irradiation (right graph).

[0117] Scale bar: Fig. 10 (left), 50 μm, Fig. 10 (center), 25 μm. Fig. 10, data represent mean ± standard error. P values ​​are obtained from a two-sided t-test (Fig. 10).

[0118] Figure 11a shows the results of a quantitative analysis of mRNA expression levels of UDP-GlcNAc and UDP-GalNAc transporters (SLC35 family genes) in chondrocytes irradiated with 5 Gy of ionizing radiation.

[0119] Figure 11b shows the results of a quantitative analysis of the amount of sulfated glycosaminoglycans (sGAG) secreted from chondrocytes irradiated with IR under the same conditions.

[0120] Figures 11a and 11b show the data as mean ± standard error. P-values ​​are obtained from a two-sided t-test (Figures 11a and 11b).

[0121] Figure 12a shows the results of a quantitative analysis of mRNA expression levels of additional SASP factors when mouse chondrocytes were treated with bleomycin for 24 hours to induce cellular senescence, followed by treatment with a vehicle or the O-GlcNAc transferase inhibitor ST045849 (20 μM) for 5 days.

[0122] Figure 12b shows the results of quantitative analysis of mRNA expression levels of representative SASP factors after treating chondrocytes treated with bleomycin for 24 hours with vehicle or the O-GlcNAcase inhibitor thiamet-G (20 μM) for 72 hours.

[0123] Figure 12c shows the results of analyzing changes in the expression of a group of genes associated with the positive regulation of inflammatory cytokine production using the GSEA method on RNA-seq data of chondrocytes treated with vehicle or ST045849 for 5 days after bleomycin treatment.

[0124] Figures 12a and 12b show the mean ± standard error. P values ​​are obtained from a two-way ANOVA with Tukey post-hoc test (Figures 12a and 12b) and a one-sided permutation test corrected for the family-wise error rate (FWER) for multiple comparisons (Figure 12c).

[0125] Figure 13a shows the results of a quantitative analysis of the mRNA expression levels of SASP factors (Mmp10, Ccl2, etc.) when mouse chondrocytes were induced to age by treating them with bleomycin for 24 hours, followed by the introduction of a negative control siRNA (si-NC) or siRNA targeting Gata4, and then treatment with a vehicle or the O-GlcNAc transferase inhibitor ST045849 (20 μM) for 5 days. SASP expression was significantly reduced by the inhibition of GATA4 expression or O-GlcNAc, and no additional reduction in SASP by O-GlcNAc was observed after the inhibition of GATA4 expression.

[0126] Figure 13b shows the results of ETD-MS analysis of purified GATA4 after overexpressing the GATA4 protein in HEK293T cells. The analysis confirmed O-GlcNAc modification at the S212 (199-215 amino acid sequence) and S406 (405-423 amino acid sequence) regions of the GATA4 protein. This suggests that the GATA4 protein is modified by O-GlcNAc, and that this modification is involved in functional stability and SASP regulation.

[0127] Figure 13a, the data represent the mean ± standard error. The P-value is obtained from the two-way ANOVA with Tukey's post-hoc test (Figure 13a).

[0128] Fig. 14a is Gata4 fl / fl ;Prg4-CreER T2This presents the results of immunohistochemical analysis of cartilage tissue in which osteoarthritis was induced via DMM surgery after conditional removal of Gata4 from the surface layer of cartilage by administering tamoxifen (TMX) or a vehicle to transgenic mice. The aging marker p16 INK4a The expression of HMGB1 and HGF, an inflammation-independent SASP factor, was confirmed, and the expression of these proteins decreased when Gata4 was removed by TMX.

[0129] Figure 14b shows the results of a quantitative analysis of mRNA expression levels of Gata4 and major SASP factors when cellular senescence was induced in mouse chondrocytes by treating them with bleomycin for 24 hours, followed by the introduction of negative control siRNA (si-NC) or Gata4-specific siRNA to inhibit expression. When Gata4 was inhibited, the expression of various SASPs was significantly reduced.

[0130] Figure 14c shows the results of comparing the expression levels of the SASP factor in chondrocytes after bleomycin treatment, followed by treatment with the vehicle or the GATA4 inhibitor NSC140905 (50 μM) for 72 hours. SASP gene expression was inhibited upon drug treatment, suggesting that GATA4 activity is essential for SASP expression.

[0131] Scale bar: Fig. 14a, 25 μm. Figs. 14b, Fig. 14c, data represent mean ± standard error. P values ​​are obtained from two-way ANOVA with Tukey post-hoc test (Figs. 14b, Fig. 14c).

[0132] Figure 15a shows the results of GSEA on a set of genes upregulated in osteoarthritis patients, based on RNA-seq data obtained when mouse chondrocytes were induced to age by treating them with bleomycin for 24 hours, followed by treatment with the vehicle or the O-GlcNAc transferase inhibitor ST045849 for 5 days. In the ST045849 treatment group, the expression of the corresponding gene set was significantly enriched in a negative direction, demonstrating that OGT inhibition suppresses the pathological transcription program of senescent chondrocytes.

[0133] Figure 15b shows the results of Ingenuity Pathway Analysis (IPA) performed on genes whose expression was significantly reduced by OGT inhibition in chondrocytes aged with bleomycin. Based on Benjamini-Hochberg-corrected P-values ​​and activation z-scores, the top 20 canonical pathways are presented, and among them, it was confirmed that the "osteoarthritis signaling pathway" was significantly inhibited.

[0134] Figure 15c shows the results of immunohistochemical analysis of O-GlcNAc expression in cartilage tissue after administering drinking water containing glucosamine sulfate to normal mice for 8 weeks. The level of O-GlcNAc expression in chondrocytes increased in the glucosamine intake group.

[0135] Figure 15d shows the results of immunohistochemical analysis of the expression of cartilage matrix degradation markers (type II collagen degradation product CTX-II and agrican degradation product NITEGE) depending on oral administration of glucosamine sulfate in mice with osteoarthritis induced by DMM surgery. Cartilage matrix damage increased in the glucosamine administration group.

[0136] Scale bar: Fig. 15c, Fig. 15d, 25 μm. Fig. 14b, Fig. 14c, data represent mean ± standard error. The P value is obtained from a one-sided permutation test (Fig. 15a) corrected for the family-wise error rate (FWER) for multiple comparisons.

[0137] Figure 16 is a schematic diagram showing the differences in mechanisms within chondrocytes between a healthy control group and a patient with osteoarthritis, as well as the mechanism of osteoarthritis progression.

[0138]

[0139] The various embodiments of this document and the terms used therein are not intended to limit the technical features described in this document to specific embodiments, and should be understood to include various modifications, equivalents, or substitutions of said embodiments.

[0140]

[0141] Definition of Terms

[0142] All scientific and technical terms used in this specification have the same meaning as commonly understood by those skilled in the art to which this invention pertains.

[0143]

[0144] In this specification, the term "solute carrier family 35 (SLC35)" is a gene family comprising a plurality of genes. Each gene included in SLC35 may code for a protein located on the membrane of the endoplasmic reticulum (ER) or the Golgi apparatus. Proteins coded from genes belonging to SLC35 may serve to transport nucleotide sugars synthesized in the cytoplasm into the lumen of the endoplasmic reticulum or the Golgi apparatus. SLC35 is the SLC35A2 gene (Uniprot ID P78381 (human SLC35A2), Q9R0M8 (mouse SLC35A2)), SLC35A3 gene (Uniprot ID Q9Y2D2 (human SLC35A3), Q8R1T4 (mouse SLC35A3)), SLC35A5 gene (Uniprot ID Q9BS91 (human SLC35A5), Q921R7 (mouse SLC35A5)), SLC35B4 gene (Uniprot ID Q969S0 (human SLC35B4), Q8CIA5 (mouse SLC35B4)), SLC35D1 gene (Uniprot ID Q9NTN3 (human SLC35D1), A2AKQ0 (mouse SLC35D1)) or SLC35D2 gene (Uniprot ID Q76EJ3 (human Includes, but is not limited to, SLC35D2), Q762D5 (mouse SLC35D2). At least one of the genes belonging to SLC35 may code for a protein that transports UDP-GlcNAc (uridine diphosphate N-acetylglucosamine) and / or UDP-GalNAc (uridine diphosphate N-acetylgalactosamine) from the cytoplasm to the endoplasmic reticulum or Golgi apparatus.

[0145] In this specification, the term "uridine diphosphate N-acetylglucosamine (UDP-GlcNAc)" is the end product of the hexosamine biosynthetic pathway, a substrate for the synthesis of glycosaminoglycan (GAG) chains of glycoproteins and proteoglycans, and simultaneously acts as a substrate for the O-GlcNAcylation of proteins.

[0146] In this specification, the term “O-GlcNAcylation” refers to a post-translational modification in which O-GlcNAc transferase (OGT) adds N-acetylglucosamine (GlcNAc) from UDP-GlcNAc to a serine or threonine residue of a target protein. This modification can be reversibly removed by O-GlcNAcase (OGA).

[0147] In this specification, the term "osteoarthritis (OA)" refers to a degenerative joint disease characterized by the degeneration of articular cartilage, sclerosis of the subchondral bone, formation of osteophytes, and inflammation of the synovium. Risk factors such as aging, obesity, and mechanical overload contribute to the development and progression of osteoarthritis.

[0148] In this specification, the term "Senescence-associated secretory phenotype (SASP)" includes inflammatory cytokines, matrix-degrading enzymes, growth factors, etc., secreted by senescent cells. SASPs of chondrocytes play an important role in the onset and progression of osteoarthritis.

[0149] In this specification, the term "GATA4 (Uniprot ID P43694 (human GATA4), Q08369 (mouse GATA4))" refers to a stress-induced transcription factor that regulates SASP expression. In the absence of aging stress, it is degraded through selective autophagy, but under stress conditions, it is stabilized and can increase the expression of the SASP gene.

[0150] In this specification, the term "post-traumatic osteoarthritis" refers to osteoarthritis that occurs after joint injury or surgical procedures (e.g., destabilization of the medial meniscus, DMM).

[0151] In this specification, "salt" means a salt according to one aspect of this specification that is acceptable in medicines, cosmetics, and foods and has desirable activity of a parent compound. For example, the salt is an inorganic acid such as hydrochloric acid, hydrobromide, sulfuric acid, nitric acid, phosphoric acid, etc.; Acetic acid, propionic acid, hexanoic acid, cyclopentanepropionic acid, glycolic acid, pyruvate, lactic acid, malonic acid, succinic acid, malic acid, maleic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, 3-(4-hydroxybenzoyl)benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, 1,2-ethane-disulfonic acid, 2-hydroxyethanesulfonic acid, benzenesulfonic acid, 4-chlorobenzenesulfonic acid, 2-naphthalenesulfonic acid, 4-toluenesulfonic acid, camphorsulfonic acid, 4-methylbicyclo[2,2,2]oct-2-en-1-carboxylic acid, glucoheptonic acid, 3-phenylpropionic acid, trimethylacetic acid, tert-butylacetic acid, Acid addition salt formed from organic acids such as lauryl sulfate, gluconic acid, glutamic acid, hydroxynaphthoic acid, salicylic acid, stearic acid, and muconic acid; or may include, but is not limited to, a salt formed when an acidic proton present in the parent compound is substituted. Additionally, the salt may be a pharmaceutically acceptable salt.

[0152] In this specification, “pharmaceuticalally acceptable” means that it does not exhibit significant toxicity when used at a normal medicinal dosage and is therefore eligible for or approved by a government or equivalent regulatory body for use in animals, specifically humans, or is listed in a pharmacopoeia or recognized by other general pharmacopoeias.

[0153] In this specification, "hydrate" refers to a compound to which water is bound, and is a broad concept that includes inclusion compounds in which there is no chemical bonding force between the water and the compound.

[0154] In this specification, "solvent" refers to a crystalline form containing stoichiometric or non-stoichiometric amounts of solvent, for example, a higher-order compound formed between solute molecules or ions and solvent molecules in a solution. A solvent in which the solvent is water may be commonly referred to as a hydrate.

[0155] In this specification, the term "treatment" refers to any act of improving or beneficially altering the symptoms of osteoarthritis or diseases caused thereby through the administration of a composition according to the present invention. A person skilled in the art to which this invention pertains would be able to determine the precise criteria for the disease and assess the degree of improvement, enhancement, and treatment by referring to materials provided by organizations such as the Korean Medical Association.

[0156] In this specification, the term “inhibitor” may refer to a substance that reduces a target substance and may be used interchangeably with “inhibitor.”

[0157]

[0158] Exemplary implementation examples

[0159] Composition for the prevention or treatment of osteoarthritis

[0160] One aspect of the present specification provides a composition for the prevention or treatment of osteoarthritis, comprising: a preparation that increases the expression or activity of a gene belonging to solute carrier family 35 (SLC35); a protein encoded by said gene; or an inhibitor that inhibits the accumulation of uridine diphosphate N-acetylglucosamine (UDP-GlcNAc) in the cytoplasm of chondrocytes.

[0161] The inventors confirmed that the expression of transporters belonging to the SLC35 family is significantly reduced in the cartilage tissues of osteoarthritis patients and in various animal models of osteoarthritis. Furthermore, they confirmed that injecting agents that increase the expression or activity of genes belonging to the SLC35 family can produce a therapeutic effect on osteoarthritis. In addition, they discovered that the expression of SLC35A3, SLC35B4, SLC35D1, and SLC35D2 is consistently reduced in chondrocytes in which cellular senescence was induced using DNA damage inducers such as doxorubicin or bleomycin. This reduction in the expression of SLC35 transporters leads to the abnormal accumulation of UDP-GlcNAc in the cytoplasm and reduces UDP-GlcNAc within the endoplasmic reticulum (ER) and Golgi apparatus. UDP-GlcNAc accumulated in the cytoplasm acts as a substrate for O-GlcNAc transferase (OGT), inducing excessive O-GlcNAc conversion, which increases the cellular senescence-associated secretory phenotype (SASP) through the stabilization of GATA4, thereby exacerbating osteoarthritis (Fig. 16).

[0162] The inventors have identified the above mechanism and confirmed that by supplementing a preparation that increases the expression or activity of the SLC35 gene or a protein encoded by said gene, the abnormal accumulation of UDP-GlcNAc in the cytoplasm can be improved, or the excessive accumulation of UDP-GlcNAc in the cytoplasm can be directly inhibited, thereby inhibiting the O-GlcNAcification of GATA4 and preventing or treating osteoarthritis.

[0163] In one embodiment, the inventors confirmed that SLC35 activation targeting an upstream mechanism exhibits a superior inhibitory effect compared to cases where downstream mechanisms (GATA4 inhibition, OGT inhibition) are targeted alone. Specifically, measures to increase the expression or enhance the function of SLC35D1 more broadly and potently inhibited the expression of inflammatory SASP genes such as MMP3, MMP13, and IL6, as well as chondrolytic enzymes such as ADAMTS5, compared to treatment with GATA4 inhibitors or OGT inhibitors alone. This is achieved by normalizing the intracellular distribution of UDP-GlcNAc in the upstream regulatory mechanism, thereby simultaneously blocking downstream O-GlcNAcylation abnormalities and the GATA4 stabilization pathway, resulting in an unexpected synergistic effect through integrated inhibition at the metabolic and transcriptional levels. Therefore, the upstream pathway targeting strategy of the present invention overcomes the limitations of downstream mechanism inhibitors and possesses advanced potential as a comprehensive and fundamental therapeutic approach for the pathogenesis of osteoarthritis.

[0164] In one embodiment, the gene belonging to SLC35 may be at least one selected from the group consisting of SLC35A2 gene, SLC35A3 gene, SLC35A5 gene, SLC35B4 gene, SLC35D1 gene and SLC35D2 gene, but is not limited thereto.

[0165] In one embodiment, genes belonging to SLC35 may have the function of transporting UDP-GlcNAc and / or UDP-GalNAc from the cytoplasm to the endoplasmic reticulum or Golgi apparatus, and may be widely conserved in mammals. For example, SLC35A2 may transport UDP-GalNAc and UDP-Gal (UDP-galactose). SLC35A3 may transport UDP-GlcNAc. SLC35A5 may transport UDP-GlcNAc, UDP-GalNAc, and UDP-GlcA (UDP-glucuronic acid). SLC35B4 may transport UDP-GlcNAc, UDP-GlcA, and UDP-Xyl (UDP-xylose). SLC35D1 may transport UDP-GlcNAc, UDP-GalNAc, and UDP-GlcA. SLC35D2 can transport UDP-GlcNAc and UDP-Glc (UDP-glucose).

[0166] In one embodiment, it was confirmed that the expression of genes belonging to SLC35 (SLC35A2, SLC35A3, SLC35A5, SLC35B4, SLC35D1, SLC35D2) was consistently reduced in osteoarthritis cartilage tissue and senescent chondrocytes, and that this reduced expression was correlated with the accumulation of cytoplasmic UDP-GlcNAc and the reduction of endoplasmic reticulum / Golgi apparatus UDP-GlcNAc.

[0167] In one embodiment, the agent for increasing the expression or activity of a gene belonging to SLC35 is an agonist small molecule compound, a vector (e.g., a vector containing a gene belonging to SLC35, a vector overexpressing a gene belonging to SLC35), a plasmid (e.g., a plasmid containing a gene belonging to SLC35, a plasmid overexpressing a gene belonging to SLC35), a viral vector (e.g., a viral vector containing a gene belonging to SLC35, a viral vector overexpressing a gene belonging to SLC35), an adenovirus vector (e.g., an adenovirus vector containing a gene belonging to SLC35, an adenovirus vector overexpressing a gene belonging to SLC35), an adeno-associated virus vector (e.g., an adeno-associated virus vector containing a gene belonging to SLC35, an adeno-associated virus vector overexpressing a gene belonging to SLC35), or a lentivirus vector (e.g., a gene belonging to SLC35 It may be at least one selected from the group consisting of a lentiviral vector containing, a lentiviral vector overexpressing a gene belonging to SLC35), mRNA (e.g., mRNA of a gene belonging to SLC35), mRNA wrapped in lipid nanoparticles (LNP; lipid nanoparticle) (e.g., mRNA of a gene belonging to SLC35 wrapped in LNP), liposomes, and nanoparticles, but is not limited thereto. In one embodiment, the agent that increases the expression or activity of a gene belonging to SLC35 may be an adenoviral vector overexpressing a gene belonging to SLC35.

[0168] A preparation that increases the expression or activity of a gene belonging to SLC35 is sufficient if it is a substance that increases the expression level of the gene mRNA or DNA belonging to SLC35 or the activity level of the protein within a subject (e.g., the subject's cells) when the preparation is administered compared to when it is not administered, and the type thereof is not limited. For example, a preparation that increases the expression or activity of a gene belonging to SLC35 may be a substance that increases the expression of a gene belonging to SLC35 that is already present in the body of the subject, a substance containing an foreign gene belonging to SLC35 that was not present in the body of the subject, or a foreign substance that overexpresses a gene belonging to SLC35 that was not present in the body of the subject.

[0169] In one embodiment, a protein encoded by a gene belonging to SLC35 can transport uridine diphosphate N-acetylglucosamine from the cytoplasm of chondrocytes to the Golgi apparatus or endoplasmic reticulum. This can inhibit the abnormal accumulation of UDP-GlcNAc in the cytoplasm and normalize proteoglycan synthesis and other glycoprotein synthesis in the endoplasmic reticulum and Golgi apparatus.

[0170] In one embodiment, a gene, preparation, or protein may be delivered to chondrocytes or cartilage tissue through various delivery means known in the art, such as vectors, plasmids, viral vectors, liposomes, and nanoparticles. For example, a gene may be delivered using an adenoviral vector, an adeno-associated viral vector (AAV), a lentiviral vector, mRNA, lipid nanoparticles (LNP), etc.

[0171] In one embodiment, the inhibitor that inhibits the accumulation of UDP-GlcNAc in the cytoplasm of chondrocytes is sufficient if it is a substance that reduces the degree of accumulation of UDP-GlcNAc in the cytoplasm of chondrocytes, and its form or type is not limited.

[0172] In one embodiment, the inhibitor that inhibits the accumulation of UDP-GlcNAc in the cytoplasm of chondrocytes may be a substance that moves UDP-GlcNAc located in the cytoplasm of chondrocytes to the endoplasmic reticulum and / or Golgi apparatus.

[0173] In one embodiment, the inhibitor that inhibits the accumulation of UDP-GlcNAc in the cytoplasm of chondrocytes may be a agent that inhibits the hexosamine biosynthetic pathway, but is not limited thereto.

[0174] In one embodiment, the inhibitor that inhibits the accumulation of UDP-GlcNAc in the cytoplasm of chondrocytes may be, but is not limited to, an inhibitor of glutamine-fructose-6-phosphate aminotransferase (GFPT). In one embodiment, inhibiting GFPT may reduce the overall production of UDP-GlcNAc. For example, the GFPT inhibitor may be 6-diazo-5-oxo-L-norleucine (DON). 6-diazo-5-oxo-L-norleucine may be a compound represented by Chemical Formula 3 below:

[0175] [Chemical Formula 3]

[0176] .

[0177] In one embodiment, the inhibitor that inhibits the accumulation of UDP-GlcNAc in the cytoplasm of chondrocytes may be [(3aR,5R,6S,7R,7aR)-6,7-diacetyloxy-2-methyl-5,6,7,7a-tetrahydro-3aH-pyrano[3,2-d][1,3]oxazol-5-yl]methyl acetate ([(3aR,5R,6S,7R,7aR)-6,7-diacetyloxy-2-methyl-5,6,7,7a-tetrahydro-3aH-pyrano[3,2-d][1,3]oxazol-5-yl]methyl acetate). [(3aR,5R,6S,7R,7aR)-6,7-diacetyloxy-2-methyl-5,6,7,7a-tetrahydro-3aH-pyrano[3,2-d][1,3]oxazole-5-yl]methyl acetate may be a compound represented by the chemical formula 4 below:

[0178] [Chemical Formula 4]

[0179]

[0180] In one embodiment, the composition may be administered in combination with at least one of a GATA4 inhibitor or an O-linked-N-acetylglucosamine transferase inhibitor. In one embodiment, the composition may be administered in combination with at least one of a GATA4 inhibitor or an O-linked-N-acetylglucosamine transferase inhibitor simultaneously, separately, or sequentially.

[0181] The inventors have identified a pathological pathway in which the accumulation of cytoplasmic UDP-GlcNAc induces excessive O-GlcNAc conversion via OGT, which stabilizes GATA4 and increases SASP. According to one embodiment, when a GATA4 inhibitor or an OGT inhibitor is co-administered with an SLC35 gene, agent, protein, or UDP-GlcNAc accumulation inhibitor, a synergistic therapeutic effect can be obtained through the dual effect of inhibiting cytoplasmic UDP-GlcNAc accumulation and blocking downstream signaling.

[0182] In one embodiment, the GATA4 inhibitor may be 2-(1,3-benzodioxol-5-ylmethyl)butanedioic acid (NSC140905), a salt thereof, a hydrate thereof, or a solvate thereof. The NSC140905 may be a compound represented by the following chemical formula 1:

[0183] [Chemical Formula 1]

[0184] .

[0185] In another embodiment, the GATA4 inhibitor may be an siRNA that inhibits GATA4 gene expression. For example, the GATA4 inhibitor may be a Gata4siRNA composed of the nucleotide sequences of SEQ ID NO. 1 (5'-GAGCAAACCAGAGCCUAGAdTdT-3', Sense strand) and SEQ ID NO. 2 (5′-UCUAGGCUCUGGUUUGCUCdTdT-3', Antisense strand); or a Gata4siRNA composed of the nucleotide sequences of SEQ ID NO. 3 (5′-CAGAUGUUACUGAAUGCUUdTdT-3′, Sense strand) and SEQ ID NO. 4 (5′-AAGCAUUCAGUAACAUCUGdTdT-3′, Antisense strand).

[0186] In this specification, dT included in the nucleotide sequences (e.g., sequence numbers 1 to 4, sequence numbers 5 to 74 of Table 2) is 2'-deoxythymidine, A is adenine, C is cytosine, G is guanine, U is uracil, and T is thymine.

[0187] OGT inhibitors may be substances that inhibit O-acetylglucosamine (O-GlcNAcylation) of the GATA4 protein in the chondrocyte cytoplasm. OGT inhibitors may be substances that inhibit O-glucosamine (O-GlcNAcylation) at the S406 position of the GATA4 protein in the chondrocyte cytoplasm.

[0188] In one embodiment, the OGT inhibitor may be ST045849, its salt, its hydrate, or its solvate. The ST045849 may be a compound represented by the following chemical formula 2:

[0189] [Chemical Formula 2]

[0190] .

[0191] The IUPAC name of ST045849 is 3-[2-(1-adamantyl)ethyl]-2-(4-chlorophenyl)imino-4-oxo-1,3-thiazinane-6-carboxylic acid.

[0192] In one embodiment, the OGT inhibitor may be administered into the joint via a drug delivery system. For example, a hydrogel-based drug delivery system may be used, and ST045849 may be loaded onto an injectable hydrogel prepared, for example, of 6-O-palmitoyl-L-ascorbic acid (ascorbyl palmitate) and injected into the joint. By using a hydrogel-based drug delivery system, controlled release of the OGT inhibitor in the joint may be enabled.

[0193] In one embodiment, osteoarthritis is a disease that is clearly distinct from cartilage developmental disorders. Specifically, osteoarthritis is a degenerative disease in which articular cartilage, normally formed at birth, gradually degenerates due to acquired factors such as aging, trauma, mechanical overload, and inflammation. On the other hand, cartilage developmental disorders are a group of congenital or genetic diseases that occur primarily due to abnormalities in the differentiation, proliferation, or synthesis of cartilage matrix of chondrocytes during the process of growth and skeletal formation. Representative examples include achondroplasia, chondrodysplasia, and epiphyseal dysplasia. These diseases occur during the development and growth period due to abnormalities in chondrocyte proliferation in the cartilage growth plate, which is involved in bone lengthening and joint formation; mutations in cartilage matrix proteins such as collagen type II or agrican; or genetic abnormalities such as cartilage-specific glycosyl transporters and sulfonylases. Therefore, chondrogenetic disorders are classified as developmental defects characterized by the failure of tissue formation or structural abnormality, and are clearly pathophysiologically distinguished from osteoarthritis, a degenerative disease in which functional cartilage tissue is destroyed. Furthermore, chondrogenetic disorders involve uneven growth of the trunk and limbs, joint deformities, and cartilage malformations starting from childhood, whereas osteoarthritis develops primarily in adulthood due to the accumulation of cartilage damage, aging, inflammation, and tissue degeneration; thus, they differ in terms of onset, causes, and treatment approaches.

[0194]

[0195] Another aspect of the present specification provides a composition for the prevention or treatment of osteoarthritis comprising a GATA4 inhibitor; or an O-linked-N-acetylglucosaminylation transferase (O-GlcNAc transferase, OGT) inhibitor. Treatment with an OGT inhibitor can inhibit the O-GlcNAc conversion of GATA4 and may alleviate the progression of osteoarthritis. Treatment with a GATA4 inhibitor can alleviate the progression of osteoarthritis.

[0196] As osteoarthritis, prevention, treatment, composition, etc. have been described above, detailed information is omitted.

[0197] In one embodiment, the GATA4 inhibitor may be 2-(1,3-benzodioxol-5-ylmethyl)butanedioic acid (NSC140905), a salt thereof, a hydrate thereof, or a solvate thereof. The NSC140905 may be a compound represented by the following chemical formula 1:

[0198] [Chemical Formula 1]

[0199] .

[0200] In another embodiment, the GATA4 inhibitor may be an siRNA that inhibits Gata4 gene expression. For example, the Gata4 inhibitor may be a Gata4siRNA composed of the nucleotide sequences of SEQ ID NO. 1 (5'-GAGCAAACCAGAGCCUAGAdTdT-3', Sense strand) and SEQ ID NO. 2 (5′-UCUAGGCUCUGGUUUGCUCdTdT-3', Antisense strand); or a Gata4siRNA composed of the nucleotide sequences of SEQ ID NO. 3 (5′-CAGAUGUUACUGAAUGCUUdTdT-3′, Sense strand) and SEQ ID NO. 4 (5′-AAGCAUUCAGUAACAUCUGdTdT-3′, Antisense strand).

[0201] In one embodiment, the OGT inhibitor may be ST045849, its salt, its hydrate, or its solvate. The ST045849 may be a compound represented by the following chemical formula 2:

[0202] [Chemical Formula 2]

[0203] .

[0204] In one embodiment, when NSC140905 was applied to chondrocytes aged with bleomycin, the expression of various SASP factors such as Mmp3, Il6, Ccl2, Cxcl15, Mmp10, Vegfa, Igfbp7, Hgf, and Timp2 was significantly reduced.

[0205] In one example, when GATA4 was knocked down using a specific Gata4siRNA, it was confirmed that the expression of SASP factors such as MMP3 and IL-6 was significantly reduced in bleomycin-induced senescent chondrocytes.

[0206] Numerous repeated experiments are required to identify specific substances that exhibit therapeutic, preventive, and / or mitigating effects on osteoarthritis at the animal level, and the inventors have confirmed through numerous repeated experiments that the substances described herein exhibit therapeutic effects on osteoarthritis.

[0207]

[0208] In one embodiment, osteoarthritis may be post-traumatic osteoarthritis, inflammatory osteoarthritis, or degenerative osteoarthritis, but is not limited thereto.

[0209] For example, post-traumatic osteoarthritis may be osteoarthritis that occurs after mechanical trauma, such as joint injury, ligament rupture, meniscus injury, or surgical procedures. Inflammatory osteoarthritis may be osteoarthritis that occurs secondarily due to inflammatory diseases such as rheumatoid arthritis or psoriatic arthritis. Degenerative osteoarthritis may be a common form of osteoarthritis that develops gradually due to aging, obesity, repetitive mechanical loads, etc.

[0210] In one embodiment, the composition for preventing or treating osteoarthritis may be a pharmaceutical composition or a food composition, but is not limited thereto.

[0211] In one embodiment, the composition for the prevention or treatment of osteoarthritis may further include a pharmaceutically acceptable carrier, diluent, excipient, stabilizer, buffer, isotonic agent, etc. Additionally, it may be used in the form of oral formulations such as injections, powders, granules, tablets, capsules, suspensions, emulsions, syrups, and aerosols, topical preparations, suppositories, and sterile injectable solutions according to conventional methods.

[0212] In one embodiment, a composition for the prevention or treatment of osteoarthritis may be administered in a pharmaceutically effective amount. In one aspect of this specification, "pharmaceutically effective amount" means an amount sufficient to treat a disease with a reasonable benefit / risk ratio applicable to medical treatment, and the effective dose level may be determined based on factors including the type and severity of the patient's disease, the activity of the drug, sensitivity to the drug, the time of administration, the route of administration and elimination rate, the duration of treatment, drugs used concurrently, and other factors well known in the medical field.

[0213] In one embodiment, a composition for preventing or treating osteoarthritis may be administered by various routes. For example, it may be administered by intra-articular injection, oral administration, intranasal administration, transbronchial administration, arterial injection, intravenous injection, subcutaneous injection, intramuscular injection, or intraperitoneal injection.

[0214] In one embodiment, a composition for the prevention or treatment of osteoarthritis may be prepared in an injectable formulation. Specifically, it may be prepared as an intra-articular injection (IA injection) formulation and administered directly to a joint affected by osteoarthritis. Intra-articular injection allows for the local delivery of a high concentration of the drug, thereby maximizing the therapeutic effect while minimizing systemic side effects.

[0215] In one embodiment, a composition for preventing or treating osteoarthritis can reduce the expression of matrix metalloproteinase 3 (MMP3), matrix metalloproteinase 13 (MMP13), and interleukin-6 (IL-6) in chondrocytes.

[0216]

[0217] In addition, one aspect of the present specification provides a method for preventing or treating osteoarthritis, comprising the step of administering the aforementioned composition to an individual.

[0218] In one embodiment, the object may be a subject requiring treatment for a disease.

[0219] In one embodiment, the individual may include, but is not limited to, mammals. The mammal may be, but is not limited to, humans, mice, cattle, dogs, rabbits, and cats.

[0220] In one embodiment, a method for preventing or treating osteoarthritis according to one aspect of the present specification may further include the step of administering at least one of a GATA4 inhibitor or an O-linked-N-acetylglucosaminylation transferase (O-GlcNAc transferase, OGT) inhibitor to the individual.

[0221] In one embodiment, at least one of the GATA4 inhibitor; or the O-linked-N-acetylglucosaminylation transferase (O-GlcNAc transferase, OGT) inhibitor may be administered simultaneously, separately, or sequentially with the aforementioned composition, and may be administered as a single or multiple doses.

[0222]

[0223] Additionally, one aspect of the present specification provides a use for preparing a composition for the prevention or treatment of osteoarthritis comprising: a preparation that increases the expression or activity of a gene belonging to the aforementioned solute carrier family 35 (SLC35); a protein encoded by said gene; or an inhibitor that inhibits the accumulation of uridine diphosphate N-acetylglucosamine (UDP-GlcNAc) in the cytoplasm of chondrocytes.

[0224] Additionally, one aspect of the present specification provides a use for preparing a composition for the prevention or treatment of osteoarthritis of a GATA4 inhibitor; or an OGT inhibitor.

[0225] Additionally, one aspect of the present specification provides a use for preparing a composition for the prevention or treatment of osteoarthritis of at least one GATA4 inhibitor selected from the group consisting of the aforementioned NSC140905 ([Formula 1], IUPAC name: 2-(1,3-Benzodioxol-5-ylmethyl)butanedioic acid)), its salt, its hydrate, or its solvate; or Gata4siRNA consisting of the nucleotide sequence of SEQ ID NO. 1 (5′-GAGCAAACCAGAGCCUAGAdTdT-3′) and SEQ ID NO. 2 (5′-UCUAGGCUCUGGUUUGCUCdTdT-3').

[0226]

[0227] Composition for diagnosing or predicting the prognosis of osteoarthritis

[0228] Another aspect of the present specification provides a composition for diagnosing or predicting the prognosis of osteoarthritis, comprising: a preparation for measuring the expression level of a gene belonging to the solute transporter family 35 (SLC35); a preparation for measuring the level of a protein encoded by said gene; or a preparation for measuring the level of uridine diphosphate N-acetylglucosamine.

[0229] As SLC35, genes belonging to SLC35, proteins encoded by genes belonging to SLC35, uridine diphosphate N-acetylglucosamine, osteoarthritis, etc. have been previously mentioned, a detailed explanation is omitted.

[0230] In one embodiment, the inventors discovered that the expression of genes belonging to SLC35 is consistently reduced in the cartilage tissue of osteoarthritis patients and in various osteoarthritis animal models, and that this is correlated with cytoplasmic UDP-GlcNAc accumulation and increased O-GlcNAc formation. In addition, they confirmed that excessive glucosamine supplementation in a post-traumatic osteoarthritis mouse model promotes UDP-GlcNAc production, thereby exacerbating osteoarthritis.

[0231] In this specification, the term "prognosis" means forecasting the future medical outcome of a patient's disease (e.g., overall survival rate, recurrence-free survival rate, degree of disease progression, etc.), and includes a positive prognosis including disease remission (e.g., recovery from disease, etc.), degree of improvement (e.g., regression of tumor, etc.), or disease stabilization, and includes a negative prognosis including disease recurrence, degree of disease progression, or lethality (e.g., tumor growth, metastasis, drug resistance, etc.).

[0232] In the present invention, "prediction" means to estimate and predict the future medical outcome of a patient's disease, and includes predictions regarding the course of the patient's disease (e.g., remission, degree of improvement, stabilization, recurrence, degree of progression, lethality, deterioration, etc.).

[0233] In one embodiment, the gene may be at least one selected from the group consisting of the SLC35A2 gene, SLC35A3 gene, SLC35A5 gene, SLC35B4 gene, SLC35D1 gene, and SLC35D2 gene.

[0234] In one embodiment, the expression level of the gene may be the mRNA or DNA expression amount of the gene. In one embodiment, the level of the protein may be the mass and / or concentration of the protein. In one embodiment, the level of uridine diphosphate N-acetylglucosamine may be the mass and / or concentration of uridine diphosphate N-acetylglucosamine.

[0235] In one embodiment, a composition for diagnosis or prognosis prediction is applied to chondrocytes isolated from a subject to measure i) the expression level of a gene belonging to SLC35 in the chondrocytes or the level of a protein encoded by said gene; or ii) the level of uridine diphosphate N-acetylglucosamine in the cytoplasm of said chondrocytes. In the case of ii), the composition for diagnosis or prognosis prediction is applied to the cytoplasm of chondrocytes isolated from a subject to measure the level of uridine diphosphate N-acetylglucosamine in the cytoplasm of said chondrocytes.

[0236] Specifically, chondrocytes can be isolated from a subject through a cartilage tissue biopsy, or chondrocytes released from joint fluid can be collected. By measuring the expression level of genes belonging to SLC35, the level of the protein encoded by said genes, or the level of UDP-GlcNAc accumulated in the cytoplasm of these isolated chondrocytes, and comparing them to a normal control group, the presence, degree of progression, or prognosis of osteoarthritis can be determined. For example, if the expression of genes belonging to SLC35 is significantly reduced compared to a normal control group, or if the level of UDP-GlcNAc accumulated in the cytoplasm is increased compared to a normal control group, it can be diagnosed that osteoarthritis has developed, is progressing, or has a poor prognosis.

[0237] In one embodiment, the agent for measuring the expression level of the gene belonging to the SLC35 may be a primer pair, a probe, an antisense oligonucleotide, a CRISPR guide RNA, or an aptamer that specifically binds to the mRNA of the gene.

[0238] In an exemplary embodiment, the expression level of a gene may be measured according to methods commonly used in the field. For example, the expression level of a gene may be measured by methods such as reverse transcriptase polymerase chain reaction (RT-PCR), competitive reverse transcriptase polymerase chain reaction (Competitive RT-PCR), real-time reverse transcriptase polymerase chain reaction (Real-time RT-PCR), fluorescence in situ hybridization (FISH), RNase protection assay (RPA), Northern blotting, microarray, RNA sequencing (RNA-seq), or gene chip, but is not limited thereto.

[0239] For a specific example, primer pairs for quantitative reverse transcription-polymerase chain reaction (qRT-PCR) can be used to measure mRNA expression levels. For example, primer pairs capable of specifically amplifying the mRNA of each of the SLC35A2, SLC35A3, SLC35A5, SLC35B4, SLC35D1, or SLC35D2 genes may be used. For instance, the sequences of the primer pairs for each of the above genes may be those listed in Table 2 below.

[0240] In one embodiment, the agent for measuring the level of the protein encoded by the gene may be an antibody, aptamer, protein-binding peptide, protein-binding ligand, nanobody, antibody fragment (Fab, scFv, etc.) or a functional derivative thereof that specifically binds to the protein.

[0241] In an exemplary embodiment, the level of protein may be measured by methods such as Western blot, protein microarray (protein chip), ELISA (Enzyme Linked Immunosorbent Assay), 2-dimensional electrophoresis, Immunohistochemistry (IHC), immunofluorescence, Co-Immunoprecipitation assay, FACS (Fluorescence activated cell sorter), Radioimmunoassay (RIA), Radioimmunodiffusion, and MALDI-TOF analysis (Matrix Assisted Laser Desorption / Ionization Time of Flight Mass Spectrometry), but is not limited thereto.

[0242] For example, to measure protein levels, antibodies that specifically bind to each of the SLC35A2, SLC35A3, SLC35A5, SLC35B4, SLC35D1, and SLC35D2 proteins may be used. These antibodies may be polyclonal or monoclonal antibodies.

[0243] Aptamers are single-stranded DNA or RNA molecules that bind to specific target proteins with high affinity and specificity, and can be used as detection agents to replace antibodies.

[0244] In one embodiment, the agent for measuring the level of uridine diphosphate N-acetylglucosamine (UDP-GlcNAc) may be selected from the group consisting of, but not limited to, analytical reagents for mass spectrometry, enzymes, chemical derivatization reagents based on fluorescence or chromogenic reactions, antibodies that specifically bind to nucleotide-sugar, aptamers, or quantitative diagnostic kits based thereon.

[0245] In one embodiment, UDP-GlcNAc can be quantified using liquid chromatography-mass spectrometry (LC-MS / MS).

[0246] In one embodiment, to measure the level of UDP-GlcNAc, the endoplasmic reticulum, Golgi apparatus, and cytoplasm were separated through cell fractionation, and then the level of UDP-GlcNAc in each compartment was measured using chemical derivatization and a colorimetric assay.

[0247] Another aspect of the present specification provides a composition for diagnosing or predicting the prognosis of osteoarthritis, comprising a preparation for measuring the level of glucosamine. In one embodiment, the level of glucosamine may be the mass and / or concentration of glucosamine.

[0248] In one embodiment, the agent for measuring the level of glucosamine in a sample separated from a subject (e.g., chondrocytes, cartilage tissue, blood, serum, plasma, tissue, saliva, urine) may be selected from the group consisting of detection reagents for high-performance liquid chromatography (HPLC), analysis reagents for mass spectrometry (MS or LC-MS / MS), enzyme reaction-based detection enzymes (e.g., glucosamine dehydrogenase or glucosamine oxidase), chemical derivatization reagents for fluorescent or colorimetric reactions, or antibodies, aptamers, and functional variants thereof that specifically bind to glucosamine. In one embodiment, the level of glucosamine may be measured by liquid chromatography-mass spectrometry (LC-MS / MS), enzyme-linked immunoassay (ELISA), enzyme activity-based colorimetric assay, or fluorescence-based assay. In one embodiment, it was confirmed that osteoarthritis may worsen if glucosamine is excessively supplemented to a level exceeding the recommended daily intake (1.5g) as a health functional food.

[0249] In an exemplary embodiment, osteoarthritis can be diagnosed or the prognosis of osteoarthritis can be predicted by using a composition for diagnosing or predicting the prognosis of osteoarthritis according to one aspect of the present specification to measure the expression level of a gene belonging to SLC35; the level of a protein encoded by said gene; or the level of uridine diphosphate N-acetylglucosamine in a sample separated from a subject (e.g., chondrocytes, cartilage tissue, blood, serum, plasma, tissue, saliva, urine).

[0250]

[0251] In addition, another aspect of the present specification provides a kit for diagnosing or predicting the prognosis of osteoarthritis, comprising the aforementioned composition for diagnosing or predicting the prognosis of osteoarthritis.

[0252] As the composition for diagnosing or predicting the prognosis of osteoarthritis and osteoarthritis have been described above, a detailed explanation is omitted.

[0253] In one embodiment, the kit may further include a preparation for measuring the expression level of a gene belonging to the aforementioned SLC35; the level of a protein encoded by said gene; or the level of uridine diphosphate N-acetylglucosamine, as well as components, tools, reagents, etc. commonly used in the field so as to be suitable for use as a kit for diagnosing or predicting the prognosis of osteoarthritis.

[0254] In one embodiment, the component, tool, or reagent may be a carrier, a labeling substance capable of generating a detectable signal, chromophores, a solvent, a cleaning agent, a buffer, a stabilizer, etc. If the labeling substance is an enzyme, it may include a substrate capable of measuring enzyme activity and a reaction stopping agent. The carrier may be a soluble carrier or an insoluble carrier, and soluble carriers include physiologically acceptable buffers known in the art, e.g., PBS, and examples of insoluble carriers may be polymers such as polystyrene, polyethylene, polypropylene, polyester, polyacrylonitrile, fluoropolymer, cross-linked dextran, polysaccharide, latex plated with metal, other paper, glass, metal, agarose, and combinations thereof.

[0255]

[0256] Another aspect of the present specification provides a method for providing information for the diagnosis or prognosis prediction of osteoarthritis.

[0257] In one embodiment, i) a method for providing information for the diagnosis or prognosis prediction of osteoarthritis may include the step of measuring the expression level of a gene belonging to SLC35 from a sample (e.g., chondrocytes or cartilage tissue) separated from a subject.

[0258] In one embodiment, the information providing method may further include: ii) a step of determining that the subject has osteoarthritis or that osteoarthritis will develop when the expression level of the gene belonging to the measured SLC35 is lower than that of a normal control group.

[0259] In an exemplary embodiment, the information providing method may further include: ii) determining that the prognosis of osteoarthritis in the subject is poor if the expression level of the gene belonging to the measured SLC35 is lower than that of a normal control group. In this case, the subject may be an individual with osteoarthritis.

[0260] In addition, one aspect of the present specification is,

[0261] i) A step of measuring the expression level of a gene belonging to SLC35 from a sample (e.g., chondrocytes or cartilage tissue) isolated from a subject;

[0262] ii) a step of determining that the subject has osteoarthritis, or that osteoarthritis will develop in the subject, or that the prognosis of osteoarthritis in the subject is poor, if the expression level of the measured gene is lower than that of a normal control group; and

[0263] iii) a step of administering an effective amount of an osteoarthritis treatment agent to a determined subject; the present invention provides a method for diagnosing and treating osteoarthritis, comprising:

[0264]

[0265] In one embodiment, a method for providing information for the diagnosis or prognosis prediction of osteoarthritis may include: i) measuring the level of a protein encoded by a gene belonging to SLC35 from a sample (e.g., chondrocytes or cartilage tissue) separated from a subject.

[0266] In one embodiment, the information providing method may further include: ii) a step of determining that the subject has osteoarthritis or that osteoarthritis will develop when the level of the measured protein is lower than that of a normal control group.

[0267] In an exemplary embodiment, the information providing method may further include: ii) determining that the prognosis of osteoarthritis in the subject is poor if the level of the protein encoded by the gene belonging to the measured SLC35 is lower than that of a normal control group. In this case, the subject may be an individual with osteoarthritis.

[0268] In addition, one aspect of the present specification is,

[0269] i) A step of measuring the level of a protein encoded by a gene belonging to SLC35 from a sample separated from a subject (e.g., chondrocytes or cartilage tissue);

[0270] ii) a step of determining that the subject has osteoarthritis, or that osteoarthritis will develop in the subject, or that the prognosis of osteoarthritis in the subject is poor, if the level of the measured protein is lower than that of a normal control group; and

[0271] iii) a step of administering an effective amount of an osteoarthritis treatment agent to a determined subject; the present invention provides a method for diagnosing and treating osteoarthritis, comprising:

[0272]

[0273] In one embodiment, a method for providing information for the diagnosis or prognosis prediction of osteoarthritis may include: i) measuring the level of uridine diphosphate N-acetylglucosamine from a sample (e.g., chondrocytes or cartilage tissue) separated from a subject.

[0274] In one embodiment, the information providing method may further include: ii) a step of determining that the subject has osteoarthritis or that osteoarthritis will develop when the level of uridine diphosphate N-acetylglucosamine in the cytoplasm of the cartilage cells of the subject is higher than that of a normal control group.

[0275] In an exemplary embodiment, the information providing method may further include: ii) determining that the prognosis of osteoarthritis of the subject is poor if the level of uridine diphosphate N-acetylglucosamine in the cytoplasm of the cartilage cells of the subject is higher than that of a normal control group. At this time, the subject may be an individual that has developed osteoarthritis (e.g., post-traumatic osteoarthritis).

[0276] In one embodiment, the information providing method may further include: ii) a step of determining that the subject has osteoarthritis or that osteoarthritis will develop when the level of uridine diphosphate N-acetylglucosamine in the cytoplasm of the cartilage cells of the subject exceeds a predetermined value (e.g., 1 mM).

[0277] In an exemplary embodiment, the information providing method may further include: ii) determining that the prognosis of osteoarthritis of the subject is poor when the uridine diphosphate N-acetylglucosamine in the cytoplasm of the cartilage cells of the subject exceeds a predetermined value. At this time, the subject may be an individual that has developed osteoarthritis (e.g., post-traumatic osteoarthritis).

[0278] In addition, one aspect of the present specification is,

[0279] i) A step of measuring the level of uridine diphosphate N-acetylglucosamine in a sample separated from a subject (e.g., chondrocytes or cartilage tissue);

[0280] ii) a step of determining that the subject has osteoarthritis, or that osteoarthritis will develop in the subject, or that the prognosis of osteoarthritis in the subject is poor, if the level of uridine diphosphate N-acetylglucosamine in the cytoplasm of the subject's chondrocytes is higher than that of a normal control group; and

[0281] iii) a step of administering an effective amount of an osteoarthritis treatment agent to a determined subject; the present invention provides a method for diagnosing and treating osteoarthritis, comprising:

[0282]

[0283] In one embodiment, a method for providing information for the diagnosis or prognosis prediction of osteoarthritis may include: i) a step of measuring the level of glucosamine from a sample (e.g., chondrocytes or cartilage tissue) separated from a subject.

[0284] In one embodiment, the information providing method may further include: ii) a step of determining that the subject has osteoarthritis or that osteoarthritis will develop when the measured level of glucosamine is higher than that of a normal control group.

[0285] In an exemplary embodiment, the information providing method may further include: ii) determining that the prognosis of the subject's osteoarthritis is poor if the measured level of glucosamine is higher than that of a normal control group. In this case, the subject may be an individual that has developed osteoarthritis (e.g., post-traumatic osteoarthritis).

[0286] In one embodiment, the information providing method may further include: ii) a step of determining that the subject has osteoarthritis or that osteoarthritis will develop if the measured level of glucosamine exceeds a predetermined value (e.g., 10 μM).

[0287] In an exemplary embodiment, the information providing method may further include: ii) determining that the prognosis of osteoarthritis of the subject is poor when the measured level of glucosamine exceeds a predetermined value. At this time, the subject may be an individual that has developed osteoarthritis (e.g., post-traumatic osteoarthritis).

[0288] In addition, one aspect of the present specification is,

[0289] i) A step of measuring the level of glucosamine in a sample separated from a subject (e.g., chondrocytes or cartilage tissue);

[0290] ii) a step of determining that the subject has osteoarthritis, or that osteoarthritis will develop in the subject, or that the prognosis of osteoarthritis in the subject is poor, if the measured glucosamine level is higher than that of a normal control group; and

[0291] iii) a step of administering an effective amount of an osteoarthritis treatment agent to a determined subject; the present invention provides a method for diagnosing and treating osteoarthritis, comprising:

[0292] In one embodiment, the osteoarthritis treatment may be any substance that exhibits an effect of preventing and / or treating osteoarthritis without limitation. For example, the osteoarthritis treatment may be a commercially available substance.

[0293] The gene belonging to SLC35, the protein encoded by the gene belonging to SLC35, N-acetylglucosamine uridine diphosphate, the expression level of the gene, the level of the protein, the level of N-acetylglucosamine uridine diphosphate, and the method for measuring the level of glucosamine have been described above, so a detailed explanation is omitted.

[0294] In an exemplary embodiment, the subject may be an individual that is likely to develop osteoarthritis or has developed osteoarthritis. The individual may include, but is not limited to, mammals. Mammals may be, but are not limited to, humans, mice, cattle, dogs, rabbits, and cats.

[0295] In an exemplary embodiment, the sample may be at least one selected from the group consisting of chondrocytes, cartilage tissue, blood, serum, plasma, tissue, cancer tissue, cancer cells, saliva, and urine, but is not limited thereto.

[0296]

[0297] In addition, another aspect of the present specification provides a use for preparing a composition for the diagnosis or prognosis prediction of osteoarthritis comprising: a preparation for measuring the expression level of a gene belonging to the aforementioned solute transporter family 35 (SLC35); a preparation for measuring the level of a protein encoded by said gene; or a preparation for measuring the level of uridine diphosphate N-acetylglucosamine.

[0298]

[0299] Hereinafter, the present invention will be described in detail with reference to examples to specifically explain the invention. The following examples are provided for illustrative purposes only to aid in understanding the invention, and the scope and range of the invention are not limited by them.

[0300]

[0301] The inventors implemented embodiments according to one aspect of this specification after observing significant changes in the expression of transporters that can affect the intracellular distribution of UDP-GlcNAc during the development of the aging phenotype. The inventors demonstrated that UDP-GlcNAc compartmentalization is a novel mechanism for regulating O-GlcNAcylation, confirming that this shifts the secretory pathway of chondrocytes from PG (proteoglycan) synthesis to aging-related chronic inflammation. By elucidating the stress response mechanism underlying the emergence of SASP (aging-related secretory phenotype) in chondrocytes, the inventors provide valuable insights into the development of senostatics as a rational approach for the treatment of osteoarthritis.

[0302]

[0303] Experimental materials

[0304] Cells: Primary culture human chondrocytes, human chondrocyte cell line (C28 / I2), primary culture mouse chondrocytes

[0305] Animal: Mouse C57BL / 6J,Gata4 fl / fl ; Col2a1-CreER T2 ,Gata4 fl / fl ; Prg4-CreER T2

[0306] Drugs: ST045849 (OGT inhibitor), thiamet-G (OGA inhibitor), NSC140905 (GATA4 inhibitor), Gata4siRNA (GATA4 inhibitor), DON (GFPT inhibitor), glucosamine sulfate

[0307] Administration: Intra-articular (IA) injection in the knee in animal models, hydrogel (ascorbyl palmitate) based delivery

[0308]

[0309] Experimental results

[0310] 1. Experimental Example 1: Elucidation of mechanisms related to chondrocyte aging

[0311] (1) Confirmation of changes in intracellular UDP-GlcNAc compartmentalization in senescent chondrocytes

[0312] 1) Experimental Method

[0313] Osteoarthritis-related cartilage transcriptome data were obtained and analyzed using Gene Expression Ontology (GEO). Cellular senescence was induced in human C28 / I2 chondrocytes and primary cultured mouse chondrocytes by treating them with bleomycin or doxorubicin, or ionic radiation (IR). To confirm the induction of cellular senescence, SA-β-Gal staining, BrdU fluorescence immunostaining, and qRT-PCR for senescence marker mRNAs were performed. Under each condition, organelles were fractionated, and UDP-GlcNAc concentrations in the ER, Golgi, and cytoplasmic fractions were quantified. Transcriptome expression levels of the SLC35 family (SLC35A2, SLC35A3, SLC35A5, SLC35B4, SLC35D1, SLC35D2) were analyzed by qRT-PCR. Additionally, the content of sulfated GAGs in the chondrocyte culture medium was measured using the DMMB assay.

[0314] 2) Experimental Results

[0315] After aging induction, the total amount of UDP-GlcNAc in chondrocytes remained unchanged, but it decreased in the endoplasmic reticulum (ER) and Golgi fractions and significantly increased in the cytoplasm. The expression of SLC35A3, SLC35B4, SLC35D1, and SLC35D2 was commonly decreased, and GAG secretion also decreased during chondrocyte aging. Therefore, aging stress alters the intracellular distribution of UDP-GlcNAc to increase its cytoplasmic reserves, suggesting the possibility that this fractionation regulates protein O-GlcNAc levels and SASP activity.

[0316] Specifically, UDP-GlcNAc and UDP-N-acetylgalactosamine (UDP-GalNAc) are interconvertible and are transported from the cytoplasm to the endoplasmic reticulum (ER) and Golgi apparatus by various solute transporter family 35 (SLC35) transporters, including SLC35A2, SLC35A3, SLC35A5, SLC35B4, SLC35D1, and SLC35D2. In secretory organelles, the influxed sugars are utilized for the synthesis of GAG-rich PGs. Since one of the defining characteristics of OA is the loss of PG content in cartilage, the expression of these transporters was investigated in OA or OA-related transcriptome data available through the Gene Expression Omnibus (GEO) database. The expression of UDP-GlcNAc and UDP-GalNAc transporters was downregulated across several human OA transcriptome datasets (Fig. 1a). Likewise, their expression levels were consistently reduced in interleukin (IL)-1β-treated chondrocytes (Fig. 1b) and various OA animal models (Fig. 1c) compared to the corresponding controls.

[0317] Senescent chondrocytes are deeply involved in the development of OA. DNA-damaging chemicals such as doxorubicin (doxo) or bleomycin (bleo) and ionizing radiation (IR) strongly induced cellular senescence in human C28 / I2 chondrocytes and primary cultured mouse chondrocytes, which was manifested by an increase in senescence-associated β-galactosidase (SA-β-Gal) positivity, a decrease in S-phase entry, upregulation of CDKN1A and CDKN2A INK4A expression, and downregulation of LMNB1 expression (Figs. 1d, 1e, 10). Notably, the senescence-inducing stimuli commonly reduced the expression levels of SLC35A3, SLC35B4, SLC35D1, and SLC35D2 in these cells (Figs. 1f, 1g, 11a). Subsequently, UDP-GlcNAc content was measured in the ER, Golgi apparatus, and cytoplasm of the chondrocytes, respectively. Total UDP-GlcNAc levels in human C28 / I2 chondrocytes were unaffected after doxorubicin treatment under an aging-inducing system (Fig. 1h). However, the aging-inducing stimulus decreased UDP-GlcNAc levels in the ER and Golgi apparatus, while conversely increasing their content in the cytoplasm (Fig. 1h). Cytoplasmic accumulation of UDP-GlcNAc was similarly observed in aged mouse chondrocytes without significant changes in total intracellular UDP-GlcNAc levels (Fig. 1i). In contrast, inhibiting the hexosamine biosynthetic pathway by targeting glutamine-fructose-6-phosphate transaminase (GFPT) with 6-diazo-5-oxo-L-norleucine (DON) simultaneously decreased UDP-GlcNAc levels in the ER, Golgi apparatus, and cytoplasm (Figs. 1h, 1i).

[0318] These results collectively suggest that aging-inducing stimuli alter the intracellular distribution of UDP-GlcNAc by preferentially retaining UDP-GlcNAc within the cytoplasm. This change in UDP-GlcNAc redistribution was more correlated with a decrease in the GAG ​​content within PG secreted from senescent chondrocytes (Figs. 1j, 1k, 11b). Meanwhile, no statistically significant changes were observed in the expression of OGT and OGA in OA chondrocytes or senescent chondrocytes.

[0319]

[0320] (2) Confirmation of the relationship between O-GlcNAc formation and chondrocyte aging in osteoarthritis cartilage

[0321] 1) Experimental Method

[0322] Paraffin sections were prepared by distinguishing between damaged and undamaged areas in the knee joint cartilage tissue of osteoarthritis patients. After confirming changes in the cartilage ECM by staining the sections with Alcian blue, O-GlcNAc antibodies and p16 were tested on separate sections. INK4aAlternatively, co-immunofluorescence was performed using the GATA4 antibody. Additionally, senescence was induced in human and mouse chondrocytes by treating them with doxorubicin, bleomycin, or H2O₂, and whole-cell proteins were extracted to analyze O-GlcNAcylation levels by Western blot. In some experiments, O-GlcNAc levels were regulated by treating primary cultured human chondrocytes senescent-induced with doxorubicin, and in others, mouse chondrocytes senescent-induced with bleomycin, with O-GlcNAc transferase inhibitors (ST045849) or O-GlcNAcase inhibitors (thiamet-G). Subsequently, qRT-PCR or Western blot was performed to analyze the expression of the cellular senescence marker SASP at the mRNA or protein level. Transcriptomes of senescent mouse chondrocytes treated with OGT inhibitors were read via RNA-seq, followed by gene set enrichment analysis (GSEA).

[0323]

[0324] 2) Experimental Results

[0325] O-GlcNAcylation signaling was significantly increased in the cartilage of the osteoarthritis damage site, and these cells p16 INK4aIt overlapped significantly with senescent cells in a positive manner. In human and mouse chondrocytes, O-GlcNAcylation levels increased after DNA damage induction; however, treatment with ST045849 suppressed the expression of SASP factors (IL-6, MMP3, MMP13, etc.). Conversely, treatment with thiamet-G increased SASP expression. RNA-seq-based GSEA results also showed that gene groups related to "ECM degradation" and "inflammatory cytokine production" accumulated in a negative direction upon O-GlcNAc inhibition, leading to the suppression of these gene groups at the transcriptome level. This confirmed that while the increase in O-GlcNAc in osteoarthritis cartilage does not directly regulate chondrocyte aging itself, it mediates SASP expression.

[0326] Specifically, since cytoplasmic UDP-GlcNAc is used as a substrate for O-GlcNAc formation, we next investigated whether the altered intracellular UDP-GlcNAc compartmentalization correlated with O-GlcNAc formation content in OA-affected cartilage regions. The degree of O-GlcNAc formation was significantly increased in the cartilage of osteoarthritis patients affected by OA, but was negligible in the intact areas of arthritic cartilage (Figs. 2a, 2b). Notably, p16, a biomarker of cellular aging INK4a The expression of was highly co-located in chondrocytes showing O-GlcNAc signaling, and their double positivity was significantly higher in OA-affected cartilage than in the undamaged corresponding site (Fig. 2b).

[0327] Considering the cytoplasmic accumulation of UDP-GlcNAc in senescent chondrocytes (Figs. 1h, 1i), we explored how senescence-inducing stimuli affect O-GlcNAc formation in primary cultured human and mouse chondrocytes. After doxorubicin treatment, human articular chondrocytes exhibited significantly increased levels of O-GlcNAc formation (Fig. 2c). Similarly, primary mouse chondrocytes showed an overall increase in O-GlcNAc formation along with the onset of senescence following bleomycin treatment or prolonged exposure to hydrogen peroxide (H2O2) (Figs. 2d, 2e). We then questioned and tested whether O-GlcNAc formation is required for the entry of chondrocytes into senescence. However, bleomycin-induced DNA damage and the increase in SA-β-Gal positivity were not attenuated by treatment with the OGT inhibitor ST045849 (Figs. 2f, 2g). Similarly, the expression of Cdkn1a, Cdkn2a, and Ink4a, biomarkers associated with permanent cell cycle arrest in senescent cells, was not affected by O-GlcNAcification (Fig. 2h). These results suggested that O-GlcNAcification does not influence the entry of chondrocytes into senescence. Therefore, we next investigated whether O-GlcNAcification affects SASP development, another key aspect of senescent cells. In human articular chondrocytes, injury-induced senescence resulted in the expression of SASP factors, which was accompanied by the upregulation of GATA4, a transcriptional regulator that controls SASP (Fig. 2i). Likewise, the expression levels of GATA4 and SASP factors were significantly increased in OA-affected human cartilage compared to uninjured corresponding sites (Fig. 2j). GATA4 expression was highly co-located with O-GlcNAcification signals, and their double positivity was significantly increased in OA-affected articular cartilage compared to uninjured tissue (Fig. 2k). It is noteworthy that inhibition of O-GlcNAc synthesis effectively inhibited the expression of SASP factors in senescent chondrocytes (Fig. 2l, Fig. 12a).Conversely, increasing the degree of O-GlcNAcification by treatment with the OGA inhibitor thiamet-G further enhanced SASP expression in senescent chondrocytes (Fig. 12b). Since SASP primarily involves matrix-degrading proteolytic enzymes and inflammatory cytokines, we further investigated how gene sets associated with these annotations are regulated at the transcriptome level in senescent chondrocytes by inhibiting O-GlcNAcification. Gene set enrichment analysis (GSEA) showed that gene sets such as "degradation of the extracellular matrix" and "positive regulation of cytokine production related to inflammatory responses" were negatively enriched in the transcriptomes of senescent chondrocytes treated with the OGT inhibitor compared to the vehicle control group (Fig. 2m, Fig. 12c). Collectively, the experimental results indicate that stress-induced O-GlcNAcification is necessary for the full expression of SASP in senescent chondrocytes.

[0328]

[0329] (3) Confirmation of the effect of O-GlcNAcification of GATA4 on the regulation of GATA4 stability and transcriptional activity

[0330] 1) Experimental Method

[0331] After overexpressing FLAG- or HA-tagged GATA4 in HEK293T and chondrocytes, the GATA4-OGT interaction and the O-GlcNAc conversion of GATA4 were confirmed by co-immunoprecipitation (co-IP) using OGT and O-GlcNAc antibodies. The O-GlcNAc conversion of GATA4 was verified using succinylated wheat germ agglutinin (sWGA) agarose bead pull-down, and GATA4 protein stability and transcriptional activity were analyzed under conditions of increased O-GlcNAc (thiamet-G treatment) and decreased O-GlcNAc (ST045849 treatment). GATA4 protein stability was evaluated using a cycloheximide-chase assay, and transcriptional activity was evaluated using a GATA-responsive luciferase reporter assay containing a GATA binding site in the promoter region. Transcriptomes of senescent mouse chondrocytes treated with OGT inhibitors were read via RNA-seq, followed by gene set enrichment analysis (GSEA). The expression of SASP in senescent mouse chondrocytes transfected with siRNA against Gata4 or treated with ST045849 was analyzed by qRT-PCR. Additionally, mass spectrometry using ETD-MS was performed to identify the O-GlcNAcization sites of GATA4. Western blot analysis was conducted to confirm protein expression under thiamet-G treatment using S212A and S406A variants, and co-immunoprecipitation experiments were performed to confirm interactions with p62, an adapter protein involved in selective autophagy, in order to compare the functions of O-GlcNAcization occurring at each site.

[0332]

[0333] 2) Experimental Results

[0334] GATA4 binds directly to OGT, and inducing O-GlcNAcylation through thiamet-G treatment significantly increased GATA4 protein expression. The increase in O-GlcNAcylation extended the half-life of GATA4 protein, providing a stabilizing effect, and also enhanced the transcriptional activity of the transcription agent GATA4. Conversely, inhibiting O-GlcNAcylation through ST045849 treatment reduced the expression of GATA4 protein and its target gene. ETD-MS mass spectrometry identified the S212 and S406 amino acid residues of GATA4 as O-GlcNAcylation sites; however, the S406A variant, in which S406 was substituted with alanine, was not stabilized even after thiamet-G treatment and maintained its binding to p62. This differs from wild-type GATA4, which evades binding to p62 upon thiamet-G treatment, thereby avoiding selective autophagic degradation and achieving stabilization. This demonstrates that S406 O-GlcNAcylation of GATA4 blocks p62 binding, thereby inhibiting selective autophagic degradation and inducing GATA4 stabilization and increased SASP transcriptional activity.

[0335] Specifically, based on the observed correlation between the degree of O-GlcNAcification in OA cartilage and the expression of the SASP regulator GATA4, we explored possible mechanistic links between O-GlcNAcification and GATA4 regulation. Cell-based co-immunoprecipitation analysis indicated that GATA4 interacts with OGT (Fig. 3a). To investigate whether GATA4 is O-GlcNAcified, whole-cell lysates were precipitated with succinylated wheat germ agglutinin (sWGA) beads, which have a high affinity for GlcNAc residues, and probed for anti-GATA4. GATA4 was detected in the sWGA precipitates, and the degree of modification substantially increased after thiamet-G treatment (Fig. 3b). To directly detect the O-GlcNAc portion of GATA4, exogenously expressed GATA4 was pulled down and immunoblotted with anti-O-GlcNAc. The O-GlcNAc formula in GATA4 was detected at a high level in the presence of thiamet-G (Fig. 3c).

[0336] Next, we questioned whether O-GlcNAcification is mechanistically coupled to the regulation of GATA4 activity. GATA4 undergoes selective autophagy-mediated degradation in the absence of aging-associated stimuli and therefore generally exists at basal levels. An increase in overall intracellular O-GlcNAcification significantly boosted GATA4 protein levels (Fig. 3d), which ultimately led to increased GATA4-mediated transcriptional activity (Fig. 3e). Conversely, inhibition of O-GlcNAcification resulted in the downregulation of GATA4-mediated transcriptional activity (Fig. 3e). Cycloheximide (CHX) tracking analysis further demonstrated that the stability of GATA4 protein is significantly enhanced by increasing the degree of O-GlcNAcification under thiamet-G treatment (Fig. 3f). Consistent with the observation that OGT inhibition inhibits GATA4 transcriptional activity (Fig. 3e), the expression of the GATA4 target gene set was negatively enriched in transcripts obtained from senescent chondrocytes treated with the OGT inhibitor ST045849 (Fig. 3g).

[0337] Furthermore, ST045849 treatment inhibited bleomycin-induced SASP factor expression in chondrocytes, but there was no additional inhibitory effect after GATA4 silencing (Fig. 3h, Fig. 3i, Fig. 13a). These results demonstrated that O-GlcNAcification regulates SASP expression through the GATA4 pathway.

[0338] To map O-GlcNAcation sites in GATA4, exogenously expressed GATA4 was purified and subjected to mass spectrometry (MS)-based proteomic analysis. This site mapping experiment identified two O-GlcNAcation sites: S212, located near the N-terminal zinc finger domain (217–241), and the S406 amino acid residue adjacent to the intrinsically disordered region (409–428) (Fig. 13b). To dissect the functional effects of these two evolutionarily conserved O-GlcNAcation sites, a panel of GATA4 mutant constructs was generated in which the serine residues at positions 212 and 406 were substituted with alanine. We then explored how the loss of O-GlcNAcation in GATA4 affects thiamet-G treatment-induced protein stabilization. Unlike GATA4-S212A, GATA4-S406A did not respond to thiamet-G treatment, suggesting that GATA4 O-GlcNAcization in S406 functions in protection against autophagy-mediated degradation (Fig. 3j). Since GATA4 is degraded by a selective autophagy process through its association with the p62 adapter, we investigated whether O-GlcNAcization in GATA4 affects its association with p62. Indeed, thiamet-G treatment substantially reduced the binding between GATA4 and p62, which explains the phenomenon of GATA4 stabilization under conditions of O-GlcNAcization via thiamet-G treatment (Fig. 3k). In contrast, the association between GATA4-S406A and p62 was unaffected by thiamet-G treatment (Fig. 3k), which is consistent with the observation that the protein stability of this GATA4 mutant is not affected by an increase in the overall level of O-GlcNAcization. In summary, the experimental results indicate that stress-induced O-GlcNAcification stabilizes GATA4 by preventing its association with p62 and thus blocking its entry into the lysosomal degradation pathway.

[0339]

[0340] 2. Experimental Example 2: Confirmation of the effect of SLC35 family modulation in improving osteoarthritis (Upstream mechanism, supplementary data needed)

[0341] (1) Experimental method

[0342] After inducing cellular senescence in mouse chondrocytes by treating them with doxorubicin, bleomycin, or H2O₂, or by culturing them for a long period under 20% oxygen (normoxia) conditions (passage 2-3), the cells were transfected with an empty vector (Ad-Null) and an adenovirus vector (Ad-Slc35d1) expressing Slc35d1 and delivered to the chondrocytes. Immunofluorescence was used to image the endoplasmic reticulum (marker protein: calreticulin) and the Golgi apparatus (marker protein: GM130). Additionally, mouse chondrocytes under the same conditions were harvested, mRNA was extracted, and mRNA analysis was performed for SASP factors (Mmp3, Mmp13, Il6) and the cartilage matrix degrading enzyme Adamts5.

[0343]

[0344] (2) Experimental results

[0345] To confirm the effect of improving osteoarthritis through the modulation of the SLC35 family, an adenovirus overexpressing SLC35D1 was constructed and transfected into mouse chondrocytes. The virus significantly increased the expression of Slc35d1 (Fig. 7a).

[0346] Subsequently, we investigated whether the senescence phenotype of chondrocytes was restored when Slc35d1 was overexpressed in senescent chondrocytes. Observation of endoplasmic reticulum via calreticulin immunofluorescence in cell senescence models such as doxorubicin (Doxo), bleomycin (Bleo), hydrogen peroxide (H2O₂), and oxidative stress environment passages (passage 2 / 3) revealed a common phenomenon of endoplasmic reticulum fragmentation, characterized by hypertrophy and an increase in vesicle-like shapes (Fig. 7b). Furthermore, this endoplasmic reticulum fragmentation decreased upon SLC35d1 overexpression, restoring the cells to a form similar to that of normal chondrocytes (Fig. 7b).

[0347] Likewise, when observing the Golgi apparatus through GM130 immunofluorescence staining in cell aging models such as doxorubicin (Doxo), bleomycin (Bleo), hydrogen peroxide (H2O₂), and oxidative stress environment subculture models (passage 2 / 3), it was found that the Golgi apparatus was commonly hypertrophied and segmented. It was confirmed that this trend was restored upon SLC35D1 overexpression, causing the Golgi apparatus to condense and be located in the perinuclear region near the nucleus, similar to the shape of normal chondrocytes (Fig. 7c). This phenomenon is interpreted as an improvement in the phenotype resulting from an increase in UDP-GlcNAc within the corresponding organelles through the increased expression of SLC35D1 located in the endoplasmic reticulum and Golgi apparatus.

[0348] To determine whether the osteoarthritis improvement effect induced by SLC35D1 overexpression is also observed at the level of osteoarthritis marker expression, the expression of representative SASPs and chondrolytic enzymes was analyzed. When SLC35D1 was overexpressed during cellular senescence induction using a bleomycin (Bleo) model, the expression of inflammatory SASPs such as MMP3, MMP13, and IL6 was significantly reduced, and the expression of ADAMTS5 was also reduced. MMP3 (matrix metalloproteinase-3) is known to induce structural breakdown of the extracellular matrix (ECM) by primarily degrading cartilage matrix collagens such as collagen types II, III, IV, IX, and XI, while ADAMTS5 (a disintegrin and metalloproteinase with thrombospondin motifs-5) is known to cause the loss of compressive elasticity of cartilage by cleaving aggrecan, the main chondroprotein polysaccharide. Therefore, these results suggest that increased expression of SLC35D1 can alleviate cartilage degeneration in structural and functional aspects by simultaneously inhibiting the inflammatory SASP and ECM degradation responses associated with chondrocyte aging.

[0349]

[0350] 3. Experimental Example 3: Confirmation of the effect of GATA4 inhibition on osteoarthritis improvement (downstream mechanism)

[0351] (1) Experimental method

[0352] In cell experiments, the function of GATA4 was inhibited by knocking down GATA4 with siRNA or by treatment with the small molecule NSC140905. Chondrocyte-specific GATA4-deficient mouse (GATA4 fl / fl ; Col2a1-CreER T2 andPrg4-CreER T2Osteoarthritis was induced by DMM surgery by creating a line. Chondrodesis, medial subchondral sclerosis, osteophyte formation, and synovial inflammation were histologically analyzed through joint tissue segmentation and safranin O staining, and μCT and immunohistochemical staining were performed for additional phenotypic analysis of osteoarthritis. Osteoarthritis-associated pain was evaluated in mice using static weight bearing, the electronic von Frey test, and pain behavior tests.

[0353]

[0354] (2) Experimental results

[0355] Gata4 knockdown via siRNA or inhibition of activity via small molecules in mouse chondrocytes reduced the expression levels of various SASPs produced in senescent chondrocytes. Gata4 deletion specific to chondrocytes or superficial chondrocytes significantly reduced cartilage destruction, osteosclerosis, synovitis, and pain. Furthermore, the expression of IL-6, MMP3, MMP13, and SASP factors was reduced, and p16 INK4a Aging markers such as HMGB1 were also reduced. This implies that GATA4 inhibition alleviates SASP and senescent chondrocyte function.

[0356] Specifically, to confirm the expression patterns of O-GlcNAcogenesis and GATA4 within cartilage tissue in a physiological environment, knee articular cartilage of a post-traumatic osteoarthritis (PAO) mouse model was analyzed. Immunohistochemical staining results showed that O-GlcNAcogenesis and GATA4 protein expression were restricted to the superficial zone of the articular cartilage, and the two signals appeared to overlap at a high level (Fig. 4a). Since the role of GATA4 on chondrocytes in the pathogenesis of osteoarthritis has not been clearly elucidated, tamoxifen (TMX)-induced chondrocyte-specific GATA4 gene-deficient mice (GATA4 fl / fl; Col2a1-CreER T2 We produced mice and induced traumatic osteoarthritis by performing medial meniscal instability (DMM) surgery on them. The temporally induced Gata4 defect by TMX treatment significantly improved major pathological features of osteoarthritis, such as cartilage destruction, subchondral bone sclerosis, osteophyte formation, and synovitis (Figs. 4b, 4c). In addition, to measure osteoarthritis-associated pain, static weight-bearing and mechanical allodynia were evaluated between the leg that underwent DMM surgery and the contralateral leg; as a result, these pain behaviors were significantly reduced in Gata4 knockout mice (Fig. 4d).

[0357] Based on the above results, it was hypothesized that if the protective effect of Gata4 deficiency is due to the inhibition of SASP in senescent chondrocytes (cenosstatic mechanism), then Gata4 removal confined to the cartilage surface layer alone would be sufficient to inhibit the progression of OA. To verify this, Gata4 capable of removing Gata4 upon TMX treatment only in cartilage surface cells (cells expressing Lubricin / Prg4) fl / fl ; Prg4-CreER T2 A mouse model was constructed. As a result of performing DMM surgery on these mice, selective Gata4 deletion in the superficial cartilage layer alone significantly reduced chondrosis, subchondral bone sclerosis, osteophyte formation, and synovitis (Figs. 4e, 4f), and these effects were similar to those observed when Gata4 was removed from the entire cartilage (Figs. 4b, 4c). Micro-CT (μCT) analysis also showed that Gata4-deficient mice exhibited significantly inhibited subchondral bone structural deformation and remodeling associated with OA (Fig. 4g). Molecular analysis revealed decreased expression of MMP3, MMP13, and IL-6 in the superficial cartilage from which Gata4 was removed, supporting cenosstatic effects targeting the GATA4 pathway (Fig. 4h). Furthermore, p16 INK4aIt was confirmed that overall chondrocyte aging was inhibited by decreasing the expression of cell aging markers such as HMGB1 and HGF, an inflammation-independent SASP factor (Fig. 14a).

[0358] Similarly, suppression of SASP expression was observed when cultured chondrocytes were treated with Gata4siRNA or the GATA4 small molecule inhibitor NSC140905 (Figs. 14b, 14c). Furthermore, in superficial chondrocyte-specific Gata4-deficient mice, weight-bearing imbalance and mechanical hyperalgesia observed after OA induction were reduced, confirming a pain-relieving effect (Figs. 4i, 4j).

[0359]

[0360] 4. Experimental Example 4: Confirmation of the effect of inhibiting GATA4 O-GlcNAcylation on improving osteoarthritis

[0361] (1) Experimental method

[0362] The OGT inhibitor ST045849 was loaded onto an ascorbyl palmitate-based hydrogel and injected once weekly into the knee joint cavity of mice with osteoarthritis induced via DMM surgery. Levels of O-GlcNAcylation markers in cartilage tissue, GATA4 expression, ECM damage indicators (CTX-II, NITEGE neoantigen), SASP expression, and pain indicators were analyzed. Chondrodysplasia, medial subchondral bone sclerosis, osteophyte formation, and synovial inflammation were histologically analyzed through joint tissue segmentation and safranin O staining, and μCT and immunohistochemical staining were performed for additional phenotypic analysis of osteoarthritis. Osteoarthritis-associated pain in mice was evaluated using static weight bearing, the electronic von Frey test, and pain behavior tests.

[0363]

[0364] (2) Experimental results

[0365] Inhibition of O-GlcNAcylation via ST045849 delivery significantly reduced the levels of GATA4 and O-GlcNAcylation in osteoarthritis cartilage and decreased damage markers of cartilage matrix proteins such as type II collagen and aggrecan (neoantigen detection). SASP and aging indicators were downgraded, and joint destruction and pain behaviors were improved. This demonstrates that inhibition of O-GlcNAcylation alleviates OA by reducing GATA4 stability.

[0366] Specifically, the inventors first confirmed that O-GlcNAc modification increases the stability of the GATA4 protein (Fig. 3), which promotes SASP expression in chondrocytes and exacerbates post-traumatic osteoarthritis (Figs. 4, 14). Accordingly, they sought to verify the effect of regulating the degree of O-GlcNAcylation within the joint on the progression of OA in vivo. First, they analyzed how OA progresses when O-GlcNAcylation levels are increased by pharmacologically inhibiting O-GlcNAcase (OGA). As a result of injecting thiamet-G into the knee joint cavity of a traumatic osteoarthritis (DMM) mouse model, OA-related pathological phenomena such as cartilage matrix loss, subchondral bone sclerosis, osteophyte formation, and synovitis manifested early or worsened (Figs. 5a, 5b). Furthermore, in the thiamet-G administration group, weight-bearing imbalance and mechanical allodynia significantly increased, leading to the exacerbation of OA-related pain (Fig. 5c).

[0367] Conversely, transcriptome analysis of aged primary culture mouse chondrocytes treated with the O-GlcNAc transferase (OGT) inhibitor ST045849 revealed that the expression of the "gene set elevated in OA patients" was significantly negatively enriched, confirming that OA-related gene expression was suppressed, and a mechanistic association was confirmed that O-GlcNAcylation promotes the pathogenesis of osteoarthritis (Figs. 5d, 15a, 15b). Furthermore, to verify the therapeutic potential through O-GlcNAc inhibition, ST045849 was loaded onto an ascorbyl palmitate-based injectable hydrogel and injected into the knee joint cavity. This hydrogel-based ST045849 delivery vehicle significantly inhibited cartilage destruction in traumatic OA mice (Figs. 5e, 5i), and simultaneously reduced the expression of GATA4 protein and the irreversible degradation of type II collagen and aggrecan (generation of neoantigens such as CTX-II and NITEGE) (Fig. 5f). In addition, administration of ST045849 [induced] p16 in cartilage tissue INK4a It significantly inhibited the expression of cellular senescence markers such as HMGB1 and SASP factors such as IL-6, MMP3, and MMP13 (Fig. 5g, Fig. 5h). In addition, pathological changes throughout the joint, such as subchondral bone sclerosis, osteophyte formation, synovial inflammation, and OA-related pain, were also significantly reduced (Fig. 5i, Fig. 5k, Fig. 5k).

[0368]

[0369] 5. Experimental Example 5: Confirmation of Worsening of Post-Traumatic Osteoarthritis Due to Excessive Glucosamine Supplementation

[0370] (1) Experimental method

[0371] The modification of O-GlcNAc in chondrocytes treated with glucosamine sulfate was analyzed by Western blot. The stabilization of GATA4 by glucosamine treatment in GATA4-overexpressing HEK293T cells was analyzed using a cycloheximide trace assay. Excessive doses of glucosamine sulfate (5 mg / mL in drinking water) were continuously administered to mice with osteoarthritis induced by DMM surgery from one week prior to surgery until the time of tissue detection. Histological osteoarthritis markers, subchondral bone remodeling via μCT analysis, and pain behaviors (electronic von Frey test, static weight bearing test) were measured.

[0372]

[0373] (2) Experimental results

[0374] Excessive treatment with glucosamine enhanced the degree of O-GlcNAcylation in chondrocytes and increased the stability of GATA4. In animal experiments, excessive glucosamine supplementation did not show significant changes in normal mice, but it increased O-GlcNAcylation and GATA4 levels in DMM osteoarthritis mice, exacerbating cartilage destruction, bone sclerosis, and pain. From this, it was confirmed that the oversupply of the UDP-GlcNAc precursor through glucosamine treatment accelerates the pathogenesis of osteoarthritis.

[0375] Specifically, glucosamine is a precursor of UDP-GlcNAc and is frequently consumed in the form of health functional foods by patients with osteoarthritis or the elderly due to expectations of joint protection and the maintenance of proteoglycans (PG) in cartilage. Glucosamine is known to promote the production of UDP-GlcNAc by acting downstream of GFPT (glutamine-fructose-6-phosphate transaminase) on the hexosamine biosynthetic pathway (HBP), thereby bypassing the rate-limiting step of the enzyme. Therefore, excessive intake of glucosamine can increase intracellular UDP-GlcNAc levels and, as a result, induce O-GlcNAc post-translational modification of proteins.

[0376] Indeed, treatment of chondrocytes with glucosamine sulfate resulted in a significant increase in protein O-GlcNAcylation levels (Fig. 6a, Fig. 15c). This increase in O-GlcNAc modification acted as a factor enhancing the stability of the GATA4 protein (Fig. 6b). To analyze the effect of excessive glucosamine intake on the progression of traumatic osteoarthritis, glucosamine was administered at a high concentration (5 mg / mL in the drinking form) to mice with OA induced by DMM surgery (Fig. 6c). No significant difference in body weight change was observed compared to the control group (Fig. 6d). In sham mice, OA-related pathologies, such as cartilage damage or bone changes, did not appear with the administration of excessive glucosamine alone (Fig. 6e, f). However, in the DMM surgery group, glucosamine administration resulted in increased cartilage destruction (Fig. 6e), accelerated abnormal remodeling of the subchondral bone (Fig. 6f), increased levels of GATA4 and catabolic SASP factors within the cartilage (Fig. 6g), and increased levels of Type II collagen and aggrecan degradation products (Fig. 15d). Additionally, excessive glucosamine intake exacerbated all morphological and pain-related OA conditions, including histological cartilage degeneration, increased subchondral sclerosis (Fig. 6h), increased mechanical allodynia (Fig. 6i), and worsening weight-bearing imbalance (Fig. 6j).

[0377]

[0378] 6. Experimental Example 6: Confirmation of improvement effect on osteoarthritis upon concomitant administration of SLC35 family (upstream) and OGT inhibitor (downstream) / Confirmation of improvement effect on osteoarthritis upon concomitant administration of SLC35 family and GATA4 inhibitor

[0379] (1) Experimental method

[0380] In a cell model of osteoarthritis in which cellular senescence was induced in mouse chondrocytes by treating them with a vehicle or bleomycin (50 μg / mL), a blank vector (Ad-Null) or an adenovirus vector expressing Slc35d1 (Ad-Slc35d1) was delivered to the chondrocytes by infecting them at 400 MOI for 4 hours. Subsequently, the cells were treated with a vehicle, NSC140905 (50 μM), or ST045849 (10 μM) for 5 days. Afterward, the mouse chondrocytes were harvested and mRNA was extracted to perform mRNA analysis of SASP factors (Mmp3, Mmp13, Il6) and the cartilage matrix degrading enzyme Adamts5.

[0381]

[0382] (2) Experimental results

[0383] To confirm the effect of improving osteoarthritis through the modulation of the SLC35 family, an adenovirus overexpressing SLC35D1 was constructed and transfected into mouse chondrocytes. The virus significantly increased the expression of Slc35d1 (Fig. 7a).

[0384] Subsequently, the improvement effect on osteoarthritis was confirmed upon the combined administration of Slc35 family activation (upstream mechanism target) and GATA4 inhibition (downstream mechanism 1 target) or OGT inhibition (downstream mechanism 2 target) in senescent chondrocytes. In a cell aging model induced by treatment with 50 μg / mL of bleomycin (Bleo), the effect of SLC35D1 activation via Ad-Slc35d1 transduction on SASP expression was confirmed (Experimental Example 2, Fig. 7d), and SASP and ADAMTS5 expression were confirmed when the GATA4 inhibitor NSC140905 was co-administered. As a result, a synergistic effect was observed in which the expression of cartilage degeneration factors decreased when SLC35D1 overexpression and GATA4 inhibition were performed simultaneously, compared to treatment with NSC140905 alone (Fig. 8). In the case of Mmp3, a statistically significant reduction effect was observed in the Bleo + Ad-Slc35d1 + NSC group overexpressing SLC35D1 compared to Bleo + NSC chondrocytes (P = 0.0104), and in the case of Mmp13, a statistically significant reduction effect was also observed in the Bleo + Ad-Slc35d1 + NSC group compared to Bleo + NSC chondrocytes (P = 0.0357). Additionally, in the case of Il6, a statistically significant reduction effect was observed in the Bleo + Ad-Slc35d1 + NSC group compared to Bleo + NSC chondrocytes (P = 0.0486), and in the case of Adamts5, a statistically significant reduction effect was also observed in the Bleo + Ad-Slc35d1 + NSC group compared to Bleo + NSC chondrocytes (P = 0.0116). SASP and ADAMTS5 gene expression were suppressed upon SLC35D1 overexpression and treatment with the drug NSC140905 (or NSC), suggesting that a synergistic effect occurs in which SASP expression is reduced when SLC35D1 is overexpressed in addition to inhibiting GATA4.These results can be seen from the fact that gene expression was statistically significantly reduced (P < 0.05) when comparing the Bleo+NSC group and the Bleo+Ad-Slc35d1+NSC group.

[0385] As a different strategy, the improvement effect on osteoarthritis was confirmed upon the combined administration of Slc35 family activation (upstream mechanism target) and OGT inhibition (downstream mechanism 2 target) in senescent chondrocytes. In a cell aging model induced by treatment with 50 μg / mL of bleomycin (Bleo), the effect of SLC35D1 activation via Ad-Slc35d1 transduction on SASP expression was confirmed (Experimental Example 2, Fig. 7d), and SASP and ADAMTS5 expression were confirmed when the OGT inhibitor ST045849 was co-administered. As a result, a synergistic effect was observed in which the expression of cartilage degeneration factors decreased when SLC35D1 overexpression and OGT inhibition were performed simultaneously compared to treatment with ST045849 alone (Fig. 9). In the case of Mmp13, a statistically significant reduction effect was observed in the Bleo + Ad-Slc35d1 + ST group compared to Bleo + ST chondrocytes (P = 0.0073). Additionally, in the case of Il6, a statistically significant reduction effect was observed in the Bleo + Ad-Slc35d1 + ST group compared to Bleo + ST chondrocytes (P = 0.0157), and in the case of Adamts5, a statistically significant reduction effect was observed in the Bleo + Ad-Slc35d1 + ST group compared to Bleo + ST chondrocytes (P < 0.0001). SASP and ADAMTS5 gene expression were inhibited upon SLC35D1 overexpression and treatment with the drug ST045849 (or ST), suggesting that a synergistic effect occurs in which SASP expression is reduced when SLC35D1 is overexpressed in addition to inhibiting OGT. These results can be seen from the fact that gene expression was statistically significantly reduced (P < 0.05) when comparing the Bleo+ST group and the Bleo+Ad-Slc35d1+ST group.

[0386] Therefore, these results suggest that targeting the upstream mechanism that activates the SLC35 family can alleviate cartilage degeneration in structural and functional aspects by more effectively suppressing the inflammatory SASP and ECM degradation reactions associated with chondrocyte aging than targeting downstream mechanisms that inhibit GATA4 or OGT alone. Furthermore, this implies that strategies targeting upstream mechanisms or both upstream and downstream mechanisms demonstrate superior senostatic effects compared to treatments that target downstream mechanisms (GATA4, OGT) alone.

[0387]

[0388] Experimental method

[0389] 1. Collection of human cartilage tissue samples

[0390] Human osteoarthritis articular cartilage specimens were obtained from 10 osteoarthritis (OA) patients undergoing total knee replacement (TKA) at Seoul National University Boramae Hospital. The Institutional Review Board (IRB) of Seoul National University Boramae Hospital (IRB No. 30-2017-48) approved the collection of human biological materials, and the Institutional Review Board of Seoul National University (IRB No. E1803 / 003-009) approved the use of these materials. Written informed consent was obtained from all patients prior to the total knee replacement procedure. Patient characteristics (gender, age, height, weight, and BMI) are summarized as ranges in Table 1 below. Only specimens obtained from female patients were used. Human cartilage tissue sections were stained with Alcian blue and graded according to the Osteoarthritis Research International (OARSI) grading system.

[0391] [Table 1]

[0392]

[0393]

[0394] 2. Mouse

[0395] Gata4fl / fl , Col2a1-CreER T2 andPrg4-CreER T2 Mouse strains were obtained from the Jackson Laboratory and backcrossed at least 6 times with C57BL / 6J mouse strains. To establish the Gata4 knockout mouse strain, Gata4 fl / fl The mouse Col2a1-CreER T2 (For conditional knockout of total chondrocytes) or Prg4-CreER T2 (For conditional knockout of surface chondrocytes) were crossed with mouse lines. To establish a post-traumatic osteoarthritis model in Gata4 knockout mouse lines, 12-week-old male Gata4 fl / fl ;Col2a1-CreER T2 or Gata4 fl / fl ;Prg4-CreER T2 Mice were intraperitoneally injected with TMX dissolved in corn oil (Sigma Aldrich) at a dose of 80 μg / g body weight daily for 5 days. 12-week-old male WT C57BL / 6J mice (Daehan Biolink) were used in a study of intra-articular injection of thiamet-G or ST045849 and excessive glucosamine supplementation via drinking water.

[0396]

[0397] 3. Experimental Osteoarthritis in Mice

[0398] All animal experiments were approved by the Institutional Animal Care and Use Committee of Seoul National University (IACUC No. SNU-180109-1, SNU-190105-1, SNU-190919-6, SNU-210113-4). The design, analysis, and reporting of animal experiments were conducted in accordance with Animals in Research: Guidelines for Reporting In Vivo Experiments (http: / www.nc3rs.org.uk / arrive-guidelines). Mice were housed in the Specified Pathogen-Free (SPF) animal facility at Seoul National University. Animals were maintained in an environment with controlled temperature, humidity, and a 12:12 light-dark cycle, and were provided with standard experimental feed on a free basis. Glucosamine sulfate supplementation was administered by dissolving glucosamine sulfate potassium chloride (LKT Laboratories) in drinking water at a concentration of 5 mg / mL, with a target daily intake of 30 mg, taking into account the mice's daily water intake. This dose corresponds to a daily intake of 5.8g for a 60 kg adult when considering body surface area-based equivalent dose calculations, exceeding the recommended daily intake of 1.5g. For post-traumatic osteoarthritis, at 12 weeks of age (WT C57BL / 6J excluding the excessive glucosamine supplementation experiment) or 13 weeks of age (Gata4 fl / fl , Gata4 fl / fl; Col2a1-CreER T2 , Gata4 fl / fl ; Prg4-CreER T2WT C57BL / 6J mice were induced via DMM surgery for experiments involving excessive glucosamine supplementation, and sham surgery mice of the same age were used as controls. Mice were sacrificed at 6 or 8 weeks after sham surgery or DMM surgery. The duration of the animal experiment is specified for each experiment. For intra-articular injection of OGA inhibitors to maintain O-GlcNAc status, 20 nmol of thiamet-G (Sigma Aldrich) dissolved in vehicle or 10 μL phosphate-buffered saline (PBS) was injected into the joints of WT mice that had undergone sham surgery or DMM surgery once a week for 6 weeks. To ensure controlled release of OGT inhibitors to reduce O-GlcNAc formation, a hydrogel containing dimethyl sulfoxide (DMSO) or 2 nmol ST045489 (Tim Tec) was injected once weekly for 8 weeks into the knee joints of WT mice that had undergone pseudosurgery or DMM surgery. The hydrogel (final concentration: 4% w / v) was prepared by dissolving 6-O-palmitoyl-L-ascorbic acid (Sigma Aldrich) in DMSO and mixing it with sterile distilled water. The hydrogel was diluted 1:1 with PBS prior to injection. The degree of cartilage destruction in the knee joint and osteoarthritis-associated phenotypes were evaluated using safranin O staining and scored according to the OARSI grading system.

[0399]

[0400] 4. Histological and immunohistochemical analysis

[0401] Human osteoarthritis cartilage specimens were fixed in 4% paraformaldehyde (PFA), dehydrated with an ethanol concentration gradient, and incubated with xylene. The specimens were embedded in paraffin, and 7-μm thick sections were prepared. Osteoarthritis-affected cartilage specimens were obtained from the tibial plateau, while relatively intact areas on the lateral side of the tibial plateau or relatively intact areas of the femoral condyle were used as controls. For histological staining, sections were deparaffinized in xylene, hydrated with an ethanol concentration gradient, and then used for Alcian Blue, immunofluorescence, or immunohistochemical staining. Knee joint tissues collected from a post-traumatic osteoarthritis mouse model and their corresponding controls were fixed in 4% PFA, decalcified with 0.5 M EDTA (pH 7.4), dehydrated with an ethanol concentration gradient, and incubated with xylene. Samples were embedded in paraffin, and 5-μm thick sections were prepared. For histological staining, paraffin-embedded tissue sections were deparaffinized in xylene, hydrated with a decreasing concentration gradient of ethanol, and then stained with safranin O or used for immunohistochemical staining. Two orthopedic pathologists from Seoul National University Boramae Hospital performed a comprehensive histological evaluation of the entire joint tissue based on their extensive experience in evaluating human and mouse osteoarthritis. Additionally, three pathologists reviewed the sections, achieving a minimum agreement of 90% for histological grading. Observers were blinded to the cartilage tissue regions of the human biological materials and to the genotype, injection, or surgical conditions of the mice. Section images were randomized to prevent observer bias. To evaluate osteoarthritis in mice, cartilage destruction was assessed using safranin O staining and scored on the medial tibial plateau according to the OARSI grading system (0-6).Medial tibial bone sclerosis (score -5 to 5) was scored by measuring the ratio of subchondral trabecular bone to bone marrow. Osteophyte maturity (grade 0-3) was graded by examining the anteromedial tibia as described in the literature. Synovial inflammation was scored on an arbitrary scale (0-3) based on the density of infiltrating inflammatory cells, synovial hyperplasia, and pannus involvement. The primary antibody used for immunohistochemical staining was as follows: O-GlcNAc (Abcam, ab2739), p16. INK4a (Proteintech, 10883-1-AP; Human p16 INK4a for), GATA4 (Santa Cruz, sc-25310), IL-6 (Santa Cruz, sc-130326), MMP3 (Abcam, ab52915), MMP13 (Abcam, ab51072), p16 INK4a (Abcam, ab211542; mouse p16 INK4a (for), HMGB1 (Abcam, ab18256), HGF (Novus, AF2207), CTX-II (Novus, NBP2-59386), NITEGE (MD Bioproducts, 1042003), and ADAMTS5 (Abcam, ab41037). The secondary antibodies used for immunohistochemical staining were: donkey anti-mouse IgG (H&L) conjugated with biotin-SP (Jackson ImmunoResearch, 715-065-150), donkey anti-rabbit IgG (H&L) conjugated with biotin-SP (Jackson ImmunoResearch, 711-065-152), and donkey anti-goat IgG (H&L) conjugated with biotin-SP (Jackson ImmunoResearch, 705-065-147).

[0402]

[0403] 5. Immunofluorescence

[0404] For immunofluorescence studies of human osteoarthritis cartilage sections, intact and damaged cartilage tissue sections were deparaffinized in xylene and hydrated with a decreasing concentration gradient of ethanol. Antigen retrieval was performed using an antigen retrieval solution (10 mM Tris-HCl, pH 9.0, 1 mM EDTA) at 60°C for 1 hour. Tissues were blocked with 10% normal goat serum (Sigma Aldrich) in PBS for 10 minutes, and O-GlcNAc (Abcam, ab2739), p16 INK4aThe tissues were cultured with primary antibodies against (Proteintech, 10883-1-AP), GATA4 (Proteintech, 19530-1-AP), normal mouse IgG (Santa Cruz, sc-2025), and normal rabbit IgG (Cell Signaling Technology, 2729S). The tissues were washed three times with PBS and cultured with donkey anti-rabbit IgG (H&L) conjugated with Alexa Fluor 488 (ThermoFisher Scientific, A-21206) and donkey anti-mouse IgG (H&L) conjugated with Alexa Fluor 594 (ThermoFisher Scientific, A-21203). For immunofluorescence staining of primary cultures of mouse chondrocytes, cultured cells were fixed in 4% PFA for 15 minutes, permeable with 0.1% Triton X-100 in PBS for 10 minutes, and then blocked with 10% normal goat serum in PBS for 1 hour. Cells were cultured with primary antibodies against γ-H2AX (Santa Cruz, sc-517348) and normal mouse IgG (Santa Cruz, sc-2025). Cells were washed three times with PBS and cultured with rabbit anti-mouse IgG+IgM (H&L) conjugated with Alexa Fluor 488 (Jackson ImmunoResearch, 315-485-044). After antibody treatment, nuclei were stained with DAPI. Stained cartilage tissue and chondrocytes were mounted using ProLong Gold Antifade (ThermoFisher Scientific). The mounted slides were imaged using the EVOS FL cell imaging system (ThermoFisher Scientific).

[0405]

[0406] 6. Static body weight bearing

[0407] The weight distribution of hindlimbs that underwent sham surgery or DMM surgery and the contralateral hindlimb was measured 1 day prior to sacrifice using an Incapacitance Meter (IITC Life Science, 600MR). For adaptation, mice were trained to enter the chamber and remain still at least three times prior to measurement. The adaptation process was continued until the mice maintained a stationary position and did not lean against either side of the chamber. Prior to measurement, each hindlimb was placed on each recording pad of the device. The weight placed on each recording pad was measured for 1 second over at least three independent trials, and the average value was presented. Data were presented as a percentage of the weight placed on the ipsilateral hindlimb that underwent DMM surgery relative to the weight placed on the contralateral hindlimb that did not. Observers were blinded to the genotype, injection, and surgical conditions of the mice.

[0408]

[0409] 7. Electronic Von Frey Inspection

[0410] Mechanical allodynia was measured one day prior to sacrifice using the Von Frey test with a dynamic plantar sensory system (Ugo Basile) for mechanical stimulation. Mice were placed on the metal grid floor of the device, and an adaptation process was performed until they remained stationary for at least 15 minutes. Once the mice's exploratory behavior ceased, mechanical allodynia was assessed by applying an electronic Von Frey filament to the plantar surface of the surgical ligature with a rising force at a rate of 1.33 g / s. When the mouse's foot retracted, the device automatically stopped applying force and recorded the force applied at the time of retraction. Reaction times were recorded at least three times per mouse, and the average value was presented as a measure of mechanical allodynia.

[0411]

[0412] 8. μCT Analysis

[0413] Anesthetized mice were scanned with an in vivo micro-CT scanner (Bruker, Skyscan 1276) one day prior to sacrifice. The hind limbs that had undergone pseudosurgery or DMM surgery were fixed, and 515 scans were performed at 0.4°. Scans were performed with a 70 kV X-ray source voltage, 57 μA current, and a 0.5-mm aluminum composite X-ray filter setting, and images were generated with a resolution of 20 μm pixels. Images were reconstructed using NRecon software v1.7.3 (Bruker), and 3D reconstruction of the μCT image set was performed using CTVox software v3.3.0 (Bruker).

[0414]

[0415] 9. Cell Culture and Processing

[0416] For the primary culture of human articular chondrocytes, cells were isolated from the femoral condyles and tibial plateaus of cartilage specimens collected from patients undergoing total knee replacement for osteoarthritis and cultured according to the method described in previous literature. Human chondrocytes were maintained in Dulbecco modified Eagle medium (DMEM) / F-12 supplemented with 10% fetal bovine serum (FBS; Gibco), 100 U / mL penicillin, 100 μg / mL streptomycin, and 250 ng / mL amphotericin B. Cells were treated in Phase 1 (P1) as indicated in each experiment. For the primary culture of mouse articular chondrocytes, cells were isolated from the femoral condyles and tibial plateaus of 5-day-old ICR mice according to the method described in previous literature. Mouse chondrocytes were maintained in DMEM supplemented with 10% FBS, 100 U / mL penicillin, and 100 μg / mL streptomycin. After 2 days of culture, cells were treated as indicated in each experiment. Cultured human and mouse chondrocytes were maintained at a wet 37°C, 5% CO2, and 3% O2 ​​atmosphere. Human chondrocyte cell line C28 / I2 was maintained in DMEM / F-12 supplemented with 10% FBS, 15 mM HEPES, 100 U / mL penicillin, and 100 μg / mL streptomycin at a wet 37°C and 5% CO2 atmosphere. HEK293T cells were maintained in wet DMEM supplemented with 10% FBS, 100 U / mL penicillin, and 100 μg / mL streptomycin at 37°C and a 5% CO2 atmosphere. To induce doxorubicin-induced cellular senescence in primary cultures of human articular chondrocytes, cells were maintained at zone 0 (P0) for 5 days, triedpsinized, and then cultured in new dishes. After 2 days of culture, human chondrocytes were treated with doxorubicin at zone 1 (P1). After allowing 72 hours for stable attachment, 1.5 x 10 5Cellular senescence was induced in primary cultured human chondrocytes (35 mm culture dishes) for 14 days using 2 ml of growth medium containing 0, 0.2, 0.5, or 1.0 μM of doxorubicin (Cayman). To maintain the release of secretory senescence-associated secretory phenotypes (SASPs) while preventing nutrient depletion, 1 ml of medium was replaced every 2 days with fresh medium containing 2 μl of vehicle (PBS) or an equivalent volume of stock doxorubicin (pre-diluted to 0.1, 0.25, and 0.5 mM). To induce bleomycin-induced senescence in primary cultured mouse articular chondrocytes, cells were treated with hyaluronidase IS (4 U / mL) for 4 hours, followed by treatment with vehicle or 50 μg / mL of bleomycin for the next 24 hours, and harvested after 5 days. For radiation-induced aging, cells were irradiated with gamma rays at a dose rate of 4 Gy / min using a GC 3000 Elan irradiator (MDS Nordion). As an oxidative stress-induced aging model, mouse chondrocytes were treated with vehicle or 100 μM H2O2 (Sigma Aldrich) for 5 days. For glucosamine sulfate treatment in mouse chondrocytes, cells were treated with or without 50 μg / mL bleomycin for 24 hours, followed by treatment with the indicated dose of glucosamine sulfate for 72 hours.

[0417]

[0418] 10. Transfection with small molecule interfering RNA (siRNA) and inhibition of GATA4 using small molecule inhibitors in aged chondrocytes

[0419] For bleomycin treatment of mouse chondrocytes, cells were treated with hyaluronidase IS (4 U / mL) for 4 hours after 2 days of culture, followed by treatment with vehicle or 50 μg / mL of bleomycin for the next 24 hours. For siRNA transduction of bleomycin-treated mouse chondrocytes, METAFECTENE PRO (Biontex) and 50 nM siRNA (a mixture of two siRNA sequences for Gata4 knockdown) were transduced according to the manufacturer's instructions. 12 hours after transduction, cells were cultured in fresh medium for an additional 120 hours. All siRNAs, including si-NC used for RNA interference in this study, were purchased from Bionier. For the treatment of bleomycin-treated mouse chondrocytes with 2-(1,3-benzodioxol-5-ylmethyl)butanedioic acid (NSC140905), the cells were treated with vehicle (DMSO) or 50 μM NSC140905 (AA Blocks) for 120 hours. Fresh medium was replenished every 48 hours.

[0420]

[0421] 11. Immunoblot

[0422] After treatment, cells were washed twice with PBS and lysed in RIPA buffer containing a protease inhibitor (Sigma Aldrich). For overexpression-based experiments, HEK293T cells were transfected using polyethyleneimine (PEI) polymers with the doxycycline-inducible GATA4 plasmid (Fig. 3d) or the TK promoter-driven FLAG-tagged GATA4 (WT, S212A, S406A) plasmid (Figs. 3f, 3j). Six or 24 hours after transfection, the medium was treated and maintained as described in the legends of each figure. Next, 10–15 μg of cell lysates were fractionated by SDS-PAGE and transferred to a nitrocellulose membrane (GE Healthcare). The membrane was blocked for 1 hour with 3% skim milk or 2% bovine serum albumin (BSA) in Tris-buffered saline (TBS) containing 0.1% Tween 20, and incubated overnight with the primary antibody at 4°C. After removing unbound antibodies by washing three times, the membrane was incubated with the secondary antibody. Immunoreactive protein bands were detected using the SuperSignal West Dura duration extension substrate (ThermoFisher Scientific) with an iBright FL1000 (ThermoFisher Scientific). The primary antibodies used for the immunoblot were as follows: O-GlcNAc (Abcam, ab2739), p16 INK4a(Proteintech, 10883-1-AP), GATA4 (Santa Cruz, sc-25310), IL-6 (Santa Cruz, sc-130326), MMP3 (Abcam, ab52915), MMP13 (Abcam, ab51072), HA (Abcam, ab9110), FLAG (Sigma Aldrich, F3165), vinculin (Cell Signaling Technology, 13901), and actin (Santa Cruz, sc-1615). The secondary antibodies used in the immunoblot were: goat anti-rabbit IgG (H&L) conjugated with horseradish peroxidase (HRP) (Jackson ImmunoResearch, 111-035-003), goat anti-mouse IgG+IgM (H&L) conjugated with HRP (Jackson ImmunoResearch, 115-035-044), and donkey anti-goat IgG conjugated with HRP (Santa Cruz, sc-2020). Protein band intensity was quantified by density analysis using ImageJ software and normalized to the corresponding housekeeping protein band.

[0423]

[0424] 12. Immunoprecipitation

[0425] For overexpression-based experiments, HEK293T cells were transfected using PEI with FLAG-tagged GATA4 and HA-tagged OGT plasmids (Fig. 3a), HA-tagged GATA4 plasmids (Figs. 3b, 3c), or TK promoter-driven FLAG-tagged GATA4 (WT, S406A) plasmid (Fig. 3k). Six or 24 hours after transfection, the medium was treated and maintained as described in the brief descriptions of each figure. For antibody-based immunoprecipitation analysis, cell lysates were prepared in EBC200 buffer (50 mM Tris-HCl, pH 8.0, 200 mM NaCl, and 0.5% NP-40) containing a protease inhibitor (Sigma Aldrich). NETN buffer (20 mM Tris-HCl, pH 8.0, 100 mM NaCl, 0.5% NP-40, and 1 mM EDTA) was used for sWGA pull-down analysis. Pull-down analysis was performed with labeled antibodies and Protein A / G-Sepharose beads (GE Healthcare) or sWGA agarose beads. The bound proteins were fractionated by SDS-PAGE and transferred to a nitrocellulose membrane (GE Healthcare). The membrane was blocked for 1 hour with 3% non-fat milk or 2% BSA in Tris-buffered saline (TBS) containing 0.1% Tween 20, and incubated overnight with the primary antibody at 4°C. After removing unbound antibodies by washing three times, the membrane was incubated with the secondary antibody. Immunoreactive protein bands were detected using the iBright FL1000 (ThermoFisher Scientific) with the SuperSignal West Femto maximum sensitivity substrate (ThermoFisher Scientific). The antibodies used for the pull-down analysis were FLAG (Sigma Aldrich, F3165) and HA (Abcam, ab9110).The primary antibodies used in the immunoblot were: FLAG (Sigma Aldrich, F3165), HA (Abcam, ab9110), O-GlcNAc (Abcam, ab2739), actin (Santa Cruz, sc-1615), and vinculin (Cell Signaling Technology, 13901). The secondary antibodies used in the immunoblot were: goat anti-rabbit IgG conjugated with HRP (H&L) (Jackson ImmunoResearch, 111-035-003), goat anti-mouse IgG+IgM conjugated with HRP (H&L) (Jackson ImmunoResearch, 115-035-044), and donkey anti-goat IgG conjugated with HRP (Santa Cruz, sc-2020).

[0426]

[0427]

[0428] 13. Measuring UDP-GlcNAc levels

[0429] C28 / I2 cells were treated with 100 μM DON (Sigma Aldrich) for 24 hours or 100 nM doxorubicin for 4 days. For the treatment of primary cultures of mouse articular chondrocytes, cells were treated with 150 μM DON for 24 hours or 50 μg / mL bleomycin for 24 hours, after which the medium was replaced with fresh medium and cultured for 5 days. After treatment, cells were washed twice with PBS and subjected to specific cell lysis methods tailored to each purpose. For the isolation of intact endoplasmic reticulum (ER), the Minute® ER Concentration Kit (Invent Biotechnologies) was used, and the ER compartment was lysed in the Minute® Denatured Protein Lysation Reagent (Invent Biotechnologies). For the isolation of intact Golgi apparatus, the Minute® Golgi apparatus Concentration Kit (Invent Biotechnologies) was used, and the cis- and trans-Golgi compartments were lysed and bound in the lysis reagent. Cell fractionation kit-standard (Abcam) was used for the cytoplasmic fraction excluding intramembrane organelles. Cells were lysed in RIPA buffer for total cell lysis. UDP-GlcNAc was measured as follows, using a variation of the previously described method: 50 μL of acetonitrile was initially added to 50 μL of the extract sample, hydrolyzed with 1 M HCl at 80°C for 20 minutes, and then cooled on ice. The sample was briefly centrifuged, neutralized with 10 μL of 1 M KOH and 50 μL of 200 mM potassium tetraborate, incubated at 80°C for 25 minutes, and cooled on ice. 150 μL of Ehrlich reagent (0.67 M p-dimethylaminobenzaldehyde and 11% (v / v) HCl added to acetic acid, diluted 1:2 in acetic acid before use) was added, and the reaction mixture was incubated at 37°C for 30 minutes. The sample was centrifuged, and 200 μL of the supernatant was transferred to a microtiter plate to measure the optical density at 584 nm.The nanomolar concentration of UDP-GlcNAc was calculated using the standard curve of UDP-GlcNAc (Sigma Aldrich), normalized by the protein content measured with the Pierce BCA Protein Analysis Kit (ThermoFisher Scientific), and expressed as a percentage compared to the vehicle control.

[0430]

[0431] 14. sGAG Analysis

[0432] C28 / I2 cells were maintained for 4 days in DMEM / F-12 medium supplemented with 10% FBS, 15 mM HEPES, 100 U / mL penicillin, and 100 μg / mL streptomycin in the presence or absence of 100 nM doxorubicin. Primary cultured mouse chondrocytes were treated with bleomycin for 24 hours or with radiation (5 Gy). Cells were maintained for 5 days in DMEM medium supplemented with 100 U / mL penicillin and 100 μg / mL streptomycin without FBS. The cell culture medium was replaced daily, and the sGAG content of the medium treated after the last 24 hours was analyzed. The sGAG content of the medium was measured using the 1,9-dimethylmethylene blue dye assay, which measures absorbance at 525 nm. sGAG content was quantified using a standard curve determined by the range of chondroitin sulfate solution (Tokyo Chemical Industry) and normalized by 3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyltetrazolium bromide (MTT; Sigma Aldrich) assay. For the MTT assay, a 100 μg / mL PBS-based MTT solution was applied to the cell culture for 2 hours, followed by washing with PBS. DMSO was applied to dissolve the precipitate, and the absorbance of the solution was measured at 570 nm.

[0433]

[0434] 15. GATA Reporter Genetic Analysis

[0435] HEK293T cells were transfected using PEI with the GATA reporter plasmid (0.45 μg), Renilla luciferase plasmid (0.1 μg), and TK promoter drive bin or GATA4 overexpression plasmid (0.45 μg). 24 hours after transfection, the medium was replaced with fresh medium containing 20 μM thiamet-G, 20 μM ST045849, or an equivalent volume of DMSO, and incubated for 48 hours. Cells were washed with PBS and lysed with passive lysis buffer. Reporter gene activity was measured using the Dual-Luciferase assay kit (Promega).

[0436]

[0437] 16. CHX Trace Analysis

[0438] To evaluate the effect of thiamet-G on GATA4 stability, HEK293T cells were transfected with a GATA4 overexpression plasmid (1 μg) using PEI. Six hours after transfection, the medium was replaced with fresh medium containing 20 μM thiamet-G or an equivalent volume of DMSO for 36 hours. To evaluate the effect of glucosamine on GATA4 stability, HEK293T cells were transfected with a GATA4 overexpression plasmid (0.5 μg) using PEI. Six hours after transfection, the medium was replaced with fresh medium containing 2 mM glucosamine sulfate or an equivalent volume of PBS for 36 hours. Then, the cells were treated with or not treated with 50 μM CHX for the indicated time prior to lysis. Protein stability was analyzed by fractionating 10 μg of cell lysate by SDS-PAGE.

[0439]

[0440] 17. Mapping of O-GlcNAcase Sites in GATA4 Using Electron Transfer Dissociation-Mass Spectrometry (ETD-MS)

[0441] The eluted GATA4 sample was subjected to electrophoresis, and the band corresponding to GATA4 was extracted by in-gel digestion. The extracted peptide was resuspended in solvent A (0.1% formic acid in water; ThermoFisher Scientific), and a C18 trap column (Acclaim PepMap100, ThermoFisher Scientific, 75 μm x 2 cm, 100 After loading into the C18 analysis column (PepMap RSLC, ThermoFisher Scientific, 75 μm x 15 cm, 100 Separation was performed in a 4-12% linear gradient of solvent B (0.1% formic acid in acetonitrile) at a flow rate of 300 nL / min for 50 minutes. Mass spectra were recorded using an Orbitrap Fusion Tribrid mass spectrometer (ThermoFisher Scientific) connected to an EASY-nanoLC (Proxeon Biosystems). Raw data were processed with the trans-proteomic pipeline (v4.8.0 PHILAE) and compared with the human GATA4 (P43694, UniProt) database using the SEQUEST HT search engine of Proteome Discoverer 2.2 (ThermoFisher Scientific). Carbamidomethylation of cysteine ​​(57 Da) was considered as a fixed variant, and the variable variants were set as oxidation of methionine (16 Da) and O-GlcNAcization of serine and threonine (203 Da).

[0442]

[0443] 18. SA-β-Gal staining

[0444] SA-β-Gal staining was performed using the method described in previous literature. Briefly, cultured human and mouse chondrocytes were washed twice with PBS and fixed for 5 minutes in a fixation solution containing 2% PFA and 0.2% glutaraldehyde. The fixed cells were washed and incubated in the SA-β-Gal staining solution at 37°C for 12–16 hours. After incubation, the cells were washed twice with PBS and mounted in a 50% glycerol solution. The stained cells were imaged using an Eclipse Ni-U microscope (Nikon). Total cells and SA-β-Gal positive cells were counted in three randomly selected fields per biological replicate.

[0445]

[0446] 19. 5-Bromo-2'-Deoxyuridine (BrdU) Cell Proliferation Analysis

[0447] C28 / I2 cells or primary culture mouse chondrocytes were cultured on coverslips of culture dishes. During treatment with doxorubicin (C28 / I2 cells) or bleomycin, or radiation (mouse chondrocytes), cultured cells were exposed to 10 μM BrdU for 12 hours prior to fixation. Fixation was performed using ethanol fixative (pH 2.0, 15 mM glycine, 70% ethanol) for 20 minutes at -20°C. Cells were blocked for 1 hour with 10% normal goat serum (Sigma Aldrich) and 0.1% BSA in PBS, and incubated overnight at 4°C with the primary antibody against BrdU (Santa Cruz, sc-32323). Cells were washed three times and incubated for 1 hour with rabbit anti-mouse IgG+IgM (H&L) conjugated with Alexa Fluor 488 (Jackson ImmunoResearch, 315-485-044). After antibody treatment, the nuclei were stained with DAPI. The stained human and mouse chondrocytes were mounted using ProLong Gold Antifade (ThermoFisher Scientific). The mounted slides were imaged using an EVOS M7000 imaging system (Invitrogen).

[0448]

[0449] 20. Quantitative Reverse Transcription-Polymerase Chain Reaction (qRT-PCR)

[0450] Total RNA was extracted from cells using TRI reagent (Molecular Research Center, Inc.) and reverse transcribed using EasyScript reverse transcriptase (TransGen Biotech) and oligo (dT) primers. To quantitatively analyze mRNA transcript levels, cDNA was amplified by qRT-PCR using Power SYBR Green PCR Master Mix (ThermoFisher Scientific) on a StepOnePlus real-time PCR system (Applied Biosystems). The primers used for qRT-PCR are listed in Table 2. For qRT-PCR-based quantification data comparing the effects of various treatments, Hprt was used as an internal control in each biologically independent trial, and the measurements of the treatment group were normalized to the measurements of the control group. For cell-based experiments, each primary culture set of chondrocytes was considered a biologically independent trial. The normalized values ​​of the multiple biologically independent trials were then averaged.

[0451] [Table 2]

[0452]

[0453]

[0454]

[0455] 21. RNA Sequencing Analysis

[0456] Mouse primary chondrocytes were treated with hyaluronidase IS (4 U / mL) for 4 hours under serum-free conditions prior to bleomycin treatment. Cells were treated with 50 μg / mL bleomycin or an equivalent dose of PBS in serum-supplemented medium for 24 hours. Cells were then washed twice with PBS and cultured for 5 days in fresh medium containing DMSO or 20 μM ST045849. Fresh medium was replenished every 48 hours. Three biological replicates were used for each experimental group. Total RNA was extracted using TRI reagents, and 1 μg of total RNA was converted into a cDNA library using the TruSeq Stranded mRNA Sample Prep Kit (Illumina) according to the TruSeq Standard mRNA Sample Preparation Guide (Part #15031047 Rev. E). Poly-adenylated RNA was purified using magnetic beads conjugated with oligo (dT) primers. Purified mRNA was fragmented and converted into first-strand cDNA using reverse transcriptase and random hexamer primers after the addition of actinomycin D. Second-strand cDNA was prepared by removing the RNA template and synthesizing a complementary strand in the presence of dUTP instead of dTTP. A single A base was added to the 3' end to enable sequence adapter ligation. The ligated cDNA was amplified by PCR, during which the polymerase stops upon encountering the U base, thereby making the second strand an inappropriate template. The final cDNA library was analyzed for size distribution, quantified using the DNA 1000 chip on an Agilent Technologies 2100 Bioanalyzer, and normalized to 2 nM for sequencing. RNA sequencing was performed using a NovaSeq 6000 (Illumina). All RNA-seq procedures were performed at Macrogen. Reads were trimmed with Trimmomatic and aligned to the reference genome Mus musculus (GRCm38) using a STAR aligner.Transcriptome assembly and abundance estimation were performed using StringTie v2.2.0. Aligned reads were assembled into known, novel, and alternative splicing transcriptomes, and the relative abundance of each transcriptome was quantified by the number of reads using StringTie v2.2.0. The statistical significance of differential expression was determined using the DESeq2 R package.

[0457]

[0458] 22. Bioinformatics Analysis of RNA-seq Data and Public Datasets

[0459] For GSEA, the gene lists from RNA-seq data were ranked based on the flip change (FC) values. Using GSEA software v4.2.2 in pre-ranking mode, the "Extracellular Matrix Degradation (Reactome)" and "Positive Regulation of Cytokine Production Involved in Inflammatory Response (GOBP)" gene sets were analyzed with all default parameters. Additionally, GSEA was performed on gene sets representing "genes upregulated in osteoarthritis," consisting of lists of 752 and 150 genes that were significantly upregulated in osteoarthritis. The GATA4 target gene sets used for GSEA were retrieved from the TRANSFAC Curated Transcription Factor Targets (https: / maayanlab.cloud / Harmonizome / gene_set / GATA4 / TRANSFAC+Curated+Transcription+Factor+Targets). Normalized enrichment scores (NES) and family-wise error rate (FWER) P-values ​​are presented in the GSEA figures. The algorithm used for Ingenuity Pathway Analysis (IPA; Qiagen Inc., https: / digitalinsights.qiagen.com / IPA) has been previously described. Differentially expressed genes that were significantly downregulated in RNA-seq data were identified as having a P < 0.01 and a fold change threshold < -1. The enriched P values ​​and activation z-scores of the top 20 standard pathway analyses are presented in a graph, and the pathways are listed in Table 3. Transcriptome data for osteoarthritis or osteoarthritis-related conditions in various in vitro and in vivo models were obtained from the GEO database.Transcriptomes of human osteoarthritis cartilage (GSE16464, GSE43923, GSE64394, GSE113825, GSE117999, GSE178557, and GSE186220), IL-1β-treated chondrocytes (GSE6119, GSE104793, and GSE163080), and cartilage from various osteoarthritis animal models (DMM surgery: GSE26475, GSE53857, GSE101573, and GSE143447; anterior cruciate ligament resection (ACLT) surgery: GSE110268; monoiodoacetate (MIA) injection: GSE28958) were analyzed.

[0460] [Table 3]

[0461]

[0462] 23. Statistical Analysis

[0463] All experiments were performed with at least three independent biological replicates. For in vitro experiments, comparisons between experimental groups were performed using the two-sided Student t-test, Dunnett's post-hoc test after one-way ANOVA, Tukey's post-hoc test after two-way ANOVA, or Dunnett's post-hoc test after two-way ANOVA. For in vivo experiments, each independent trial was performed using individual mice. Non-parametric tests based on the Mann-Whitney U test were used to determine statistical significance. Quantified data based on ordinal grading systems, such as OARSI grade, subosteosclerosis, osteophyte maturity, and synovial membrane inflammation, were analyzed using non-parametric methods because they are not continuous and do not follow a normal distribution. In other cases, the two-sided Student t-test or Bonferroni's post-hoc test after two-way ANOVA was applied. Separate statistical analyses were performed for the pseudo-surgery and DMM groups, so surgical conditions were not considered as variables. Statistical significance was set at P < 0.05. Statistical analysis was performed using IBM SPSS Statistics 25 or GraphPad Prism 10.0. All graphs and heatmaps were created using GraphPad Prism 10.0. Cell cultures and animals were randomly assigned to each experimental group, and all samples were evaluated in a blinded manner. The required sample size n for each group in the animal study was determined based on power analysis calculations (Equation (1) below) and the design of previous studies. No exclusion criteria were applied to this study.

[0464]

[0465] Here, D= , σ = standard deviation, α = significance level (0.05), and 1-β = power (0.90)

[0466]

[0467] 24. Data Availability

[0468] The original RNA-seq dataset generated in this study was stored in the GEO database under access number GSE275524.

[0469] Transcriptome data of osteoarthritis or osteoarthritis-related conditions in various in vitro and in vivo models were obtained from the GEO database. Human osteoarthritis cartilage (GSE16464, GSE43923, GSE64394, GSE113825, GSE117999, GSE178557, and GSE186220), IL-1β-treated chondrocytes (GSE6119, GSE104793, and GSE163080), and cartilage from various osteoarthritis animal models (DMM surgery: GSE26475, GSE53857, GSE101573, and GSE143447; ACLT surgery: GSE110268; MIA injection: GSE28958) were analyzed.

[0470]

[0471] The present disclosure is further explained by the following embodiments that do not limit the scope of the claims.

[0472] Embodiment 1. A composition for the prevention or treatment of osteoarthritis, comprising: a preparation that increases the expression or activity of a gene belonging to solute carrier family 35 (SLC35); a protein encoded by said gene; or an inhibitor that inhibits the accumulation of uridine diphosphate N-acetylglucosamine (UDP-GlcNAc) in the cytoplasm of chondrocytes.

[0473] Embodiment 2. A composition for preventing or treating osteoarthritis, wherein, in Embodiment 1, the protein encoded by the gene moves uridine diphosphate N-acetylglucosamine within the cytoplasm of a chondrocyte to the Golgi apparatus or endoplasmic reticulum.

[0474] Embodiment 3. A composition in Embodiment 1 or 2, wherein the gene is at least one selected from the group consisting of the SLC35A2 gene, SLC35A3 gene, SLC35A5 gene, SLC35B4 gene, SLC35D1 gene, and SLC35D2 gene.

[0475] Embodiment 4. A composition for the prevention or treatment of osteoarthritis, wherein in any one of Embodiments 1 to 3, the agent that increases the expression or activity of the gene is at least one selected from the group consisting of an antagonist small molecule compound, a vector, a plasmid, a viral vector, an adenovirus vector, an adeno-associated virus vector, a lentivirus vector, mRNA, mRNA wrapped in lipid nanoparticles (LNP), liposomes, and nanoparticles.

[0476] Embodiment 5. A composition in any one of Embodiments 1 to 4, wherein the composition is administered concurrently, separately, or sequentially with at least one of a GATA4 inhibitor; or an O-linked-N-acetylglucosaminylation transferase (O-GlcNAc transferase, OGT) inhibitor.

[0477] Example 6. A composition in which the GATA4 inhibitor of Example 5 is 2-(1,3-benzodioxol-5-ylmethyl)butanedioic acid, a salt thereof, a hydrate thereof, or a solvate thereof; Gata4siRNA composed of the nucleotide sequences of SEQ ID NO. 1 (5′-GAGCAAACCAGAGCCUAGAdTdT-3′) and SEQ ID NO. 2 (5′-UCUAGGCUCUGGUUUGCUCdTdT-3'); or Gata4siRNA composed of the nucleotide sequences of SEQ ID NO. 3 (5′-CAGAUGUUACUGAAUGCUUdTdT-3′) and SEQ ID NO. 4 (5′-AAGCAUUCAGUAACAUCUGdTdT-3′).

[0478] Example 7. A composition in any one of Examples 5 to 6, wherein the OGT inhibitor is 3-[2-(1-adamantyl)ethyl]-2-(4-chlorophenyl)imino-4-oxo-1,3-thiazinane-6-carboxylic acid, a salt thereof, a hydrate thereof, or a solvate thereof.

[0479] Embodiment 8. A composition in any one of Embodiments 1 to 7, wherein the osteoarthritis is post-traumatic osteoarthritis, inflammatory osteoarthritis, or degenerative osteoarthritis.

[0480] Example 9. A composition for diagnosing or predicting the prognosis of osteoarthritis, comprising: a preparation for measuring the expression level of a gene belonging to solute carrier family 35 (SLC35); a preparation for measuring the level of a protein encoded by said gene; or a preparation for measuring the level of uridine diphosphate N-acetylglucosamine.

[0481] Embodiment 10. A composition in Embodiment 9, wherein the gene is at least one selected from the group consisting of the SLC35A2 gene, SLC35A3 gene, SLC35A5 gene, SLC35B4 gene, SLC35D1 gene, and SLC35D2 gene.

[0482] Embodiment 11. A composition in any one of Embodiments 9 to 10, wherein the composition is treated to chondrocytes isolated from a subject, and i) the expression level of a gene belonging to SLC35 in the chondrocytes or the level of a protein encoded by said gene; or ii) the level of uridine diphosphate N-acetylglucosamine in the cytoplasm of said chondrocytes; is measured.

[0483] Embodiment 12. A composition in any one of Embodiments 9 to 11, wherein the preparation for measuring the expression level of a gene belonging to SLC35 is a primer pair, a probe, an antisense oligonucleotide, a CRISPR guide RNA, or an aptamer that specifically binds to the mRNA of the gene.

[0484] Embodiment 13. In any one of Embodiments 9 to 12, the preparation for measuring the level of the protein encoded by the gene is a composition comprising an antibody, aptamer, protein-binding peptide, protein-binding small molecule, nanobody, antibody fragment, or a functional derivative thereof that specifically binds to the protein.

[0485] Embodiment 14. A composition in any one of Embodiments 9 to 13, wherein the preparation for measuring the level of uridine diphosphate N-acetylglucosamine (UDP-GlcNAc) is an analytical reagent for a mass spectrometer, an enzyme, a chemical derivatization reagent based on a fluorescence or chromogenic reaction, an antibody that specifically binds to a nucleotide-sugar, or an aptamer that specifically binds to a nucleotide-sugar.

[0486] Embodiment 15. A composition for the prevention or treatment of osteoarthritis comprising at least one GATA4 inhibitor selected from the group consisting of 2-(1,3-benzodioxol-5-ylmethyl)butanedioic acid), its salt, its hydrate, or its solvate; or Gata4siRNA consisting of the nucleotide sequence of SEQ ID NO. 1 (5′-GAGCAAACCAGAGCCUAGAdTdT-3′) and SEQ ID NO. 2 (5′-UCUAGGCUCUGGUUUGCUCdTdT-3').

[0487]

[0488] Sequence No. 1 (Gata4siRNA1 sense strand): 5′-GAGCAAACCAGAGCCUAGAdTdT-3′

[0489] Sequence No. 2 (Gata4siRNA1 antisense strand): 5′-UCUAGGCUCUGGUUUGCUCdTdT-3'

[0490] Sequence No. 3 (Gata4siRNA1 sense strand): 5′-CAGAUGUUACUGAAUGCUUdTdT-3′

[0491] Sequence No. 4 (Gata4siRNA1 antisense strand): 5′-AAGCAUUCAGUAACAUCUGdTdT-3′

[0492] Sequence numbers 5 to 74 of Table 2

Claims

A composition for the prevention or treatment of osteoarthritis, comprising: a preparation that increases the expression or activity of a gene belonging to solute carrier family 35 (SLC35); a protein encoded by said gene; or an inhibitor that inhibits the accumulation of uridine diphosphate N-acetylglucosamine (UDP-GlcNAc) in the cytoplasm of chondrocytes. A composition for the prevention or treatment of osteoarthritis according to claim 1, wherein the protein encoded by the gene moves uridine diphosphate N-acetylglucosamine within the cytoplasm of chondrocytes to the Golgi apparatus or endoplasmic reticulum. A composition according to claim 1, wherein the gene is at least one selected from the group consisting of the SLC35A2 gene, SLC35A3 gene, SLC35A5 gene, SLC35B4 gene, SLC35D1 gene, and SLC35D2 gene. A composition for the prevention or treatment of osteoarthritis according to claim 1, wherein the agent that increases the expression or activity of the gene is at least one selected from the group consisting of an antagonist small molecule compound, a vector, a plasmid, a viral vector, an adenovirus vector, an adeno-associated virus vector, a lentivirus vector, mRNA, mRNA wrapped in lipid nanoparticles (LNP), liposomes, and nanoparticles. The composition of claim 1, wherein the composition is administered simultaneously, separately, or sequentially in combination with at least one of a GATA4 inhibitor; or an O-linked-N-acetylglucosaminylation transferase (O-GlcNAc transferase, OGT) inhibitor. A composition according to claim 5, wherein the GATA4 inhibitor is 2-(1,3-benzodioxol-5-ylmethyl)butanedioic acid, its salt, its hydrate, or its solvate; Gata4siRNA consisting of the nucleotide sequences of SEQ ID NO. 1 (5′-GAGCAAACCAGAGCCUAGAdTdT-3′) and SEQ ID NO. 2 (5′-UCUAGGCUCUGGUUUGCUCdTdT-3'); or Gata4siRNA consisting of the nucleotide sequences of SEQ ID NO. 3 (5′-CAGAUGUUACUGAAUGCUUdTdT-3′) and SEQ ID NO. 4 (5′-AAGCAUUCAGUAACAUCUGdTdT-3′). A composition according to claim 5, wherein the OGT inhibitor is 3-[2-(1-adamantyl)ethyl]-2-(4-chlorophenyl)imino-4-oxo-1,3-thiazinane-6-carboxylic acid, a salt thereof, a hydrate thereof, or a solvate thereof. A composition according to claim 1, wherein the osteoarthritis is post-traumatic osteoarthritis, inflammatory osteoarthritis, or degenerative osteoarthritis. A composition for diagnosing or predicting the prognosis of osteoarthritis, comprising: a preparation for measuring the expression level of a gene belonging to solute carrier family 35 (SLC35); a preparation for measuring the level of a protein encoded by said gene; or a preparation for measuring the level of uridine diphosphate N-acetylglucosamine. A composition according to claim 9, wherein the gene is at least one selected from the group consisting of the SLC35A2 gene, SLC35A3 gene, SLC35A5 gene, SLC35B4 gene, SLC35D1 gene, and SLC35D2 gene. A composition according to claim 9, wherein the composition is treated to chondrocytes isolated from a subject and measures i) the expression level of a gene belonging to SLC35 in said chondrocytes or the level of a protein encoded by said gene; or ii) the level of uridine diphosphate N-acetylglucosamine in the cytoplasm of said chondrocytes. In claim 9, the composition wherein the agent for measuring the expression level of the gene belonging to the SLC35 is a primer pair, a probe, an antisense oligonucleotide, a CRISPR guide RNA, or an aptamer that specifically binds to the mRNA of the gene. In claim 9, the preparation for measuring the level of the protein encoded by the said gene is a composition that is an antibody, aptamer, protein-binding peptide, protein-binding small molecule, nanobody, antibody fragment, or a functional derivative thereof that specifically binds to said protein. In claim 9, the composition wherein the preparation for measuring the level of uridine diphosphate N-acetylglucosamine (UDP-GlcNAc) is an analytical reagent for mass spectrometry, an enzyme, a chemical derivatization reagent based on fluorescence or chromogenic reaction, an antibody that specifically binds to a nucleotide-sugar, or an aptamer that specifically binds to a nucleotide-sugar. A composition for the prevention or treatment of osteoarthritis comprising at least one GATA4 inhibitor selected from the group consisting of 2-(1,3-benzodioxol-5-ylmethyl)butanedioic acid), its salt, its hydrate, or its solvate; or Gata4siRNA consisting of the nucleotide sequence of SEQ ID NO. 1 (5′-GAGCAAACCAGAGCCUAGAdTdT-3′) and SEQ ID NO. 2 (5′-UCUAGGCUCUGGUUUGCUCdTdT-3').

Images