Application of targeted H3K18la-AKR1C2 axis drug combination in preparation of drug for improving TNFi drug resistance

Abstract

The invention relates to the technical field of biological medicines, and discloses an application of a targeted H3K18la-AKR1C2 axis drug combination in preparation of a drug for improving TNFi drug resistance. The drug combination comprises a first active component and a second active component, the first active component is an inhibitor targeting H3K181a and / or an inhibitor targeting AKR1C2, and the second active component is TNFi. The drug combination can effectively improve the secondary TNFi drug resistance of patients with refractory rheumatoid arthritis by inhibiting the anti-apoptosis phenotype of rheumatoid arthritis fibroblast-like synovial cells and reducing the TNF-beta secretion level. The invention further provides a pharmaceutical composition containing the active ingredients and pharmaceutically acceptable auxiliary materials, the dosage form can be selected from an injection, an oral preparation or an external preparation, the administration mode is combined administration or sequential administration, a new strategy is provided for precise treatment of TNFi drug resistance, and the treatment blank in the field of regulation and control of TNFi drug resistance by a metabolism-epigenetic axis is filled.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, and in particular relates to the application of combined drug therapy targeting the H3K18la-AKR1C2 axis in the preparation of drugs that improve TNFi resistance. Background Technology

[0002] Rheumatoid arthritis (RA) is an autoimmune disease based on chronic inflammation. Although the use of biologics and small molecule targeted drugs has significantly improved the treatment landscape for RA, some patients still do not respond well to multiple antirheumatic drugs, including biologics. These patients are referred to as Difficult-to-treat rheumatoid arthritis (D2T-RA).

[0003] The treatment bottleneck for D2T-RA is closely related to drug resistance, especially secondary resistance to tumor necrosis factor-alpha inhibitors (TNFi). TNFi are the first-line drugs for RA patients who have failed conventional synthetic disease-modifying antirheumatic drugs (csDMARDs). In clinical applications, approximately 20% of patients experience loss of efficacy after 24 weeks of treatment, and this proportion can rise to 30%-40% in long-term follow-up. Drug resistance not only leads to drug failure and continued disease progression, increasing the incidence of joint deformities by 3-5 times and reducing quality of life scores by more than 40%, but also causes a surge in medical costs due to repeated changes in treatment regimens—the average annual treatment cost for D2T-RA patients is 2-3 times higher than that for ordinary RA patients, which has become a pressing clinical challenge that needs to be overcome.

[0004] However, current research on TNFi resistance mechanisms, both domestically and internationally, is still in its early stages, and the underlying mechanisms remain unclear, severely hindering the development of precision treatment strategies for D2T-RA. Existing treatments still rely on a trial-and-error approach, lacking targeted interventions based on resistance mechanisms. Only about 50% of resistant patients achieve partial remission after switching medications, and there is currently no clear strategy to reverse existing TNFi resistance.

[0005] Metabolic-epigenetic interactions play a central role in immune regulation and drug resistance. Fibroblast-like synoviocytes (FLS) and immune cells in rheumatoid arthritis (RA) are under high metabolic stress, creating a lactate-rich microenvironment. Lactate, as a histone modification substrate, participates in the regulation of immune function and mediates drug resistance through the metabolic-epigenetic axis. However, how energy metabolism regulates the expression of drug resistance proteins through epigenetic remodeling remains unclear. More importantly, there are currently no targeted reversal strategies targeting the metabolic-epigenetic axis in clinical practice. This research gap provides a crucial target for breakthroughs in the treatment of D2T-RA and highlights the urgent clinical need to explore the mechanism by which this pathway regulates TNFi resistance. Summary of the Invention

[0006] The purpose of this invention is to overcome the shortcomings of existing technologies, such as the unclear mechanism of TNFi resistance and the lack of precise targeted intervention strategies for the resistance mechanism. This invention provides an application of combined drug therapy targeting the H3K18la-AKR1C2 axis in the preparation of drugs to improve TNFi resistance. Based on the core mechanism of lactate-mediated metabolic-epigenetic interaction regulating TNFi resistance, this invention clarifies that lactate can regulate histone H3K18 lactation (H3K18la), further regulating AKR1C2 expression and mediating abnormalities in TNFβ-related signaling pathways, ultimately triggering the lactate-H3K18la-AKR1C2-TNFβ-resistance axis regulatory mechanism. Using this as a target, a combined drug therapy regimen is designed to achieve precise reversal of TNFi resistance in D2T-RA patients, providing a highly efficient targeted synergistic treatment plan. This fills the clinical treatment gap in regulating TNFi resistance through the metabolic-epigenetic axis and provides a new technical pathway for precise stratified treatment of D2T-RA.

[0007] This invention provides the application of combined drug therapy targeting the H3K18la-AKR1C2 axis in the preparation of drugs that improve TNFi resistance.

[0008] Furthermore, the combination therapy comprises a first active ingredient and a second active ingredient; the first active ingredient is an inhibitor targeting H3K18la and / or an inhibitor targeting AKR1C2, and the second active ingredient is TNFi.

[0009] Furthermore, the improvement of TNFi resistance is for patients with refractory rheumatoid arthritis, wherein the TNFi resistance is secondary TNFi resistance.

[0010] Furthermore, the inhibitors targeting H3K18la are histone lactation modification inhibitors, including dichloroacetic acid, lactate dehydrogenase inhibitors, mitochondrial function regulators, and other small molecule compounds that can reduce the level of H3K18la modification.

[0011] Furthermore, the inhibitor targeting AKR1C2 is selected from AKR1C2 gene silencing agents, AKR1C2 protein activity inhibitors, and AKR1C2 antibodies, specifically including siRNA, shRNA, glycyrrhizin-based small molecule inhibitors, or anti-AKR1C2 monoclonal antibodies.

[0012] Furthermore, the inhibitor targeting H3K18la includes dichloroacetic acid, and the inhibitor targeting AKR1C2 includes glycyrrhizin.

[0013] Furthermore, the combined medication improves TNFi resistance by inhibiting the anti-apoptotic phenotype of RA-FLS cells and reducing TNF-β secretion levels.

[0014] Furthermore, the administration method of the combined medication is either combined administration or sequential administration.

[0015] The present invention also provides a pharmaceutical composition for improving TNFi resistance, comprising the first active ingredient, the second active ingredient, and pharmaceutically acceptable excipients described above.

[0016] Furthermore, the dosage form of the pharmaceutical composition is an injection, an oral preparation, or a topical preparation.

[0017] The present invention also provides a pharmaceutical composition for improving TNFi resistance, comprising the first active ingredient, the second active ingredient, and pharmaceutically acceptable excipients described above.

[0018] Compared with the prior art, the beneficial effects of the present invention are:

[0019] 1. Clarifying the core mechanism of drug resistance and filling a technological gap: This invention is the first to systematically reveal the molecular mechanism of secondary TNFi resistance in D2T-RA patients mediated by the "lactate-H3K18la-AKR1C2 axis". It clarifies that H3K18la specifically regulates AKR1C2 transcription and that AKR1C2 drives drug resistance through a dual pathway of "abnormal TNF-β secretion + RA-FLS anti-apoptosis". This solves the core pain point of the existing technology in not clearly explaining the mechanism of TNFi resistance and provides a clear target direction for drug resistance intervention.

[0020] 2. Synergistic Effects Reversing Drug Resistance and Significant Therapeutic Efficacy: The combined treatment regimen of "H3K18la inhibitor / AKR1C2 inhibitor + TNFi" achieves multi-target synergistic effects—inhibiting the epigenetic activation of AKR1C2 by H3K18la and directly blocking the AKR1C2-mediated drug resistance pathway. Compared to TNFi alone, it significantly reduces the proliferation, migration, and invasion of RA-FLS, decreases TNF-β secretion, and reverses the anti-apoptotic phenotype. Animal experiments have confirmed that this combined regimen effectively improves joint swelling and reduces bone destruction. Clinical sample data show that it can restore the sensitivity of D2T-RA patients to TNFi treatment, overcoming the limitations of existing single-target TNF-α therapies.

[0021] 3. Strong targeting and enhanced safety: This invention focuses on the unique "abnormal activation of the lactate-H3K18la-AKR1C2 axis" characteristic of D2T-RA patients. The selected inhibitors have been verified through cell and animal experiments to specifically target drug resistance-related pathways with minimal impact on normal synovial cells and body metabolism. Compared to the traditional "trial and error" approach of changing drugs, this method is more precise, reduces unnecessary drug exposure, lowers the risk of adverse reactions, and improves the safety and tolerability of clinical treatment.

[0022] 4. Facilitating Precision Stratified Treatment and Reducing Medical Costs: The H3K18la, AKR1C2, and lactate levels involved in this invention are all detectable biomarkers that can be used to screen high-risk individuals for TNFi resistance in advance, providing a basis for developing individualized combination therapy regimens in clinical practice. This characteristic avoids treatment failure and waste of medical resources caused by the indiscriminate use of TNFi, promoting the transformation of D2T-RA treatment from experience-based trial and error to precise stratification, significantly reducing the risk of patient disability and the social medical burden. Attached Figure Description

[0023] Figure 1 A clinical sample validation diagram of lactate metabolism and protein lactation levels in patients with rheumatoid arthritis (RA); Figure 1 The graph in center A shows a comparison of lactate levels between patients with osteoarthritis (OA) and rheumatoid arthritis (RA). Figure 1 Image B shows the immunoblotting detection and quantitative analysis of pan-lactated protein (PAN-kla) in the synovial tissue of the trauma control group (TC) and RA patients. Figure 1 The middle image (C) shows the pathological morphology of the synovial tissue stained with hematoxylin and eosin (HE) in patients with TC and RA. Figure 1 In the middle D, the immunohistochemical staining images of pan-lactated protein in the synovial tissue of patients with TC and RA are shown; *p<0.05, p<0.01, *p<0.001, the same below;

[0024] Figure 2This is an in vivo experimental verification diagram of how dichloroacetic acid (DCA) improves the arthritis phenotype in a rat model of collagen-induced arthritis (CIA). Figure 2 The diagram in section A is a schematic diagram of the modeling, drug administration, and experimental cycle of the CIA model rats; Figure 2 Figure B shows the statistical analysis of the changes in arthritis scores over time in rats in the control group (Ctrl), CIA model group, DCA treatment group, and rotenone (ROT) treatment group. Figure 2 The graph in middle C is a statistical analysis of the changes in the thickness of the rat paw pads over time in each group; Figure 2 The middle D shows the appearance of the paws of rats in each group on day 21 (d21) and day 41 (d41) after modeling, as well as a comparison of the bone structure of the paws detected by Micro-CT imaging.

[0025] Figure 3 This image serves as a validation diagram of the pathological findings of synovial tissue and the expression of pan-lactated protein in a rat model of collagen-induced arthritis (CIA) induced by dichloroacetic acid (DCA). Figure 3 In the middle A, the pathological morphology of the synovial tissue of rats in the control group (Ctrl), CIA model group, DCA treatment group and rotenone (ROT) treatment group is obtained by hematoxylin-eosin (HE) staining. Figure 3 Image B shows the morphology of cartilage matrix stained with Safranin O-Fix Green in the synovial tissue of rats in each group; Figure 3 Figure C shows the immunoblotting detection and quantitative analysis of pan-lactated protein (PAN-kla) in the synovial tissue of rats in each group;

[0026] Figure 4 This is a verification diagram showing the synergistic effect of lactate (LA) and TNF-α and the effect of dichloroacetic acid (DCA) intervention on the function of fibroblast-like synovial cells (RA-FLS) in rheumatoid arthritis. Figure 4 Image A in the middle shows representative images of scratch healing, cell migration, and invasion experiments of RA-FLS in different treatment groups; Figure 4 Figure B shows a quantitative analysis of the RA-FLS scratch width at different time points for each group. Figure 4 The graph in C is a quantitative analysis of the migration and invasion capabilities of RA-FLS in each group;

[0027] Figure 5 This figure serves as a validation of the synergistic effect of lactate (LA) and TNF-α and the influence of dichloroacetic acid (DCA) intervention on the expression of inflammatory factors and histone lactation modification in fibroblast-like synovial cells (RA-FLS) of rheumatoid arthritis. Figure 5 Figure A shows the quantitative analysis of IL-1 and IL-6 mRNA expression in RA-FLS from different treatment groups; Figure 5 Figure B shows the immunoblotting detection and quantitative analysis of RA-FLS pan-lactated protein (PAN-kla) and histone H3K18 lactation (H3K18la) in different treatment groups.

[0028] Figure 6 Immunoblotting detection and quantitative analysis of multiple histone lactation modification sites in synovial tissue of trauma control group (TC) and rheumatoid arthritis (RA) patients;

[0029] Figure 7 This diagram validates the expression of histone H3K18 lactation (H3K18la) in clinical samples, animal models, and cell experiments. Figure 7 Image A shows the immunohistochemical staining of H3K18la synovial tissue from the trauma control group (TC) and patients with rheumatoid arthritis (RA). Figure 7 Image B shows the immunoblotting and quantitative analysis of H3K18la in the synovial tissue of rats in the control group (Ctrl), collagen-induced arthritis (CIA) model group, dichloroacetic acid (DCA) treatment group, and rotenone (ROT) treatment group. Figure 7 In the middle C, the immunoblotting detection and quantitative analysis of H3K18la fibroblast-like synovial cells (RA-FLS) in different treatment groups of rheumatoid arthritis are shown.

[0030] Figure 8 This is a combined analysis of the effects of TNF-α combined with lactate (LA) treatment on the binding mode of histone H3K18 lactation (H3K18la) and the transcriptome in rheumatoid arthritis fibroblast-like synovial cells (RA-FLS). Figure 8 In the middle A, the heatmap and density peak diagram of the binding abundance of H3K18la in the transcription start site (TSS) region of RA-FLS in the TNF-α group and TNF-α+LA group are obtained by CUT&Tag experiment. Figure 8 In Figure B, the RNA-seq volcano plot of differentially expressed RA-FLS genes after TNF-α+LA treatment is shown.

[0031] Figure 9 A diagram illustrating the screening and validation of target genes regulated by histone H3K18 lactation (H3K18la). Figure 9 In the middle A section, there is a Venn diagram integrating the results of CUT&Tag experiments and RNA-seq, as well as a list of core target genes obtained through screening. Figure 9 Figure B shows the qPCR verification of AKR1C2, C3AR1, and SLC2A12 gene mRNA expression in fibroblast-like synovial cells (RA-FLS) of rheumatoid arthritis in the TNF-α group and the TNF-α+lactic acid (LA) group. Figure 9 Figure C shows the Western blot (WB) detection and quantitative analysis of AKR1C2 protein expression in the two RA-FLS groups;

[0032] Figure 10 IGV genome browser validation diagram for histone H3K18 lactation binding signal in the promoter region of target genes;

[0033] Figure 11 This is a clinical validation graph showing the correlation between lactate and AKR1C2 levels and resistance to TNF inhibitors (TNFi) in D2T-RA patients. Figure 11 Figure A shows a comparative analysis of plasma lactate levels between non-D2T-RA patients and D2T-RA patients. Figure 11 Figure B shows a comparative analysis of plasma AKR1C2 protein levels in the two groups of patients. Figure 11 The bar chart in the middle (C) shows the resistance rates of various antirheumatic drugs (MTX, TNFi, IL6i, TOF) at different AKR1C2 expression levels. Figure 11 The figure in D is a comparative analysis of synovial fluid lactate levels between non-D2T-RA patients and TNFi-resistant D2T-RA patients; Figure 11 Figure E shows a comparative analysis of AKR1C2 protein levels in the synovial fluid of the two groups of patients.

[0034] Figure 12 A model for predicting the risk of resistance to TNF inhibitors (TNFi) in D2T-RA patients and a validation diagram of synovial tissue. Figure 12 In the middle section, A is a nomogram constructed by integrating indicators such as VAS score, lactate level, and AKR1C2 level, which is used to quantitatively predict the risk of TNFi resistance in D2T-RA patients; Figure 12 Image B shows a comparison of immunohistochemical staining of histone H3K18 lactation (H3K18la) and AKR1C2 protein in the synovial tissue of TNFi-resistant D2T-RA patients and ordinary D2T-RA patients.

[0035] Figure 13 A graph validating the levels of inflammatory factors and AKR1C2 function in D2T-RA patients; Figure 13 Figure A shows a comparative analysis of TNF-β levels in plasma and synovial fluid between non-D2T-RA patients and D2T-RA patients resistant to TNF inhibitors (TNFi). Figure 13 Figure B shows a comparative analysis of TNF-α levels in plasma and synovial fluid between the two groups of patients. Figure 13 Image C represents the immunoblotting detection and quantitative analysis of AKR1C2, BCL2, and BAX protein expression in rheumatoid arthritis fibroblast-like synovial cells (RA-FLS) after AKR1C2 silencing (si-AKR1C2). Figure 13 Figure D shows the quantitative analysis of AKR1C2, TNF-α, and TNF-β gene mRNA expression in RA-FLS after AKR1C2 silencing. Detailed Implementation

[0036] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0037] Example 1: Verification of Lactate Accumulation and Abnormal Activation of Pan-Lactation Modification in the Synovial Microenvironment of RA Patients

[0038] 1.1 Experimental Methods

[0039] Sample collection: Patients with osteoarthritis (OA), rheumatoid arthritis (RA), and trauma control (TC) were included. Joint fluid (100 cases each in the OA group and RA group) and synovial tissue (10 cases each in the OA group, RA group, and TC group) were collected. All samples were ethically approved and informed consent was obtained from the patients.

[0040] Sample processing: The joint fluid was centrifuged to obtain the supernatant, flash-frozen in liquid nitrogen, and stored at -80℃; the synovial tissue was divided into three parts, which were used for paraformaldehyde fixation, protein extraction, and preparation for later use.

[0041] Lactate level detection: The lactate test kit was used, and the absorbance value was detected by an enzyme-linked immunosorbent assay (ELISA) reader according to the instructions. The lactate concentration in the synovial fluid was calculated based on the standard curve.

[0042] Detection of pantothenic acid modification (PAN-kla):

[0043] Western blot analysis: Total protein was extracted from synovial tissue, and after BCA quantification, SDS-PAGE electrophoresis, membrane transfer, and blocking were performed. The membrane was incubated with PAN-kla primary antibody at 4°C overnight, and then with secondary antibody at room temperature for 1 hour. The membrane was then analyzed by ECL chemiluminescence method.

[0044] IHC detection: Paraffin sections of synovial tissue were dewaxed to water, antigen was repaired, and then incubated with PAN-kla primary antibody. DAB staining was performed, followed by hematoxylin counterstaining, and the sections were mounted with neutral resin. The sections were observed under a microscope and scored according to the percentage of positive cells and the intensity of staining.

[0045] 1.2 Experimental Results

[0046] Compared with the OA group, the lactate level in the synovial fluid of RA patients was significantly higher ( Figure 1 A) suggests abnormal accumulation of lactic acid metabolism in the synovial microenvironment of RA; compared with the TC group, the PAN-kla level in the synovial tissue of RA patients was significantly increased ( Figure 1 (BD) confirmed the abnormal activation of synovial lactation modification in RA patients, establishing a preliminary association between metabolic abnormalities and epigenetic modification activation.

[0047] Example 2: In vitro and in vivo verification of lactation modification-mediated RA inflammation amplification and joint bone destruction

[0048] 2.1 Experimental Methods

[0049] (1) Cell experiments:

[0050] Cell culture: RA fibroblast-like synovial cells (RA-FLS) were isolated and cultured in a medium containing 10% fetal bovine serum and incubated at 37°C in a 5% CO2 incubator.

[0051] Grouping treatment: control group, lactate alone group (10mM), TNF-α alone group (10ng / mL), lactate + TNF-α combination group, lactate + TNF-α + DCA group (20mM, H3K18la inhibitor).

[0052] Functional assays: Scratch assay and Transwell assay were used to detect cell migration and invasion capabilities; CCK8 assay and EdU assay were used to detect cell proliferation capabilities; RT-qPCR was used to detect the expression levels of IL-1β and IL-6 mRNA; and Western blotting was used to detect PAN-kla expression.

[0053] (2) Animal experiments:

[0054] Model establishment: SPF-grade male Wistar rats were used to establish a collagen-induced arthritis (CIA) model by intradermal injection of bovine type II collagen and a complete Freund's adjuvant emulsion mixture, and boosted immunization on day 7.

[0055] Grouped administration: The patients were divided into a control group, a CIA model group, a DCA treatment group (200 mg / kg / d by gavage), and a ROT treatment group (10 mg / kg / d by gavage, H3K18la promoter), and were administered the drugs continuously for 21 days.

[0056] Indicator testing: Arthritis scores and footpad thickness were measured every 3 days; at the experimental endpoint, Micro-CT scans were performed to assess bone destruction, and HE staining and Safranin-Fix-Green staining were used to assess joint pathological damage; Western blotting was used to detect H3K18la levels in synovial tissue (see experimental procedure). Figure 2 A).

[0057] 2.2 Experimental Results

[0058] Cellular experiments: Lactic acid alone can promote RA-FLS migration, proliferation and invasion, and upregulate PAN-kla levels; combined treatment with lactate and TNF-α further amplifies the cell activation phenotype and significantly activates IL-1β and IL-6 mRNA transcription; DCA can completely inhibit this synergistic effect. Figure 4 , Figure 5 ).

[0059] Animal experiments: DCA gavage significantly reduced H3K18la levels in synovial tissue and improved arthritis scores, joint swelling, and bone destruction in the CIA model; ROT treatment group showed increased H3K18la levels and significantly worsened arthritis symptoms. Figure 2 BD Figure 3 AC).

[0060] Conclusion: Lactic acid directly drives the amplification of RA inflammation and joint destruction by regulating lactation modification, confirming that this abnormality is a key link in the pathogenesis of RA.

[0061] Example 3 Screening of histone lactation modification sites: Validation of specific enrichment of H3K18la in RA

[0062] 3.1 Experimental Methods

[0063] Sample preparation: Synovial tissue was collected from RA patients and TC group, synovial tissue from CIA model, and RA-FLS.

[0064] Western blot (WB) detection: Total protein was extracted from each sample, quantified by BCA, and then subjected to electrophoresis, membrane transfer, and blocking. Specific antibodies for each protein lactation site (H2BK16la, H3K9la, H3K14la, H3K16la, H3K18la, H4K12la) were added and incubated overnight at 4°C. After secondary antibody incubation, the membrane was developed, and the expression differences at different sites were analyzed.

[0065] IHC detection: H3K18la staining was performed on synovial tissue sections from RA patients and TC group, and expression localization and intensity were observed under a microscope and semi-quantitative scoring was performed.

[0066] 3.2 Experimental Results

[0067] The level of H3K18la in RA synovial tissue was significantly elevated, with a much larger variation than at other histone lactation sites. Figure 6 In the synovial tissue of the CIA model and in RA-FLS, the H3K18la modification level was also significantly upregulated ( Figure 7 AC), identifying H3K18la as a key epigenetic regulatory molecule for RA.

[0068] Example 4: Verification of the transcriptional regulatory mechanism of H3K18la targeting and binding to the AKR1C2 promoter.

[0069] 4.1 Experimental Methods

[0070] Cell treatment: RA-FLS cells were treated with a combination of TNF-α (10 ng / mL) and lactate (10 mM) and then collected for subsequent experiments.

[0071] CUT & Tag Detection: Relying on Wuhan Fraser Gene Information Co., Ltd., cell nuclei were isolated and bound to magnetic beads. H3K18la primary antibody, secondary antibody and pA-Tn5 transposase were added. After enzyme digestion, DNA was extracted, amplified by PCR and sequenced.

[0072] RNA-seq detection: Total RNA was extracted from processed cells, rRNA was removed and fragmented, cDNA library was constructed by reverse transcription, and differentially expressed genes were analyzed by high-throughput sequencing.

[0073] Verification experiment:

[0074] ChIP-qPCR: Detection of the binding of H3K18la to the AKR1C2 promoter.

[0075] WB and RT-qPCR were used to detect the mRNA and protein expression of AKR1C2 in the control group, TNF-α group, lactate group, and TNF-α+lactic acid group.

[0076] 4.2 Experimental Results

[0077] CUT&Tag showed that H3K18la was enriched in the TSS region of the gene ( Figure 8 A); RNA-seq screened differentially expressed genes ( Figure 8 B); Combined CUT&Tag and RNA-seq analysis screened 19 differentially expressed genes directly regulated by H3K18la. H3K18la specifically binds to the promoter regions of AKR1C2, C3AR1, and SLC2A12 genes. Combined treatment with lactate and TNF-α significantly upregulated the mRNA and protein expression of AKR1C2. Figure 9 AC), and the upregulation was the most significant. IGV verified the binding signal of H3K18la to the AKR1C2 promoter ( Figure 10 This reveals the "lactic acid-H3K18la-AKR1C2" regulatory axis.

[0078] Example 5: Validation of lactate-H3K18la-AKR1C2 axis-specific activation and drug resistance association in D2T-RA patients.

[0079] 5.1 Experimental Methods

[0080] Sample collection: D2T-RA patients and non-D2T-RA patients were included. Plasma and synovial fluid samples were collected from 100 patients and synovial tissue samples from 10 patients respectively. The patients' response to TNFi treatment was recorded.

[0081] Indicator Testing:

[0082] Lactate levels: The concentration of lactate in plasma and synovial fluid was detected by LC-MS.

[0083] AKR1C2 levels: AKR1C2 protein concentration in plasma and synovial fluid was detected by ELISA; AKR1C2 mRNA expression in synovial tissue was detected by RT-qPCR.

[0084] H3K18la level: The expression level of H3K18la in synovial tissue was detected by Western blotting and IHC, and its co-expression with AKR1C2 was analyzed.

[0085] Drug resistance prediction model construction: Integrating lactate concentration, H3K18la modification abundance, AKR1C2 level and clinical indicators, a multi-factor nomogram model was constructed.

[0086] 5.2 Experimental Results

[0087] The concentrations of lactate and AKR1C2 in plasma and synovial fluid of D2T-RA patients were significantly higher than those in the non-D2T-RA group, and the plasma AKR1C2 level was positively correlated with the TNFi resistance rate. Figure 11 AE).

[0088] The levels of H3K18la and AKR1C2 in the synovial tissue of D2T-RA were significantly higher than those in the non-D2T-RA group, and their co-expression was significantly enhanced. Figure 12 B).

[0089] The constructed nomogram model can effectively predict the resistance risk of D2T-RA to TNFi. Figure 12 A) confirmed that the "lactic acid-H3K18la-AKR1C2 axis" is directly related to the clinical drug resistance phenotype.

[0090] Example 6: Validation of the dual mechanism of AKR1C2-mediated TNFi resistance

[0091] 6.1 Experimental Methods

[0092] (1) Cell line construction:

[0093] Knockdown cell line: RA-FLS was transfected with siRNA, and the AKR1C2 silencing efficiency was verified by RT-qPCR and WB 48 hours later.

[0094] Overexpression cell lines: RA-FLS cells were infected with lentivirus, and fluorescence expression was observed under a fluorescence microscope. Stable cell lines were selected using puromycin.

[0095] (2) Mechanism verification:

[0096] Inflammatory factor detection: ELISA and RT-qPCR were used to detect the expression of TNF-α and TNF-β in the AKR1C2 knockdown / overexpression group and the combined TNFi group.

[0097] Apoptosis detection: Western blot was used to detect the expression of Bcl-2 and BAX proteins; TUNEL staining and Annexin V-FITC / PI flow cytometry were used to detect the apoptosis rate.

[0098] TNF-β intervention experiment: TNFi was added after different concentrations of TNF-β were used to treat RA-FLS. The sensitivity of cells to TNFi was detected by CCK8 and EdU assays.

[0099] (3) In vivo verification:

[0100] CIA model grouping: divided into CIA group, CIA+TNFi group, CIA+TNFi+DCA group, and CIA+TNFi+AKR1C2 inhibitor (Liquiritin) group.

[0101] Indicator testing: assess arthritis score, joint swelling and bone destruction; detect the levels of inflammatory factors in serum and synovial tissue.

[0102] 6.2 Experimental Results

[0103] In TNFi-resistant D2T-RA patients, the levels of TNF-α and TNF-β in plasma and synovial fluid were elevated, with the increase in TNF-β being significantly greater than that in TNF-α. Figure 13 AB).

[0104] After knocking down AKR1C2, the decrease in TNF-β mRNA was much greater than that of TNF-α, Bcl-2 expression decreased, BAX expression increased, and the anti-apoptotic phenotype of RA-FLS was reversed. Figure 13 CD).

[0105] Exogenous TNF-β can reduce the sensitivity of RA-FLS to TNFi; the combined use of DCA or AKR1C2 inhibitors with TNFi is more effective than TNFi alone in treating CIA models.

[0106] Conclusion: AKR1C2 drives TNFi resistance through a dual mechanism: on the one hand, it promotes abnormal TNF-β secretion, forming an inflammatory escape pathway that TNFi cannot target, and continuously amplifying the synovial inflammatory cascade; on the other hand, it regulates the Bcl-2 / BAX apoptosis balance, enhancing the anti-apoptotic ability of RA-FLS cells and maintaining their abnormal cell survival advantage, ultimately leading to TNFi treatment failure. This mechanism systematically reveals the core molecular mechanism of metabolic-epidemiological interaction regulating TNFi resistance in D2T-RA patients.

[0107] It will be apparent to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above, and that the invention can be implemented in other specific forms without departing from the spirit or essential characteristics of the invention. Therefore, the embodiments should be considered in all respects as exemplary and non-limiting, and the scope of the invention is defined by the appended claims rather than the foregoing description. Thus, it is intended that all variations falling within the meaning and scope of equivalents of the claims be included within the present invention.

Claims

1. Application of combination therapy targeting the H3K18la-AKR1C2 axis in the preparation of drugs to improve TNFi resistance.

2. The application according to claim 1, characterized in that, The combination therapy comprises a first active ingredient and a second active ingredient; the first active ingredient is an inhibitor targeting H3K18la and / or an inhibitor targeting AKR1C2, and the second active ingredient is TNFi.

3. The application according to claim 1, characterized in that, The improvement of TNFi resistance is for patients with refractory rheumatoid arthritis, and the TNFi resistance is secondary TNFi resistance.

4. The application according to claim 2, characterized in that, The inhibitors targeting H3K18la are histone lactation modification inhibitors, including dichloroacetic acid, lactate dehydrogenase inhibitors, mitochondrial function regulators, and other small molecule compounds that can reduce the level of H3K18la modification.

5. The application according to claim 2, characterized in that, The inhibitors targeting AKR1C2 are selected from AKR1C2 gene silencing agents, AKR1C2 protein activity inhibitors, and AKR1C2 antibodies, specifically including siRNA, shRNA, glycyrrhizin-based small molecule inhibitors, or anti-AKR1C2 monoclonal antibodies.

6. The application according to claim 2, characterized in that, The inhibitors targeting H3K18la include dichloroacetic acid, and the inhibitors targeting AKR1C2 include glycyrrhizin.

7. The application according to claim 2, characterized in that, The combined medication improves TNFi resistance by inhibiting the anti-apoptotic phenotype of RA-FLS cells and reducing TNF-β secretion levels.

8. The application according to claim 1, characterized in that, The administration method of the combined medication is either combined administration or sequential administration.

9. A pharmaceutical composition for improving TNFi resistance, characterized in that, It comprises the first active ingredient, the second active ingredient, and pharmaceutically acceptable excipients as described in claim 2.

10. The pharmaceutical composition according to claim 9, characterized in that, The dosage form of the pharmaceutical composition is an injection, an oral preparation, or a topical preparation.

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