Application of ITGB1-ISG15 signal channel inhibitor in preparation of medicine for preventing and treating myocardial fibrosis

Abstract

The invention discloses application of an ITGB1-ISG15 signal channel inhibitor in preparation of a medicine for preventing and treating myocardial fibrosis, and relates to the technical field of cardiovascular disease prevention and treatment and molecular targeted therapy. It is found for the first time that in myocardial fibrosis, integrin beta1 (ITGB1) and interferon stimulated gene 15 (ISG15) are synergistically up-regulated and combined, and the ISG15 competitively inhibits ubiquitination of ITGB1 through ubiquitination-like modification (ISGpolarization) of ITGB1, so that endocytosis and protein degradation are reduced, ITGB1 is stabilized in cell membranes, the interaction time of ITGB1 and upstream ligands is prolonged, downstream signals are continuously activated, and myocardial fibrosis is inhibited. And fibrotic gene transcription and collagen deposition are induced. By inhibiting expression of ITGB1 and ISG15, the phenotype of myocardial fibrosis can be reduced, and si-ITGB1 and si-ISG15 are knocked down to generate a synergistic effect, so that the treatment effect is enhanced.

Description

Technical Field

[0001] This invention relates to the field of cardiovascular disease prevention and molecular targeted therapy, and in particular to the application of ITGB1-ISG15 signaling pathway inhibitors in the preparation of drugs for the prevention and treatment of myocardial fibrosis. Background Technology

[0002] Myocardial fibrosis is a common pathological basis for many cardiovascular diseases, including hypertensive heart disease, ischemic / metabolic cardiomyopathy, and HFpEF / HFrEF, and is also a decisive factor in disease progression. Essentially, it involves the continuous activation of myocardial fibroblasts / myofibroblasts, and the excessive synthesis, deposition, and cross-linking of extracellular matrix (ECM) such as type I / III collagen, leading to increased matrix stiffness, decreased diastolic compliance, and increased electromechanical heterogeneity and risk of arrhythmias. Current drugs (ACEI / ARB, ARNI, β-blockers, MRA, SGLT2 inhibitors, etc.) mainly alleviate fibrosis progression indirectly through the regulation of the neuro-humoral axis and the improvement of hemodynamics and metabolism, but their ability to reverse cross-linked ECM is limited. Existing imaging techniques (such as cardiac MRI T1 mapping / ECV, LGE, ultrasound strain) and serum markers (PICP, PIIINP, CITP, Galectin-3, sST2, MMP / TIMP ratio, etc.) are not sensitive enough to effectively distinguish between "early, active fibrosis-promoting signal enhancement" and "static residual scars", and lack biomarkers that can specifically reflect the persistence of upstream adhesion-mechanical transduction and specific post-translational modification rearrangements. Previous studies have largely focused on downstream or broad-spectrum signaling nodes such as TGF-β / SMAD, MMP, CTGF, Galectin-3, inflammation, and epigenetic regulation. However, these studies suffer from insufficient target selectivity, potential side effects, and the risk of triggering compensatory activation of alternative signaling pathways. Direct inhibition of adhesion mechanopathies such as FAK / Src, YAP / TAZ, and Rho / ROCK carries the risk of disrupting system homeostasis. Integrin β1 (ITGB1), a key "entry point" for adhesion and mechanical signals in multi-α subunit pairing, has been observed to be upregulated in stress and injury models, but its membrane residence time and pathological stabilization mechanisms at the post-translational modification level remain unclear. The ISGylation process mediated by interferon-stimulated gene 15 (ISG15) relies on a cascade reaction consisting of the E1 activator UBA7, the E2 ligase UBE2L6, and the E3 ligase HERC5 (human) / HERC6 (mouse), and is negatively regulated by the specific ISG15 deactivator USP18. ISGylation has been shown to affect substrate protein stability, complex assembly, and competition with ubiquitination in antiviral, tumor immunology, and inflammatory contexts. However, there are no publicly available reports establishing a causal chain between ISGylation and ITGB1 ubiquitination-endocytosis-degradation fate competition, membrane residence prolongation, sustained amplification of upstream adhesion signals, and transcriptional activation of fibroblast genes. Furthermore, there is a lack of composite diagnostic techniques that simultaneously detect ITGB1 activation / conformation or downstream phosphorylation status, ISGylation occupancy at specific sites, and ubiquitination levels.

[0003] Systematic screening and application of small molecules, antibodies, bispecific / multispecific antibodies, interfering peptides, oligonucleotides, PROTACs, de-ISGylation (USP18) modulators, or agents promoting ITGB1 reubiquitination and degradation around the ITGB1-ISG15 binding / ISGylation axis are lacking. There are currently no specific protocols for early risk stratification, treatment window determination, and real-time efficacy monitoring using this axis. Therefore, existing technologies suffer from: a lack of upstream pathologically specific "adhesion signal stabilization" cutoff points; a lack of quantifiable methods for detecting the ITGB1 ISGylation / ubiquitination competition ratio and membrane residency dynamics; and the absence of precise intervention strategies and corresponding companion diagnostic methods for this post-translational modification competition event. This gap highlights the need to develop a novel anti-fibrotic targeting and detection system centered on the ITGB1-ISG15 axis to meet the urgent technological demands for early identification, stratified intervention, and reversal of myocardial fibrosis.

[0004] Therefore, existing technologies still need improvement and development. Summary of the Invention

[0005] In view of the shortcomings of the prior art, the purpose of this invention is to provide the application of ITGB1-ISG15 signaling pathway inhibitors in the preparation of drugs for the prevention and treatment of myocardial fibrosis, and to provide a new strategy for the prevention and treatment of myocardial fibrosis.

[0006] The technical solution of the present invention is as follows: Firstly, it provides the application of ITGB1-ISG15 signaling pathway inhibitors in the preparation of drugs for the prevention of myocardial fibrosis.

[0007] Secondly, it provides the application of ITGB1-ISG15 signaling pathway inhibitors in the preparation of drugs for treating myocardial fibrosis.

[0008] Optionally, the ITGB1-ISG15 signaling pathway inhibitor includes: an ITGB1 inhibitor and / or an ISG15 inhibitor.

[0009] Optionally, the ITGB1 inhibitor is a small molecule, nucleic acid, gene editing system, protein, or polypeptide that specifically interferes with ITGB1 expression.

[0010] Optionally, the ISG15 inhibitor is a small molecule, nucleic acid, gene editing system, protein, or polypeptide that specifically interferes with ISG15 expression.

[0011] Optionally, the ITGB1 inhibitor is a siRNA that specifically interferes with the ITGB1 gene, and its nucleotide sequence is shown in SEQ ID NO.1.

[0012] Optionally, the ITGB1 inhibitor is GLPG0187.

[0013] Optionally, the ISG15 inhibitor is a siRNA that specifically interferes with the ISG15 gene, and its nucleotide sequence is shown in SEQ ID NO.5.

[0014] Thirdly, a drug for preventing myocardial fibrosis is provided, the drug comprising an inhibitor of the ITGB1-ISG15 signaling pathway.

[0015] Fourthly, a drug for treating myocardial fibrosis is provided, said drug comprising an inhibitor of the ITGB1-ISG15 signaling pathway.

[0016] Beneficial Effects: This invention is the first to discover that integrin β1 (ITGB1) and ISG15 are synergistically upregulated and bind in the heart and myocardial fibroblasts of a mouse model of myocardial fibrosis. ISG15 competitively inhibits the ubiquitination of ITGB1 through ISGylation, reducing endocytosis and protein degradation, stabilizing ITGB1 on the cell membrane, prolonging its interaction time with upstream ligands, and continuously activating downstream signaling such as ERK, inducing the transcription of pro-fibrosis genes such as COL1A1, COL3A1, ACTA2, CTGF, and LOX, and collagen deposition. Inhibiting the expression of ITGB1 and ISG15 can reduce the myocardial fibrosis phenotype, and the dual knockdown of si-ITGB1 and si-ISG15 produces a synergistic effect, enhancing the therapeutic effect. Based on this mechanism, this invention provides the application of an inhibitor that blocks the interaction between ITGB1 and ISG15 in the preparation of drugs for the prevention and treatment of myocardial fibrosis. This target strategy has the following advantages: (1) it can locate key upstream nodes of the adhesion-signal amplification chain with high specificity and can be intervened in the early stage of the disease; (2) it can achieve the "destabilization" and rapid cut-off of abnormal signals by restoring the normal ubiquitination-endocytosis turnover of ITGB1; (3) it can be synergistic with existing standard treatments such as ACEI / ARB, β-blockers, MRA, and SGLT2 inhibitors, and has the complementary synergistic potential of reducing the burden and inhibiting the excessive activation of neurohumoral function. Attached Figure Description

[0017] Figure 1 This figure shows the effect of upregulation of ITGB1 expression in myocardial fibrosis and the improvement of profibrotic phenotype by its inhibition.

[0018] Figure 2 This is a graph showing the upregulation of ISG15 expression in cardiac fibrosis samples and its direct and high-affinity binding characteristics with ITGB1.

[0019] Figure 3 This is a diagram showing the results of inhibiting ISG15 to reduce the activation of cardiac fibroblasts.

[0020] Figure 4This is a diagram showing the results of ITGB1 stabilization driving increased chromatin accessibility to profibrosis genes and synergistic transcriptional amplification (RNA-seq and ATAC-seq integration).

[0021] Figure 5 This is a diagram showing the synergistic inhibitory effect of dual knockdown of si-ITGB1 and si-ISG15 on the transcription of profibrosis genes. Detailed Implementation

[0022] This invention provides the application of ITGB1-ISG15 signaling pathway inhibitors in the preparation of drugs for the prevention and treatment of myocardial fibrosis. To make the objectives, technical solutions and effects of this invention clearer and more explicit, the invention is further described in detail below.

[0023] This invention relates to a comprehensive technical solution that focuses on the balance of post-translational modifications related to integrin β1 (ITGB1) and ISG15 (the competition between ITGB1 ISGylation and its ubiquitination) to intervene in the persistence of adhesion / mechanical signals in cardiomyogenic fibroblasts, and to conduct stratified diagnosis, dynamic monitoring, targeted intervention, and efficacy evaluation. The overall concept of this technical solution is to focus on whether ISGylation occurs at the lysine residues of the ITGB1 cytoplasmic tail, and to influence the membrane residence time of ITGB1 and its mediated FAK→ERK cascade signal output by detecting and regulating the relative levels of this type of ISGylation and ubiquitination, thereby providing a tool for identifying the active stages of myocardial fibrosis and optimizing intervention strategies.

[0024] To achieve the above concept, the present invention provides the following composable modules and implementation methods that work together (each module can be used individually or deployed in combination, and it is not necessary for all of them to exist at the same time): I. Site Identification and Molecular Tools Module (1) Lysine residues in ITGB1 that may undergo ISGylation can be identified by immunoprecipitation (IP) combined with high-resolution mass spectrometry (LC-MS / MS) under set stimulation conditions (such as mechanical stretching and low levels of type I interferon), and corresponding K→R site-directed mutants, truncated mutants or marker (Flag / His / HA / Biotin site) fusion constructs and wild-type controls can be constructed.

[0025] (2) The mutants, truncated forms or marker (Flag / His / HA / Biotin site) fusion constructs are used for: functional comparison, membrane residence monitoring, downstream signal intensity analysis and cell model establishment for screening inhibitors.

[0026] (3) It can further provide the ITGB1 tail peptide (containing candidate lysine) in synthetic or recombinant form as a substrate for in vitro interaction or screening platform.

[0027] II. Competitive Post-Translation Modification Detection Module (1) Tandem immunoprecipitation (first capture ITGB1, then distinguish ISG15 or ubiquitin tag), ratio quantitative mass spectrometry (SILAC, TMT) or nearest neighbor linkage (PLA), dual FRET / bioluminescent resonance energy transfer probes can be used to achieve synchronous or sequential reading of the relative levels of ITGB1 ISGylation and ubiquitination.

[0028] (2) The ITGB1 membrane residence half-life was calculated by combining pH-sensitive staining or pHluorin-labeled endocytosis tracking, surface biotinylated pulse-tracking, real-time fluorescence recovery (FRAP) or endocytosis inhibitor / promoter treatment.

[0029] (3) Based on the above results, establish the correlation parameters between the "ISGylation / ubiquitination ratio" and the membrane residence index and signal output index to provide input for comprehensive scoring.

[0030] III. Conformation and Signal Activation Detection Module (1) Use monoclonal antibodies or nanobodies that recognize the ITGB1 activation conformation (which can be prepared by phage display, library screening or known sequence) in combination with flow cytometry, high content imaging (HCA) or confocal microscopy to improve the ability to quantify conformational state.

[0031] (2) Simultaneously collect pFAK / total FAK, pERK / total ERK, and ECM synthesis and cross-linking related indicators (ACTA2, COL1A1, CTGF, FN1, POSTN, TGFB1) to obtain mRNA and protein levels.

[0032] (3) Multivariate regression, random forest, gradient boosting or other machine learning models can be introduced to generate fiber-promoting signal-driven index.

[0033] IV. Screening and Verification Platform (1) Molecular / biochemical layer: AlphaScreen, TR-FRET, surface plasmon resonance (SPR), biolayer interference (BLI) or microcalorimetry (ITC) are used to detect the binding or complex formation of the ITGB1 tail peptide with ISG15, HERC5, UBE2L6, etc.

[0034] (2) Cell layer: a dual reporter model containing ITGB1-ISG15 nearest neighbor probe + pFAK reporter system, used for preliminary screening of candidate inhibitors.

[0035] (3) 3D / mechanical layer: hydrogels with different stiffness, collagen matrix shrinkage experiments, atomic force microscopy (AFM) elasticity measurement and stretch bioreactor.

[0036] (4) In vivo validation: stress overload (TAC), post-myocardial infarction, mineralocorticoid / salt load, or other fibrosis-related models, combined with tissue staining (Masson, Sirius red, etc.), ultrasound or MRI (including ECV) evaluation.

[0037] V. Candidate Intervention Forms (each form can be independent or in combination) (1) Small molecules: By using structural prediction (AlphaFold2, molecular dynamics), pharmacophore model and docking screening, potential pockets at the tail of ITGB1, active or interacting interfaces of HERC5 / UBE2L6 / UBE1L are obtained to mask ISGylation sites or inhibit ISGylation cascade.

[0038] (2) Interfering peptide / cyclic peptide / stapled peptide: mimics the tail sequence of ITGB1 or its key fragments, including cell-penetrating sequences and stabilization modifications, and is used to compete for ISG15 or E3 recognition.

[0039] (3) Antibody / bispecific or multispecific molecules: one arm binds to activate ITGB1, and the other arm blocks ISG15 from approaching or recruiting ISGylation functional modules (such as fused with USP18 domain or its functional fragment).

[0040] (4) PROTAC / Molecular glue: connect HERC5 (or UBE2L6) binding ligands and E3 linker ligands (such as CRBN, VHL, IAP) to construct degradation inducers, or use molecular glue to promote the self-aggregation and degradation of target enzymes.

[0041] (5) Oligonucleotides: siRNA, shRNA, ASO or antisense oligodeoxynucleotides targeting UBE1L, UBE2L6, HERC5, other regulatory factors or ITGB1 related regulatory elements.

[0042] (6) CRISPR interference / activation, base editing or Prime Editing: target downregulation of ISGylation cascade expression, or replace specific lysine in ITGB1 with arginine to limit ISGylation.

[0043] (7) Enhanced USP18 function: engineered USP18 mutants (increase stability / localization ability) or small molecules / linkers can localize USP18 to the nearest neighbor of ITGB1; PROTAC can also be used to improve the stability level of USP18.

[0044] (8) Aptamer: recognizes surface markers (FAP, PDGFRα, etc.) of cardiomyocytes and acts as a directional carrier or conjugates with nucleic acids / small molecules.

[0045] (9) Nano / carrier systems: pH or ROS responsive polymer nanoparticles, lipid nanoparticles (LNPs, targeting peptides or ligand modified), engineered exosomes, hybridization carriers (exosome-LNPs), self-assembled peptide nanofibers, used to deliver the above small molecules, nucleic acids, editing systems, proteins or peptides.

[0046] (10) Combination strategy: Select sequential or combined strategies such as “site blockade + metabolic improvement”, “ISGylation removal + anti-crosslinking (LOXL2 inhibition)”, and “enzyme cascade degradation + structural repair” based on FIS dynamics and structural indicators.

[0047] VI. Companion Diagnostics and Reagent Kits (1) The kit may include: a solid-phase antibody that captures the activated conformation of ITGB1; an antibody (or a dual-labeled secondary antibody) for detecting ISG15 and ubiquitin tags; standards (recombinant or chemically simulated ISGylated ITGB1 fragments); internal control or quality control (stable isotope labeled) materials; detection reagents (HRP / fluorescent substrate); data analysis software or cloud module (to realize raw signal import, indicator standardization, FIS calculation, and threshold determination).

[0048] (2) It can be expanded to support the detection of ISGylated ITGB1 related indicators in tissue sections, peripheral blood-derived fibroblast-rich clusters or extracellular vesicle graded components.

[0049] Specifically, embodiments of the present invention provide the application of ITGB1-ISG15 signaling pathway inhibitors in the preparation of drugs for the prevention of myocardial fibrosis.

[0050] This invention provides the application of ITGB1-ISG15 signaling pathway inhibitors in the preparation of drugs for treating myocardial fibrosis.

[0051] In some embodiments, the ITGB1-ISG15 signaling pathway inhibitor includes: an ITGB1 inhibitor and / or an ISG15 inhibitor.

[0052] In some embodiments, the ITGB1 inhibitor is a small molecule, nucleic acid, gene editing system, protein, or polypeptide that specifically interferes with ITGB1 expression.

[0053] In some embodiments, the ISG15 inhibitor is a small molecule, nucleic acid, gene editing system, protein, or polypeptide that specifically interferes with ISG15 expression.

[0054] In some embodiments, the ITGB1 inhibitor is a siRNA that specifically interferes with the ITGB1 gene, and its nucleotide sequence is shown in SEQ ID NO.1.

[0055] In some embodiments, the ITGB1 inhibitor is GLPG0187 (CAS: 1320346-97-1).

[0056] In some embodiments, the ISG15 inhibitor is a siRNA that specifically interferes with the ISG15 gene, and its nucleotide sequence is shown in SEQ ID NO.5.

[0057] This invention provides a drug for preventing myocardial fibrosis, the drug comprising an inhibitor that blocks the interaction between ITGB1 and ISG15.

[0058] This invention provides a drug for treating myocardial fibrosis, the drug comprising an inhibitor that blocks the interaction between ITGB1 and ISG15.

[0059] In some embodiments, the drugs for preventing and / or treating myocardial fibrosis also include standard heart failure drugs (ACEI / ARB, ARNI, SGLT2i, β-blockers).

[0060] The present invention will be further described below through specific embodiments.

[0061] In the examples, unless otherwise stated, all other reagents are commercially available products.

[0062] "Profibrotic exogenous ligands" (hereinafter referred to as "profibrotic stimuli") refer to exogenous stimulating factors that can induce extracellular matrix (ECM) protein deposition in cardiac fibroblasts (or fibroblasts in myocardial tissue) and upregulate myofibroblast phenotypic markers (such as αSMA). In specific embodiments, recombinant THBS1 protein (rhTHBS1) is used as a representative example; this ligand can be replaced with other stimuli that have similar profibrotic signal activation effects and can upregulate / maintain ITGB1-related adhesion and FAK–ERK (or MAPK) pathway activity, including but not limited to: TGFβ1, CTGF, AngII, matrix fragments containing RGD motifs, mechanical stretch stimulation, matrix stiffness enhancement (≥10 kPa) or their functionally equivalent variants and combinations thereof.

[0063] The "ITGB1 inhibition" described in this invention can be achieved through one or more of the following methods: (1) small interfering RNA (siRNA) / shRNA / ASO targeting ITGB1; (2) integrin antagonists (examples: GLPG0187, cyclic RGD peptide, specific or multivalent blocking antibodies, nanobodies, functional fragments, mimic peptides); (3) compounds that affect the competitive binding or covalent modification process of ITGB1 with its ligands / regulatory factors (including ISG15); (4) synergistic therapeutic effect of dual siRNA knockdown of ITGB1 and ISG15.

[0064] Example 1: The effect of ITGB1 upregulation in myocardial fibrosis and its inhibition on improving the pro-fibrotic phenotype. (1) Materials 1) Experimental animals: C57BL / 6J male mice, 8-10 weeks old, SPF grade; used for tissue-level verification.

[0065] 2) Primary cardiomyocytes: ventricular tissue from 1-3 day old newborn mice was isolated, purified by differential adhesion, and used from the 2nd to 4th passages (P2-P4).

[0066] 3) Pro-fibrotic stimulation: Recombinant THBS1 (rhTHBS1, purity >95%, endotoxin-free) working concentration 500 ng / mL.

[0067] 4) si-RNA: si-ITGB1; negative control si-NC; final concentration of all was 50 nM.

[0068]

[0069] 5) GLPG0187 (CAS: 1320346-97-1): Integrin broad-spectrum antagonist, stock in 10 mM DMSO, dilute before use; working concentration 5 μM.

[0070] 6) Antibodies: anti-ITGB1 (abcam, ab183666), anti-COLIA1 (proteintech, 14695-1-AP), anti-COLIII (proteintech, 22734-1-AP), anti-αSMA (abcam, ab124964), anti-GAPDH (proteintech, 60004-1) (validated for WB and / or IF).

[0071] 7) Others: Standard culture medium, transfection reagent, RIPA lysis buffer (containing protease / deubiquitinase inhibitor), BCA kit, SDS-PAGE reagent, PVDF membrane, ECL substrate, DAPI.

[0072] (2) Method 1) Animal grouping (Example 2 group): Mice were randomly divided into a blank control group (PBS) and a fibrosis-promoting stimulation group (rhTHBS1) (n≥5); ITGB1 expression in the hearts of mice was detected after administration of the drugs.

[0073] 2) Cell grouping (Example 4 groups): Unstimulated control (Control); fibrosis-promoting stimulation group (rhTHBS1); ITGB1 expression downregulation group (rhTHBS1 + si-ITGB1); ITGB1 function inhibitor treatment group (rhTHBS1 + in-ITGB1 (GLPG0187)).

[0074] 3) siRNA transfection: When the cells reach 60-70% confluence, si-ITGB1 or si-NC are transfected using liposomes. After 6 h of transfection, the medium is replaced and incubated for 24 h before adding rhTHBS1.

[0075] 4) Small molecule inhibition: GLPG0187 was added 1 h before rhTHBS1 (final concentration 5 μM, final concentration of DMSO ≤0.1%).

[0076] 5) Stimulation and sampling: rhTHBS1 treatment for 24 h was used for Western Blot (WB); 48 h (or 24-48 h) was used for immunofluorescence.

[0077] 6) Western Blot: After lysis of each well, quantify the protein by loading 20-30 μg of protein; after transfer, incubate overnight at 4°C with primary antibody, then at room temperature with secondary antibody for 1 h, and develop with ECL; quantify using ImageJ grayscale and normalize GAPDH.

[0078] 7) Immunofluorescence: 4% (w / v) paraformaldehyde fixation for 15 min → 0.1% (w / v) Triton X-100 permeabilization for 5 min → 5% (w / v) BSA blocking for 1 h → primary antibody overnight at 4℃ → secondary antibody at room temperature (25℃) for 1 h → DAPI nuclear staining; laser confocal microscopy (63× oil immersion) to determine the mean fluorescence intensity (MFI) of COLI and αSMA.

[0079] (3) Results 1) The rhTHBS1 treatment group (exogenous stimulation) upregulated the protein level of ITGB1 in the heart compared with the control group. Figure 1 (A)

[0080] 2) In the rhTHBS1 treatment group, the protein expression levels of ECM and fibrosis markers (COLI, COLIII, αSMA) were upregulated compared to the control group. si-ITGB1 or in-ITGB1 (GLPG0187) intervention significantly reduced these indicators and improved fibrosis. Figure 1 (CE).

[0081] 3) Immunofluorescence showed enhanced COLI and αSMA signals in the rhTHBS1-treated group, and decreased fluorescence intensity after si-ITGB1 or in-ITGB1 (GLPG0187) intervention. Figure 1 (Middle F).

[0082] (4) Conclusion As demonstrated in this embodiment, profibrotic stimulation (e.g., rhTHBS1) induces upregulation of ITGB1, accompanied by enhanced ECM protein and fibrotic phenotype. This profibrotic phenotype can be reversed by gene knockdown of ITGB1 or pharmacological inhibition of ITGB1 and its downstream pathways using GLPG0187. Other ligand and integrin inhibition strategies with similar effects can be equivalently substituted.

[0083] Example 2: Screening of ISG15 and structural prediction and kinetic stability assessment of its complex with ITGB1 (1) Materials 1) ITGB1 binding protein screening (Co-IP + LC-MS / MS) 1.1) Antibodies: Anti-ITGB1 (abcam, ab183666, used for immunoprecipitation), and isotype IgG negative control (CST7074P2).

[0084] 1.2) Capture medium: Protein A / G magnetic beads.

[0085] 1.3) Lysis / washing reagents: mild nonionic lysis buffer; protease inhibitor; PBS.

[0086] 1.4) Digestion and sample preparation: DTT, iodoacetamide (IAA), sequencing-grade trypsin, C18 desalting column.

[0087] 1.5) Mass spectrometry platform: a nanoliter reversed-phase liquid chromatography and high-resolution mass spectrometry system (Q Exactive / Exploris 480) using data-dependent acquisition (DDA) mode.

[0088] 2) Public database data: GEO dataset GSE116250.

[0089] 3) Experimental animals (same as in Example 1): C57BL / 6J male mice, 8-10 weeks old, SPF grade; used for tissue-level verification.

[0090] 4) Computing platform: AlphaFold3 (multimer mode); GROMACS 2022.05.

[0091] 5) Force field and solvent: Charmm36-feb2021 force field; TIP3P water model; Na+ / Cl- to adjust the system to neutral and 0.15 M ionic strength.

[0092] 6) Analysis and visualization tools: PyMOL (interface contact, hydrogen bond / hydrophobicity display); GROMACS built-in tools (rms, rmsf, ​​hbond, etc.); gmx_MMPBSA (MM / PBSA binding energy calculation); scripts (Python / R, data preparation and plotting).

[0093] (2) Method 1) Screening of ITGB1-related interacting proteins (Co-IP + LC-MS / MS) 1.1) Sample source: Mouse myocardial tissue or primary myocardial fibroblast lysate treated with control (PBS) and rhTHBS1 (under the same conditions as in Example 1).

[0094] 1.2) Lysis and pre-cleaning: Lysis was performed in an ice bath for 30 min, followed by centrifugation at 12,000 g to remove debris; the supernatant was then pre-cleaned for 1 h with Protein A / G magnetic beads.

[0095] 1.3) Immunoprecipitation: Add anti-ITGB1 antibody or IgG negative control to the pre-cleaned lysate and incubate overnight at 4°C by rotation; then add magnetic beads and continue incubation for 2 h.

[0096] 1.4) Washing: Mild nonionic lysis buffer → high salt (300 mM NaCl) buffer → low salt (150 mM NaCl) buffer → PBS (3-5 times each), rapid operation at low temperature.

[0097] 1.5) Elution and digestion: Mild elution (low pH or SDS-PAGE loading buffer). The eluted products are reduced (DTT) and alkylated (IAA) before being digested with trypsin in gel or in solution (enzyme to substrate ratio 1:50 ~ 1:100, overnight at 37°C).

[0098] 1.6) LC-MS / MS: After separation by nanoliter liquid chromatography, the peptide mixture was analyzed by Orbitrap mass spectrometry (full scan resolution ≥60,000), and secondary mass spectra were acquired using DDA mode.

[0099] 1.7) Inclusion criteria: Fold change (rhTHBS1 vs control) ≥1.5 or ≤0.67; adjusted FDR <0.05; identified in ≥70% of biological replicates.

[0100] 1.8) Candidate priority: Integrating (a) fold change and significance; (b) known association with immune-related signals in the literature; (c) coupling with potential integrin function. ISG15 was significantly enriched in the volcano plot and was therefore the preferred candidate.

[0101] 2) ISG15 expression in population samples retrieved from public transcriptome data: The dataset GSE116250 was obtained from the NCBI GEO database. This dataset contains left ventricular tissue RNA-Seq data (RPKM values) from 14 non-heart failure (NF) controls, 37 patients with dilated cardiomyopathy (DCM), and 13 patients with ischemic cardiomyopathy (ICM). ISG15 expression data were extracted, and the Mann-Whitney U test was used to compare the differences between the heart failure group (DCM and ICM combined) and the NF group, with a significance level set at p<0.05.

[0102] 3) Animal grouping (same as in Example 1): blank control group (PBS); fibrosis-promoting stimulation group (rhTHBS1); ISG15 levels in mouse hearts were measured after administration of the drugs.

[0103] 4) Complex prediction (AlphaFold3): 4.1) Sequence proteins: ITGB1 protein (10587-H08H1, Sino Biological) and ISG15 protein (12729-HNAE1, Sino Biological).

[0104] 4.2) AlphaFold3 (multimer mode) generates 5 models, and selects the one with the highest combined score of ipTM + pTM as the initial conformation for subsequent MD.

[0105] 4.3) PyMOL Visualization: Annotation of interface hydrogen bonds, salt bridges, and hydrophobic contacts. Representative interface residues include LYS-8, SER-50, GLY-51, ALA-53, GLN-55, ASP-76, ASP-246, SER-247, ALA-285, GLY-286, ILE-319, ALA-320, and GLU-347.

[0106] 5) Molecular dynamics simulations: 5.1) Software and version: GROMACS 2022.05.

[0107] 5.2) Force field and water model: Charmm36-feb2021 + TIP3P.

[0108] 5.3) System construction: The solvent box was ≥1.2 nm away from the outermost atoms of the protein; Na+ / Cl- was added to neutralize and the salinity was adjusted to 0.15 M.

[0109] 5.4) Energy minimization: Steepest descent method for 50,000 steps until the maximum repulsion is <1,000 kJ / mol / nm (or the default convergence threshold is reached).

[0110] 5.5) Equilibrium: NVT (100 ps) → NPT (100 ps), temperature 310 K (V-rescale), pressure 1 bar (Berendsen).

[0111] 5.6) Production simulation: 100 ns, free run, step size 2 fs; Verlet neighborhood list, updated every 10 steps; truncation 1.2 nm; trajectory output every 10 ps.

[0112] 5.7) Analysis: Main chain RMSD / RMSF (gmx rms, gmx rmsf); interface hydrogen bonds (gmx hbond).

[0113] 5.8) Combining free energy and energy decomposition: gmx_MMPBSA (MM / PBSA model, uniformly sampling frames in the 20-100 ns interval after time, with an interval of 100-200 ps).

[0114] 5.9) Hotspot residues: sorted and screened by energy contribution (ΔG decomposition, electrostatics + van der Waals).

[0115] (3) Results 1) Interaction profiles identified by LC-MS / MS after ITGB1 immunoprecipitation showed enrichment of various proteins related to cellular stress / immune modification; among them, ISG15 was significantly upregulated under rhTHBS1 stimulation, as highlighted in the volcano plot ( Figure 2 (A)

[0116] 2) Under exogenous pro-fibrotic stimulation (rhTHBS1), ISG15 expression was upregulated in the heart of mice. Figure 2 (B)

[0117] 3) ISG15 expression was increased in cardiac tissue in patients with heart failure (dilated cardiomyopathy (DCM) and ischemic heart disease (ICM)) compared to non-heart failure (NF) individuals. Figure 2 (C)

[0118] 4) AlphaFold3 predicts that ITGB1 (VWFA domain) forms a stable interface with ISG15, and shows key residues (LYS-143, GLN-55, ASP-246, GLY-286, ASP-76, ALA-53, SER-247, ALA-285, GLY-51, SER-50, LYS-8, ILE-319, ALA-320, GLU-347, etc.) Figure 2 (D).

[0119] 5) In the 100 ns simulation of the complex, the ITGB1-ISG15 complex (with ISG15) maintained low fluctuations, while the ITGB1 standalone system (without ISG15) showed a rise in the RMSD plateau and a larger drift, indicating that the conformation was more unstable without the constraint of ISG15. Figure 2 (E).

[0120] 6) The RMSD of the interfacial residues in the complex is lower than that of the corresponding positions in the ITGB1 system alone, further supporting the stabilizing effect. Figure 2 (E).

[0121] (4) Conclusion Under exogenous profibrotic stimulation, the expression of ISG15 and its binding level with ITGB1 in the heart were significantly upregulated. Furthermore, ISG15 can form a stable multi-point interaction interface with the VWFA domain of ITGB1, significantly reducing ITGB1 conformational fluctuations and endowing the complex with high binding energy stability, suggesting that ISG15 participates in the regulation of ITGB1-related signal transduction processes through structural stabilization.

[0122] Example 3: High affinity binding of ISG15 to ITGB1 and its competitive inhibition of ITGB1-ubiquitin binding (1) Materials 1) Proteins and reagents: ISG15 protein (12729-HNAE1, Sino Biological); Ubiquitin protein (HY-P701839, MCE); ITGB1 protein (10587-H08H1, Sino Biological).

[0123] 2) BLI related: Ni-NTA biosensor, Octet series instruments, binding buffer (PBS + 0.01% (w / v) Tween-20 + 1% (w / v) BSA).

[0124] (2) Method 1) BLI affinity determination: His-ITGB1 was loaded onto the Ni-NTA sensor, and binding / dissociation was measured with different concentrations of ISG15 (6.25-100 nM gradient) or ubiquitin (62.5-1000 nM gradient) (900 s each). The binding sensing curve was globally fitted using a 1:1 binding model to obtain the binding rate constant (kon), dissociation rate constant (koff), and equilibrium dissociation constant (KD).

[0125] 2) BLI competition experiment: Under a fixed high concentration of ubiquitin (1000 nM), ISG15 (0-100 nM) was added in increasing concentrations, and the rate of change of the maximum response value (Rmax) of ITGB1 binding to ubiquitin was recorded to evaluate the competitive inhibition effect.

[0126] 3) Quantitative and statistical analysis: All BLI experiments included ≥3 independent replicates. Kinetic parameters (kon, koff, KD) were obtained from the instrument's software. Data are expressed as mean ± standard error (SEM).

[0127] (3) Results 1) Based on ISGylation as a ubiquitin linker, the role of ISG15 in the binding of ITGB1 to ubiquitin and its ubiquitination-related prerequisites was investigated.

[0128] 2) BLI showed that ITGB1 had a higher affinity for ISG15 than for ubiquitin (KD=8.03 nM vs 92.9 nM), the difference mainly due to a faster binding process and a slower dissociation process. Figure 2 (F and G).

[0129] The BLI kinetic measurements showed that the equilibrium dissociation constant (KD) for ITGB1 with ISG15 was 8.03 nM, while the KD for ITGB1 with ubiquitin was 92.9 nM. This indicates that, under equilibrium conditions, to achieve the same receptor (ITGB1) occupancy, the required free concentration of ISG15 is much lower than that of ubiquitin. In other words, ITGB1 has a significantly higher affinity for ISG15 than for ubiquitin (approximately 11-fold higher; a lower KD indicates higher affinity). More specifically, the corresponding binding rate constants (kon) are 8.5 × 10⁻⁶ and 8.5 × 10⁻⁶, respectively. 4 M -1 s -1 (ITGB1-ISG15) and 8.5×10 3 M -1 s -1 (ITGB1-ubiquitin). A higher binding rate constant (kon) indicates a higher effective collision frequency and faster binding process per unit concentration. Furthermore, the dissociation rate constants (koff) for both are 6.82 × 10⁻⁶.-4 s -1 (ITGB1-ISG15) and 8.30×10 -4 s -1 (ITGB1-ubiquitin). A smaller dissociation rate constant (koff) indicates slower dissociation and a more stable complex after formation. The data above show that the affinity of ITGB1 to ISG15 is approximately 11 times higher than that to ubiquitin, an improvement resulting from both a faster binding rate and a slower dissociation rate. Therefore, the ITGB1-ISG15 complex is more efficient and stable in formation than the ITGB1-ubiquitin complex.

[0130] 3) Competition experiments showed that as the concentration of ISG15 increased, the binding signal of ITGB1 to ubiquitin gradually decreased, exhibiting a competitive inhibition characteristic. Figure 2 (H).

[0131] (4) Conclusion ISG15 forms a nanomolar-level high-affinity stable complex with ITGB1, exhibiting significantly higher affinity than ubiquitin. ISG15 competitively / blocks ubiquitin binding sites on ITGB1, leading to a decrease in ITGB1 ubiquitination levels and reduced degradation. Therefore, binding to ISG15 prolongs the ITGB1 half-life, promoting its stability in the THBS1-induced environment and sustaining pro-fibrotic signaling. Blocking the ISG15-ITGB1 interaction restores ITGB1 ubiquitination and accelerates degradation, reducing the myocardial fibrosis phenotype.

[0132] Example 4: ISG15 knockdown weakens fibrotic stimulation-induced cardiomyocyte activation and ECM generation (1) Materials 1) Primary cardiac fibroblasts (same as in Example 1): Newborn mouse primary cardiac fibroblasts (P2–P4).

[0133] 2) Pro-fibrotic stimulation (same as in Example 1): Recombinant THBS1 (rhTHBS1) 500 ng / mL.

[0134] 3) si-RNA: si-ISG15; negative control si-NC (same as in Example 1); final concentration of all was 50 nM.

[0135]

[0136] 4) Antibodies: Anti-COL 1A1 (proteintech, 14695-1-AP), Anti-COL III (proteintech, 22734-1-AP), Anti-α-SMA (abcam, ab124964), Anti-ITGB1 (abcam, ab183666), Anti-GAPDH (proteintech, 60004-1).

[0137] 5) qPCR related: TRIzol, reverse transcription kit, SYBR Green Master Mix, specific primers (COL1A1, COL III, ACTA2 (α-SMA), ITGB1, si-ISG15, GAPDH).

[0138] 6) Immunofluorescence: 4% (w / v) paraformaldehyde, 0.1% (w / v) Triton X-100, BSA (w / v) blocking solution, secondary antibody (Alexa Fluor 488 / 594), DAPI.

[0139] 7) Others: Transfection reagent (same as in Example 1), RIPA lysis buffer (containing protease and deubiquitin / deISG15 enzyme inhibitor), BCA kit, SDS-PAGE / ECL system, ImageJ.

[0140] (2) Method 1) Transfection: When the cells reach 60-70% confluence, transfect them with si-ISG15 or si-NC respectively. Change the medium 6 h after transfection; continue culturing for 24 h and then add rhTHBS1 stimulation (0 h time).

[0141] 2) Western Blot: lysis, quantification, loading 20-30 μg sample; overnight incubation at 4℃, ECL imaging; quantification ratio (target / GAPDH).

[0142] 3) Immunofluorescence: After cell treatment, cells were fixed with 4% paraformaldehyde, permeabilized with 0.1% Triton X-100, blocked with 5% BSA, and then incubated sequentially with primary antibody and fluorescent secondary antibody, followed by DAPI staining of the nuclei. Images were acquired using a laser confocal microscope, and the mean fluorescence intensity (MFI) of COL1A1 and the coverage of α-SMA positive stress fibers were analyzed using ImageJ software.

[0143] (3) Results 1) Knockdown efficiency: si-ISG15 reduced ISG15 mRNA and protein by about 75-85% (p<0.001), did not significantly affect baseline ITGB1 transcription, but slightly reduced ITGB1 protein (~15-20%, p<0.05), suggesting that it mainly acts on the stability level (consistent with Example 3).

[0144] 2) Fibrosis-related protein levels (WB): rhTHBS1 group: COL1A1, COL III, and ACTA2 (α-SMA) were upregulated compared to the control; rhTHBS1+si-ISG15 group: the above genes were decreased compared to the rhTHBS1 group. Figure 3 AC (Chinese)

[0145] 3) Immunofluorescence: rhTHBS1 stimulation promoted the formation of abundant α-SMA stress fibers and enhanced COL1A1 deposition in cells. Knockdown of ISG15 significantly inhibited both the assembly of α-SMA stress fibers and COL1A1 deposition. Figure 3 (D).

[0146] (4) Conclusion ISG15 knockdown significantly weakened rhTHBS1-induced upregulation of ECM genes (COL1A1, COL III) and myofibroblast marker (α-SMA) proteins in cardiomyocytes, reducing collagen deposition and α-SMA stress fiber formation. This example validates at the cellular functional level that ISG15 is a key cofactor in amplifying pro-fibrotic signals, and its knockdown can serve as a strategy to inhibit abnormal activation of cardiomyocytes and excessive ECM production.

[0147] Example 5: ITGB1-ISG15 complex stabilizes downstream signaling and synergistically opens pro-fibrosis gene chromatin, promoting transcriptional activation. (1) Materials 1) Primary cardiac fibroblasts (same as in Example 1): Newborn mouse primary cardiac fibroblasts (P2–P4).

[0148] 2) siRNA (same as in Example 1): si-ITGB1, negative control si-NC.

[0149] 3) RNA-seq reagents: total RNA extraction (TRIzol), mRNA enrichment or rRNA removal reagents, library preparation kit (strand specific).

[0150] 4) ATAC-seq reagents: Tn5 transposase kit, nuclear separation buffer, and two-end high-fidelity PCR reagents.

[0151] 5) Sequencing platform: Illumina NovaSeq (PE150 or PE50 / 75, RNA-seq target ≥30-40M cleanreads / sample; ATAC-seq ≥50M).

[0152] 6) Computational analysis environment: Linux server; software fastp, STAR, featureCounts, DESeq2, Bowtie2, SAMtools, picard, MACS3, bedtools, deeptools, HOMER / MEME Suite, chromVAR, ClusterProfiler, R (≥4.2), pyGenomeTracks.

[0153] 7) Validation experiment: qPCR (primers for ACTA2, COL1A1, CTGF, FN1, POSTN, TGFB1, ITGB1 and GAPDH), other general reagents are the same as in Example 4.

[0154] (2) Method 1) ATAC-seq sequencing and analysis: 5 × 10⁵ samples were taken for each condition. 4 -8×10 4Cardiac fibroblasts were gently washed with cold PBS and then lysed on ice to preserve nuclear integrity before immediately undergoing a Tn5 transposition reaction (37°C, 30 min). The transposition products were quantified using real-time qPCR monitoring to determine the minimum number of PCR cycles required for adequate amplification (avoiding over-amplification and sequence bias). After sequencing, the library underwent quality control: fragment length distribution should exhibit a typical gradient of <150 bp nucleosome-free fragments and single / double nucleosomes (~200 / ~400 bp). Non-Redundant Fraction (NRF), PCR Bottleneck Coefficient (PBC), and Fraction of Reads in Peaks (expected >0.2-0.3) were calculated to assess complexity and signal-to-noise ratio. Sequences were aligned to a reference genome using Bowtie2 (--very-sensitive) to filter low-quality alignments (MAPQ <30), mitochondrial reads, and PCR repeats. MACS3 (parameters: --shift -37 --extsize 73 -q 0.01) was used to call up the peaks of each sample and merge them to generate a union peak set to construct the design matrix. After reading count, RPKM or CPM normalization was used to generate a bigWig trajectory for visualizing differential accessibility (the track can be further smoothed or deep normalized for cross-sample comparison). The accessibility changes obtained from differential analysis (calculating log2FC and multiple test FDR) lay the foundation for subsequent transcription integration.

[0155] 2) RNA-seq sequencing and analysis: Parallel samples were prepared into strand-specific libraries using rRNA removal or poly(A)+ enrichment. After Illumina sequencing, STAR alignment was performed, and gene layer counts were obtained using featureCounts. Differential expression analysis was conducted using DESeq2 (judgment threshold |log2FC| ≥ 1 and FDR < 0.05). The ATAC differential results were integrated with the RNA-seq upregulated gene set for multi-omics analysis: First, the promoter window was defined as 1,000 bp upstream of the TSS to +100 bp downstream. The presence of significantly upregulated differential ATAC peaks within this window was counted (log2FC_accessibility ≥ 0.58, FDR < 0.05). Genes simultaneously satisfying "enhanced promoter accessibility + transcriptional upregulation" were grouped into a co-activation set. This set was then enriched using KEGG / GO with ClusterProfiler, and Benjamini-Hochberg correction was applied (FDR < 0.05 was considered significant) to elucidate the epigenetic and transcriptional co-amplification pathway network driven by ITGB1 stabilization. If necessary, motif (HOMER) and chromVAR analyses can be added to this analysis to link open peaks with transcription factor activity.

[0156] 3) Multi-omics collaboration: Genes that are significantly upregulated in RNA-seq and have a significant open peak in their promoter region (TSS± 1.5 kb) in ATAC-seq are defined as "ITGB1-dependent co-activation gene set".

[0157] 4) qPCR: RNA was extracted with TRIzol, reverse transcribed, and subjected to the 2^-ΔΔCt method (internal reference GAPDH).

[0158] (3) Results 1) RNA-seq detection: The rhTHBS1 (pro-fibrosis stimulation) group significantly upregulated fibrosis / ECM / adhesion-related genes compared to the control group; most genes could be reversed after si-ITGB1. Figure 4 (B)

[0159] 2) Pathway enrichment: The reversed upregulated genes were enriched in fibrosis-related pathways such as Focal adhesion, ECM-receptor interaction, regulation of actin cytoskeleton, MAPK signaling, TGF-β signaling, and Adherens junction. Figure 4 (C)

[0160] 3) ATAC-seq detection: The overall proximal accessibility distribution of the promoter in the rhTHBS1 (profibrosis-stimulating) group shifted to the right, indicating enhanced chromatin opening; si-ITGB1 reduced this proportion, resulting in a significant reversal of the enhancement. Figure 4 (D).

[0161] 4) Multi-omics joint analysis: Genes closely related to fibrosis simultaneously satisfy transcriptional upregulation + enhanced promoter accessibility, including: ACTA2, COL1A1, CTGF, FN1, POSTN, TGFB1 ( Figure 4 (E).

[0162] 5) Representative gene verification: qPCR and RNA-seq results were consistent; pro-fibrotic stimulation upregulated multiple pro-fibrotic genes, including ACTA2, COL1A1, CTGF, FN1, POSTN, and TGFB1; si-ITGB1 significantly reduced the upregulation magnitude. Figure 4 (Middle F).

[0163] (5) Conclusion Profibrotic stimuli (e.g., THBS1) trigger a broad transcriptional program of profibrotic genes via ITGB1, accompanied by enhanced accessibility of proximal chromatin to the corresponding promoters. ITGB1 knockdown leads to widespread reverse transcription and upregulation of accessibility, suggesting that ITGB1 is a core node driving this epigenetic transcriptional reprogramming. Echoing the aforementioned Examples 2 (structural stability), 3 (ISG15 binding inhibits ubiquitination, prolonging ITGB1 half-life), and 4 (functional phenotype suppression), this example provides omics-level evidence for the mechanistic chain of "stable ITGB1-ISG15 complex → synergistic interaction between transcription factors and coactivators → sustained high transcription of profibrotic genes."

[0164] Example 6: Synergistic inhibitory effect of dual knockdown of si-ITGB1 and si-ISG15 on transcription of profibrosis genes (1) Materials 1) Cells: Newborn mouse primary cardiomyocytes (P2–P4, same as in Example 1).

[0165] 2) Pro-fibrotic stimulation: Recombinant THBS1 (rhTHBS1, same as in Example 1) 500 ng / mL.

[0166] 3) siRNA: si-ITGB1 (same as in Example 1), si-ISG15 (same as in Example 4).

[0167] 4) qPCR related: TRIzol, DNase I (to remove genomic contamination), reverse transcription kit (with gDNA remover step), SYBR Green Master Mix; primers for (mouse): ACTA2, COL1A1, CTGF, FN1, POSTN, TGFB1, ITGB1, ITGB1, ISG15, GAPDH.

[0168] 5) Others: Transfection reagents, endotoxin-free water, serum-free Opti-MEM (transfection period), conventional culture media, DNase / RNase-free consumables.

[0169] (2) Method 1) Experimental grouping and treatment (4 groups, all stimulated in the THBS1 background): Group A: THBS1 (si-NC); Group B: THBS1 + si-ITGB1; Group C: THBS1 + si-ISG15; Group D: THBS1 + si-ITGB1 + si-ISG15.

[0170] siRNA transfection was performed when cells reached 60-70% confluence. The culture medium was replaced with fresh complete medium 6 hours after transfection. Cells were cultured for another 24 hours, then stimulated with rhTHBS1 for 24 hours before collecting cells to extract RNA.

[0171] 2) qPCR: Total RNA was extracted using the TRIzol method, and genomic DNA was digested with DNase I and reverse transcribed into cDNA. qPCR was performed using SYBR Green reagent. The relative expression levels of each gene mRNA were calculated using the si-NC group as the calibration sample (GAPDH was the internal reference gene) using the 2^(-ΔΔCt) method.

[0172] 3) Knockdown efficiency and specificity: qPCR was used to verify that the reduction in ITGB1 and ISG15 mRNA was ≥70%; double knockout required confirmation that both genes met the criteria simultaneously. 4) Statistical analysis: Univariate ANOVA + Tukey; p < 0.05 significant; multigene analysis can be corrected using FDR.

[0173] (3) Results 1) Knockdown efficiency verification: Both single and double knockdown can effectively and specifically downregulate the target gene mRNA level (efficiency >70%), and the knockdown efficiency of the two siRNAs in the double knockdown group is not significantly different from that in the single knockdown group. Figure 5 (A, B)

[0174] 2) Gene expression detection: Fibrosis-promoting stimulation significantly upregulated profibrosis genes ACTA2, COL1A1, CTGF, FN1, POSTN, and TGFB1; si-ITGB1 and si-ISG15 both reduced the upregulation magnitude. Figure 5 (CH).

[0175] 3) Synergistic effect verification: Dual knockdown of si-ITGB1 and si-ISG15 significantly reduced the upregulation of profibrosis genes, with a reduction greater than that of single gene knockdown. Figure 5 (CH).

[0176] (4) Conclusion In the context of THBS1-induced profibrosis, knockdown of either ITGB1 or ISG15 alone reduced the transcription of typical profibrotic genes (ACTA2 / COL1A1 / CTGF / FN1 / POSTN / TGFB1). The dual knockdown of si-ITGB1 and si-ISG15 produced a synergistic effect, supporting a complementary mechanism: these results demonstrate at the functional transcriptional level that the "ITGB1-ISG15 interaction is the synergistic center of the persistent profibrotic program." This synergy suggests that future research could consider: a combination of small molecule / antibody blocking of ITGB1 ligand binding and interference at the ISG15-ITGB1 binding interface; or low-dose dual-target intervention to achieve antifibrotic effects similar to or even better than high-dose single-target intervention, potentially reducing side effects.

[0177] The images involved in the above embodiments are as follows: Figure 1 The results of Example 1 demonstrate the upregulation of ITGB1 in myocardial fibrosis and the improvement of pro-fibrotic phenotype by its inhibition. A shows a comparison of ITGB1 protein expression in mouse hearts under exogenous pro-fibrotic stimulation (rhTHBS1) and a blank control (PBS) condition, showing that ITGB1 expression is upregulated in mouse hearts under exogenous pro-fibrotic stimulation (rhTHBS1). B is a schematic diagram of the groupings: control, pro-fibrotic stimulation group, ITGB1 expression downregulation group (si-ITGB1), and ITGB1 function inhibitor treatment group (in-ITGB1), illustrating that reducing ITGB1 expression or blocking its function can inhibit subsequent abnormal activation. CE is Western blotting quantification: COLIII, COLI I, and αSMA levels increased under pro-fibrotic stimulation; both si-ITGB1 and ITGB1 inhibitors significantly reduced the expression of these fibrosis-related proteins. F represents immunofluorescence staining and quantification: the fluorescence signals of COL1A1 and αSMA were enhanced in the profibrotic stimulation group, while they decreased in the si-ITGB1 and ITGB1 inhibitor groups, supporting the involvement of ITGB1 in driving profibrotic remodeling.

[0178] Figure 2The results of Examples 2 and 3 demonstrate the expression of ISG15 in cardiac fibrosis samples and its direct high-affinity binding to ITGB1. In Example A, ISG15 is significantly enriched in the volcano plot. Example B compares ISG15 protein expression in mouse hearts under exogenous pro-fibrotic stimulation (rhTHBS1) and a blank control (PBS) condition, showing that ISG15 expression is upregulated in mouse hearts under exogenous pro-fibrotic stimulation (rhTHBS1) condition. C compares the expression of ISG15 in cardiac tissues of patients with heart failure (dilated cardiomyopathy (DCM) and ischemic heart disease (ICM)) and those without heart failure (NF), showing that ISG15 expression is increased in cardiac tissues of patients with heart failure (DCM and ICM) compared to those without heart failure (NF). D shows the molecular docking and molecular dynamics (MD) results: ISG15 forms a stable interface with the VWFA domain of ITGB1; key residues mainly include GLY-51, SER-50, ALA-53, GLN-55, ASP-76, LYS-8, LYS-143, ASP-246, SER-247, ALA-285, GLY-286, ILE-319, ALA-320, and GLU-347, mediating hydrogen bonding / electrostatic / hydrophobic interactions. E shows the 100 ns MD results. Simulated main-chain RMSD results: The trajectory fluctuation of the complex bound to ISG15 is lower than that in the ligand-free (or ISG15-free) state, suggesting an interface stabilization effect. F represents the biolayer interference (BLI) kinetics: ITGB1 binding to ISG15 KD ≈ 8.03 nM (Kon 8.50 × 10⁻⁶). 4 M -1 s -1 Koff 6.82×10 -4 s -1 It exhibits high affinity and rapid binding. G is the BLI result: ITGB1 binds to ubiquitin at a KD ≈ 92.9 nM (Kon 8.50 × 10⁻⁶). 3 M -1 s -1 Koff 8.30×10 -4 s -1 The affinity of ISG15 is significantly lower than that of ISG15. These results support the view that ISG15 can preferentially occupy the ITGB1-related binding interface compared to ubiquitin. H is a competition experiment: as the concentration of ISG15 increases, the binding response of ITGB1 to ubiquitin decreases stepwise, proving that ISG15 competitively occupies the same or overlapping interfaces, restricting ubiquitin binding and thus reducing the possibility of subsequent ubiquitination.

[0179] Figure 3The results of Example 4 demonstrate that inhibiting ISG15 can reduce the activation of cardiac fibroblasts. AC represents Western blotting quantification: COLIII, COL1A1, and αSMA levels increased under pro-fibrotic stimulation; si-ISG15 significantly reduced the expression of these fibrosis-related proteins. D represents immunofluorescence staining and quantification: the fluorescence signals of COL1A1 and αSMA were enhanced in the pro-fibrotic stimulation group and decreased in the si-ISG15 group, supporting the involvement of ISG15 in driving pro-fibrotic remodeling.

[0180] Figure 4 The results of Example 5 demonstrate that ITGB1 stabilization drives chromatin accessibility and transcriptional co-amplification of pro-fibrotic genes (RNA-seq and ATAC-seq integration). A illustrates the experimental procedure: cardiac fibroblasts underwent omics analysis under three conditions: control, exogenous pro-fibrotic stimulation, and pro-fibrotic stimulation + si-ITGB1, resulting in homologous integration of ATAC-seq (chromatin opening) and RNA-seq (transcription products). B shows the overall changes in the transcriptional profile: pro-fibrotic stimulation induced widespread upregulation; ITGB1 knockdown caused most upregulated genes to revert to their previous levels. C shows KEGG enrichment: ITGB1-regulated upregulated pathways were concentrated in Focal adhesion, ECM-receptor interaction, regulation of actin cytoskeleton, MAPK signaling, TGF-β signaling, adherens junction, and Cardiac muscle. Contraction, etc.; ITGB1 knockdown significantly weakened the activity of these signaling axes. D is chromatin accessibility: pro-fibrotic stimulation broadly enhanced the proximal opening of promoters; this enhancement was partially or significantly reversed under si-ITGB1 conditions. E and F are integrated analyses that identified typical pro-fibrotic factors (ACTA2, COL1A1, CTGF, FN1, POSTN, TGFB1) showing that promoter accessibility increased synchronously with mRNA levels; ITGB1 knockdown inhibited this co-amplification.

[0181] Figure 5 The results from Example 6 demonstrate the synergistic inhibitory effect of dual knockdown of si-ITGB1 and si-ISG15 on the transcription of profibrotic genes. In Example 6, gene knockdown efficiency was verified by qPCR in sections A and B. A represents the ITGB1 mRNA level after transfection with si-ITGB1; B represents the ISG15 mRNA level after transfection with si-ISG15. CH represents the relative mRNA expression levels of key profibrotic genes in each treatment group: C for ACTA2, D for COL1A1, E for CTGF, F for FN1, G for POSTN, and H for TGFB1.

[0182] In summary, the core advantage of this invention lies in: (1) The pathogenic chain of the "ITGB1-ISG15 competitive post-translational modification axis" in myocardial fibrosis was identified and defined: ISG15 binding / ISGylation competitively inhibits ITGB1 ubiquitination → prolongs membrane residence → maintains the continuous output of fibrosis-related signals such as FAK / Src, MAPK / Rho-ROCK / YAP-TAZ, etc. → drives epigenetic (increased chromatin openness) and amplification of profibrotic genes. Previous literature has not established this competitive relationship and its causal closed loop in the continuous activation of myocardial fibroblasts.

[0183] (2) Innovation in interface structure and dynamic stability: For the first time, AlphaFold multimer prediction + molecular dynamics + energy decomposition is used to lock the characteristic interface and key hot residues of ITGB1 and ISG15, providing an irreplaceable design basis and target for developing precise intervention strategies such as "interface blocking agents" and "interface mimic peptides".

[0184] (3) Overcoming the limitations of single inhibitors, providing multi-level reversible regulatory strategies: including (i) blocking binding (antibodies / peptides / small molecules, etc. disrupt the ITGB1-ISG15 binding interface); (ii) inhibiting the ISGylation cascade (targeting ISGylation cascade reaction enzymes); (iii) enhancing de-ISGylation (USP18 localization / activation); (iv) redirecting the ubiquitination degradation of ITGB1 (PROTAC, molecular glue); (v) gene editing (editing key ISG15 binding sites or ubiquitination sites on ITGB1); (vi) combination / sequential therapy (intervening in the formation of a functional closed loop of "interface + de-ISGylation + ECM crosslinking inhibition").

[0185] (4) A novel perspective for drug design based on upstream "post-translational modification competition": Unlike strategies that directly inhibit downstream kinases (such as FAK, Src) or broadly block ECM, this invention targets the upstream pathological stability "switch," namely the post-translational modification competition process of ITGB1. This intervention strategy theoretically has the characteristics of earlier intervention, higher specificity, and more reversibility, and is expected to significantly reduce the risk of side effects on normal tissue homeostasis while effectively inhibiting the progression of fibrosis.

[0186] The applications and extensions of this invention include: A. Purpose Classification (Pharmaceutical Use) 1) The reagents that regulate (inhibit or relieve) ITGB1 ISGylation are used to prevent or treat myocardial fibrosis, HFpEF / HFrEF, fibrosis-related arrhythmias or decreased ventricular compliance.

[0187] 2) Measuring the ITGB1 ISGylation / ubiquitination ratio is used for: risk stratification of early active myocardial fibrosis, efficacy monitoring, recurrence prediction, and determination of the treatment window.

[0188] 3) Use the FIS index to guide the intervention dosage, sequential regimen, or timing of discontinuation of the ITGB1-ISG15 axis.

[0189] B. Composition / Formulation 1) Contains: interface blocking antibody (or single-domain / bispecific) + USP18 activator or localization enhancer.

[0190] 2) Interface mimic peptide (cyclic peptide or stapled peptide) + si-UBE2L6 (or si-HERC5).

[0191] 3) Small molecule ISGylation inhibitor + PROTAC (targeting residual overstability ITGB1).

[0192] 4) ITGB1 site-specific editing vector (CRISPR base editor) + ECM crosslinking inhibitor (such as LOXL2 inhibitor).

[0193] 5) Any combination of the above plus standard heart failure medications (ACEI / ARB, ARNI, SGLT2i, β-blockers) forms a synergistic regimen.

[0194] C. Molecules and their engineered variants 1) Antibodies / nanobodies / bi- or multi-specific antibodies targeting the ITGB1 activation conformation or ISG15 binding interface, including humanized and Fc-modified versions (extending half-life or reducing effector function).

[0195] 2) Interface blocking peptides, competitive peptides (including D-amino acids, cyclized, stapling, PEGylated, lipopeptidated, CPP fusion) and their pharmaceutically acceptable salts.

[0196] 3) Small molecules or PROTAC / molecular gels (including linker arms and E3 ligand series variants) that regulate UBE1L / UBE2L6 / HERC5 / USP18.

[0197] 4) Gene editing construction: ITGB1 specifies Lys→Arg (or multi-site combination) expression vector and AAV / nano delivery system.

[0198] 5) USP18 targeting fusion protein (USP18 or its active domain + ITGB1 binding fragment / nanobody / membrane localization signal).

[0199] 6) Aptamer-drug or aptamer-nucleic acid conjugates, targeting receptors on the surface of cardiomyocyte fibroblasts (FAP, PDGFRα, DDR2, etc.).

[0200] 7) Computationally designed and optimized interfacial small molecules (including their stereoisomers, tautomers, prodrug forms, and metabolically stable derivatives).

[0201] D. Diagnostic and Companion Testing 1) An integrated kit for simultaneous detection of ITGB1 (total amount + activated conformation + ISGylated + ubiquitination) (including standards and calibration curve algorithm).

[0202] 2) A method for quantitative detection of ITGB1-ISG15 neighboring cells and modified cells / tissues using FRET / BRET / PLA dual-signal probes.

[0203] 3) A method for constructing an early activity score by calculating the ISGylation / ubiquitination ratio and membrane residence half-life (surface biotin pulse tracking or pHluorin labeling).

[0204] 4) The computer program, cloud platform, or embedded software that generates the FIS index (covering data cleaning, feature normalization, risk stratification, and efficacy trend prediction), its usage methods, and storage media.

[0205] E. Treatment strategies and administration regimens 1) "Induced destabilization window" therapy: first short-term interface blockade (reducing membrane residence) → followed by ECM dismantling / tissue reconstruction (such as MMP regulation or anti-crosslinking) → maintenance period low-frequency USP18 enhancement.

[0206] 2) "Biphasic combination" scheme: immediate inhibition of ISGylation + delayed period to promote ubiquitination (PROTAC) to prevent restabilization.

[0207] Individualized adjustment: High FIS index → ​​priority interface blockade; if the decrease does not reach the threshold → add enzyme cascade inhibition; enter the monitoring period after reaching the low risk threshold.

[0208] It should be understood that the application of the present invention is not limited to the examples above. Those skilled in the art can make improvements or modifications based on the above description, and all such improvements and modifications should fall within the protection scope of the appended claims.

Claims

1. Application of ITGB1-ISG15 signaling pathway inhibitors in the preparation of drugs for the prevention of myocardial fibrosis.

2. Application of ITGB1-ISG15 signaling pathway inhibitors in the preparation of drugs for the treatment of myocardial fibrosis.

3. The application according to claim 1 or 2, characterized in that, The ITGB1-ISG15 signaling pathway inhibitors include: ITGB1 inhibitors and / or ISG15 inhibitors.

4. The application according to claim 3, characterized in that, The ITGB1 inhibitor is a small molecule, nucleic acid, gene editing system, protein, or polypeptide that specifically interferes with ITGB1 expression.

5. The application according to claim 3, characterized in that, The ISG15 inhibitor is a small molecule, nucleic acid, gene editing system, protein, or polypeptide that specifically interferes with ISG15 expression.

6. The application according to claim 3, characterized in that, The ITGB1 inhibitor is a siRNA that specifically interferes with the ITGB1 gene, and its nucleotide sequence is shown in SEQ ID NO.

1.

7. The application according to claim 3, characterized in that, The ITGB1 inhibitor is GLPG0187.

8. The application according to claim 3, characterized in that, The ISG15 inhibitor is a siRNA that specifically interferes with the ISG15 gene, and its nucleotide sequence is shown in SEQ ID NO.

5.

9. A drug for preventing myocardial fibrosis, characterized in that, The drug for preventing myocardial fibrosis contains an inhibitor of the ITGB1-ISG15 signaling pathway.

10. A drug for treating myocardial fibrosis, characterized in that, The medication for treating myocardial fibrosis contains an inhibitor of the ITGB1-ISG15 signaling pathway.

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